CN115721671A - Method for extracting and separating effective components of liquorice root in full-industrial-chain cooperation manner - Google Patents

Method for extracting and separating effective components of liquorice root in full-industrial-chain cooperation manner Download PDF

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CN115721671A
CN115721671A CN202211015511.1A CN202211015511A CN115721671A CN 115721671 A CN115721671 A CN 115721671A CN 202211015511 A CN202211015511 A CN 202211015511A CN 115721671 A CN115721671 A CN 115721671A
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李玉山
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Abstract

A licorice active ingredient full-industrial chain synergy clean extraction separation method, the invention divides the licorice whole plant into licorice root, stem; the method comprises the steps of extracting liquorice leaves and liquorice seeds in a segmented whole industrial chain, and comprises the following steps: (1) Extracting squalene, protein, polysaccharide, oil and other components from Glycyrrhrizae radix seed; (2) Extracting volatile components, chlorophyll, tea polyphenols, flavone, polysaccharide, etc. from the licorice leaves; (3) Extracting radix Glycyrrhizae to obtain polyphenol, glycyrrhizin, licoflavone, liquiritigenin dextran, liquiritigenin, protein, lignin, cellulose, etc. In the raw material processing process, the method of the invention needs to convert byproducts, leftovers and the like into useful substances besides main products, and the components in the liquorice are numerous, so that the method extracts and separates the components with practical application values at present. The method of the invention does not simply 'eat and press completely', but changes the traditional extraction and separation from 'extensive type' to 'intensive type', fully utilizes each part of the raw materials and makes the best use of the materials. The 'eating dry squeezing out' is the value orientation of resource comprehensive utilization, but the cyclic economy is never the 'eating dry squeezing out', and the method for cooperatively and cleanly extracting and separating the effective components of the liquorice is a method for changing wastes into valuables and turning harm into good for wastes generated in the production process while pursuing the 'eating dry squeezing out' of the resources, so that the aim of comprehensive utilization is fulfilled, the development of the cyclic economy is promoted, the resource saving is realized, and the environment-friendly economic mode is realized.

Description

Method for extracting and separating effective components of liquorice root in full-industrial-chain cooperation manner
Technical Field
The invention relates to a full-industrial-chain synergistic clean extraction and separation method for effective components of liquorice.
Background
Licorice (Glycyrrhiza radix et rhizoma) is a dried root and rhizome of Glycyrrhiza plants in Leguminosae (Leguminosae), and is a preferred product for regulating various traditional Chinese medicines in China.
1. Chemical composition
The national Chinese herbal medicine assembly states that licorice root and rhizome contain glycyrrhizin, i.e. 6-14%, glycyrrhizic acid is hydrolyzed to generate a molecule of glycyrrhetinic acid and two molecules of glucuronic acid, and contains a small amount of glycyrrhizin, aglycone glycyrrhizin, i.e. glycyrrhizin and glycyrrhizin (glycyrrhetin), isoliquiritin (iso-liquiritin), dihydroxyglycyrrhetinic acid (dihydroglycyrrhetinic acid), glycyrrhetinic acid (glycyrrhidin), i.e. 3', 6-diisopenten-2 ',4', 5-trihydroxyisoflavan, glycyl alcohol (glycyrrhol), 5-O-methyl glycyl alcohol (5-O-methyl glycyl), isoglycyl alcohol (iso-glycyl), furthermore, mannitol (nite), glucose 3.8%, sucrose 2.4-6.5%, malic acid, betulinic acid (betulinic acid), nicotinamide, asparagine (nicotinic acid, asparagine (asparagine), and bio-cyclodextrin (beta/g), and other small amount of life starch.
In the Chinese medicine dictionary, it is recorded that the root and rhizome of licorice contains glycyrrhizin, which is potassium and calcium salt of glycyrrhizic acid. Further comprises liquiritin (liquiritin), liquiritigenin (liquiritigenin), isoliquiritin (isoliquiritin), isoliquiritigenin (isoliquiritigenin), neoliquiritin (neoliquiritin), neoisoliquiritin (neoisoliquiritin), etc.
The chemical components of licorice, glycyrrhiza glabra, glycyrrhiza inflata, glycyrrhiza glabra and Glycyrrhiza yunnanensis are recorded in the Chinese materia medica. And (1) the licorice root and the rhizome mainly contain triterpenoid saponin. Glycyrrhizin (glycyrrhizin) is a sweet component of licorice, and is potassium salt and calcium salt of glycyrrhizic acid (glycyrrhizic acid) produced by combining 1 molecule of 18 beta-glycyrrhetic acid (18 beta-glycyrrhetic acid) and 2 molecules of glucuronic acid (glucuronic acid). Other triterpene saponins are: uralen glycyrrhizin (uralsaponin) A, B and glycyrrhizin (lichociaponin) A3, B2, C2, D3, E2, F3, G2, H2, J2, K2.
And contains flavonoids: glycyrrhizin (liquiritigenin), liquiritin (liquiritin), isoliquiritin (isoliquiritin), neoliquiritin (neoliquiritin), neoisoliquiritin (neoisoliquiritin), liquiritin (licocidin), licopin (licocidin), licoricone (licorone), formononetin (formononetin), 5-O-methyl glycyrrhizin (5-O-methylicoridin), liquiritin-4 '-apigenin-glucoside [ liquiritin-4' -qpifour-azo-acyl (1 → 2) glucopyranoside, apioligenin ], liquiritin-7, 4 '-diglucoside (liquiritin-7, 4' -diglucoside), neolanoside II (vicenin II), i.e.e.6, 8-diglucoside, apigenin-4 '-diglucoside (apigenin → isoglucosid), apigenin-4' -apigenin-2 '-isoglucosinoloside (apigenin → 2), apigenin-4' -apigenin → isoglucosidin (apigenin). Also contains a coumarin compound: glycyrrhiza-coumarins (glyco-arim), glycyrrhizin (glycorol), isoglycyrrhizin (isoglycyrol), glycyrrhiza-coumarins-7-methyl ether (glycorin), neoglycanol (neoglycyrol), glycyrrhiza-pyranosium-coumarins (lipopyranosium), and Glycyrrhiza-coumarone (liporimarinone). And also contains alkaloid: 5,6,7,8-Tetrahydro-4-methylquinoline (5,6,7,8-Tetrahydro-methylquinoline), 5,6,7,8-Tetrahydro-2,4-dimethylquinoline (5,6,7,8-Tetrahydro-2,4-dimethylquinoline) 4-, 3-methyl-6,7, 8-tetrahydropyrrolo [1,2-a ] pyrimidin-3-one (3-methyl-6,7, 8-tetrahydropyrrolo [1,2-a ] pyrimidin-3-one). Further comprises licobenzofuran (licobenzofurane), also known as liconeolignan (licoolignan), beta-sitosterol, n-tricosane (n-tricosane), n-hexacosane (n-hexacosane), n-heptacosane (n-heptacosane), etc. In addition, it contains licorice dextran GBW, three kinds of neutral licorice polysaccharide (glycyrrhin) UA, UB and UC with reticuloendothelial activity, several kinds of polysaccharide (polysaccharide) GR-2a, GR-2IIb, GR-2IIC and polysaccharide GPS, etc. The leaves of licorice contain flavone compounds: new Zealand vitexin-II, narcissin (narcissin), nicotiflorin (nicotiflorin), rutin (rutin), isoquercitrin (isoquercitin), astragalin (astcmlibagalin), uralen (uralenol), neoUralen (neralenol), uralen (uralin), quercetin (queralen), quercetin-3,3'-dimethyl ether (queretin-3, 3' -dimethyl ether), uralen-3-methyl ether (uralol-3-methyl ether), uralen (uralene), quercetin (queretin), etc.
Also contains glatirneoside (uronoside). The aerial part of Glycyrrhrizae radix is separated to obtain scopoletin (scopoletin), formononetin, luteolin (lupiwighteone), sigmoidin (sigmoidin) B and gancamone (gancanonin) A, B, C, D, E, L, M, N, O, P, Q, R, S, T, U, and V. (2) The roots and the rhizomes of Glycyrrhiza glabra contain glycyrrhizin, and besides glycyrrhizic acid and 18-beta glycyrrhetinic acid obtained by separation, various triterpenoids are obtained: 18 α -hydroxyglycyrrhetinic acid (18 α -hydroxyglycyrrhetic acid), 24-hydroxyglycyrrhetinic acid (24-hydroxyglycyrrhetic acid), 24-hydroxy-11-deoxyglycyrrhetinic acid (24-hydroxy-11-deoxyglycyrrhetinic acid), 11-deoxyglycyrrhetinic acid (11-deoxyglycyrrhetinic acid), 3 β -hydroxyolea-11, 13 (18) -diene-30-oic acid [3 β -hydroxyolean-11, 13 (18) -dien-30-oic acid ] glycyrrhetin (glycodeoxyglycyrrhetin), glycyrrhetinate (glycofuroide), isoglycyrrhetinate (isoqueribraride), glycyrrhetinide (deoxyglycyrolide), 21 α -hydroxyisovaleride (21 α -hydroxyisobutyrolide), glycyrrhetinic acid (epoxyoride), and the like. And contains flavone components: glabroside (liquiritin), glabrigenin (liquiritigenin), liquiritin (isoliquiritin), isoliquiritin (isoliquiritin), neoliquiritin, isoliquiritin-4 '-apigenin (licuraside, licazid), isoliquiritin-4-apigenin [ neocuroside, isoliquiritin-4-apigenin (1 → 2) glucopyranoside ], glabranin (glabernin), glabrol (glatirol), glabridin (glatiridin), glabranone (glaberrone), glabrene (glatirenone), 7,2' -dihydroxy-3 ',4' -methylenedioxyisoflavone (7-methylxanthone-7-methylxanthone (7-methylxanthone-2-methylxanthone), isoxanthosine (7-methylxanthone-7-methylxanthone (7-methylxanthone-2), etc.
Also contains Glycyrrhiza glabra essence (Liqcoumarin), water-soluble polysaccharide and pectin (pectin). Separating aerial part of Glycyrrhiza glabra to obtain 18 beta-glycyrrhetinic acid, 18 alpha-glycyrrhetinic acid (18 alpha-glycyrrhetic acid), namely ursolic acid (uraemic acid), and various flavonoid compounds: quercetin, isoquercitrin (isoquercitin), quercetin-3-biglucoside (quercitin-3-glucoside), kaempferol (kaempferol), astragalin, saporin (saponarin), liquiritin, isoliquiritigenin, genkwanin (genkwanin), kaempferol-3-biglucoside (kaempferol-3-glucoside), etc. And 9.7 percent of polysaccharide, wherein the water-soluble polysaccharide is 1.6 percent. (3) The radix Glycyrrhizae Uralensis contains triterpene sweet, glycyrrhetinic acid-3-apioglycor glucuronide (apioglycyrrhizin), and glycyrrhetinic acid-3-arabino glucuronide (araboglycyrrhizin). Other triterpene components are: 18 beta-glycyrrhetinic acid, 11-deoxyglycyrrhetinic acid, glatiramer glycyrrhizin A3, G2, H2, etc. And contains flavonoid components: licopin, liquiritin, isoliquiritigenin, isoliquiritin, formononetin, 4',7-dihydroxyflavone (4', 7-dihydroflavone), licoflavone (licolavone) A, licopin-4 '-apioglucoside, isoliquirigenin-4' -apioglucoside, licotophanone (licohalcone) A, B, C, D, licochalcone (echinatin), glabrolone, etc.
Also contains diaryl propylene diketone components: 5 '-isopentenylglycyrrhetine (5' -prenyllicodione), glycyrrhetine (glycyrrhdione) A and B, and glycyrrhetine (glyinflanin) A, B, C and D. The distending fruit licorice dione A and the distending fruit tannin A are the same substance and contain beta-sitosterol. (4) the glabrous greenbrier rhizome contains triterpenes components: glycyrrhizic acid, glabrene lactone, and the like. And contains flavonoid components: glycyrrhizin, isoliquiritin, glycitin (glyasprin) A, B, C, D, ursolic acid bamboo (kumatakinin), flavonolicin (topazolin), licoisoflavone (licoisoflavone) B, semilicoisoflavone (semilicoisoflavone) B, licoisoflavanone (licoisoflavanone), 3'- (gamma, gamma-dimethylallyl) trivitamin ketone [3' (gamma, gamma-dimethylallyl) -kievitone ], glycitein, glycitin (licooflavan) A, 1-methoxyphenanthrenefoliol (1-methoxyfiifolin). And the ingredients of the fragrant bean essence are as follows: glycyrrhiza essence, isoglycyrrhiza essence (isoglycyrrhiza essence), glycyrrhizin, licocoumarone, water soluble polysaccharide and pectin. (5) the root and rhizome of the yellow licorice contain triterpenes: glycyrrhizic acid, uralisin A and B, glycyrrhetin (glyerysiponin), and flavonoid component: glycyrronide, formononetin, liquiritin, isoglucosides, glycyrhizin-4 '-apioglucoside, isoglycyrhizin-4' -apioglucoside, choerospondin (choerospondin), daidzein (sophoraflavone) B, schaftoside (schaftoside), trifoligenin (isoviovi-osidin), scirpol-3-O-glucoside (medicarpin-3-O-glucoside), new Zealand vitexin II, beta-sitosterol (beta-sitosterol), daucosterol (daucosterol) and phlorizic acid (phlorotic acid). (6) the Yunnan licorice root contains triterpenes. Hydrolyzing the total saponin to obtain Yunnan glycyrrhetinic saponin D (glyunnanpro-sapogenin D), yunnan glycyrrhetin A (glyunnansapogenin) B (glyunnansapogenin), C (glycogenin), E (glycogenin), G (glycogenin), H (glycogenin acid), and beta-sitosterol. Also contains flavonoid components: isoliquiritigenin, 4', 7-dihydroxyflavone, 7-methoxy-4' -hydroxyflavone (7-methoxy-4 ' -hydroxyflavone), and 7-methoxy-4' -hydroxyflavonol (7-methoxy-4 ' -hydroxyflavone). It has also been reported that triterpene saponins: the pharmaceutical composition comprises (a) yunnanoside (yunnanoside) A1, B1, C1, D1, E2 and F2, and alkaloid: hypaphorine (hypaphorine).
Xuzhou eosin and the like extracts and separates 6 flavonoids and 3 acetylated derivatives from the root of licorice (Glycyrrhiza uralensis Fisch.), namely 4' -O-methyl glabridin, hispaglabridin B, glabridin, liquiritigenin, isoliquiritigenin, liquiritin, 4' -O-diacetylisoliquiritigenin, 4-O-acetyl isoliquiritigenin and 4' -O-acetyl liquiritigenin respectively (the plant resources and environments report, 2020, 29, 32-41). The Liu Yuan and the like are separated from 80% ethanol extract of Glycyrrhiza uralensis Fisch to obtain 2 flavonoid compounds which are respectively: (2S) -liquiritigenin-4 '-O-beta-D-glucose- (1 → 6) -O-beta-D-glucoside, and (2R) -liquiritigenin-4' -O-beta-D-glucose- (1 → 6) -O-beta-D-glucoside (pharmacy newspaper, 2017, 52, 948-951). Separating the licorice dreg flavone extract by Zhangjuan, etc. to obtain 6 flavone substances of swollenin I inflacoside-marin A, dehydrohydroxychalone A, licochalcone A, isoapigenin glycyrrhizide, apigenin apioside liquiticide and naringin (Chinese medicine science and technology, 2009, 16, 127-128). Zhangjuan et al identified 8 total flavonoids from the residue of swollen licorice, 2',4' -trihydroxychalcone, licochalcone D, licochalcone A, 4' -hydroxy-2 ", 2" -dimethylpyrano [5 ", 6,7] flavone, licoflavone C, glabrene, licoflavone B and kanzonolE E (modern medicine and clinic, 2012, 27, 558-561). Zhang Qihai et al isolated 15 prenylflavonoids from the root of Glycyrrhiza uralensis Fisch (Glycyrrhiza uralensis), identified as Liquiridine, ulexine A, sophoronol A, lupalbigenin, 8-prenylluteone, isodorone, 1-methoxy-phaseolin, glycyrrone, glyuralin A, glycyrrhizin, glabridin, ganonin F, 1-phaseolinisoflavan, G ancanon G, and licosoflavonone C (Chinese patent medicine, 2021, 43, 670-675). The ethanol extract of liquorice is separated by the queen seedling to obtain 2 new compounds which are coumarin compounds and flavanone glycoside compounds crotoliquiritin respectively, and 42 known compounds comprise 16 isoflavone compounds which are respectively: formononetin, barpisoflavone A, gliricidin, pedunculoside A, daidzein, formononetin, orobol, licoisoflavone A, northwest licoisoflavone, genistein, wistoside, licoricone, licoisoflavone B, liconin G, isoangostone A and indian pterocarpus indicus glycoside, 6 flavanoid compounds, respectively: liquiritin, apioside liquiritin, liquiritigenin-7-O-beta-D-furanapitosyl-4' -O-beta-D-glucopyranoside, liquiritigenin, naringenin and glabrol, 2 isoflavanone compounds are: licorice isoflavanone and licorice isoflavanone, 2 flavonoids are: 7,4' -dihydroxyflavone and licoflavone, 5 flavonol compounds, respectively: isolicoflavonol, licoflavonol, kaempferol, 7,4' -dihydroxyflavonol and galangin, and 5 chalcone compounds are: tetrahydromethoxyhalcone, isoliquiritigenin, 2',4',3,4, alpha-pentahydroxychalcone, licochalcone B and butein, and 3 coumarins: carthamus tinctorius coumestrol B, glycyrrhizin and glycyrrhin, 1 pterosins compound santalin, 1 stilbene compound glycyrrhetin and 1 other compound 4-isopentenylphenol (Shandong university, 2020 academic thesis). Separating chemical components of cultivated Glycyrrhrizae radix to obtain glabrene (glabrone), formononetin (formononetin), p-hydroxybenzoic acid (p-hydroxybeniconic acid), licochalcone A (licochalcone A), 6, 7-dihydroxycoumarin (6, 7-dihydroxycoumarin), licoflavone A (licolavone A), mevalon-3-O-glucoside (medicarpin-3-O-glucoside), formononetin (ononin), liquiritin-4' -apigenin (1-2) -glucoside), and isoliquiritin-glucose apigenin (licarpe 654) (Chinese herbal medicine, 2005, 36, 652-654). Queen et al, isolated and purified 18 monomeric compounds from ethyl acetate fraction of Glycyrrhiza uralensis leaf, named as licotilbene A, licotilbene B, licouranol A, licouranol B, α ' -dihydroxy-3, 5,4' -trihydroxy-4,5' -diacetentenylstilbene, glycopyranoline B, luteolin, diosmetin, calycosin, scopoletin, quercetin-3,4' -dimethyl ether (quercetin-3, 4' -dimethy ether), licochalcone (echinatin), 3',4' -dimethyl quercetin (3 ',4' -dimethy ether), quercetin-3, 3' -dimethyl quercetin (3, 3' -dimethy ether), quercetin (quercitin-A), quercetin (Quercetin-beta), quercetin (Quercetin-O), quercetin (Quercetin-beta), and Quercetin-O (Quercetin-beta). Gunn et al separated 10 flavonoids from the licorice 70% ethanol extract, which were identified as 4',6, 7-trihydroxy-2 ' -methoxychalcone, 3',4',5, 7-tetrahydroxy-8- (3-hydroxy-3-methylbutyl) -isoflavone, isoliquiritigenin, isoliquiritin, licochalcone sweet pea phenol, formononetin, 2 (S) -3',5', 7-trihydroxy flavanone, choeroside, 4',7-dihydroxyflavone (Chinese herbal medicine, 2019, 50, 5187-5192). Plum and the like are 9 compounds separated and identified from 95% ethanol extract of liquorice pomace of the fig. tumefaciens by volume fraction, and the 9 compounds are licochalcone A (licochalcone A), 2',4' -trihydroxychalcone (2 ',4' -trihydroxychalcone), (-) alpha, 2',4' -tetrahydroxydihydrochalcone ((-) alpha, 2',4' -tetrahydroxydihydrochalcone), licochalcone C (licochalcone C), licochalcone D (licochalcone D), echinacone (echinatatin), kanzonol E, docosan (docosylfanfeate) and glycyrrhetinic acid (glycyrrhetic acid) (Shenshikonic university report, shen 2011, 28, 368-370-379). Zhao Sen Ming, etc. are separated from the ethanol extract of licorice dregs to obtain 10 compounds, which are respectively: formononetin, hemiglycyrrhiza isoflavone B, 5, 7-dihydroxy-4 ' -methoxyisoflavone-7-O-beta-D-glucopyranoside, formononetin, isoerythrinin A, genistein, 2',4' -dihydroxy-4-methoxychalcone, 4' -dihydroxy-2 ' -methoxychalcone, medicagine, liquiritigenin (proceedings of Guangdong pharmaceutical sciences university, 2019, 35, 614-618). Isolation of p-hydroxybenzoic acid (p-hydroxybenic acid), liquiritigenin-7,4 '-diglucoside (liquiritigenin-7, 4-diglucoside), liquiritigenin-4' -O-apiosyl- (1 → 2) glucoside (liquiritigenin-apiosyl (1 → 2) glucoside), isoliquiritigenin apigenin (licura), isoliquiritin (isoliquiritin), liquiritigenin (liquiritigenin), isoliquiritigenin (isoliquiritigenin), formononetin (formononetin), liquiritin (liquiritin), licochalcone (echinatatin) (food industry science, 2019, 40, 229-232). The doline is obtained by separating glabrene T, glabridin, glyasprin C, glycyrrhetin, isolicoflavonol, licoisoflavone A, licoisoflavone B, hemilicoisoflavone B, formononetin, and liquiritigenin from Glycyrrhiza glabra ethyl acetate part (Chinese herbal medicine, 2018, 49, 4780-4784). Zhangqi et al isolated and identified 11 compounds from licorice, including p-hydroxybenzylmalonic acid, liquiritin, neoliquiritin, isoliquiritin, neoisoliquiritin, naringenin-7-O-glucoside, formononetin, apioside liquiritin, apioside isoliquiritin, liquiritigenin and isoliquiritigenin (Chinese pharmacy, 2017, 26, 15-19). The custard-shimeqi separated and identified the structures of 13 flavonoid components and 1 coumarin component from the licorice dregs, and identified as licoflavone B, glaberone, semiicoflavone B, licoflavone A, licohalcone A, formononetin, liquiritin, isoliquiritinigenin, licolavone C, liquiritin, kumatakinin A, licoribride and neoglycyclol (university of traditional Chinese medicine, nanjing, 2016. Academic paper). The powerful herb of Bibusta is used for separating 12 compounds from ethanol extract of overground part of liquorice and respectively identified as (2S) -3'- (2-hydroxy-3-methylibout-3-enyl) -4',5, 7-trihydroxy-dihydroflavanone, pinocembrin, sigmoidin B (3), licolavanone, 6-isopentenylnaringenin, pinobanksin, galangin, genistein, pratensein, kaempferol-3-O-beta-D-rutinoside, rutin, alpha '-dihydro-3,5,3' -trihydroxy-4 '-methoxy-5' -isopentenyl-stilbene (Chinese herbal medicine, 2016, 47, 21-25). Royal jelly and the like are extracted from 95% and 70% ethanol of root of Glycyrrhiza uralensis of Hangjinqi of inner Mongolia to obtain 19 compounds which are respectively identified as 3-methylkaempferol, genistein, naringenin, licoisoflavone A, licoriinone, isoformononen, hemiglycyrrhiza isoflavone B, 3' -isopentenyl genistein, luteolin, licoflavonol, ruby lupulin, licoisoflavone B, isolicoflavonol, glycyronin H, glabrene A, glycexine, coarse glabridin D, coumarol and 7-methyl lupin isoflavone (Chinese herbal medicines, pea, 45, 31-36). The surrogans are separated from the ethyl acetate extraction part of glycyrrhiza inflata to obtain 5 compounds which are respectively identified as formononetin (formononetin), isoliquiritigenin (isoliquiritigenin), viburnine aglycone (dihydroisoliquiritigenin), alpha-glycyrrhetinic acid (alpha-glycyrrhetinic acid) and beta-glycyrrhetinic acid (beta-glycyrrhetinic acid) (Anhui medicine, 2013, 17, 1121-1123). Rong Qing et al separated and purified the ethyl acetate extract of Glycyrrhiza uralensis 70% ethanol extract, and separated 14 compounds, which were identified as 3, 7-dimethyl licoflavonol, glycyronin I, glycyrolone, 8-methyl ledum, 2 '-hydroxyisolupagenin, isoeugenol, dehydrocoarse glabrene D, glycyrolin, glycyrol, licochalcone B, isoangu stone A, glycyronin G, 5,7,4' -trihydroxy-6, 8-diisopentenyl isoflavone (Chinese herbal medicine, 2012, 43, 1886-1890). The chemical components of the licorice residue are separated by using Liufen and the like to obtain 9 known compounds which are respectively identified as betulinic acid (betulinic acid), glabrene (glabrone), licoflavone C (licolavone C), licoflavone B (licolavone B), 3-carbonyl glycyrrhetinic acid (3-oxo-18 beta-glycyrrhetic acid), formononetin (formononetin), licoflavone (licoflavone), beta-sitosterol and daucosterol (daucosterol) (Chinese journal of pharmaceutical chemistry, 2011,2l, 312-314). The chemical components of cultivated licorice were studied by royal biont and the like, and isoliquiritigenin (isoliquiritigenin), licochalcone (echinatatin), licochalcone B (licohalcone B), liquiritigenin (liquiritigenin), 4',7-dihydroxyflavone (4', 7-dihydroflavanone), liquiritin (liquiritin), and isoliquiritin (isoliquiritin) (journal of northwest pharmacology, 2004, 19, 252-253) were isolated. Eight compounds isolated from Shanxi licorice of Xuancheng et al were identified, six of which: sucrose (sucrose), 4',5,7-trihydroxy-8-prenylflavone (4', 5, 7-trihydroxy-8-prenylflavone), formononetin (isogonin), 4',7-dihydroxyflavone (4', 7-dihydroflavonone), liqiritin, 3- β -hydroxy-11-oxo-agent olean-12-en-30-carboxy-3-O- β -D-glucuronic acid n-butyl ester (1 → 2) - β -D-glucuronic acid n-butyl ester glycoside, named diaquinase methyldi-n-butyl ester (dinbuthyl uronin emulsifiers) (natural product research and development, 2000, 12, 18-22). The chloroform extract of Glycyrrhiza glabra (Glycyrrhizayunnanensis) of Gaoyang et al further separated 6 compounds, which were identified as: formoterol (hemoptocarpin), mestranol (medicarpin), formononetin (formononetin), 4'-methoxy-4-hydroxychalcone (4' -methoxy-4-hydroxychalcone), and Yunnan liquiritigenin B (glyunnasapogenin B), and also beta-sitosterol (Chinese herbal medicine, 1994, 25, 507-508+51 3) can be obtained. Identification of the structures of five additional compounds obtained from the extract of Glycyrrhiza inflata Bat, root and rhizome of Glycyrrhiza inflata, identified as daucosterol, licochalcone A, beta-sitosterol, isoformononetin and 4',7-dihydroxyflavone (4', 7-dihydroxy-flavone), respectively, by Zhongkun et al, after diafiltration with 95% ethanol (1993, 5, 1-4). A95% ethanol extraction fraction of Yunnan licorice was studied by Gaogongying et al, and 4 compounds were separated from the ethyl acetate fraction, i.e., isoliquiritigenin (iso-iquiritigenin), 4',7-dihydroxyflavone (4 ', 7-dihydroxyflavone), 7-methoxy-4' -hydroxyflavone (7-methoxy-4 ' -hydroxyflavone), and 7-methoxy-4' -hydroxyflavonol (7-methoxy-4-hydroxyflavone) (proceedings of Beijing medical university, 1993,2, 302). Gioshi et al separated 7 flavonoid aglycones, 7 phenolic acids and flavonoid glycosides from dried leaves of Glycyrrhiza uralensis Fisch (Glycyrrhiza uralensis Fisch), and identified the separation and identification of two prenylated flavonoids as 3,5,7,4' -tetrahydroxy-3 ' -methoxy-6-prenylated flavone and 5,7,3',4' -tetrahydroxy-8-prenylated flavanone, the former named as Licorionin P-3 ' -A (pharmaceutical science, 1993, 28, 623-625). Lianghong et al isolated 8 compounds, 5 flavonoids from Glycyrrhiza squamula F ranch —) produced in Henan: isoliquiritigenin, echinocandin (echinatatin), formononetin (formononetin), 4',7-dihydroxyflavone (4', 7-tetrahydroxyflavone), avermectin (afrormosin), and also daucostero (daucostero) and sucrose (sucrose) (proceedings of Beijing university of medical science, 1992, 24, 399-400). Five compounds were isolated from the roots and rhizomes of Glycyrrhiza glabra (Glycyrrhiza pallidiflora Maxim) by Zealand et al, the compound 4-hydroxy-2, 4 '-dimethoxychalcone was named Glycyrrhizachalcone (glycallichalcon e), others were 4' -O-methyl-coumestrol, glutamate acetylate (n-acetylglutamicaced), formononetin (formononetin) and beta-sitosterol (beta-sitosterol), respectively (Proc. Rev., 1992, 27, 748-751). The 10 chemical components of Liu Qin and the like obtained from the root and rhizome of liquorice which is produced in the Jinta of Gansu are respectively identified as: formononetin, liquiritigenin-4' -O- [ D-beta-D-furanapiosyl (1 → 2) ] -beta-D-glucopyranoside, isoliquiritigenin-4-O- [ D-beta-D-furanapiosyl (1 → 2) ] -beta D-glucopyranoside, choerospondin, isoflorisin, new Zealand vitexin II, urarel glycyrrhizin A, daucosterol and phlorizin (Journal of Integrated Plant Biology,1991, (04), 314-322). Zeng Lu et al isolated 7 compounds from Glycyrrhiza glabra (Glycyrrhiza aspera pall.) root produced in Xinjiang, which were glycyrrhizic acid (Glycyrrhiza acid), liquiritin (liquiritin), isoliquiritin (isoliquiritin), glycyrrhizin (glycorol), isoliquiriphenol (isolycyrro l), glycycoumarin (glycocoumarin) and isoglycycoumarin (Journal of integrated Plant Biology,1991, (02), 124-129). The five monomers are obtained by separating roots and rhizomes of Glycyrrhiza pallidiflora Maxim (Glycyrrhiza glabra) in Yangtze river and the like, are determined to be beta-sitosterol, macedonic acid methyl ester and 21-dehydro-mac edonic acid methyl ester and are named as methyl echinoate (pallidifloric acid methyl ester) and 5-hydroxy-4-methoxy isoflavone and are named as glabrene (pallidiflorin) (pharmaceutical science, 1990, 25, 689-693). Liu et al, from Glycyrrhiza glabra Glycyrrhiza euryca P.C.Li (G.Korshinsky Kai non Grig.) root and rhizome of Glycyrrhiza in Leguminosae, four flavonoid glycosides were isolated, formononetin-7-O- [ D- β -D-furanosyl (1 → 2) ] - β -D-glucopyranoside (formononetin-7-O- [ D-apo- β -D-furanosyl (1 → 2) ] - β -D-glucopyranoside) is a new natural product, named xanthosine (glycyroside), among others, glycyrrhizin (liquiritin), isoliquiritin (isoliquiritin) and schatophoroside (schaftoside) (Proc. Natl. Acta., 1989, 24, 531-525). Poplar et al, isolated 1 component from Glycyrrhiza inflata (Glycyrrhiza inflata Bat., gansu) in 1 part, 8 of which are flavonoids, namely licochalcone A, licochalcone B, licoflavone, liquiritin, liquiritigenin, isoliquiritigenin, formononetin and 4',7-dihydroxyflavone (4', 7-dihydroflavanone), and 3 of which are triterpenes, namely glycyrrhizic acid (glycyrrhetic acid), glycyrrhetic acid (glycyrrhetic acid) and 11-deoxyglycyrrhetic acid (11-deoxyglycyrrhetic acid) and beta-sitosterol (beta-sitosterol) (journal of 182, plant 176, biol 176).
Zhengyunfeng etc. separates and purifies the water extract of Glycyrrhiza glabra (Glycyrrhiza glabra L.), 10 triterpene saponin compounds are respectively identified as: 3 beta-O- [ beta-D-glucopyranosyl- (1 → 2) -beta-D-glucopyranosyl]-30β-O- β-Dglucuronpyranosyl-oleanane-11-oxo-12(13)-ene、3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-gl ucuronpyranosyl]-30 β -O- α -L-hydroxyanisole-olenane-11-oxo-12 (13) -en-22 β,30-diol, uronsapon C, lichorice-saponin A3, lichoriceaponin P2, 22 β -acetoxy-glycyrrhizin, macedoside A, 29-hydroxy-glycyrrhizin, lichorice-saponin G2 and glycyrrhizin (Proc. Rev. Pharman, 2021, 56, 289-295). <xnotran> 14 , , , -6' - , -6 ″ - , -6',6 ″ - , -6 ″ - , -6' - , -6' - , -6 ″ - , -6 ″ - -6' - , -6' - -6 ″ - , -6',6 ″ - , , ( , 2016, 29, 31-36). </xnotran> The cold crystal is separated from n-butanol fraction of Glycyrrhrizae radix to obtain 14 compounds, which are respectively identified as macedoside E, 22 beta-acetyl uracyl glycyrrhizin C, glycyrrhizic acid, uracyl glycyrrhizin F, glycyrrhizin G2, 22 beta-acetyl glycyrrhizin, methyl glycyrrhizin, naringenin, isoliquiritigenin, formononetin, liquiritin, isofloridin, apioside (Chinese herbal medicine 2015, 46, 1576-1582). 11 glycyrrhetinic triterpenoid saponins are separated from the azalea widmanoensis taking glycyrrhetin glycrd as a research object and are respectively compounds such as glycyrrhetin, glycyrrhetin s aponin, 18 a-glycyrrhetin saponin, macedoside, 29-hydroxy-glycidyl-ricin, glycyrrhetin, 24-hydroxy-lactic-saponin, 22 beta-acetoxy-glycidyl-ricin and glycyrrhetin (2015 university of Nanjing medicine). The Mustelus griseus Pietschmann is separated from 75% ethanol extract of waste residue of Glycyrrhrizae radix produced in Xinjiang to obtain 5 compounds The plants are identified as sophorocoumarin C, 3 beta-O-p-hydroxy-trans-cinnamoyl-oleanolic acid, 3-carbonyl glycyrrhetinic acid, 3 beta, 18-O-isopropylidene-8 (14), 15-iso-dexterpinolene, 3 beta, 18-O-isopropylidene-7, 15-iso-dexterpinolene (Chinese herbal medicine, 2015, 46, 174-177). The toviamin is separated from 50% ethanol extract of Glycyrrhiza uralensis Fisher to obtain 14 saponin compounds which are respectively identified as uralenin C, uralenin, licorice-s aponin, uralenin, 22 beta-acetoxy-glycyrrhizin, 24-hydroxy-licorin-saponin, licorice-saponin), 22 beta-acetoxy-glycyrrhizate, 3 beta-O- [ beta-D-glucopyranosyl- (1 → 2) -beta-D-glucopyranosyl]Glycyrrhetino, araboglycyrrazin, lichorice-saponin, glycyrrhizic acid, monoglucuronic acid glycyrrhetinic acid (Chinese herbal medicine, 2013, 44, 1552-1557). Picrorhiza et al isolated 10 compounds from root of Yunnan licorice (Glycyrrhiza yunnanensis), identified 9 as pentacyclic triterpenoids: 3 beta, 21a, 24-trihydroxy-olean-12-ene-29-carboxylic acid, 3 beta, 21 alpha-dihydroxy-olean-12-ene, 3 beta 0, 21a, 24-trihydroxy-olean-12-ene, betulinic acid, 23-hydroxybetulinic acid, oleanolic acid, 3 beta-hydroxy-16-oxo-11, 13 (18) -diene-30-carboxylic acid, 24-hydroxyglycyrrhetin, 21 alpha-acetoxy friedelan-3-one and beta-daucosterol (reports in pharmacology, 1995, 30, 27-33). Zhang Ruyi et al, from the root of Glycyrrhiza inflata (Glycyrrhiza inflata Bat.), separated seven new triterpene saponins, identified as: glycyrrhetinic acid-3-O-beta-D-6 '-ethyl-glucuronopyranosyl- (1 → 2) -beta-D-6' -n-butyl-glucuronopyranoside and glycyrrhetinic acid-3-O-beta-D-6 '-n-butyl-glucuronopyranosyl- (1 → 2) -beta-D-6' -ethyl-glucuronopyranoside, named as oncomelanin II and VI, respectively (Journal of Chinese pharmaceutical Sciences,1995, (03), 113-116). Plum tree friend is obtained by extracting dried root of Glycyrrhiza uralensis (Glycyrrhiza uralensis) with methanol under ethanol-water distribution, and subjecting water phase to μ -Bondapak C 18 Performing column chromatography to obtain component 3, and processing with SiO 2 Separating and purifying by column and Zorbax ODS column HPLC chromatography to obtain 10 new oleanane type triterpene oligoglycosides, simultaneously separating glycyrrhizic acid and several known flavonoids, and determining triterpene oligosaccharide saponinLicorice root saponin A 3 、B 2 And C 2 (foreign medicine (TCM and TCM handbook), 1994, (04), 34-35). Yuying et al isolated 9 compounds from Glycyrrhiza inflata (Glycyrrhiza inflata bat.) of gansu, 5 of which are triterpenoids: methyl glycyrrhetate (methyl glycyrrhetate), glycyrrhetinic acid acetylate (glycyrrhetic acid acetate), glycyrrhetinic acid (glycyrrhetic acid) uraloside B (uralsaponin B) and glycyrrhizic acid (glycyrrhetic acid), 4 flavonoids: liquiritigenin, isoliquiritigenin, liquiritin, and isoliquiritin (isoliquiritin) (university of Beijing medical science, 1990, 22, 283-284+ 286). Zhang Ruyi et al separated three saponins compounds from the rhizome of Glycyrrhiza uralensis Fisch (Glycyrrhiza uralensis Fisch), 3 beta-hydroxy-11-oxidized-olean-12-ene-30-carboxylic acid-3-O-beta-D-glucuronosyl (1 → 2) -beta-D-glucuronosyl glycoside, named as Ural glycyrrhizin A, 3 beta-hydroxy-11-oxidized-olean-12-ene-30-carboxylic acid-3-O-beta-D-glucuronosyl (1-3) -beta-D-glucuronosyl glycoside, named as Ural glycyrrhizin B (pharmaceutical report, 1986, 21, 510-515). Five crystals are separated from roots of liquorice (glycyrrhiza uralensis Fisch) produced in northern Shaanxi by Cheng Yong et al and are respectively identified as: beta-sitosterol (beta-sitosterol), formononetin (formononetin), glycyrrhetin (lichicidin), licoricone (licoricocone) and liconeolignan (licoleolignan) (proceedings of pharmacy, 1983, 18, 45-50). The pelargonium graveolens is obtained from the root of Glycyrrhiza uralensis (Glycyrrhiza glabra var. Ty-pica) by six compounds: liquiritigenin (liquiritigenin), liquiritin (l iquitin), rhamnose liquiritin (rhamno-liquiritin), isoliquiritigenin (isoliquiritigenin), isoliquiritin (i soliquititin), and rhamnose isoliquiritin (rhamno-isoliquiritin) (foreign medical references & pharmaceutical handbook, 1974, (04), 239-240).
Extraction and analysis of Majunyi and other volatile chemical components of the leaves of the glycyrrhiza inflata are mainly nonadecane, nonacosane and wrightrene, (1, alpha 2, beta 5 alpha) -2, 6-trimethyl-bicyclo [3, 1] heptane, 2,6, 11-trimethyl-dodecane, 1-chloro-octadecane, (E) -acetic acid-3, 7-dimethyl-2, 6-octadiene-1-ester, eicosane, 1-eicosadiene, (-) -E-pinane, methylnonanone and the like (Chinese modern application medicine, 2007, 24, 1-4). Licorice volatile components were studied by Liangyong et al, and 32 kinds of components were identified, among the tested substances, 8 kinds of alcohol, phenol, ester compounds such as 2, 6-bis (1, 1-dimethylethyl) -4-methylphenol (20.16%), dimethyl phthalate (6.82%), 8 kinds of acid compounds such as n-hexadecanoic acid (8.33%), n-octadecanoic acid (4.68%), 14 kinds of hydrocarbon compounds such as n-nonadecane (4.71%), n-octadecanol (4.16%), n-heptadecane (4.02%), and 2 kinds of ketone compounds (J.Med., 2005, 20, 3-5). The chemical components of the lindera glaucescens leaf volatile oil are analyzed by Majunyi and the like, 61 compounds are confirmed, the chemical components mainly comprise nonadecane (12.89%), eicosane (12.23%), 1-heptadecene (12.05%), 2,6, 11-trimethyl-dodecane (7.54%), octadecane (7.46%), E-pinane (5.54%), docosane (5.30%), tricosane (4.92%), hexadecyl-ethylene oxide (3.09%) and the like, and the content of the 9 compounds accounts for 71.02% of the total volatile oil (China journal of pharmacy, 2005, 40, 1534-1536). Separating chemical components of aerial parts of cultivated Glycyrrhrizae radix by Baihong et al to obtain 7 aliphatic compounds, which are respectively identified as undecanoic acid-2-p-hydroxy ethyl ester (undecanoic acid,2- (4-hydroxyphenyl) ethyl), 1-docosanoic acid glyceride (2, 3-dihydroxyphenyl), 1-tetracosanoic acid glyceride (2, 3-dihydroxyphenyl), 1-docosanoic acid-2, 3-isopropylidene glyceride (docosanoic acid), (2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl ester ], 1- (22-hydroxydocosanoic acid) glyceride (docosanocarboxylic acid,22-hydroxy-,2, 3-dihydroxypyraster), 1- (24-hydroxytetracosanoic acid) glyceride (tetracosaenoic acid,24-hydroxy-,2, 3-dihydroxypyraster) and palmitic acid (palmitatic acid) (journal of northwest pharmacology, 2005, 20, 59-61). Analysis of the chemical components of the glycyrrhiza inflata essential oil by Majunyi shows that the chemical components mainly comprise 3-methylheptane (8.27%), 4-methylheptane (7.95%), 2-methylheptane (7.38%), heptane (6.99%), octane (6.45%), 2, 4-dimethylhexane (6.22%), 3-ethylpentane (5.39%), 3-methylhexane (5.17%), 2-methylhexane (4.52%), methylcyclohexane (4.33%), 2, 3-dimethylhexane (4.17%), 2, 5-dimethylhexane (3.23), 2, 3-dimethylpentane (3.00) and the like, and the content of the thirteen compounds accounts for 73.67% of the total amount of the essential oil (modern traditional Chinese medicine research and practice, 2005, 19, 32-35). Zhang Ji et al analyzed the chemical composition of Glycyrrhiza spinosa root mainly as ethyl linoleate (32.77%), ethyl palmitate (1.02%), 2,3, 7-trimethyl-quinidine (6.49%), 5-methyl-heneicosane (5.74%), tricosane (3.80%), 1-cyclohexylnonene (3.70%), eicosane (3.63%), ethyl stearate (3.59%) (J. Pharmacol., 2002, 37, 902-904).
Bafang is separated from root and rhizome of Gansu cultivated licorice to obtain 4',3 "-dihydroxy-5-methoxy-4", 5 "-dimethyl- [2, B ] -furan-cou mestan, isolicopyranochrome, neoglycyrrhizin, honghua astragalus coumestrol B, grisium, glycycoumarin, 7,2',4 '-trihydroxy-5-methoxy-3-coumarins, liquiritigenin, hemiglycyrrhizal isoflavone B, licoisoflavone B, 7-O-methyl lupin isoflavone, isoangustone A, gliicone, calycosin, licoisoflavone A, isoliquiritigenin, licochalcone B, 3',4 '-tetrahydroxy-2-methoxychalcone, 6-isopentenyllicoricolone-3-methyl ether, licoflavonol, kaempferol C, (R) -zhou grass, glycyrol, 2014' -tetrahydroxy-2-methoxychalcone, and betulinic acid 201olazine I (university of Phaeophysochloa, biophysco I). Van-Yuhong is separated from liquorice leaves to obtain 15 compounds, wherein three new isopentenyl dihydro stilbene compounds are respectively named as glycyrrhiza stilbene A (glycyrrhizalbene A), glycyrrhiza hydropyraben stilbene (glycyrrhizalbilbene) and glycyrrhiza stilbene B (glycyrrhizalbebene B), and other compounds are known and are respectively identified as alpha, alpha '-dihydro-3,5, 4' -trihydroxy-4,5'-diisopentenyl stilbene (alpha, alpha' -dihydro-2,3,5,4'-trihydroxy-4,5' -diisopentenyl stilbene), alpha '-dihydro-3,5,3',4'-tetrahydroxy-2,5' -diisopentenyl stilbene (alpha, alpha '-dihydro-3,5,3',3,3,3, 4'-tetrahydroxy-2,5' -diisopropenylstilbene), 6-prenyleriol (6-prenyleriodytol), 5 '-prenyleriol (5' -prenyleriodytol), 6-prenylquercetin-3-methyl ether (6-prenylquercetin-3-methyl ether), 5'-prenylquercetin (5' -prenylquercetin), 6-prenylquercetin (6-prenylqueretin), 3'-prenylnaringenin (3' -prenylnaringenin), sigmoidin C, 8- [ (Z) -3-hydroxymethyl-2-butenyl ] -eriodynol (8- [ (Z) -3-hydroxymethy-2-butenyl ] -eriodytol, quercetin-3-methyl ether (quercetin-3-methyl ether), the separated compounds include 5 dihydrostilbene compounds, 6 dihydroflavonoids compounds and 4 flavonols (university of inner Mongolia, academic paper of 2018). The 90% ethanol extract of the overground part of the Ural licorice is separated and purified by week powerful, and the structures of the 90% ethanol extract are identified as (2S) -3'- (2-hydroxy-methyl-3-enyl) -4',5, 7-dihydroxy-dihydroflavanone, pin ocembrin, sigmoidin B, licolavanone, 6-phenylnaringenin, pinobanksin, galanin, genistein, pratensein, kaempferol-3-O-beta-D-rutinoside, rutin, alpha '-dihydroxy-3, 5,3' -dihydroxy-4 '-methoxy-5' -isopentenyl-stilbene and D-1-O-methylitol respectively; the 90% ethanol extract of aerial part of glycyrrhiza inflata is separated into 2- (3-methyl-butyl) -3,5,4'-trihydroxy-bibenzyl, (2S) -6- [ (E) -3-hydroxymethyl-butyl ] -3',4',5, 7-tetrahydro-dihydroflavone, 5,7,3',4 '-tetrahydro-8- (3', 3 '-dimethylnaphthyl) -flavone 5,7,3',4 '-tetrahydro-6- (3', 3 '-dimethylnaphthyl) -flavone, 8-phenylnaphthyl, 6-phenylnaphthyl, 5, 7-dihydroxyphenyl-8- (3', 3 '-dimethylnaphthyl) -flavone, 5, 7-dimethylnaphthyl-3' -dimethylnaphthyl-flavone, 5,3 '-dimethylnaphthyl-3' -dihydronaphthyl-flavone (university of Takara Shuzo). Liuyuchen adopts 95% ethanol water solution and 50% ethanol water solution to extract liquorice, 33 compounds are separated and identified, and the compounds are respectively tetracosanoic acid, beta-sitosterol, 5-O-methyl glycyrrizine, 1-methoxyficoll, docosanol, betulinic acid, glycitein, oleanolic acid, galangin, caffeic acid docosanol, neoliquiritigenin, liquirinin H, isoliquiritigenin, liquiritigenin, luteolin, 3R-donkey carnosol, echinacone, glycycoumarin, glycyrumarin, 7,2',4' -trihydroxy-5-methoxy-3-aromicin, glycyrrone, isoliquiritigenin, glycyrol, formononetin, glycyrhizic acid, griseolus, formononetin, liquiritin, apioside, glycyrrhizic acid, apioside, astragaloside B and trematose E (Pharmacol, J. 1255, 1255-1251255, 1255-1251251255-1251255-1251251255-1255-one). The Liangjun takes dried roots of Glycyrrhiza spinosa as raw materials, and beta-sitosterol and santalin are extracted and separated (Cissus of Qizihaer medical college, 2009, 30, 978-979). Shironghuo separates 17 compounds from root and rhizome of Glycyrrhiza pallidiflora Maxim (Glycyrrhiza pallidiflora Maxim) of Glycyrrhiza of Leguminosae, and determines the structures of 13 compounds, which are respectively: posterior santalin (homoptococcarinn), 5 α -stigmast-3, 6-dione (5 α -Stigmastane-3, 6-dione), β -sitosterol (β -sitosterol), 4',7-dimethoxyisoflavone (4', 7-dimethoxyisolavone), betulinic acid (betulinic acid), medakamin (medicarpin), isoliquiritigenol (isoglabrosol), 10-methoxymedakamin (10-methoxy-medicarpin), 9-methoxycoumestrol (9-methoxycoumestrol), 21 α -hydroxy-3-oxooleana-11, 13 (18) -diene-29-oic acid, formononetin (formononetin), macetic acid (maculotherein), daucosterol (daucosterol) (university of traditional Chinese medicine, 2001). From roots of Yunnan licorice (Glycyrrhiza yunnanensis), 13 compounds were isolated by Pepperwort et al, and the structures of two of the compounds were identified as methylated mevastatin and 5-hydroxy-7-methoxyflavanone, which was named Yunmanning (natural products research and development, 1994, 6-8). 7 compounds are firstly separated from petroleum ether and dichloromethane extracts of dried roots and stems of glycyrrhiza rotundifolia and the like, and are identified as follows: n-pentacosane, n-hexacosanic acid, 7,4 '-dihydroxyflavone, isoliquiritigenin, 4-hydroxy-2, 4' -dimethoxychalcone, liquiritin and sucrose (proceedings of the university of Henan university (Nature science edition), 1994, 22, 48-50). Lianghong has identified five triterpenoids, namely betulinic acid, methyl ester of maquintic acid, glycyl alcohol B, olean 13 (18) -ene-22 alpha-chloro-3 beta, 24-diol (olean-13 (18) -ene-22 alpha-C1-3 beta, 24-diol) and round fruit sapogenin (squasapogenol) (pharmaceutical report, 1993, 28, 116-121) from the roots of Glycyrrhiza glabra (Glycyrrhiza squamosa Franch.).
2. Pharmacological action
2.1 Effect on digestive System
2.1.1 anti-ulcer Dian Van Ye and other reported main active ingredients of licorice and its prescription can eliminate helicobacter pylori by inhibiting gastric acid and pepsin secretion, promote gastric mucosa mucus secretion, protect and repair gastric mucosa, reduce gastric mucosa injury, promote granulation tissue growth of ulcer focus, accelerate ulcer healing, improve gastric mucosa blood flow and microcirculation, and prevent and treat gastric ulcer in different degrees, and has good anti-ulcer effect (Henan Chinese medicine, 2019, 39, 951-954). Glycyrrhizin has obvious protective effect on rat experimental ulcer formed by histamine and pylorus ligation, and can obviously reduce the ulcer incidence rate caused by pylorus blockage of the rat. Zhaoyuan et al reported that glycyrrhiza polysaccharide has inhibitory effect on capillary inflammatory infiltration and gastric ulcer of both ethanol type and reserpine type, and has obvious dose-effect relationship (traditional medicine of Asia-Pacific, 2015, 11, 12-14). Lishengwei and the like report that the liquorice total flavonoids can relieve aspirin-induced GES-1 injury through the ways of promoting proliferation, resisting apoptosis, resisting oxidative stress and the like, have a protective effect on aspirin-injured gastric mucosal epithelial cells of a human body, and the mechanism of the liquorice total flavonoids is probably related to the inhibition of an ERK1/2 signal pathway (Chinese general medicine, 2018, 21, 3975-3975). 2.1.2 influence on gastric acid secretion Zhang Yan reports that glycyrrhizic acid can increase the capability of generating NO in vivo, and NO has strong gastric mucosa protection effect and can prevent or obviously reduce various experimental gastric mucosa injuries. The German Rea Krausse in 04 years verifies that the glycyrrhizic acid has a certain inhibiting effect on the growth of H.pyrori through experiments. The liquorice fluid extract can directly adsorb gastric acid by intragastric administration, and can reduce gastric acid for normal dogs and rats with experimental ulcer.
2.1.3 spasmolytic effect on gastrointestinal smooth muscle flavone glycosides contained in licorice root clinically used in rabbit and guinea pig in vitro intestinal canal of the suppression, reduce contraction times, reduce tensity, and have spasmolytic effect on intestinal smooth muscle spasm in vitro caused by barium chloride and histamine.
2.1.4 liver protection function Wanggu reports that isoliquiritigenin has good killing function on two kinds of liver cancer cells, and the mechanism can regulate and control MAPK/STAT 3/NF-kB signal channel by up-regulating the generation of intracellular active oxygen, thereby causing the liver cancer HepG2 cells to generate cell cycle arrest and reclamation apoptosis (university of eight agricultural crops in Helongjiang, 2020 academic thesis). Glycyrrhiza polysaccharide pair CCl reported in Chendongxue et al 4 The induced acute liver injury of the mouse has a certain protection effect (Chinese pharmacy 2016, 27, 1322-1325). Zhana et al reported that glycyrrhetinic acid has significant antitumor activity on liver cancer in vitro and in vivo, and deserves further research and development (J. Chinese Experimental & formulary, 2015, 21, 37-41). Glycyrrhizin can significantly prevent CCl 4 The increase of glutamic pyruvic transaminase of poisoned rats can also reduce the accumulation of triglyceride in liver. Glycyrrhetinic acid can strongly inhibit CCl 4 Can generate free radicals and lipid peroxide, and inhibit Ca formation 2+ Cellular damage caused by influx into the cell.
2.1.5 effects on bile secretion Liuzhong et al report that the addition of liquiritigenin to histidine-tryptophan-ketoglutarate solution can significantly reduce the liver injury score, reduce lactate dehydrogenase leakage and increase bile secretion, while after reperfusion, the liver stored in the histidine-tryptophan-ketoglutarate solution containing liquiritigenin has significantly reduced HMGB1, TLR4 and NF- κ B expression and hepatocyte apoptosis, and significantly reduced TNF- α and IL-6 contents in perfusate with the addition of GL (journal of medical research, 2019, 48, 52-57). The glycyrrhizin can increase bile secretion of the rabbit with bile duct transfusion fistula, and has an inhibiting effect on bile red rise of the rabbit after bile duct ligation.
2.2 Effect on the cardiovascular System
2.2.1 anti-arrhythmic Xieshanrong et al report that glycyrrhetinic acid has significant anti-arrhythmic effect on rat and mouse (pharmaceutical guide, 2004, 23, 140-142). The glycyrrhizin has exciting effect on isolated toad heart, and has obvious antagonistic effect on acetylcholine and physostigmine, and obvious synergistic effect with epinephrine.
2.2.2 lipid-lowering action and antiatherosclerotic glycyrrhizin have certain effect of lowering cholesterol in blood for experimental hypercholesterolemia of rabbits and hypertension patients with elevated cholesterol. Glycyrrhetinic acid salt has effects of reducing blood cholesterol, lipoprotein and beta-lipoprotein triglyceride for hyperlipemia rat and experimental atherosclerosis rabbit.
2.2.3 anti-myocardial ischemia, heart failure Mahanning, etc. reported that glycyrrhetinic acid has protective effect on the heart function of CA/CPR rats, and the action mechanism of glycyrrhetinic acid is related to the inhibition of oxidative stress and the down regulation of Caspase 3/Bax/Bcl-2 apoptosis signal pathways (Chinese medicine pharmacology and clinic, 2019, 35, 28-33). Anyhuan albizzia et al reported that isoliquiritigenin has an effect of resisting myocardial ischemia reperfusion injury, and its effect may be related to its antioxidant, anti-inflammatory and anti-apoptotic effects (proceedings of the university of rockriver (nature science edition), 2016, 34, 343-348). ZhengLei et al reported that lowering intracellular reactive oxygen species level may be one of the effective mechanisms of glycyrrhizic acid and glycyrrhetinic acid in relieving heart failure, and glycyrrhizic acid and glycyrrhetinic acid may be important effective components of licorice in intervening heart failure (J.I.T. combined with cardiovascular and cerebrovascular diseases, 2016, 14, 21-25).
2.3 Effect on the respiratory System
Zhang et al reported that glycyrrhizin could improve lung function and alleviate fibrosis in silicosis mice (proceedings of Anhui medical university, 2022, 57, 121-125). Zhang Yu et al reported that glycyrrhetinic acid can improve the general condition of mice with acute radiation-induced lung injury, reduce inflammatory response of lung tissues and improve collagen deposition (Chinese clinical pharmacology and therapeutics, 2015, 20, 629-633). The glycyrrhiza extract and the glycyrrhiza mixture can cover inflamed pharyngeal mucosa after being orally taken and relieve the irritation of inflammation to the pharyngeal mucosa, thereby playing the role of relieving cough, the glycyrrhetinic acid has obvious central cough relieving effect, the hydrogen succinate of the glycyrrhetinic acid is orally taken, the cough relieving effect is similar to codeine, and the glycyrrhetinic acid has weaker protection effect on bronchospasm caused by substances such as 5-hydroxytryptamine and the like.
2.4 Effect on the Central nervous System
2.4.1 neuroprotective effect Jiangjimei et al reported that glycyrrhizin may have neuroprotective and anti-inflammatory effects on diabetic patients (university of Chinese pharmacy, 2020, 51, 711-717). Wanglin et al reported that administration of glycyrrhizin via the tail vein was effective in reducing cerebral cortical neuronal damage in rats with status epilepticus, and that its neuroprotective effect was dose-dependent, with a mechanism probably related to its inhibition of the expression of serum and cerebral cortical inflammatory factor HMGB1 (J. Apoplexia and neurological disorders, 2017, 34, 893-896). The traditional Chinese medicine glabridin is reported by the Yan Zhao and the like to play a role in protecting the learning and memory capacity of the mouse with the Parkinson disease caused by the 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine through inhibiting a protein kinase signal channel, resisting oxidation, relieving inflammation and the like (Chinese medical journal, 2017, 97, 2050-2054). The swertia arborescens et al report that the extraction part of the total flavonoids of licorice has good anti-depression pharmacological activity on rat depression behavior caused by chronic unpredictable stress, and can play a role in protecting hippocampal nerve regeneration damage caused by stress under a larger dose (pharmacy report, 2012, 47, 1612-1617). Studies of Shaosyang and the like report the influence of liquiritin on inflammation and neuron apoptosis in spinal cord injury mice, and liquiritin can reduce the spinal cord injury of the mice by inhibiting inflammation and neuron apoptosis through MAPK pathway inhibition (2022, 51, 518-523 & lt 547 & gt, university of Chinese medicine).
2.4.2 antipyretic action Glycyrrhetinic acid and glycyrrhizin have antipyretic action on febrile rat, mouse and rabbit, respectively, glycyrrhetinic acid 40mg/kg is injected into abdominal cavity to defervesce fever of febrile rat, equivalent to 600mg/kg sodium salicylate, and has no cooling effect on normal body temperature rat.
2.4.3 weight peak of sedation and the like reports that the extraction part of the liquorice total flavonoids has the function of resisting mice PTZ induced convulsion (world composite medicine, 2017, 3, 41-43+ 47).
2.5 corticoid-like actions
2.5.1 mineralocorticoid action extractum glycyrrhizae, glycyrrhizin and glycyrrhetinic acid have the action of promoting sodium and water retention in healthy people and various animals, which is similar to the action of mineralocorticoid deoxycorticosterone, can cause edema and blood pressure rise after long-term application, but can also be used for treating mild Addison's disease, and can obviously enhance and prolong the action of Corson.
2.5.2 glucocorticoid-like action Glycyrrhetinic acid etc. can make thymus gland of rat atrophy and adrenal weight increase, and also has glucocorticoid-like action such as jaundice resisting action and immunosuppression action etc.
2.6 Effect on the urogenital System
Glycyrrhizic acid and its sodium salt intravenous injection enhance theophylline diuresis, can inhibit rabbit experimental vesical calculus formation, can inhibit estrogen from to adult animal uterus growth effect, glycyrrhizin has antidiuretic action to rat, glycyrrhetinic acid and its salt have obvious antidiuretic action too. Liuwenjing and the like report that glycyrrhetinic acid has a protective effect on early and middle stage renal fibrosis of a kk-Ay diabetes model mouse, and possibly influences the expression of renal fibrosis transformation factors TGF-beta 1, lamininl, E-cadherin and alpha-SMA fibrosis related protein, so that the development of early and middle stage diabetic renal interstitial fibrosis is delayed (Yunnan J.Med.Med.Med.Med.J., 2020, 41, 72-78).
2.7 Effect on immune function
Research on the influence of glycyrrhiza inflate polysaccharide Gi P-B1 on mouse spleen lymphocyte proliferation and induction of cytokines such as IL-2, IL-13, TNF-alpha and the like by using thermomela miji and the like, wherein Gi P-B1 has an immunopotentiation effect (Chinese medicine, pharmacology, 2016, 34, 1647-1649). Wangyue et al reported that glycyrrhiza inflata polysaccharide has positive regulation effect on specific immunity and non-specific immunity of mice and dose effect relationship (food research and development, 2016, 37, 41-43).
2.7.1 antiallergic action A complex (Lx) is extracted from Glycyrrhiza uralensis Fisch, and Lx has obvious protective effect on anaphylactic shock of mice and also has the ability of inhibiting antibody production. Zhengxiuqiang et al discusses the effect of licorzinc in the treatment of diabetic periodontal disease, and the treatment with licorzinc can effectively improve the patient's immune function, reduce bacterial invasion, and promote the recovery of periodontal tissue, and is worthy of clinical application (New world of diabetes, 2016, 19, 83-84).
2.7.2 influence on non-persistent heterosexual immune function Robotic et al report that Glycyrrhiza polysaccharide has multiple biological activities of immunoregulation, anti-tumor, anti-oxidation, antivirus, etc., and has multiple functions of improving animal production performance and promoting immunity in animal production (Chinese feed, 2022, (07), 6-12). The study of quality girl and the like shows that the glycyrrhiza inflata polysaccharide can activate a TLR4/MyD 88/NF-kB signal channel, promote the expression of related genes, enable NF-kB to enter cell nucleus to regulate the transcription level of related cytokines, and further activate the immune function of macrophages (natural product research and development, 2021, 33, 2073-2081).
2.7.3 Effect on specific immune function the effect of glycyrrhizic acid on polyclonal antibody production was studied using an in vitro antibody production system, where glycyrrhizic acid at a certain concentration significantly increased antibody production, adherent cells were isolated from human peripheral blood mononuclear cells, and after culturing with glycyrrhizic acid at various concentrations, mononuclear cells were added to the culture supernatant to investigate the effect on PWM stimulation induced antibody production.
2.8 antiviral action
2.8.1 anti-HIV Effect glycyrrhizin exerts the effect of destroying HIV cells (HIV) in test tubes by recovering T helper cells, 0.5mg/mL glycyrrhizin inhibits the proliferation of HIV by more than 98%, and new polyphenols in Glycyrrhiza glabra exhibit proliferation inhibitory effect on HIV cells at low concentration compared to glycyrrhizin.
2.8.2 action against other viruses Glycyrrhiza polysaccharide has obvious activity against vesicular stomatitis virus, adenovirus type 3, herpes simplex virus type 1, vaccinia virus, etc., and can remarkably inhibit the occurrence of cytopathic effect, so that the cells cultured by the tissue are protected. Glycyrrhizin has inhibitory effect on varicella-zoster virus in test tube, and has inhibitory concentration of 0.55mg/mL on human fetal fibrosis infected by varicella-zoster virus (VZV) belonging to herpes virus group. Glycyrrhetinic acid appears to have a specific effect on herpes simplex virus.
2.9 antibacterial, anti-inflammatory action
The Glycyrrhrizae radix extract and sodium glycyrrhetinate have inhibitory effect on Staphylococcus aureus, bacillus tuberculosis, escherichia coli, amoeba and trichomonad in vitro, and Glycyrrhrizae radix has antiinflammatory effect like phenylbutazone or hydrocortisone, and its antiinflammatory components are glycyrrhizin and glycyrrhetinic acid. Glycyrrhetinic acid has inhibitory effect on cotton ball granuloma and formaldehyde foot swelling subcutaneous granulomatous inflammation of rat, and glycyrrhizin and glycyrrhetinic acid have inhibitory effect on stage I, II and III of inflammatory reaction. Licoflavone has effects of inhibiting mouse carrageenan edema and inhibiting sensitive cells from releasing chemical transfer substances. Glycyrrhetinic acid and glycyrrhizin have antipyretic effect on febrile rat, mouse and rabbit. Zhang li Peng et al reported that glabridin, a traditional Chinese medicine, alleviates inflammation by inhibiting p38MAPK/ERK signal pathway and resisting oxidation, and plays a role in protecting rat ARDS caused by LPS (J.Zhonghua Med., 2016, 96, 3893-3897).
2.10 detoxification action
Glycyrrhizin has a certain detoxification capacity to food poisoning, drug poisoning and in vivo metabolite poisoning, the detoxification mechanism is that glycyrrhizin has an adsorption effect to toxicants, glucuronic acid generated by glycyrrhizin hydrolysis can be combined with toxicants, and glycyrrhizin has an adrenocortical hormone-like effect to enhance the detoxification capacity of livers. Glycyrrhizin or its calcium salt has strong detoxifying effect on white larynx toxin and tetanus toxin, and also has detoxifying effect on some allergic diseases, animal experimental hepatitis, tetrodotoxin and snake venom.
2.11 antitumor Effect
Wanshansan et al reported that licochalcone A has effects of inhibiting proliferation and promoting apoptosis on human brain glioma U251 cell, and its mechanism may be related to the down-regulation of beta-catenin mRNA, cyclin D1 mRNA, ki-67 and Bcl-2 expression levels (Chinese contemporary medicine, 2022, 29, 4-7). Huangshang et al reported that glabridin can inhibit proliferation and invasion of Hela cells and promote apoptosis, and the action mechanism of glabridin is probably related to the inhibition of the activation of Wnt/beta-catenin signal channel (modern tumor medicine, 2020, 28, 3289-3293). Guo Junyu et al reported that isoliquiritigenin can enhance the sensitivity of nasopharyngeal carcinoma nude mouse transplanted tumor to radiotherapy, and the action mechanism of isoliquiritigenin may be related to the inhibition of the expression level of hypoxia inducible factor-1 alpha (HIF-1 alpha) and Vascular Endothelial Growth Factor (VEGF), thereby inhibiting the generation of tumor blood vessels (Hebei medicine, 2016, 38, 3704-3707). Wangjiu et al describe the anti-tumor effect of LCA and its molecular mechanism, and provide reference for further clinical research (modern oncology, 2016, 24, 3870-3874). Wangli et al reported that Glycyrrhiza polysaccharide has significant anti-tumor effect, and the anti-tumor mechanism of Glycyrrhiza polysaccharide is probably related to the increase of IL-7 secretion by small intestine mucous epithelial cells (Tianjin Chinese medicine, 2016, 33, 373-377). Glycyrrhizic acid can generate morphological change on mouse Ehrlich Ascites Carcinoma (EAC) and rat ascites hepatoma cells, can inhibit subcutaneously transplanted Jitian sarcoma, and glycyrrhizic acid monoammonium salt has inhibitory effect on mouse ehrlich ascites carcinoma and sarcoma. Glycyrrhetinic acid has effect in inhibiting Oberling Guerin myeloma transplantation of rat, and sodium glycyrrhetinate has effect in inhibiting mouse Ehrlich carcinoma (EAC) and sarcoma-45 cell growth. The liquiritin can generate morphological changes on rat ascites liver cancer and mouse ehrlich ascites cancer cells. The glycyrrhizin can remarkably inhibit mouse skin cancer caused by euphorbia ester diterpenoid alcohol p-xylene anthracene (DMBA).
2.12 anticancer Effect
2.12.1 inhibition of hepatoma carcinoma cell Chenoporing reports that licochalcone A can enhance the self-oxidation resistance of the hepatoma carcinoma cell and promote the death and apoptosis of the hepatoma carcinoma cell (Jilin university, 2018). Muhe Buli, abelia cuminum cyminum and the like discuss the inhibition activity of total flavonoids of Glycyrrhiza glabra (Glycyrrhiza glabra L) and monomer components glabridin (Gb) on the proliferation of human liver cancer Bel-7402 cells, and the human liver cancer Bel-7402 cells have certain sensitivity on the anticancer activity of the total flavonoids of Glycyrrhiza glabra (university of Xinjiang medical science, 2013, 36, 1744-1748). Zhouyou 261077 and the like report that glabridin can influence the dryness of liver cancer stem cells through a Hedgehog signal pathway so as to inhibit the proliferation of Hep3B cells (Zhongnan pharmacy, 2020, 18, 1483-1487).
2.12.2 inhibition of cervical cancer cell Wang Yongbo screens out isoliquiritigenin, 3-methoxy isoliquiritigenin, 3-hydroxy isoliquiritigenin and 3,4' -dihydroxy-4-methoxy chalcone which have obvious apoptosis promoting effect on SiHa cell and HeLa cell of cervical cancer, licochalcone compounds which have obvious inhibition activity and apoptosis promoting effect on SiHa cell and HeLa cell and lay an important foundation for screening out effective candidate drugs with cervical cancer resistance from isoliquiritigenin derivatives (Xinjiang medical university, 2015 academic paper). Yaojian et al reported that the antiproliferative and proapoptotic properties of glycyrrhizic acid on cervical cancer cells may be caspase activity in intracellular and extracellular pathways activated by inducing mitochondrial membrane potential damage, and glycyrrhizic acid can be used as an auxiliary means for preventing and controlling cervical cancer (Chinese modern application medicine, 2020, 37, 2727-2733). The methoxylation modification of licochalcone B reported by Mirex Guli, buying Timine and the like can obviously improve the anti-cervical cancer activity (the food safety quality detection bulletin, 2019, 10, 6217-6222).
2.12.3 inhibition of breast cancer cell Yuan Yong et al reported that licochalcone A can induce apoptosis in human breast cancer MDA-MB-231 cells by increasing intracellular ROS levels, decreasing MMP-induced mitochondrial dysfunction and endoplasmic reticulum stress (J. Chinese Experimental formulary, 2021, 27, 95-100). Chen Ke et al reported that liquiritigenin can inhibit the growth of human breast cancer MCF-7 cells, and the effect is related to the promotion of apoptosis and the induction of autophagy (J.Gen.J.J.Med., 2013, 22, 1466-1470). Jichao et al reported that isoliquiritigenin plays an important role in apoptosis of siHa cells, and the action mechanism of isoliquiritigenin may reduce the cell biological characteristics of cervical cancer and the expression of mTOR/P70S6K level so as to inhibit cell proliferation and migration (Chinese maternal and child care, 2021, 36, 2630-2633).
2.12.4 inhibition of Lung adenocarcinoma cell Glycyrrhetinic acid can inhibit experimental myeloma and ascites hepatoma of white mouse, and inhibit ehrlich ascites carcinoma of white mouse. Royal et al reported that licochalcone A could effectively inhibit proliferation and migration of human lung adenocarcinoma A549 cells, and the mechanism thereof may be related to the regulation of p-Akt and p-GSK-3 beta expression level by licochalcone A (Cizizahal medical college, 2017, 38, 2853-2856). Xuwangting utilizes a lung cancer A549 cell line to explore a potential anticancer molecular mechanism of 18 beta-Gly for lung cancer for the first time, and provides a certain theoretical basis for a traditional Chinese medicine extract to treat tumors (university of eight agricultural reclamation in Heilongjiang, 2021).
2.12.5 inhibition of gastric cancer cell Wanghuifeng and the like reports that glycyrrhizin inhibits proliferation, adhesion and migration of gastric cancer cell BGC-823 cells by regulating cell cycle, and the action mechanism of glycyrrhizin is probably related to Wnt/beta-catenin signal pathway (China's journal of digestion, 2015, 23, 2868-2873). Dibutylperbate and the like report that glycyrrhetinic acid inhibits gastric cancer cell SGC7901 proliferation and induces apoptosis by down-regulating NF-kB signal pathway protein expression (Chinese journal of clinical pharmacology, 2019, 35, 1902-1904+ 1908).
2.12.6 inhibition of intestinal cancer cells, sun Wei et al reported that glycyrrhetinic acid can inhibit the proliferation of colorectal cancer LoVo cells and reduce the invasive ability, and the mechanism of these effects is related to the inhibition of NF-kB protein expression (J.Gen.Physiol., 2022, 38, 37-40). Liyajing et al reported that Glycyrrhiza polysaccharide reduces myotube atrophy induced by C26 colon cancer cells co-cultured with RAW264.7 macrophage by inhibiting STAT3 signaling pathway (proceedings of university of medicine, shanghai, 2021, 35, 45-53). Sunyu et al reported that licochalcone can prevent colon cancer from occurring or alleviate pathological changes from multiple indexes by inhibiting the release of colonic cell inflammatory factors, and licochalcone is expected to be a novel candidate drug for preventing and treating colon cancer (J. China national medicine, 2020, 26, 29-31).
2.12.7 inhibition of tongue cancer cell von Breliance et al reported that glycyrrhizin can inhibit the proliferation and invasion ability of human tongue cancer Cal-27 cell line in vitro and promote apoptosis, and the mechanism may be realized by up-regulating the expression of Bax and p-caspase-3 and down-regulating the expression of Bcl-2 (modern medicine, 2020, 48, 747-751).
2.12.8 report that glycyrrhizin has the effect of inhibiting the growth of prostate cancer cell strain PC3, and the action mechanism of glycyrrhizin is probably related to the regulation of cyclin D1 and P21 protein expression and PI3K/AKT signal pathway activation by glycyrrhizin (J.Clin and pathological conditions, 2018, 38, 2308-2313).
2.13 resistance to oxidation
Zhang Fuxin et al determines the antioxidant activity of licorice total flavonoids through the clearance rate of DPPH free radical, superoxide anion free radical and hydroxyl free radical, and licorice flavonoids have better antioxidant and immunological activity (Chinese veterinary science, 2019, 39, 1180-1183). Kangxuefang et al reported that the aerial part of licorice fat-soluble total flavonoids has strong antioxidant capacity (round-the-clock Chinese medicine, 2016,9, 567-570). Van Yu culvert and the like report that isoliquiritigenin and glabridin have an inhibiting effect on tyrosinase activity and can be used for developing and utilizing effective components in waste licorice residues as plant source whitening additives (Shizhen national medicine, 2013, 24, 2649-2652). Zhangcai et al report that addition of glycyrrhiza polysaccharide in basal feed can reduce piglet mortality, improve coat development, increase blood glucose level, and enhance organism antioxidant capacity, thereby improving piglet health status (proceedings of northwest university of agriculture and forestry (Nature science edition), 2022, 50, 10-16).
2.14 other effects
The influence of glycyrrhetinic acid on the auditory function of the inner ear of the guinea pig is researched, after the glycyrrhetinic acid is injected into the guinea pig by 100mg/kg, the amplitude of cochlear microphonic potential and auditory nerve action potential caused by short sound is increased, and the reaction threshold of the auditory nerve action potential is reduced, which indicates that the glycyrrhetinic acid has the function of improving the auditory function of the inner ear of the guinea pig. Glycyrrhizic acid and glycyrrhetinic acid both have obvious inhibition effect on acetylcholinesterase and compete-non-competitive mixed inhibition effect on acetylcholinesterase. Wu-Yangtze research shows that LA has an anti-aging effect, and the discovery provides further important evidence for improving the medicinal value of liquorice (Stone river university, 2020 academic thesis).
3. Preparation method
3.1 Glycyrrhiza oil
The method adopts the traditional solvent method to extract the liquorice grease, the optimal extraction process conditions are that the extraction temperature is 70 ℃, the liquid-material ratio is 20mL/g, the extraction time is 5 hours, and the extraction rate of the liquorice grease reaches 4.49 percent; identifying 19 fatty acids by GC-MS, of which 4 fatty acids were predominant, palmitic acid (19.15%), oleic acid (4.92%), linoleic acid (59.5%), linolenic acid (11.16%); the mass fraction of saturated fatty acid is 23.31%, the mass fraction of unsaturated fatty acid is 76.69%, wherein the mass fraction of monounsaturated fatty acid is 5.71%, the mass fraction of diunsaturated fatty acid is 59.83%, and the mass fraction of triunsaturated fatty acid is 11.16% (Chinese food and oil science, 2018, 33, 102-106). The optimal conditions for extracting Glycyrrhrizae radix essential oil by steam distillation method such as DOUCANGNING are ultrasonic power of 270W, extraction time of 6h, glycyrrhrizae radix solution concentration of 5%, and Glycyrrhrizae radix essential oil extraction rate of 0.209% (China regulation) Monosodium glutamate, 2016, 41, 109-112+ 128). Guo Yao et al extracted the volatile oil from Glycyrrhiza uralensis produced in Gansu by steam distillation, and the results showed that the volatile oil contains 132 kinds of components, mainly acids, alcohols and alkanes, and a small amount of lipids and olefins (reported by Gansu Kaishao, 2013, 18, 37-41). The noble peach and the like are treated by ultrasonic treatment for 40min at 50 ℃ by taking petroleum ether as an extraction solvent and adopting an ultrasonic method, the extract yield is 0.78%, and the squalene content in the extract is 0.33% (journal of Liaoning traditional Chinese medicine, 2011, 38, 2047-2049). The optimal ultrasonic-assisted extraction process of squalene in liquorice, such as Gaoyao peach and the like, is determined to be that the ratio of material to liquid is 1: 15, the soaking time is 10 hours, the ultrasonic time is 50min, the ultrasonic temperature is 50 ℃, the 26 components are separated and identified in the coarse fat of liquorice, and the squalene content is 0.8% (Chinese journal of Experimental and formulation science, 2011, 17, 5-8). The content of squalene in the extraction product with acetone as entrainer is up to 0.47%, and the total extraction rate of squalene from radix Glycyrrhizae by supercritical fluid extraction is 0.018% (Hubei agricultural science, 2011, 50, 3600-3601). Soxhlet extraction with petroleum ether as a solvent, qualitative detection by thin layer chromatography, and quantitative analysis by High Performance Liquid Chromatography (HPLC) to test squalene in Glycyrrhrizae radix seed and root, wherein the content of squalene in Glycyrrhrizae radix seed and root is low, and the determined chromatographic conditions are as follows: c18 column, mobile phase acetonitrile-methanol (60: 40, v/v), detection wavelength of 210nm, flow rate of 2mL/min, and column temperature of 30 ℃ (university of Beijing, 2010, 32, 123-126). Supercritical CO for Wangmai 2 The extraction technology is used for extracting the licorice seed oil, the optimal extraction process is determined, volatile components and fatty acid components of the licorice seed oil are researched by a GC-MS method, solid matters obtained after oil extraction of the licorice seed oil are used as raw materials for extracting flavone, 16 volatile components are firstly identified from the glycyrrhiza uralensis seeds, dibutyl phthalate and alkane compounds are used as main components, 5 fatty acid components are simultaneously identified in a separating way, two unsaturated fatty acids, namely linoleic acid and d-linolenic acid, which have various activities and are necessary for human bodies are used as main components, and the relative percentage contents are respectively 24.3 percent and 25.51 percent; extracting volatile oil from stem and leaf of Glycyrrhrizae radix by steam distillation, and analyzing the volatile oil by GC-MS methodThe 41 volatile components are separated and identified for the first time, and terpene compounds and polycyclic aromatic hydrocarbon with various pharmacology and biological activities are taken as main components, so that a foundation is laid for further research on pharmacology and drug effect; ethanol is used as entrainer and supercritical CO is adopted 2 The method extracts flavonoids compounds in the stems, leaves and the solid after oil extraction of liquorice, and the result shows that the content of total flavonoids in the liquorice leaves is more than that of the solid after oil extraction of seeds and more than that of the stems, and the content of flavonoids in the leaves reaches 2.78 percent, thereby providing a basis for further developing and utilizing liquorice resources (northeast forestry university, 2005).
3.2 pigment of Liquorice Stem and leaf
Extracting isoflavonoids from licorice leaves at dawn, separating the isoflavonoids from chlorophyll, and determining an optimal process for separating the isoflavonoids from the chlorophyll by using HPD100 type macroporous resin: when the ratio of the column width to the column height is about 1: 4, the mass ratio of the polyamide blend (1: 2) to the adsorption macroporous resin is 1: 5, and the isoflavone and the chlorophyll can be completely separated by 50% ethanol 2BV and 90% ethanol 4BV, so that the isoflavone sample A content is 10%, and the chlorophyll adsorbed by the dichloromethane desorption resin is obtained (Ningxia university, 2005). A method for continuously extracting chlorophyll and isoflavone from stem and leaf of Glycyrrhrizae radix by use of Liu Steel comprises adding ethanol, distilling under reduced pressure, recovering solvent, mixing with polyamide when the material liquid becomes viscous, vacuum drying, loading onto macroporous resin column, gradient eluting with water and ethanol, collecting positive eluate, concentrating under reduced pressure, and recovering solvent to obtain crude isoflavone; percolating the macroporous resin percolation column eluted with ethanol with dichloromethane, concentrating, and recovering dichloromethane to obtain dark green ethanol solution, i.e. chlorophyll (CN 1328274C).
3.3 Glycyrrhiza Polyphenol
Miao Yongmei, etc. uses the dry powder of the callus of the glycyrrhiza glabra as the material, and the optimal process for extracting the total phenol is that the liquid-material ratio is 50: 1, the concentration of ethanol is 60 percent, the temperature is 70 ℃, and the time is 25min (proceedings of the academy of science and technology of Anhui, 2021, 35, 77-82). Shenwei and the like take liquorice as raw materials, and polyphenol substances are extracted by adopting an ultrasonic-assisted ethanol extraction method, wherein the optimal extraction process parameters of the liquorice polyphenol are as follows: the concentration of ethanol is 70%, the ratio of material to liquid is 1: 42g/mL, the ultrasonic time is 66min, the ultrasonic power is 250w, and the extraction rate of polyphenol is 4.97% (food technology, 2021, 46, 233-239).
3.4 Glycyrrhiza alkaloids
Lijie et al determined the optimal extraction process parameters of licorice alkaloid are: the ethanol concentration is 87%, the extraction time is 90min, the extraction temperature is 53 ℃, the feed-liquid ratio is 1: 20 (g/mL), and the liquorice alkaloid yield can reach 0.3659mg/g (Chinese food additive, 2018, (04), 128-133). The root alkaloid components of the main medicinal liquorice, namely uralis (G.uralensis), glycyrrhiza glabra (G.inflata), glycyrrhiza inflate (G.palliadiflora) and glycyrrhiza spinosa (G.pallidiflora) are analyzed and researched by Wangyongarmy, and the alkaloid components are quinoline derivatives and isoquinoline derivatives, and the total content is 0.29 percent on average (northwest plant science, 2001, (06), 211-214). Soaking licorice powder in sulfuric acid for extraction, regulating the pH value of the extract to 3-6, adding enzyme for reaction for 8-12 hr to obtain enzymolyzed extract, adding potassium hydroxide and ammonia water to regulate pH value to 6-9, standing for precipitation, discarding supernatant, washing the precipitate with water, stoving to obtain coarse licorice alkaloid product, dissolving the coarse licorice alkaloid product in ethanol, filtering to eliminate insoluble matter, decolorizing the filtrate with active carbon while eliminating impurity, heating to 60-80 deg.c, adding acid to the filtrate for reaction, standing for 6-10 hr to obtain precipitate, and filtering to obtain similar white alkaloid (CN 112457352A). Liangxin 2815630-95% alcohol-water solution is used as extraction solvent, soaking and reflux extraction are carried out to extract a licorice sample, and licorice extract is obtained after filtration and concentration, and the obtained licorice extract is subjected to liquid-liquid extraction treatment to obtain licorice alkaloid compound (CN 111228326A).
3.5 glycyrrhizin
3.5.1 multistage countercurrent extraction of Roughage and the like adopts multistage countercurrent extraction of glycyrrhizic acid under the optimal conditions of 70 ℃ of extraction temperature, 60 min of single-stage extraction time, 6 of liquid ratio and 5 of extraction stage number, wherein the extraction rate of the multistage countercurrent extraction is higher than that of cold soaking at room temperature for 44.3h, 40min of ultrasonic extraction, 4h of Soxhlet extraction and 54min of microwave extraction (Beijing university of Industrial science), 2011, 29 and 33-37).
3.5.2 resin method (1) D-101 macroporous resin Korean philosophy adopts D-101 as adsorption resin, the maximum adsorption capacity is 171.2 (mg glycyrrhizic acid/g resin), the desorption rate is 85.22%, and the optimization conditions in the adsorption and separation process are as follows: the concentration of the upper column liquid is 9mg/mL, the pH value is 6, the desorption is carried out by adopting 4 BV10% ethanol and the flow rate of 1BV/h, the purity of the glycyrrhizic acid is 81.72%, and the purity reaches 90% after the glycyrrhizic acid is decolored by activated carbon (the university of Ringchun, 2009 academic paper). D101 macroporous adsorption resin is selected for the Royal Robinia (Royal) Kuntze, the sample loading is 3BV, the pH of a sample solution is 6.0,3BV is eluted by 70 percent ethanol, the yield of glycyrrhizic acid is 61.98 percent, the content of glycyrrhizic acid reaches 65.69 percent, the glycyrrhizic acid is refined by recrystallization, the content of glycyrrhizic acid reaches 93.80 percent, and the total yield is 46.31 percent (Tianjin scientific and technical university, academic paper of 2019). D101 resin is adopted in the Sunxinghua, the pH value is 6.5, the sample loading concentration is 10mg/ml, the flow rate of a stock solution is 2BV/h, the adsorption quantity of glycyrrhizin is the maximum, and the optimal desorption conditions are as follows: eluting with 1.5BV 50% ethanol solution to obtain glycyrrhizin content of 35.23% (university of Hebei science and technology, 2014 academic paper). (2) The optimum dynamic adsorption condition of HPD722 for HPD series resin beam cloud is as follows: the sample loading amount is 7BV, the concentration of the sample loading liquid is 6.5mg/mL, the pH of the sample loading liquid is 7.0, the flow rate of the sample loading liquid is 3BV/h, and the optimal dynamic desorption conditions are as follows: the optimum concentration of the ethanol in the eluent is 70%, the extracted glycyrrhizic acid crude product with the purity of 30.07% is purified, the purity of the purified glycyrrhizic acid can reach (70.65 +/-0.62)%, and the yield can reach (80.50 +/-0.51)% (university of Tianjin science and technology, academic thesis in 2019). Removing impurities from high-grade snow rock with HPD400 type macroporous resin, 30% ethanol, and eluting with 50% ethanol to obtain Glycyrrhrizae radix total saponin part with total saponin content of more than 50% (2011, 27, 78-81). The HPD-300 type resin adopted by Zhuzhongjia and the like has better adsorption performance and desorption effect, and the purification process comprises the steps of enabling the mass concentration of a sample solution to be 0.2g of crude drug/mL, enabling the sample amount to be 5BV, and eluting 6BV with 50% ethanol (Chinese patent medicine, 2011, 33, 341-343). (3) LX-60 resinkanglii and the like are based on a non-polar macroporous adsorption resin LX-60 sold in the market, and have more excellent adsorption performance (the adsorption capacity is improved by 33.6 percent and reaches 91.70 mg/g) by introducing chloromethyl, sulfonic acid group and carboxyl group into the macroporous adsorption resin, while the desorption capacity is relatively reduced (the desorption capacity is reduced by 83.6 percent and reaches 10.03 mg/g) (Chinese brewing, 2016, 35, 113-118). (4) ADS-17 resin Sunxinghua adopts ADS-17 resin, pH value is 6.0, sample concentration is 10mg/mL, stock solution flow rate is 2BV/h, glycyrrhizin adsorption capacity is maximum; the optimal desorption conditions are as follows: eluting with 4BV 10% ethanol to obtain glycyrrhizin 65.07%; a series of resins are synthesized by a suspension polymerization method, after dynamic adsorption is completed, gradient elution is carried out, ethanol solution is firstly used for pre-washing to remove a part of impurities, then glycyrrhizin is eluted by ethanol solution containing sodium hydroxide solution, the purity of the purified glycyrrhizin can reach 40-50%, glycyrrhizin transformation reaction is carried out under the condition of sodium hydroxide backflow, and the result shows that the purity of the transformed 18-alpha glycyrrhizin is 87.66% and the yield is 44.16% (university of Hebei science and technology, 2014 academic paper). (5) The optimum process conditions for purifying glycyrrhizic acid by AB-8 type macroporous resin such as AB-8 macroporous resin Sunwaotao and the like are that the pH of a sample solution is 6.0, the mass concentration of the sample solution is 5.89mg/mL, the eluent is 50% ethanol solution, the purity of the glycyrrhizic acid is 52.3%, and the purity after recrystallization is 76.0% (food science, 2013, 34, 93-97). The Lujiao wine adopts a process route of establishing macroporous adsorption resin for separating and purifying glycyrrhizic acid, which is as follows: selecting AB-8 macroporous adsorption resin, wherein the concentration of a sample solution is 1.7 mg/mL, the flow rate of the sample solution is 2BV/h, the elution flow rate is 7BV/h, and the volume of an eluent is 8BV, adopting a gradient elution method, removing impurities by using 20% ethanol, and collecting 50% ethanol eluent as a product, wherein the purity of glycyrrhizic acid is 61.94% (Beijing university of chemical industry, academic thesis in 2010). The royal jelly and the like adopt AB-8 macroporous absorbent resin, the maximum sample loading of the licorice medicinal material is 0.75g medicinal material/mL resin, the best eluent is 50% ethanol, and the elution rate is 68.81%; the maximum sample loading amount of the D-101 liquorice medicinal material is 0.75g medicinal material/mL resin, the optimal elution solvent is 60% ethanol, and the elution rate is 64.67%; h-103, the maximum sample loading of the licorice medicinal material is 0.5g medicinal material/mL resin, the optimal elution solvent is 60% ethanol, and the elution rate is 51.18% (Chinese traditional medicine journal, 2006, 31, 295-297). (6) The technological conditions of refining glycyrrhizic acid with X-5 macroporous resin such as X-5 macroporous resin Geoqiong, etc. are that the concentration of glycyrrhizic acid aqueous solution is 8mg/mL, pH value is 6, adsorption flow rate is 2BV/h, and 90% ethanol is used for elution, and the purity of glycyrrhizic acid can reach 95.2% (Chinese patent medicine, 2006, 28, 794-796). (7) Screening the macroporous resin XDA by XDA macroporous resin Friedel-crafts and the like, wherein the optimization conditions are as follows: the pH value of the sample liquid is 5, the glycyrrhizic acid concentration is 1.5mg/mL, the total flavone concentration is 0.75mg/mL, the sample flow rate is 3BV/h, the eluent adopts 1.5BV/h 45% ethanol as the best, and 80% ethanol containing 1% sodium hydroxide is used for eluting at the flow rate of 1.5BV/h to obtain the glycyrrhizic acid, the yield of the total flavone is 69.7%, the glycyrrhizic acid yield is 52%, and the purity is 65.5% (modern traditional Chinese medicine research and practice, 2004, (S1), 45-50).
3.5.3 solid phase extraction column Liangxinji5634, etc. adopts hydrophilic SPE column, the glycyrrhizic acid extract is applied to the balanced SPE column, 2-20 times of column volume 50-80% (v/v) ethanol solution is used for leaching, finally, 4-10 times of column volume 20-80% (v/v) ethanol solution is used for eluting, one of hydrochloric acid, formic acid and trifluoroacetic acid is added into eluent, the final volume concentration is 0.1-10%, and the eluent is freeze-dried after rotary evaporation and concentration to obtain crude glycyrrhizic acid (CN 107759655A).
3.5.4 adsorbing the leaching solution of radix Glycyrrhizae with strongly basic ion exchange fiber column, eluting with water, ethanol or their mixture and water solution with concentration of 0.5-6.0 mol/L -1 The hydrochloric acid is prepared by the volume ratio of 2-10: 1, elution is carried out by 1-4 BV water, then elution is carried out by 2-12 times of column volume of eluent, eluent is collected, the elution temperature is 10-90 ℃, the flow rate is 0.2-10.0 BV.h -1 And drying the eluate to obtain purified extract powder (CN 102304165B) containing glycyrrhizic acid of Glycyrrhrizae radix.
3.5.5 reverse micelle Liu Hui et al fully mix nonionic surfactant micelle solution with glycyrrhizic acid leach liquor, adjust pH of mixed solution to 1.5-3.0 with inorganic acid, then heat to cloud point temperature and carry on the phase separation extraction, glycyrrhizic acid enriches in the micelle phase that nonionic surfactant forms at this moment, isolate micelle phase and use for reverse extraction, adopt water to dissolve the micelle phase obtained, adjust pH of mixed solution to 6.0-8.0 with inorganic base, heat to cloud point temperature phase separation again, obtain aqueous phase solution of enriching glycyrrhizic acid, aqueous phase solution obtained is precipitated by inorganic acid, get crude glycyrrhizic acid after drying (CN 101260137B).
3.5.6 molecular imprinting wuweibao, etc., using glycyrrhizic acid, different monomers (methacrylic acid, hydroxyethyl methacrylate and 4-vinylpyridine) and a cross-linking agent (divinylbenzene and ethylene glycol dimethacrylate) as raw materials, in a mixed solution of acetonitrile and N, N-dimethylformamide, using azobisisobutyronitrile as an initiator, preparing a series of Molecular Imprinting Polymers (MIPs) by a precipitation polymerization method, wherein in a 5g/L glycyrrhizic acid solution, the equilibrium adsorption amount of the MIPs to the glycyrrhizic acid can reach 398.5mg/g, the imprinting factor is 3.73, a licorice extract is refined by using a Molecular Imprinting Solid Phase Extraction (MISPE) technology, the mass fraction of the glycyrrhizic acid in the licorice extract is increased from 11.27% to 73.10%, and the recovery rate is 86.87% (chemical refinement, 2018, 35, 1859-1865+ 1870). The glycyrrhizic acid molecularly imprinted polymer is prepared by glycyrrhizic acid serving as a template molecule, hydroxyethyl methacrylate serving as a monomer, ethylene glycol dimethacrylate serving as a cross-linking agent, azodiisobutyronitrile serving as an initiator and N, N-dimethylformamide serving as a pore-forming agent, and the adsorption effect of the glycyrrhizic acid molecularly imprinted polymer in a methanol/water (2/8, v/v) solution is the best, the adsorption equilibrium concentration of the glycyrrhizic acid MIP is 2.0mM, the adsorption equilibrium time is 10min, and the maximum adsorption amount is 0.40 mmol/g.
3.5.7 the best extraction process condition is adopted for the basic solvent EtkAcc: adopting a hot reflux extraction mode, taking 0.1% ammonia water solution as a solvent, extracting for 2 times at a solid-liquid ratio of 1: 10 and a temperature of 85 ℃ for 3h, wherein the highest yield of glycyrrhizic acid reaches 9.05%;
the optimal purification process conditions are as follows: the glycyrrhizic acid is purified by an acid precipitation process, the concentration multiple is 6 times, the pH value is 1.5, the precipitation is 12 hours, the content of the glycyrrhizic acid crude product is 5.18 percent, and the yield reaches 4.08 percent (university of Lanzhou Ringchu, academic thesis of 2021 year). The Wu Si Jia and the like adopt the optimal extraction conditions as follows: the ammonia concentration is 0.64%, the ethanol concentration is 62%, the reflux time is 2.06h, and the liquid-solid ratio is 11.2: 1 (J.Med.Med.Med., 2019, 34, 1719-172). NaHCO 0.1% by von Neue et al 3 -50% etoh as solvent, the best conditions for extracting glycyrrhizic acid from glycyrrhiza uralensis is: the material-liquid ratio is 1: 10, the ultrasonic temperature is 90 deg.C, the ultrasonic time is 150min, and the extraction rate of glycyrrhizic acid pure product can reach 5.19% (human body)Study, 2013, 25, 35-38). Extracting Glycyrrhrizae radix with ammonia alcohol-complex enzyme method at enzymolysis temperature Tr =40 deg.C, complex enzyme dosage V = 25mL, solid-to-liquid ratio r = 1: 20, extraction time T =1.5h, leaching solution pH =8, extraction frequency n =3, extraction temperature T =45 deg.C, and leaching solution V (CH) 3 CH 2 OH)∶V(H 2 O) = 7: 3 ammonia water dosage V (NH 3. H2O) =0.6%, the extraction rate of the glycyrrhizic acid can reach 87.56%, and the extraction amount is 13.15 mg/g (university of Ranchun Ridgeon, academic paper 2014). Liyuhui and the like extract the glycyrrhizic acid in the glycyrrhiza glabra, and the optimal extraction conditions are as follows: 60% ethanol solution containing 0.6% ammonia water is used as extractant, the ratio of material to liquid is 1: 20 (w/v), the extraction is carried out for 2h under the condition of 75 ℃, and the yield of glycyrrhizic acid is 5.08% + -0.03% (research and development of natural products, 2011, 23, 547-550+ 546). The optimum extraction conditions of glycyrrhizic acid adopted by the Wanghai peak and the like are as follows: the extraction temperature is 40.28 deg.C, the extraction time is 5.84h, the concentration of ammonia water is 0.41%, the concentration of ethanol is 16.78%, and the extraction rate of crude glycyrrhizic acid reaches 66.40% (Chun university of Changchun, science edition, 2010, 31, 66-69). Zhang Jersen and the like take dark skinned liquorice produced by endosymus as a raw material, alcohol ammonia solution is used as a solvent, the microwave power is 250w, the microwave time is 7 min, the material-liquid ratio is 1: 15, the yield of the glycyrrhizic acid crude product is 12.9%, and the purity is 31.3% (food research and development, 2009, 30, 101-103). The best extraction process of glycyrrhizic acid comprises the following steps: reflux-extracting with 30% ethanol solution containing 0.45% ammonia for 3 times, adding 4 times of solvent for 1 time, reflux-extracting for 1.0h for 1 time, adding 3 times of solvent for 1.5 h for 2 and 3 times, respectively, concentrating the extractive solution, adjusting pH to 1.5, and drying the precipitate under reduced pressure to dryness (Liaoning chemical industry, 2009, 38, 620-622 +677). The best preparation process comprises reflux-extracting the medicinal materials with 13 times of 50% ethanol solution containing 0.5% ammonia for 3 times, adding 7 times of solvent for reflux-extracting for 30min for the 1 st time, reflux-extracting for 15min for the 2 nd and 3 rd times respectively with 3 times of solvent, concentrating the extractive solution to 1: 2 (medicinal material: medicinal liquid) (g/mL), adjusting pH to 1, precipitating, and drying under reduced pressure to dryness (Chinese patent medicine, 2007, 29, 686-689).
3.5.8 the acidic solvent Gaoshixiong takes wild licorice in Xinjiang as raw material, glycyrrhizic acid is extracted by using an ultrasonic-hydrogen peroxide method, hydrogen peroxide and acetic acid are added in the ultrasonic extraction process to form a peroxyacid system, the optimal extraction process is that the power is 124w, the extraction temperature is 68 ℃, the hydrogen peroxide content is 8.7 percent, the extraction time is 2h, and the maximum extraction rate after secondary extraction is 18.91 percent (2020, 41, 14-18 in food industry)
3.5.9 ultrasonic-assisted Nizhifeng et al uses wild licorice in Xinjiang as raw material, and the optimum technological conditions for ultrasonic extraction are 180w of extraction power, 70 ℃ of extraction temperature, 75min of extraction time, 1: 20 (g/mL) of solid-to-liquid ratio, 0.10 μm of membrane pore diameter, 0.1MPa of operation pressure and 30 ℃ of liquid medicine (2020, 41, 122-125 in food industry). The Luyuan determines the optimal process conditions for ultrasonic strengthening and leaching glycyrrhizic acid in liquorice: the leaching temperature is 60 ℃, the ultrasonic electric power is 125w, the ultrasonic frequency is 20kHz, the solvent is an ethanol solution with the volume fraction of 40%, the liquid-solid ratio is 400: 4mL/g, the concentration of glycyrrhizic acid in the balanced leaching solution is 1.536mg/mL, the ethanol solution with the volume fraction of 50% is used for recrystallization for 2 times, and the purity of the licorice crystal can reach 91.24% (Harbin engineering university, academic paper of 2012). The plum kindergarten and the like adopt the optimal process conditions of ultrasonic enhanced extraction: the temperature is 30 ℃, the ultrasonic electric power density is 80w/cm < 2 >, the ultrasonic action time is 90min, the acidification pH value is 1.0, and the average yield is 12.20% (scientific report of Gansu province, 2010, 22, 53-57).
3.5.10 microwave-assisted Chenyi and the like adopt a new microwave-assisted extraction process to extract glycyrrhizic acid from liquorice under the optimal process conditions: the solvent is a mixed solution of 50wt% ammonia and 30wt% ethanol, the solid-liquid ratio is 1: 12, the microwave power is 560w, the microwave time is 30s, the microwave frequency is 3 times, the licorice has fine powder particles, and the glycyrrhizic acid yield is 9.54% (Jiangxi chemical industry, 2006, (03), 66-68). The optimal process conditions for microwave-assisted extraction reported by taimen are as follows: adding mixed extractant (1/2 volume of 10% ethanol and +1/2 volume of 0.5% ammonia water), heating for 3 times with microwave power of 550w, wherein the heating time is 20s, and the average yield after optimization is 10.77% (scientific report of Gansu, 2010, 22, 53-57).
3.5.11 steam explosion technique Zhoumuna utilizes steam explosion to prepare licorice sweetening agent, promote glycyrrhizic acid to remove glucoside and convert it into high sweetener monoglucuronic acid glycyrrhetinic acid and high value product glycyrrhetinic acid, the extraction yield of glycyrrhizic acid and its conversion product after steam explosion reaches the maximum value within 2h extraction time, compared with raw materials, the extraction equilibrium time is shortened by more than 80%, and the diffusion coefficient D value is increased by more than 2 times, show that the extraction efficiency of glycyrrhizic acid and its conversion product after steam explosion is obviously enhanced (Tianjin science and technology university, academic paper of 2019).
3.5.12 Membrane technique Niugu over-determined the optimum refining conditions for glycyrrhizic acid as follows: the membrane aperture is 0.10 μm, the operating pressure is 0.10MPa, the liquid medicine temperature is 30 deg.C, and the purity of the refined glycyrrhizic acid product reaches 85.32% (Beijing, 2019, china university of Petroleum). The influence of pH change of the liquid medicine extract on the microfiltration process of the traditional Chinese medicine water extract by the inorganic ceramic membrane in the research of Pangyan and the like is adjusted, the pH value of the licorice water extract is adjusted to be 2, 3, 4, 5, 6, 7 and 8, and 0.2 mu m Al is respectively filtered under the conditions of constant temperature, pressure and membrane surface flow rate 2 O 3 The inorganic ceramic membrane is used for measuring the membrane stable flux and the glycyrrhizic acid transfer rate of index components of the liquorice water extract with different pH values, the membrane stable flux is increased remarkably around pH4, the membrane stable flux is the largest at pH7, the glycyrrhizic acid content is the highest, and the glycyrrhizic acid transfer rate at pH8 is the highest (world science and technology research and development, 2008, 30, 709-710).
3.5.13 optimal extraction conditions of glycyrrhizic acid such as Tanga acuta by organic solvent extraction are as follows: 60% of ethanol, naHCO 3 The addition of 0.4 percent, the liquid-material ratio of 15: 1, the extraction time of 2.5h, the extraction temperature of 60 ℃ and the average extraction rate of 33.27 percent (Shaoyang academy of sciences, version 2019, 16, 52-59). The optimum extraction process conditions of glycyrrhizic acid in Ningxia salt pond Ural licorice root studied by old soldiers and the like are as follows: the concentration of ethanol is 50%, the solid-liquid ratio is 1: 50, the extraction time is 4h, the temperature is 50 ℃, the extraction amount of glycyrrhizic acid in liquorice is 121.0073mg/g, and the extraction rate is 86.58% (Guangzhou chemical, 2018, 46, 53-55). The optimum process conditions for extracting glycyrrhizic acid by ultrasonic such as Shenhui and the like are that the ethanol concentration is 70%, the ultrasonic time is 30min, the material-liquid ratio is 2.8g/L, and the glycyrrhizic acid content is 8.34% (2016, 44, 167-170 in agricultural science of Anhui).
3.5.14 Ultrafiltration-complexation extraction technique Zhongbo, etc. adopts ultrafiltration-complexation extraction technique to prepare glycyrrhizic acid, and the optimum complexation extraction process conditions of the obtained glycyrrhizic acid are as follows: the pH value of the ultrafiltrate is 2, the extracting agent is trialkyl phosphine oxide (TRPO) -sulfonated kerosene (5: 95), the volume ratio of an organic phase to a water phase is 1: 1, the average extraction rate of glycyrrhizic acid reaches 99.2%, the optimal glycyrrhizic acid back-extraction process condition is that 22.5mmol/L sodium hydroxide aqueous solution is used as a back-extracting agent, the volume ratio of the organic phase to the back-extracting agent is 1: 1, the single back-extraction rate of glycyrrhizic acid reaches 98.8%, and the total glycyrrhizic acid transfer rate is 98.1% (Chinese herbal medicines, 2019, 50, 1323-1327).
3.5.15 enzymolysis litting adopts cellulase and pectinase to extract glycyrrhizic acid, the enzymolysis temperature is 40 ℃, the enzymolysis pH is 4.5, the enzyme addition is 5mL, the extraction temperature is 80 ℃, the feed-liquid ratio is 1: 15, and when the ethanol concentration is 60%, the extraction rate is 92.4% (Tianjin scientific and technical university, academic thesis in 2019). When the eucrypti takes Ural liquorice as a raw material and the liquorice raw material is 1g, the optimal process conditions in the extraction stage of the compound enzyme method are as follows: the ratio of the complex enzyme (pectinase: cellulase) is 1: 3 (U/U), the dosage of the complex enzyme is 175U/g.substrate, the pH is 5.5, the time is 1h, the temperature is 50 ℃, the granularity of the raw material is 40 meshes, and the ratio of the raw material to the liquid is 1: 25 (g/mL) (Tianjin university of science and technology, academic thesis in 2019). The technological conditions for extracting glycyrrhizic acid by using the optimized compound enzyme method of Longjiapeng and the like are as follows: adding 250mL of complex enzyme into 40g of liquorice powder, carrying out enzymolysis at 40 ℃, adding 70% ethanol according to the material-liquid ratio of 1: 20 (m/m), adjusting the pH value to 8, leaching for 1.5h, leaching for 3 times at 45 ℃ (the solvent volume ratio is 5: 4), wherein the extraction rate of the liquorice can reach 85.02% (pharmacy in China, 2014, 25, 626-629).
3.5.16 foam separation of tourmaline and the like, a liquorice-ephedra compatible foam separation process is developed, when the temperature is 40 ℃, the gas volume flow is 100 ml.min -1 Initial concentration of glycyrrhizic acid is 0.2 g.L -1 When the mass ratio of the glycyrrhizic acid to the liquorice and the ephedra is 5: 3, the obtained glycyrrhizic acid has the enrichment ratio and the recovery rate of 8.34 and 62.5 percent respectively, and the enrichment ratio of the glycyrrhizic acid is improved by 121.7 percent compared with the glycyrrhizic acid in the liquorice separated by independent foam (2016, 30, 1053-1059 in the chemical engineering report of colleges and universities). The optimum process conditions for separating glycyrrhizin from Suyan peach and the like by adopting intermittent foam are pH4, the mass concentration of glycyrrhizic acid in the feeding liquid is 0.23mg/mL, the air inlet speed is 600mL/min, the feeding volume is 1000mL,the recovery rate of glycyrrhizic acid in the foam phase is 91.9%, the enrichment ratio is 6.4, and the mass fraction of glycyrrhizic acid is 32.3% (Chinese herbal medicines, 2007, 38, 365-368).
3.5.17 flocculation Zhang Jianwei etc. research xi electric potential to the influence of licorice water extract flocculation effect, the flocculation rate of the liquid medicine is that the absolute value of xi electric potential of the system is lowest, reach the highest when closest to isoelectric point, the best technological condition for licorice water extract flocculation is that chitosan dosage is 0.347g/L, pH value is 6, temperature is 30 ℃, the xi electric potential of the system is-1.67 mV under this condition, closest to isoelectric point in all test groups, the flocculation rate of the liquid medicine is as high as 95.29%, xi electric potential of the raw liquid before flocculation is-14.78 mV, average particle size of particles in the liquid medicine is 17.12 μm, xi electric potential of supernatant after flocculation is-1.67 mV, average particle size of particles is 6.77 μm, change of xi electric potential changes particle behavior in the sol system, and finally influences flocculation rate of the liquid medicine, during licorice water extract process, xi electric potential has important influence on liquid medicine, the absolute isoelectric value is closer to isoelectric point, the more the lower the xi electric potential is provided for flocculation rate of the liquid medicine (the flocculation process is more powerful, therefore, the flocculation rate of the flocculation process is provided for Chinese herbal medicine) because the flocculation condition 1469-1469.
3.5.18 simulation of Mobile Longjiapene adopts MAR mixed bed of LZ-49+ LZ-51+ LZ-54+ LZ-66 with mass distribution ratio of m (LZ-19) to m (LZ-54: mLZ-66= 1: 1, and the absorption recovery rate F =80.00% of MAR to glycyrrhizin, desorption rate D =88.00%, absorption qa =80.15mg/g, desorption rate qe =70.53mg/g, purity can reach more than 85.00%; under the optimal process conditions of LZ-50+ LZ-59, the adsorption rate F of the adsorption of the mixed bed on licoflavone is 75.00%, the desorption rate D is 89.23%, the adsorption amount qa is 15.24mg/g, the desorption amount qe is 13.60 mg/g, and the purity can reach more than 80.00%, the optimal process conditions of adsorption of the mixed bed on stevioside with the best separation effect are that the pH of an adsorption solution is 6, the adsorption temperature is 35 ℃, the adsorption is 135min, the desorption solution is 60% ethanol, the desorption temperature is 40 ℃, and the purity of the obtained stevioside product can reach more than 92% (a university of Lanzhou Marigy, 2014 academic paper).
3.5.19 the electrostatic field synergistic ultrasonic method is used for extracting the Yangerfu and the like by adopting the electrostatic field synergistic ultrasonic method, and the most extraction conditions are as follows: the liquid-solid ratio is 30mL/g, the extraction time is 29min, the ultrasonic electric power is 100w, the static voltage is 9kV, and the extraction rate of glycyrrhizic acid is 11.02% (applying acoustics, 2014, 33, 160-166). The electrostatic field is introduced into the ultrasonic extraction of Chenvivu, so that the target extraction rate can be obviously improved, and the optimal technological parameters of glycyrrhizic acid in liquorice are obtained as follows: the solid-liquid ratio is 30mL/g, the extraction time is 28.92min, the ultrasonic power is 100w, the electrostatic voltage is 9.01kV, and the extraction rate predicted value of glycyrrhizic acid is 11.0764% (university of south China, university of academic 2012).
3.5.20 aqueous two-phase System Suchen et al adopts polyoxyethylene ether AEO-7/sodium phosphate aqueous two-phase system to extract glycyrrhizic acid, and the suitable conditions are as follows: when the volume fraction of AEO-7 is 10%, na 3 PO 4 The mass concentration of the glycyrrhizic acid is 47.50g/L, the pH value is 2, the extraction time is 1h, the extraction rate of the glycyrrhizic acid can reach 99.30%, and the distribution coefficient reaches 317.90 (food science and technology, 2014, 39, 237-241). Wu Jiang et al adopts two aqueous phases to extract glycyrrhizic acid from K 2 HPO 4 Adding a liquorice concentrated solution into a double-aqueous-phase extract consisting of EO60PO40 and distilled water, uniformly mixing, centrifuging for 10-15 min at the speed of 3000-6000 r/min, separating an upper phase and a lower phase, heating the upper phase containing glycyrrhizic acid in a constant-temperature water bath at the temperature of 45-60 ℃ for 0.5-2 h, and separating the lower phase to obtain glycyrrhizic acid (CN 108033991A).
3.5.21 extraction of pughua etc. extracting glycyrrhizic acid extract with ethyl acetate-dichloromethane mixed solvent, ethyl acetate-dichloromethane mixed solvent and ethyl acetate-dichloromethane mixed solvent in equal volume, regulating pH of mixed solution to 7.0-10.0, collecting water phase solution, adding active carbon to decolorize, regulating pH to 1.0-3.0, centrifuging, collecting precipitate, drying to obtain glycyrrhizic acid (CN 104262448B). The preparation method comprises the steps of purifying glycyrrhizic acid from leaching liquor by adopting an organic extractant, adjusting the pH value of the glycyrrhizic acid leaching liquor to 1.5-6.0, extracting by using a neutral phosphorus extractant or an alcohol extractant, enriching the glycyrrhizic acid in an organic phase, performing back extraction on the organic phase by adopting water, adjusting the pH value of an aqueous solution to 6.0-12.0 to obtain an aqueous phase solution enriched with the glycyrrhizic acid, adding active carbon to perform decoloration treatment on the aqueous phase solution enriched with the glycyrrhizic acid, and performing acid precipitation and drying to obtain refined glycyrrhizic acid (CN 1203085C).
3.5.22 the optimum process conditions for extracting glycyrrhizic acid at normal temperature under ultrahigh pressure, such as Zhang Jun, and the like, under ultrahigh pressure: the extraction time is 15min, the volume fraction of ethanol is 49.50%, the liquid-solid ratio is 13: 1, the extraction pressure is 382MPa, and the average extraction rate of glycyrrhizic acid is 14.67% (food science, 2009, 30, 75-79). 3.5.23 negative pressure cavitation method for extracting isoliquiritigenin from licorice by using supercritical carbon dioxide extraction technology, and negative pressure cavitation method for extracting glycyrrhizic acid from solid after supercritical extraction, and its optimum technological conditions are: 0.3% diluted ammonia water is used as solvent, the liquid-solid ratio is 10: 1, negative pressure cavitation extraction is carried out for 15min, the extraction rate of the glycyrrhizic acid is 2.0%, and the content is 6.6% (university of northeast forestry, 2005 academic paper).
3.6 Total Licorice flavones
3.6.1 macroporous resin adsorption method (1) HPD type macroporous resin Dongzhuang reports that the optimum purification process for HPD-100 type macroporous resin is: the concentration of the sample solution is 1.62mg/mL, the pH of the sample solution is 5, the sample loading speed is 1BV/h, the sample loading amount is 3.0BV, the desorption process is 70wt% ethanol elution, the eluent speed is 2BV/h, the elution volume is 2BV, the content of the total flavonoids in the overground part of the liquorice is improved to (36.5 +/-0.48)% from (6.8 +/-0.37%), and the total flavonoids content is improved by about 5.4 times (university of Anhui, 2021 academic thesis). The suitable conditions for adsorbing and desorbing licoflavone of HPD300 resin of inexpensive Yijun et al are: the concentration of the sample loading solution is 2.0mg/mL, the sample loading flow rate is 1.5BV/h, the sample loading amount is 2BV, the elution is carried out by 80% ethanol, the elution rate is 1.5BV/h, the dosage of the eluent is 3BV, and the purity of the obtained flavone is improved by more than 2 times compared with that before the purification (2013, 20, 49-52, chinese medicinal information journal). The optimal process for purifying licoflavone by HPD-BJQH macroporous resin reported by Han Asia male and the like comprises the following steps: the sample loading concentration is 2.15mg/mL, the sample loading amount is 9 BV, the pH of the sample loading solution is 5, the sample loading speed is 3BV/h,70% ethanol elution is carried out, the elution speed is 2BV/h, the elution volume is 3BV, and the purity of the purified total flavone is improved to 36.11% from the original 9.68% (Jilin traditional Chinese medicine, 2014, 34, 82-86). Zhanglin reports HPD-400 resin dynamic adsorption separation of light sweet The proper sample concentration of the pralidine extract is 0.845 mg/mL -1 The sample loading flow rate is 3 BV.h -1 The elution solvent is 70 percent ethanol, and the elution flow rate is 2 BV.h -1 The purity of glabridin can be improved from 4.5% to 21.8% (university of Henan science and technology, 2011 academic thesis). (2) The HZ-835 type macroporous resin Konwei and the like adopt HZ-835 type macroporous resin with the optimal process conditions as follows: the pH value of the loading liquid is 4.5, the mass concentration is 2mg/mL, the maximum loading amount of 7g of macroporous resin is not more than 40mg, 21.0mL of ethanol solution with the mass fraction of 10% and 10.5mL of ethanol solution with the mass fraction of 20% are sequentially used for washing the resin column, then 31.5mL of ethanol with the mass fraction of 70% is used for eluting, the flow rate in the adsorption-desorption process is 1mL/min, the yield of the macroporous resin after purification is 55.82%, the purity is 15.76%, which is 3.31 times of that before purification, the macroporous resin is used for adsorption after liquid-liquid extraction, and the purity can reach 35.80% (Xuzhou institute of engineering academy (Nature science edition), 2020, 35, 19-27). (3) ADS-7 type resin swiftlet et reports that the most suitable condition for purifying flavone by ADS-7 type resin is as follows: the pH of the sample solution is 7, the sample concentration is 8mg/ml, the sample flow rate is 2.5BV/h, the sample loading amount is 5BV, the concentration of ethanol for elution is 80%, the elution rate is 3BV/h, the dosage of eluent is 6BV, the content of the three batches of total flavone samples after purification is respectively improved to 48.59%, 47.38% and 45.26% from the original 10.32% (Shaanxi Chinese medicine, 2019, 40, 1471-1476). The optimum process for enriching 6 flavonoid components in licorice by using ADS-7 type macroporous resin is: the pH value of the sample is 4.5, the mass concentration of the sample is 0.20g/mL, the sample loading amount is 1.0g/g, the volume flow of the sample loading is 0.6mL/min, 83% ethanol with 6.7BV is eluted at the volume flow of 1.0mL/min, and the recovery rate of 6 flavonoid components after purification is 86% (Chinese herbal medicines, 2016, 47, 1118-1125). ADS-7 type macroporous adsorption resin is used for purifying licoflavone, the optimal sample concentration is 1.2mg/ml, the eluent is ethanol with the volume fraction of 5BV being 0.70, the flavone content of the purified flavone powder is 47.1%, and the refining rate is 211.2% (Anhui agricultural science, 2011, 39, 8364-8366). The Liuxiana determines the optimal adsorption and desorption process conditions of the resin ADS-F8: adsorbing pH7, adsorbing temperature 20 deg.C, flow rate 1.5mL/min, desorbing 15% ethanol solution 100mL, pre-eluting to remove impurities Then, 80% ethanol is used as an eluent, the flow rate is 1.5mL/min, the adsorption rate of the isoliquiritigenin is 75.69%, the desorption rate is 80.1%, the recovery rate of the isoliquiritigenin is 60.6%, the purity is improved from 3.55% to 27.77%, and the purity is improved by 7.82 times (northeast forestry university, 2006 academic paper). (4) D101 macroporous adsorption resin for Kantong selects D101 macroporous adsorption resin for refining the crude product of licoflavone, the sample loading is 2BV, the pH of the sample solution is 6.18, the ethanol concentration of the eluent is 60 percent, the using amount is 4BV, the yield after purification is 63.3 percent, and the flavone content reaches 66.8 percent (Tianjin scientific and technical university, academic paper of 2019). The conditions of the process for separating and purifying licoflavone by a Dengli report D101 macroporous adsorption resin method are as follows: a40% ethanol aqueous solution with the pH value of 7 is selected as a loading solution of licoflavone, the loading concentration is 0.9mg/mL, the loading speed is 2BV/h, 40% ethanol aqueous solution is firstly used for washing off part of impurities, and then 5BV 80% ethanol aqueous solution is used for elution, the elution speed is 4BV/h, and the licoflavone content is 40.3% (university of Lanzhou Lijian, 2011 academic paper). 10mL of glabrous greenbrier rhizome extract of Aoming Chao et al is put on a D101 type macroporous resin column at the speed of 2BV/h, 3BV water and low-concentration ethanol solution are used for elution to remove impurities, then 4BV90% ethanol is used for elution, the elution speed is 3BV/h, the glabridin percentage content in the eluate of 90% ethanol can reach about 45%, and the glabridin recovery rate is 67.79% (the ninth national discussion on medicinal plants and botanical pharmacology symposium in 2010). (5) The AB-8 type macroporous resin Lihongli and the like report that the preferable process conditions of the AB-8 type macroporous resin are as follows: the mass concentration of the sample liquid is 2.128 mg/mL -1 The pH of the sample solution is 5.42, the loading amount of the resin is 7mL/g, the loading speed is 1.5BV, the sample solution is sequentially eluted by 2BV water and 60 percent ethanol at 4BV, the purity of the total flavone is improved from 9.08 percent to 34.02 percent, and the yield is 74.40 percent (the university of traditional Chinese medicine in Hubei, 2017, 19, 39-42). The AB-8 type macroporous adsorption resin purification optimum process conditions reported by Luziming et al are as follows: the mass concentration of the sample solution is 0.2g/mL, the maximum sample amount per mL of resin is 2mL, the adsorption flow rate is 0.5BV/h, the pH of the sample solution is 6,6BV water is eluted and decontaminated at the flow rate of 1BV/h, 4BV 70% ethanol is eluted at the flow rate of 1BV/h, and the purity of the licorice total flavonoids in the eluate is 38% (J.J.Chinese Experimental Pathology, 2012, 18, 24-27). The best resin for screening Fuyujie et al is AB-8 para-isoliquiritigeninThe optimal adsorption and desorption technological parameters comprise that the adsorption pH is 5, the room temperature is realized, the flow rate is 1.5BV/h, the solution treatment capacity is 5BV, the eluent is 70% ethanol solution, the flow rate is 1BV/h, the dosage of the eluent is 4.5BV, the recovery rate of the isoliquiritigenin is 76.7%, the purity is improved from 2.02% to 29.1%, and the purity is improved by 14.4 times (ion exchange and adsorption, 2006, 22, 315-322). (6) SZ6 resin macroporous resin Lihongli reports that the SZ6 resin macroporous resin purified total flavone is sequentially eluted by 2BV water and 4BV 60% ethanol according to the mass concentration of 2.128 mg.mL < -1 >, the sample loading volume of 5BV and the sample loading volume flow of 1.5BV/h, the total flavone mass is improved from 9.08% to 34.42%, the purity is improved by 3.8 times, and the yield is 74.40% (Beijing university of traditional Chinese medicine, 2016 academic paper). (7) SP825 macroporous adsorbent resin Liujia reports that the optimal process for separating and purifying licoflavone by SP825 macroporous adsorbent resin comprises the following steps: SP825 macroporous adsorbent resin, wherein the pH value of the loading solution is 3, the loading concentration is 4.22mg/mL, the ethanol concentration of the eluent is 80%, the elution flow rate is 2mL/min, and the dosage of the eluent is 100g licoflavone crude extract, and the elution needs 15mL 80% ethanol (university of Lanzhou, academic paper of 2012). The Zhangxidong adopts SP825 resin which is more suitable for extracting licoflavone, a chromatography column with the diameter-height ratio of 15mm multiplied by 350mm is selected, wet-process sample loading is adopted, the licoflavone solution with the concentration of 2.5mg/mL and the flow rate of 3.0mL/min is fed with the total volume of 800mL is fed with ethyl acetate at the flow rate of 1.0mL/min for elution until the total volume is 360mL, the purity of the licoflavone can reach nearly 70 percent, and the final recovery rate can reach 83.5 percent (Tianjin university, 2009 academic paper). (8) XAD type macroporous resin xu Qing Lian et al report that XAD-16 macroporous resin separates licoflavone optimum adsorption condition: the initial mass concentration of the licoflavone feed liquid is 0.96 mg/mL, the loading amount is 20mg/g wet resin, the pH value of the feed liquid is 2.0, the desorbent is 80% ethanol, the desorption pH value is 14, the adsorption rate is 76.58%, the desorption rate is 61.84%, and the content of the licototal flavone is 55.10% (proceedings of Henan university of industry (Nature science edition), 2010, 31, 49-52). The XDA-1 macroporous adsorption resin reported in Li Xianchun et al has the optimized conditions for separating the licorice total flavone and glycyrrhizic acid as follows: the pH of the sample solution is 5, the glycyrrhizic acid concentration is 1.5 mg/mL, the total flavone concentration is 0.75mg/mL, the sample flow rate is 3BV/h, the eluent adopts 1.5BV/h 45% ethanol as the best, and then 1% sodium hydroxide-containing ethanol is used Eluting with 80% ethanol at a flow rate of 1.5BV/h to obtain glycyrrhizic acid with total flavone yield of 69.7%, glycyrrhizic acid yield of 52%, and purity of 65.5% (modern Chinese medicine research and practice, 2004, (S1), 45-50). (9) DA201 type macroporous adsorption resin Zhang Yuyan and the like adopt DA201 type macroporous adsorption resin to optimize the process conditions that sample liquid with the concentration of 0.6 time of stock solution with the volume of 7 times of the resin column is loaded, the flow rate is 2.5 BV/h, the adsorption time is 6h, and the adsorption time is 12BV is eluted by 70 percent of ethanol, and the yield of the active part of liquorice is 4.18 percent, wherein the active part contains 78.40 percent of glycyrrhizic acid and 17.51 percent of liquiritin (Chinese modern Chinese medicine, 2008, 10, 29-31). (10) The best adsorption and desorption conditions of HP-20 type macroporous adsorption resin are reported by the HP-20 type macroporous adsorption resin Zrui and the like, the maximum recovery rate of licoflavone in the licorice extract is 7.28%, and the content of licoflavone is 53.5% (Beijing university school (Nature science edition), 2006, 20, 20-22). (11) Celite resin Chen legimine and the like report that the Celite resin has high adsorption quantity to glabridin, is easy to elute, has good separation effect, has higher recovery rate than a common method, and has purity of more than 50 percent (Xinjiang traditional Chinese medicine, 2014, 32, 63-66). (12) Dissolving polyvinylpyrrolidone with the molecular weight of 5000-22000 in water to obtain a liquid resin extracting solution, wherein the content of the polyvinylpyrrolidone in the liquid resin extracting solution is 100-600 mg/L, crushing raw material glycyrrhiza glabra, wherein the proportion of coarse grass with filaments larger than 3mm in the crushed glycyrrhiza glabra is less than 10%, adding the crushed glycyrrhiza glabra into the liquid resin extracting solution, leaching for 24h, filtering, leaching once again, filtering and combining the extracting solutions, wherein the mass ratio of the glycyrrhiza glabra to the liquid resin extracting solution is 1: 5-10, adding a calcium chloride solution into the extracting solution until the calcium concentration is 0.02-0.06 mol/L, standing until a precipitate is separated out, filtering, adding an ethyl acetate aqueous solution into the precipitate, adding acid to adjust the pH value of the solution to 2.0, extracting, dissolving, standing for layering, taking an ethyl acetate layer, carrying out reduced pressure concentration on the ethyl acetate layer solution, and carrying out vacuum drying to obtain the glabridin with the content of more than or equal to 40% (CN 104072512B). 3.6.2 ion exchange chromatography separation Liqingtan et al report that the optimal separation process for separating glycyrrhizic acid and licoflavone by D941 is: the sample loading mass concentration is 1.0 mg/mL, the volume is 2.70L, the volume flow is 12.5mL/min, B The alcohol elution volume fraction is 70%, the volume is 1.80L, the volume flow rate is 12.50mL/min, the NaOH elution concentration is 0.50mol/L, the volume is 0.6L, the volume flow rate is 5.0mL/min, the purity of the prepared glycyrrhizic acid is 76.53%, the yield is 2.09%, the purity of the licoflavone is 67.33%, and the yield is 2.68% (university report of Dalian industry, 2019, 38, 24-28).
3.6.3 optimal separation technique for separating licoflavone from polyamide resin Liujia by using polyamide column is 30-60 mesh polyamide, pH value of sample liquid is 6, material-liquid ratio (licoflavone crude extract: polyamide) is 32.48 mg: 1g, and sample concentration is 3.215 mg.mL -1 Eluting with 70% ethanol at 3 mL/min -1 The eluent dosage is 100g of licoflavone crude extract, and 11mL of 70% ethanol is needed for elution (university of Lanzhou, academic paper of 2012). The optimal conditions for purifying licoflavone by polyamide resin chromatography are as follows: the concentration of the sample solution is 3.51mg/mL, the pH value is 10.0, 70% ethanol is used for eluting at the flow rate of 1.00mL/min, and the purity of the licoflavone can reach 49.75% through two times of chromatography (Guangdong agricultural science, 2011, 38, 85-87). Lijunsong, etc. is enriched by using polyamide adsorption column, and its purification process condition is that the licorice polyamide ratio is 2: 1 (g/g), resin diameter-height ratio is 1: 7, and it is eluted by using 70 ethyl alcohol whose column volume is 5 times that of said column, and the licorice flavone elution rate is about 90, and its content measurement average sample-adding recovery rate is 98.81 (Chinese patent medicine 2007, 29, 997-1000). Sun Yu and the like are subjected to crude extraction by using water extraction, alcohol extraction, chloroform extraction and other modes, and then purified and refined by using macroporous resin, polyamide column chromatography and other methods, so that the licochalcone A content of the obtained licochalcone purified product is 14.87% and 16.5% respectively (Xinjiang traditional Chinese medicine, 2019, 37, 45-47). The Zhengyunfeng and the like adopt optimized process conditions as follows: the pH of the liquid medicine is 7.0, and the mass concentration of liquiritin in the liquid medicine is 1.296 g.L -1 Loading the sample with volume of 3BV, eluting the adsorbed resin with water, 10%, 20% and 30% ethanol respectively, collecting the 20% ethanol eluate, recovering the solvent, and increasing the purity of liquiritin from 4.86% to 88.5% (J.CHINESE MEDICINE, 2013, 38, 3902-3906). Liyu can be purified by polyamide (60-80 mesh) chromatographic column (20 mm × 300 mm) with sample concentration of 25mg/mL and sample flow rate of 2mL/min, and eluting with 40% ethanol, and collecting 50% ethanolConcentrating part of the powder under reduced pressure to obtain thick paste, and freeze drying to obtain brick red powder with glabridin purity of 53.4% (university of south Jiangnan, 2011 academic paper).
3.6.4 organic solvent extraction of Triton et al takes hairy roots of Glycyrrhiza uralensis Fisch as raw material, the highest extraction rate of total flavonoids from hairy roots of Glycyrrhiza uralensis Fisch is 1.25% under the conditions of solvent of 75% propylene glycol, material-liquid ratio of 1 g: 15mL, ultrasonic temperature of 50 ℃ and ultrasonic time of 40min, the highest content of isoliquiritin in hairy roots of Glycyrrhiza uralensis Fisch is 3, and the highest content of total flavonoids, liquiritin and glabridin in hairy roots of Glycyrrhiza glabra Fisch is 2022, 50, 155-162 of Jiangsu agricultural science.
3.6.5 subcritical fluid extraction Wuhao and the like take licorice residues as raw materials, research on subcritical water extraction of licorice total flavonoids and oligosaccharides is carried out, the extraction conditions are that the grain diameter of the licorice residues is 250-425 mu m, the liquid-solid ratio is 15mL/g, extraction is carried out for 135min at 185 ℃, the extraction rate of the licorice total flavonoids is 2.81%, and the extraction rate of the oligosaccharides is 9.6% (Nanjing university school news (Nature science edition), 2021, 43, 25-31). Extracting 3-4 h with aqueous solution with pH value of 9-9.5, wherein the material-liquid ratio of glycyrrhiza glabra to the aqueous solution is 1: 8-1: 10, separating grass residue and extracting solution, retaining filtrate, repeatedly extracting for 4-5 times, drying the extracted grass residue for later use, placing the grass residue in a high-pressure extraction kettle, extracting with subcritical fluid 1, 2-tetrafluoroethane (R134 a), collecting the extraction product under the process conditions of extraction pressure of 10-12 MPa, extraction temperature of 45-50 ℃, extraction time of 80-90 min, dissolving the extraction product in 90-99% methanol or ethanol, adding water to dilute to 40-60% alcohol concentration, placing in a refrigerator of 0-5 ℃ for standing for 2-6 h, separating supernatant, decolorizing the supernatant with 10-20% silica gel decolorizing sand, decolorizing at decolorizing temperature of 30-40 ℃, preserving heat for 0.5-2 h, filtering and separating silica gel for decolorization, leaving supernatant for later use, purifying with supernatant resin, concentrating supernatant fluid with decolorizing sand of 10-20% of supernatant weight of supernatant into white sample (CN 2-55: 11255 h), and concentrating supernatant fluid sample (CN 2-2 h).
3.6.6 cloud point extraction palace sealSubjecting glycyrrhizin in Glycyrrhrizae radix to cloud point extraction with nonionic surfactant Triton X-100, adding Triton X-100 (5 g/100 mL), subjecting to cloud point extraction at 75 deg.C for 30min, and subjecting the glycyrrhizin obtained by extraction with surfactant Triton X-100 to average content of 0.307mg g -1 (strait pharmaceuticals, 2019, 31, 33-35). Tween 60 is selected as the optimal active agent for the Qian ginger and the like, the optimal process conditions are that the volume fraction of ethanol is 30%, the liquid-solid ratio is 35mL/g, the extraction time is 70s, the mass concentration of Tween 60 is 0.01g/mL, the microwave power is 390w, and the yield of total flavonoids is 4.30% (Nanjing university of industry science report (Nature science edition), 2020, 42, 671-676). The beta-CD microwave synergistic extraction of flavone from licorice root in Ningguang et al has the optimal extraction conditions: the liquorice, beta-CD (mass ratio) is 1: 0.8, the microwave power is 200w, the material-liquid ratio is 1: 30 (g/mL), the microwave extraction time is 150s, the volume fraction of ethanol is 60%, and the optimized result of a response surface method test is as follows: microwave power is 200w, microwave extraction time is 145 s, and feed-liquid ratio is 1: 32 (g/mL) (2016, 37, 114-118+173 for research and development of food). The prince sword takes sodium dodecyl sulfate as a surfactant, and adopts an ultrasonic microwave-assisted micelle extraction method to extract licoflavone, and the optimal technological parameters are as follows: the mass fraction of the sodium dodecyl sulfate is 2%, the liquid-material ratio is 21, the microwave power is 832w, the time is 10min, and the extraction rate of the licoflavone reaches 3.65%. (northeast forestry university, 2020 academic thesis).
3.6.7 purification of crude flavone by metal complex method Queen sword, the optimal technological parameters are as follows: the concentration of licoflavone is 2mg/mL, the mass ratio of licoflavone to calcium chloride is 1: 0.3, the pH of the solution is 10, the purity of the crude product of licoflavone is 63.56%, the recovery rate is 77.27%, and the contents of licochalcone and isoliquiritigenin are 0.81% and 1.46%, respectively (northeast forestry university, 2020 university paper).
3.6.8 ultrasonic extraction of Yuanqin and the like takes the total extraction amount of 4 flavonoid components of apioside liquiritin, apioside isoliquiritin and formononetin as an investigation index, the optimized extraction process is to take 50% ethanol as an extraction solvent, add 50mL of the medicinal material into 0.200g of the medicinal material and perform ultrasonic extraction for 50min, wherein the extraction amounts of apioside liquiritin, apioside isoliquiritin and formononetin are respectively 10.7330, 27.7849, 3.4419 and 0.4291mg/g, and the average total extraction amount of the 4 flavonoid components is 42.3889mg/g (pharmacy of China, 2019, 30 and 355-359). The best extraction process for extracting the liquorice total flavonoids by adopting an ultrasonic-assisted method by taking liquorice as a raw material and an alkaline ethanol solution as an extractant comprises the following steps: the liquid-solid ratio is 11, the volume fraction of ammonia water is 1.0%, the volume fraction of ethanol is 66%, the ultrasonic time is 20min, the ultrasonic temperature is 74 ℃, and the extraction rate of the licorice total flavonoids is 2.15% (Beijing university of chemical industry, school news, nature science edition, 2016, 43, 66-72). The optimum conditions for extracting the total flavonoids in the liquorice by adopting an ultrasonic method such as Jiahui are as follows: the volume fraction of ethanol is 70.5%, the ultrasonic time is 20min, the ultrasonic power is 328W, the liquid-material ratio (mL: g) is 20.8: 1, and the average yield of total flavonoids is 4.31% (university of Jilin, academic edition, 2008, 38 (S2), 293-298) Wangsun uses ultrasonic-assisted extraction technology of licoflavone to optimize as follows: the ethanol concentration is 64.38%, the extraction time is 21h, the ultrasonic power is 112.42W, the liquid-material ratio is 30.15: 1, and the maximum extraction rate is 6.56% (university of Hefei industry, academic paper of 2019).
3.6.9 Membrane filtration Zhu Yinhua and the like, wherein the optimal extraction conditions are 24 times of 0.75% ammonia water, the extraction is carried out for 3 times, each time is 60min, the average extraction rates of glycyrrhizic acid and liquiritin are respectively 98.3% and 72.3%, and the optimal ultrafiltration process parameters are as follows: under the conditions that the aperture of the inorganic ceramic membrane is 10nm, the pressure is 0.12MPa and the temperature is 25 ℃, the average retention rates of glycyrrhizic acid and liquiritin are respectively 99.3 percent and 98.9 percent, and the average impurity removal rate is 23.3 percent (Chinese herbal medicines, 2016, 47, 4173-4178). Under the conditions that the operation pressure is 0.6MPa, the operation time is 30min, the operation temperature is 25-30 ℃ and the dilution multiple of a crude extract is 3 times, the content of total flavonoids in licorice dregs purified by an ultrafiltration membrane technology is obviously increased, the average content is increased by 2.15 times, and the purity is increased by 25.12 percent (chemical and biological engineering, 2013, 30, 47-48).
3.6.10 three liquid phase flotation of Weiyun, etc. adopts a three liquid phase flotation method for separation and enrichment, 10mL of ethyl acetate is used as an upper phase, 10mL of PEG1000 aqueous solution (w: w = 1: 1) is used as a middle phase, parameters such as solution pH, salt concentration, nitrogen flow rate, etc. are adjusted to ensure that liquiritin, apioside liquiritin and glycyrrhizic acid are enriched in a PEG phase, and Wularg glycyrrhizin is enriched in an ethyl acetate phase, and then preparative high performance liquid chromatography is adopted to sequentially separate and purify liquiritin, apioside liquiritin, glycyrrhizic acid and Wularg glycyrrhizin from flotation products (30 th academic annual meeting abstract collection of national society in 2016).
3.6.11 two-aqueous phase extraction of Chenlijuan and the like adopts the optimal extraction process that the mass concentration of ammonium sulfate is 0.40g/mL, the volume fraction of ethanol is 70%, the solid-liquid ratio is 1: 25, the extraction time is 3min, the microwave power is 300W, the average extraction rate of the total flavonoids of licorice is 2.038%, and the two-aqueous phase extraction rate is 94.46% (pharmacy 2016, 27, 525-528 in China). Liangshiying and the like take liquorice as raw materials, and the optimal process conditions for extracting and separating the total flavonoids of the liquorice by coupling ultrasonic and an ethanol-ammonium sulfate aqueous two-phase system are as follows: the feed-liquid ratio is 1: 35, the alcohol-water ratio is 0.7: 1, the mass concentration of ammonium sulfate is 0.25g/mL, the extraction time is 40min, and the yield of the licorice total flavonoids is 1.96% (food research and development, 2015, 36, 101-104). PEG/(NH) is adopted by Liaoping et al 4 ) 2 SO 4 Double aqueous phase system extraction of effective components from licorice (chemical research and application, 2005, 17, 230-232)
3.6.12 microbial fermentation Xuchunshan, etc. adopts enzyme method and microbial method to pretreat licorice root dregs, the synergistic effect of cellulase and xylanase is best, the flavone yield of Phanerochaete chrysosporium treated 12d is highest and reaches 45.38mg/g (food industry science and technology, 2015, 36, 222-226). The theory optimal combination of Zhangqin and other methods for treating flavone yield by adopting Phanerochaete Chrysosporium (PC) and cellulose decomposition fungus Q59 is as follows: inoculating 0.5mL of PC (polycarbonate) bacteria and Q592mL into every 10g of licorice residue, wherein the fermentation temperature is 30 ℃, the water content of a fermentation material is 61%, the concentration of ammonium tartrate serving as a nitrogen source is 0.4g/L, the fermentation time is 3d, and the predicted flavone yield is 1.59%; the theoretical optimal combination of the yield of the flavonoids treated by the coriolus versicolor and the Q59 comprises the following steps: inoculating 5 coriolus versicolor blocks with the diameter of 0.6cm into each 10g of licorice residue, inoculating Q592mL, fermenting at 28 ℃, wherein the water content of the fermented material is 67%, the nitrogen source concentration is 0.1g/L, the fermentation time is 3d, and the predicted flavone yield is 1.25% (food science and technology, 2012, 37, 229-232). The flavone yield of plum-British guest is 0.89% and 0.87% after fermentation with white rot fungus and cellulose decomposing fungus, respectively, which is higher than that of flavone obtained by direct ethanol extraction method The yield is improved by 34.85 percent and 31.82 percent; the white rot fungi and the cellulose decomposition fungi are mixed and fermented, the flavone yield reaches 1.32 percent, and compared with the ethanol direct extraction method, the flavone yield is improved by 100 percent and is respectively 48.31 percent and 51.72 percent higher than the single-fungus fermentation of the white rot fungi and the cellulose decomposition fungi (2010, 31, 156-159 percent of food research and development). The optimum enzyme extraction process for extracting free total flavone from licorice residue by cellulase and pectinase wall-breaking technology comprises the following steps: the solid-liquid ratio (mass-volume ratio) is 1: 50, 50 U.mL -1 The dosage of cellulase is 0.8mL (per gram of licorice root residue), 120U g -1 30mg of pectinase (per gram of licorice root residue), 95 percent of ethanol by mass, 50 ℃, 3 hours of enzymolysis time and 4.5 of pH value (chemical and biological engineering, 2008, 25, 49-51). The technological parameters of the process for extracting the isoliquiritigenin in the liquorice by the cellulose hydrolysis method of the Liuxiana are as follows: pH7 (natural water), enzyme concentration of 0.4mg/mL, enzyme catalysis time of 48h, enzyme solution volume to raw material of 8: 1 (mL/g), shaking incubator (shaking speed) of 100r/m, isoliquiritigenin extraction rate of 2.98 per mill, isoliquiritigenin content in extract of 3.32%, after hydrolysis of licorice raw material by enzyme method, the optimized technological conditions of ethanol ultrasonic extraction of isoliquiritigenin are as follows: extracting with 80% ethanol at a liquid-solid ratio of 10: 1 for 10min under ultrasonic conditions for 3 times with an extraction rate of 3.11% and isoliquiritigenin content in the extract of 3.55% (university of northeast forestry, 2006 academic paper). Gujie uses amygdalase to hydrolyze liquiritin to obtain crude liquiritigenin, and uses Sephedex LH-20 column to separate and purify to obtain liquiritigenin with purity more than 97% (Nanjing university of traditional Chinese medicine, 2014 academic paper), and carries out mixed bacteria solid fermentation pretreatment on licorice dregs to make flavone easier to extract, and takes eutectic solvent (such as ethylene glycol and tetrapropylammonium bromide) suitable for separating licorice dregs flavone, and finally obtains liquiritigenin with purity up to 95% through separation and purification (application publication No. CN 114591283A). Crushing liquorice, sterilizing, adding water, mixing uniformly to obtain a solid fermentation culture medium, wherein the ratio of the dry weight of the liquorice to the volume of the water is 1g to (1.0-1.2) mL, inoculating mutagenized aspergillus niger strains in the solid fermentation culture medium, then carrying out solid fermentation culture, and carrying out liquiritigenin enrichment on microorganisms (application publication No. CN 108588140A).
3.6.13 microwave extraction of Yulei, etc. with licorice root and rhizome as material, the best extraction process for extracting flavonoid from licorice root is microwave treatment strength of 600W for 3min, ethanol concentration of the extracted liquid is 70%, material-to-liquid ratio is 1: 25g/mL, and flavonoid yield is 61.17mg/g (northern horticulture, 2014, (07), 130-132). Malbiose and the like take ethanol solution as solvent to extract liquiritin in liquorice in a microwave-assisted manner, and the optimal process conditions are as follows: the microwave radiation time is 40min, the material-liquid ratio is 1: 12 (g: mL), the ethanol concentration is 40%, and the microwave power is 640W (Spectroscopy laboratory, 2009, 26, 1409-1412).
3.6.14 ultrahigh pressure simultaneous extraction Vanli and the like adopt the optimal ultrahigh pressure extraction conditions as follows: the extraction pressure of 60% ethanol is 500MPa, the extraction time is 3min, the material-liquid ratio is 1: 40 (g/mL), and the extraction rates of glycyrrhizic acid and glabridin in Glycyrrhiza glabra L.can respectively reach 49.84mg/g and 1.05 mg/g (food technology, 2013, 38, 214-218).
3.6.15 supercritical CO 2 The extraction temperature of Zhao De Sheng and the like is 50 ℃, the extraction pressure is 32MPa, the extraction time is 1.5h 2 The extraction effect is best when the flow is 20kg/h and the entrainer is 90% ethanol (Jiangsu agricultural science, 2012, 40, 213-215). The optimum technological parameters adopted by the Paiyije and the like are as follows: adopting 40-60 mesh raw material, 80% ethanol as entrainer, extracting for 1.5h at extraction pressure of 30.0MPa and extraction temperature of 50 deg.C with CO 2 Flow 10kg h -1 The separation pressure is 5.8MPa, the separation temperature is 40 ℃, the extraction rate of the liquorice total flavonoids is 2.09%, and the content is 5.42% (plant research, 2007, 27, 372-375). The optimum process conditions for the extraction of the crude extract of the liquorice and the glabridin by Rong, hongcong and the like are as follows: the extraction temperature is 45 deg.C, the extraction pressure is 25MPa, the static extraction time is 60min, the crude extraction yield is 1.259%, wherein the glabridin yield is 0.009% (applied to chemical engineering, 2018, 47, 1166-1169).
3.6.16 the best extraction conditions of the royal bell by the rapid solvent extraction method are as follows: the extraction time is 7min, the extraction temperature is 120 ℃, the extraction times are 1 time, and the ethanol concentration is 70% (university of Catharan, 2011 academic paper).
3.6.17 alkaline alcohol solution Liyue will select the best extraction conditions for extracting glabridin and glycyrrhizic acid from ammonia alcohol solution: 60% ethanol solution containing 0.6% ammonia water as extractant, with a material-to-liquid ratio of 1: 20 (w/v), extraction at 80 deg.C for 2h, yield of glabridin of 0.238%, yield of glycyrrhizic acid of 5.08%, content of glabridin of extracted product of 6.30mg/G, and content of glycyrrhizic acid of 0.134G/G (university of south Jiangnan, academic thesis of 2011). The Yanghui and the like adopt the optimal process conditions that: the solvent is a mixed solution with the mass ratio of alkaline to alcohol of 1: 2 (NaOH concentration: 0.4mol/L; ethanol concentration: 75%), the solid-liquid ratio is 1: 30, the reflux time is 3h, the reflux temperature is 95 ℃, and the flavone content can reach 4.210% (Jiangxi chemical industry, 2006, (03), 90-92). Zhao 31054, radium, etc. adopts ammonia ethanol as the best extraction solvent, 60% ethanol of 0.3% ammonia water in 5 times of the amount of the medicinal materials is heated and refluxed for extraction for 4 times, 2h each time, the purity of purified glycyrrhizic acid is higher than 85%, the total extraction rate is 43%, the content of purified liquiritin is higher than 15%, and the total extraction rate is 67% (pharmacy in China, 2009, 20, 426-429).
3.6.18 high-voltage pulse electric field Zhaojing brightness, etc. adopts optimum extraction process and electric field strength is 20kv cm -1 The pulse number is 10, the ratio of material to liquid is 1: 10, the liquiritin extracted from the liquorice reaches 4.5 percent, and the paste rate reaches 17.982 percent (the modern traditional Chinese medicine research and practice, 2010, 24, 50-52).
3.6.19 Multi-walled carbon nanotubes Zhaohai et al, which use oxidized Multi-walled carbon nanotubes (o-MWCNTs) have greater adsorption capacity for two compounds than for original Multi-walled carbon nanotubes (r-MWCNTs), especially for isoliquiritigenin, o-MWCNTs have greater adsorption capacity for isoliquiritigenin and Liquiritigenin than for r-MWCNTs, and the desorption efficiency is better, the desorption rates for isoliquiritigenin and Liquiritigenin are 48.57% and 32.86%, respectively, while r-MWCNTs have only 24.56% and 17.46% (Journal of Chinese medicinal Sciences,2010, 19, 443-450).
3.6.20 simulated moving bed Wangshaoyan et al, in which octadecylsilane chemically bonded silica (ODS) is used as a stationary phase and V (ethanol) = V (water) = 30: 70 is used as a mobile phase, a Simulated Moving Bed Chromatography (SMBC) system is used for continuously separating liquiritin and liquiritigenin in licoflavone, and the SMBC system is provided with an elution zone I:1 chromatographic column, refining zone II:2 chromatographic columns; the adsorption zone III,2 chromatographic columns, zone I is independent, the eluent of zone I is ethanol, and the result shows that: selecting SMBC operation conditions close to the bottom edge of the triangle, obtaining liquiritin at an extract outlet, wherein the HPLC purity is higher than 70.44%, the yield is higher than 95.17%, simultaneously obtaining liquiritigenin at an extract outlet, the HPLC purity is higher than 73.10%, the yield is higher than 90.74%, SMBC can effectively separate liquiritin and liquiritigenin, and the HPLC purity of two products is higher than 96% after liquiritin extract and liquiritigenin raffinate are respectively concentrated and recrystallized (fine chemical engineering, 2017, 34, 1260-1264). The Liquirolith and the like adopt a simulated moving bed chromatography technology to purify liquiritin, and the optimal conditions of liquid-solid extraction of raw materials are as follows: water is used as an extraction solvent, the mass concentration of raw materials is 40g/L, the raw materials are extracted for 2.0h in a constant-temperature water bath at 90 ℃, the simulated moving bed chromatography is performed at the sample injection flow rate of 0.1mL/min, the elution flow rate of 1.5mL/min, the extraction flow rate of 1mL/min, the switching time of 2021min, the mass concentration of a sample is 0.2g/mL, the mobile phase V (ethanol):V (water) = 15: 85, and the working mode is as follows: under the condition of 1-1-2 (namely the number of chromatographic columns of an elution zone, a rectification zone and an adsorption zone in the three-zone simulated moving bed chromatography is respectively 1,1,2), the fine separation of the liquiritin can be realized (fine chemical engineering, 2005, 22, 912-915).
3.6.21 flash extraction method adopted by Deng Yimei adopts flash extraction method with short extraction time and less solvent consumption, the optimal extraction process of orthogonal experimental method is that solid-liquid ratio is 1: 40, ethanol concentration is 70%, extraction time is 6min, and the optimal process conditions of response surface method are as follows: the ethanol concentration is 70.2%, the liquid-solid ratio is 40.3: 1, the extraction time is 5min, and the extraction rate of total flavonoids under the process condition is as high as 2.91% (university of China and south, 2008 academic paper)
3.6.22 semi-bionic extraction of Chengni celery and the like adopts the semi-bionic extraction process conditions: the pH value of the water for decocting the three times is 5.88, 7.50 and 8.90 in sequence, the total time for decocting the three times is 3.93h, and the technological conditions of an SBE method are determined by combining the practical industrial production: the pH value of the water for decocting the three is 6.0, 7.5 and 9.0 in sequence; the decoction time is 2, 1 and 1h (Chinese medicinal materials, 2007, 30, 598-601) in sequence.
3.6.23 high-speed countercurrent chromatography high bud, etc. to separate four licorice flavonoids, namely licoflavonol, liquiritigenin, formononetin and licorice isoflavone A, from crude extract of licorice produced in Xinjiang through high-speed countercurrent chromatography, wherein a solvent system is n-hexane-ethyl acetate-methanol-water (1: 2: 1), an upper phase is used as a stationary phase, a lower phase is used as a mobile phase, a main machine rotates clockwise, the rotating speed is 850rpm, the flow rate is 4.0mL/min, the detection wavelength is 260nm, and the licoflavonol, the liquiritigenin 8mg, the formononetin 12mg and the licorice isoflavone A10 mg are obtained from 400mg of crude extract of licorice through 160min separation, and the purity is 96.3%, 95.7%, 98.5% and 98.8%, respectively (Sichuan chemical industry, 2007, 10, 34-37 50). The Wangqiao' e adopts HSCCC to separate, purify and prepare licochalcone A and swollenin A, the adopted solvent system is n-hexane-chloroform-methanol-water (5: 6: 3: 2), the upper phase is stationary phase, the lower phase is mobile phase, and the separation parameters of HSCCC are as follows: the working temperature is 25 ℃, the rotating speed of a main machine is 800r/min, the flow rate of a mobile phase is 1.0mL/min, the purity of licochalcone A and the purity of licocarvacrol A obtained by one-time separation are 95.0% and 97.8% respectively, the licochalcone A and the licocarvacrol A are purified continuously by an HSCCC method, a solvent system adopted for purification is n-hexane-chloroform-methanol-water (1.5: 6: 3: 2), the upper phase is a stationary phase, the lower phase is a mobile phase, the flow rate of the mobile phase is 1.0mL/min, the working temperature is 25 ℃, the rotating speed of the main machine is 800r/min, and the purity of the licochalcone A and the licocarvacrol A after purification is 99.1% and 99.6% respectively (university of Xiamen, 2004 scientific position). Yuanqiang et al roughly separated the licorice ethyl acetate extract by polyamide column, further separated the 30% ethanol eluate by high speed counter current chromatography, the two-phase solvent system used was ethyl acetate-water (5: 5, v/v), the rotation speed was 850rpm, the flow rate was 2.0mL/min, the detection wavelength was 254nm, and 8.7mg of glycyrrhizin and 4.2mg of formononetin were obtained from 50mg of 30% ethanol eluate, the purity was 99.5% and 97.3% respectively (research and development of natural products, 2011, 23, 1148-1150). The ethyl acetate extract of licorice is first separated through polyamide column chromatography to obtain component Fr1, which is further separated through high speed countercurrent chromatography, and the two-phase solvent system includes ethyl acetate-water (5: 5, v/v), upper phase as the stationary phase and lower phase as the mobile phase, and has rotation direction of clockwise main machine, rotation speed of 850r/min, flow rate of 2.0mL/min, detection wavelength of 254nm, and 30.3 mg of formononetin with purity of 99.3% obtained from 80mg of component Fr1 (Shandong science, 2010, 23, 18-21). Crushing licorice residue, ultrasonic extracting with 95 vol% ethanol, filtering, concentrating to dry to obtain coarse licorice residue extract, and high speed countercurrent chromatography with solvent system comprising dichloromethane, acetone, n-butanol and water in the volume ratio of 7 to 5 to 8 to 4, with the upper phase as the mobile phase and the lower phase as the stationary phase; dissolving the crude extract of licorice residue with mixed solvent of upper phase and lower phase at equal volume ratio to obtain sample solution, dissolving proper amount of licorice residue in 1, 2-propylene glycol at flow rate of 3mL/min, 25 deg.C and rotation speed of 850r/min, adding water in four times of 1, 2-propylene glycol volume, shaking gently, standing at 4 deg.C, collecting precipitate, washing with 4 deg.C pre-cooled acetone, repeating crystallization for 1 time, washing with 4 deg.C pre-cooled acetone, and blow-drying to obtain glabridin (application publication No. CN 110551137B).
3.6.24 best scheme for extracting total flavone from radix Glycyrrhizae, such as royal jelly by alkali extraction, is as follows: 20g of licorice residue powder, 20.8g of calcium hydroxide, 20: 1 of liquid-solid ratio, 2.5 of pH value of acid precipitation, 2 hours of extraction time, and 2.56 percent of maximum extraction rate of total flavonoids of licorice (proceedings of Zhengzhou academy of engineering, 2004, 25, 61-63). 3.6.25 Complex extraction Sunxianthus and the like, and the isoliquiritin is prepared by extracting and back-extracting from the liquorice ultrafiltrate, and the optimal complex extraction conditions are that the extraction rate of trialkyl phosphine oxide (TRPO) -sulfonated kerosene (7: 93) on the isoliquiritin reaches 97.60 percent, the optimal back-extracting agent is 0.26 percent of sodium hydroxide aqueous solution, and the back-extracting rate of the isoliquiritin reaches 95.40 percent (Chinese herbal medicines, 2019, 50, 4920-4924). The optimum complex extraction process conditions of Zhongbo et al by using liquiritin are TRPO-sulfonated kerosene (9: 91), the pH value of liquiritin ultrafiltrate is 4, the volume ratio of organic phase to aqueous phase is 1: 1, the average extraction rate of liquiritin reaches 99.6%, and the back extraction process researches show that under the condition that the volume ratio of organic phase to back extraction agent is 1: 1, 17.5mmol/L sodium hydroxide aqueous solution is the optimum back extraction agent, the back extraction rate of liquiritin is 99.3%, and the total transfer rate of liquiritin is as high as 98.9% (Chinese herbal medicines, 2019, 50, 1323-1327'). In Tianqing et al, a trialkyl phosphine Oxide (TRPO) petroleum ether solution is used as an extraction organic phase, water-soluble licoflavone is extracted and separated from a liquorice leaching liquor, the total flavone extraction rate is gradually reduced along with the increase of a pH value within the range of pH 5-8, the extraction rate of glycyrrhizic acid is rapidly reduced along with the increase of the pH value within the range of pH 5-6, the total flavone extraction rate is almost reduced to 0 above pH6, the total flavone extraction rate is reduced along with the increase of a phase ratio, the extraction rate of the total flavone is rapidly improved along with the increase of an extractant concentration, the extraction rate of the total flavone is reduced along with the increase of temperature, the reaction of extracting the flavone is an exothermic reaction, the extraction ratio of the TRPO extracting liquiritin is 3, and the separation of the water-soluble licoflavone and glycyrrhizic acid can be realized by a solvent extraction method (proceedings of the process engineering, 2007,7, 496-500).
3.6.26 flash extraction of Xuezjun et al the optimal conditions for flash extraction of licorice total flavone from licorice residue are 90% ethanol, material to liquid ratio (g/mL) is 1: 40, extraction time is 3min, rotation speed is 16000r/min, 35.71mg of liquiritin is separated from 90mg of licorice total flavone, purity can reach 94.7%, and yield is 87.8% (Chinese patent medicine, 2016, 38, 72-76).
3.6.27 Ionic liquid extraction Lixiayue, etc. the influence of the alkalinity and hydrophobicity of the ionic liquid on the extraction performance of glabridin is examined, and the alkaline hydrophobic ionic liquid [ Hmim [ ]][N(CN)2]The extraction rate of the glabridin is the highest and can reach 87.27 percent (the university of stone river, the science edition, 2015, 33 and 145-148). Selective hydrophobic ionic liquid [ C4mim ] of plum-snow organ and the like][PF6]The optimal extraction process conditions for extracting glabridin are as follows: phase volume ratio of 1: 2.5 (v/v), pH 7, extraction temperature of 45 deg.C, extraction time of 30min, extraction rate of glabridin of 85.49%, and ionic liquid regeneration of 2 mol.L -1 The recovery rate of the glabridin obtained by using a mixed solution of sodium hydroxide and absolute ethyl alcohol as a back extraction agent is more than 90 percent, the ionic liquid is recycled for 5 times, and the extraction rate is not obviously reduced (2013, 25, 169-173). Mixing temperature response type ionic liquid and Glycyrrhrizae radix powder, extracting, separating solid and liquid, collecting supernatant, adding sodium hydroxide solution, heating to 65-80 deg.C to obtain liquiritin-containing ionic liquid phase and glycyrrhizic acid-containing water phase, and separating water phase and ionic liquid phase (application publication No. CN 113603737A). Mixing Glycyrrhrizae radix powder with pure ionic liquid, ultrasonic extracting, centrifuging, and collecting supernatant to obtain optimal ionic liquid [ C8MIM ]BF4, the best extraction conditions are: the solid-liquid ratio is 28.31mL/g, the extraction time is 32.77min, the extraction temperature is 92.60 ℃, the soaking time is 9.83h, and the obtained productThe four compounds isoangustone A, glycycoumarin, glycyrrexate and glycyrrexate in the product have content of 45.6, 346.9, 214.9 and 224.5 μ g/g respectively (application publication No. CN 110028472A).
3.6.28 the supported liquid membrane plum is characterized in that the highest extraction rate of [ Hmim ] [ N (CN) 2] alkaline ionic liquid to glabridin can reach 87.27%, the [ Hmim ] [ NTf2] is selected as membrane solution, a flat PVDF hydrophobic membrane is selected as a base membrane, the supported liquid membrane is prepared by adopting a pressurization method, the feasibility and the process conditions of separating and purifying the glabridin by the supported liquid membrane are researched, the influence of the aperture, the rotating speed, the concentration of raw material liquid and the concentration of reverse solution on the extraction of the glabridin is examined, and the result shows that: when the pore diameter is 0.22 mu m, the rotating speed is 260rpm, the concentration of the glabridin in the raw material liquid is 0.25mg/mL, the concentration of the sodium hydroxide on the back extraction side is 0.1mol/L, and the extraction time is 20h, the highest extraction rate and the highest recovery rate are 85.65 percent and 85.29 percent respectively, which indicates that the ionic liquid supported liquid membrane can be used for glabridin extraction (the university of Hezi, 2014 university paper).
3.6.29 eutectic solvent propionic acid and choline chloride are mixed and stirred to prepare eutectic solvent, the mixture is extracted under the action of ultrasound, the mixture is filtered and the extract is collected to obtain glabridin crude extract, the obtained crude extract is extracted by using an extractant ethyl acetate, an extractant phase is subjected to gradient rotary evaporation, dissolved by ethanol, and macroporous resin is selected for separation and purification to obtain glabridin extract; adding an ethanol solution into the eutectic solvent for sedimentation and centrifugation to obtain sediment and supernatant, and collecting the sediment to obtain xylooligosaccharide; performing rotary evaporation on the supernatant until no ethanol exists to obtain a concentrated solution, adding deionized water for sedimentation, centrifuging to obtain a precipitate and a supernatant, and collecting the precipitate as lignin; the supernatant was further passed through a reverse osmosis membrane to remove water, and the eutectic solvent was recovered (application publication No. CN 114685523A). Mixing choline chloride and propionic acid, stirring at room temperature for 30-60 min to obtain an eutectic solvent, adding the extraction solvent consisting of the eutectic solvent and water into licorice root residue, heating to 50-90 ℃ for extraction to obtain an extraction solution and a solid residue of the flavonoid compounds in the licorice root residue, washing the solid residue with pure water to be neutral, drying, adding the solid residue into a buffer solution containing cellulase for enzymolysis to obtain a glucose solution, washing the solid residue after enzymolysis to be neutral with pure water, drying, putting the solid residue into a high-pressure reaction device, adding distilled water for cooking, filtering and drying to obtain the licorice root residue protein feed (application publication No. CN 114657226A).
3.7 Glycyrrhiza polysaccharide
Zhang XIAO JING adopts alkali-extracted polysaccharide, ethanol precipitation with ethanol, deproteinization by sevag method, pigment removal by AB-8 macroporous adsorbent resin, dialysis by 1000D dialysis bag, and vacuum freeze drying to obtain Glycyrrhrizae radix residue alkali-extracted crude polysaccharide with polysaccharide content of 67.82 + -0.21% and yield of 2.33%, and purifying by DEAE and Sephadex G-150 step by step to obtain polysaccharide content of 93.29 + -0.25% and yield of 0.17%, and is named as AGP; AGP molecular weight is 2.89 × 106Da by high performance liquid chromatography; monosaccharide component analysis shows that AGP is composed of rhamnose, arabinose, xylose, mannose, glucose and galactose at a relative molar ratio of 1: 2.33: 2.85: 0.69: 3.05: 1.54; periodic acid oxidation and smith degradation, fourier infrared spectroscopy, methylation, nuclear magnetic resonance and other analysis methods show that the AGP glycosidic bond is connected by (1 → 6) linked glucose as a main chain, (1 → 4) linked xylose, (1 → 5) linked arabinose, (1 → 6) linked galactose, (1 → 3) linked rhamnose, (1 → 3, 6) linked mannose as a branch chain, and (1 →) linked glucose as a terminal (Tianjin scientific university, 2020 academic thesis). The ficus pumila produces water-soluble crude glycyrrhiza glabra polysaccharide with the polysaccharide content of 51.09 percent, the crude glycyrrhiza glabra polysaccharide is purified by Sephadex G-200 to obtain pure glycyrrhiza glabra polysaccharide which is named as GPS, the purity of the polysaccharide is 91.00 percent, the yield of the polysaccharide is 0.33 percent, the molecular weight of the crude glycyrrhiza glabra polysaccharide is 1.96 multiplied by 103kDa, the result shows that the specific rotation of the GPS is +20 degrees, the polysaccharide consists of arabinose, glucose and galactose, the molar ratio of the arabinose, the glucose and the galactose is 1: 4.048: 3.174, the glycosidic bond connection mode of the GPS is that the (1 → 4) linked glucose, the (1 → 6) linked galactose and the (1 → 3, 6) linked glucose are used as main chains, and the (1 → 3) linked galactose, the (1 → 5) linked arabinose and the T tail end glucose are used as branched chains (Tianjin scientific university, 2019 academic position paper). Extracting crude polysaccharide from old orange by water extraction and alcohol precipitation, extracting root and rhizome of Glycyrrhiza inflata with water extraction and alcohol precipitation, separating and purifying by ion exchange column chromatography and gel column chromatography to obtain 3 kinds of crude polysaccharide with relative molecular weight of more than 2.0 × 10 6 Homogeneous acid of DaAnd the 3 polysaccharide components are acid heteropolysaccharides consisting of arabinose (Ara), rhamnose (Rha), galactose (Gal) and galacturonic acid (Gal A) in different molar ratios, the main chain of the polysaccharide consists of 1,4-Galp A and 1,2-Rhap residues, the branched chain consists of 1, 5-linked Araf and 1, 3-linked Galp, and the branched branch point is positioned at the O-4 position of the Rhap residue (food safety quality testing bulletin, 2017,8, 4651-4658). Separating and purifying a water-soluble polysaccharide (GIP-2) obtained from Glycyrrhiza inflata InflaBat. Of Xinjiang province by Rena, kasimu and the like, removing protein by degreasing, reflux extraction, ethanol precipitation and Sevag methods, extracting crude polysaccharide from a Glycyrrhiza inflata medicinal material, dialyzing, separating and purifying by DEAE-52 ion exchange chromatography, sepharose CL-6B and Sephadex G-50 gel column chromatography to obtain a water-soluble polysaccharide (GIP-2), the Glycyrrhiza inflata polysaccharide GIP-2 is yellowish white powder, has no sweet taste, is easily dissolved in water, has obvious absorption peak at 192nm under UV detection, has no absorption peak at 260 nm and 280nm, proves that the tested object is polysaccharide, does not contain nucleic acid and protein, and has IR analysis results of 3393, 2932, 1616, 1423 and 1101cm -1 It shows a typical polysaccharide absorption peak, GIP-2 has a molecular weight > 2000kDa, and is mainly composed of glucose, arabinose and galactose at a molar ratio of 3.3: 11.7: 1.0 (Waxi journal of pharmacy, 2008, 23, 448-450). Quality girl is extracted from the glycyrrhiza inflata by a water extraction and alcohol precipitation method to extract crude polysaccharide, after dialysis, the crude polysaccharide is subjected to DEAE cellulose ion exchange column chromatography fractionation, gel chromatography fillers such as Sephadex, sepharose and the like are adopted for each fraction to be separated and purified, and the purity and the average molecular weight are determined by an HPGPC method; extracting the glycyrrhiza inflate with water, precipitating with ethanol, and removing proteins and pigments by Sevag method and repeated alcohol washing to obtain crude polysaccharide; repeatedly separating and purifying by DEAE-52 ion exchange column chromatography and Sepharose CL-6B and Sephadex G-50 gel column chromatography to obtain 13 homogeneous polysaccharide components from the plant for the first time, and separating and purifying to obtain 13 homogeneous polysaccharides, wherein Gi-A1 and Gi-A2 are neutral arabinogalactan, gi-A3 and Gi-A4 are neutral arabinogalactan, gi-B1, gi-C1 and Gi-D1 have typical RGI type pectic polysaccharide structure characteristics, and its main chain comprises 1 → 4 connected alpha-D-GalpA composed of unbranched smooth region and unbranched smooth regionBranched hair zones consisting of alternating 1, 2-linked α -L-Rhap and 1, 4-linked α -GalAp junctions, with branches at the 4-position of Rha, the others comprising 2 neutral heteropolysaccharides and 4 acidic heteropolysaccharides (university of Xinjiang medical, 2008 academic paper). The optimal extraction conditions of the glycyrrhiza polysaccharide determined by the He Pedun et al are as follows: setting ultrasonic power at 500w, liquid-material ratio at 20: 1 (mL/g), ultrasonic time at 60min, and temperature at 60 deg.C, and performing monosaccharide component analysis on Glycyrrhiza polysaccharide by GC/MS to obtain monosaccharide components of artificial and wild Glycyrrhiza polysaccharides mainly comprising mannose and glucose, and further comprising arabinose, ribose, xylose, galactose, glucuronic acid and galacturonic acid, which indicate that artificial and wild Glycyrrhiza polysaccharides are both acidic polysaccharides, and finally separating and purifying the Glycyrrhiza polysaccharide by Sepharose CL-6B chromatography column to obtain two polysaccharide components of artificial Glycyrrhiza polysaccharide with relative molecular weights of 212 ku and 25.1ku, and two components of wild Glycyrrhiza polysaccharide with relative molecular weights of 34.1ku and 0.1ku (2016, 37, 72-76 for food research and development).
3.7.1 combination of trichloroacetic acid and Sevage method and deproteinization method, dongzhuang adjusted the pH of the glycyrrhizic acid filtrate to neutral, concentrated 10 times the extract, ethanol concentration 80%, precipitated 16h, glycyrrhiza polysaccharide yield 4.11%, content 31.10%, purification process using trichloroacetic acid and Sevage method combined, obtained according to the solid-to-liquid ratio of 1: 5, trichloroacetic acid concentration 7%, stirred 0.5h, filtered and precipitated to obtain Glycyrrhiza polysaccharide white, content 38.50%, yield 89.00%, in Sevage method, chloroform-n-butanol solution amount was 0.2 times of the original solution, shaken 30min, deproteinized 4 times, filtered and dried to obtain Glycyrrhiza polysaccharide average content 46.90%, yield 84.10% (university of Anhui, 2021 academic paper).
3.7.2 ultrasonic-assisted Chaimeiling and the like take the traditional processed liquorice as raw materials, and the optimal process conditions for extracting the glycyrrhiza polysaccharide are as follows: the grinding particle size is 127 mu m, the liquid-material ratio is 19.32mL/g, the ultrasonic temperature is 65 ℃, the ultrasonic time is 30.2min, and the polysaccharide yield is 10.867% (food industry science and technology, 2021, 42, 192-200). The Tianyanhua and the like take liquorice in Gansu Taoism as raw materials, and the optimal technological conditions for extracting the liquorice polysaccharide are as follows: the temperature is 70 ℃, the time is 85min, the liquid-material ratio is 13: 1, the ultrasonic power is 600w, the average extraction yield of polysaccharide is 4.23%, GCP2 is reductive polysaccharide mainly comprising alpha-glycosidic bond, is light yellow white powder, is easily soluble in water, mainly comprises arabinose, glucose and galactose, and has the molar ratio of 0.166: 5.56: 1.60 (science and technology of food industry, 2017, 38, 296-302). The optimal extraction process conditions of the glycyrrhiza polysaccharide adopted by chenfengqing and the like are as follows: when the lambda max =488nm, the particle fineness is 0.25-0.5 mm, the ultrasonic treatment is carried out for 25min, the material-liquid ratio reaches 1: 25 (vol), and the extraction temperature is 30 ℃, the extraction content of the glycyrrhiza polysaccharide reaches 60.23mg/L and the glycyrrhiza polysaccharide content reaches 4.818% (Chinese brewing, 2009, (01), 138-139+ 142).
3.7.3 anion exchange Liuyuena etc. takes crude polysaccharide obtained by pretreating and water extracting licorice root powder as raw material, anion exchange crystal gel medium prepared by grafting dimethylaminoethyl methacrylate to polyacrylamide crystal gel matrix, and carries out rapid chromatographic separation on licorice polysaccharide, wherein the anion crystal gel medium can adsorb licorice polysaccharide, the chromatographic separation is rapid, deionized water is used as base liquid for bed column balance, flushing and eluent preparation, the flow rate is 1 cm.min -1 Under the condition, the adsorption capacity of the crystal gel medium to the glycyrrhiza polysaccharide reaches 670 mu g/mL -1 Crystal gel (journal of chemical engineering in colleges and universities 2015, 29, 1507-1512). Extracting the sesbania root for 2 times by using 15 times of water solution in 80 ℃ water bath, extracting for 2h each time, concentrating the extracting solution to 2/5 of the original volume, adding 98% ethanol of 4 times of the volume of the concentrated solution, precipitating for 2 times by using a trichloroacetic acid method according to an optimal deproteinization method, centrifuging to remove the precipitate, removing pigments by using 0.4% of activated carbon according to an optimal decoloring process, performing low-temperature freeze drying in an optimal drying mode to obtain the average content of the polysaccharide in the liquorice of 1.43%, and separating the polysaccharide component of the liquorice by using anion exchange resin DEAE-52 column chromatography to obtain 4 series polysaccharides GPS1, GPS2, GPS3 and GPS4. Performing Sephadex G-100 column chromatography, dialysis and freeze-drying on the GPS1 fraction to obtain a white GPS1 polysaccharide pure product, and measuring the relative molecular mass of the pure product to be 66KDa (university of science and technology in Huazhong, academic paper of 2007).
3.7.4 macroporous resin inexpensive Yijun, etc. adopting HPD-722 resin to adsorb the glycyrrhiza polysaccharide with a rate of 73.25% and a desorption rate of 86.59%, suitable for purifying the glycyrrhiza polysaccharide, and the optimal separation conditions of the glycyrrhiza residue polysaccharide are as follows: the concentration of the glycyrrhiza polysaccharide in the sample loading liquid is 4.12mg/mL, the sample loading amount is 2BV, the sample loading flow rate is 2BV/h, the eluent is 50% ethanol, the elution flow rate is 3BV/h, the eluent dosage is 3BV, and the purity of the glycyrrhiza polysaccharide is improved to 51.65% from 7.64% before purification under the optimal condition (the scientific report of the university of Stone river (Nature edition), 2015, 33, 351-356). Wuyu discovers that the protein and pigment contents of purified polysaccharide are remarkably reduced by treatment of D301T and S-8, the purity of polysaccharide after dialysis can reach more than 80%, the protein and pigment contents of polysaccharide after resin dynamic adsorption treatment can be reduced by 75% and 55% at most, the purity of polysaccharide is improved by about 4%, polyvinyl alcohol microspheres are activated and aminated to prepare resin, the adsorption performance and the purification of polysaccharide are researched, the optimal adsorption condition is 10mg/mL of liquorice polysaccharide, the flow rate is 12BV/h, the sample loading amount is about 2BV (BV is the number of resin volume), the treatment amount is obviously higher than that of cellulose resin, when the treatment amount is 1BV, the protein content is 0.24%, the polysaccharide color is similar to white, the polysaccharide purity is improved by 30% and 71%, and when the treatment amount is 2BV, the polysaccharide purity is improved by 10% and reaches 54% (Tianjin university, 2009 scientific thesis). In 9 different types of macroporous adsorption resins, huangshen et al have better adsorption and desorption performances of LSA-5B type macroporous adsorption resin, and the dynamic adsorption rate of the macroporous adsorption resin on the glycyrrhiza polysaccharide is 56 percent and the dynamic desorption rate of the macroporous adsorption resin is 99 percent (Shizhen Chinese and medical drugs, 2007, 18, 2620-2621). Ultrasonic extraction of radix astragali et rhizoma Rhei Franch etc. to extract Glycyrrhiza polysaccharide, alcohol-separating concentrated Glycyrrhiza polysaccharide solution with 4 times of 95% ethanol twice, removing protein by Sevege method for 4 times, dialyzing with dialysis bag for 12h, and purifying with LSA-5B type macroporous resin, wherein the optimum process comprises loading solution concentration of 2mg/mL, adsorption rate of 2BV/hr, eluent of 55% ethanol solution, elution rate of 3BV, and eluent dosage of 3BV (CN 101492510).
3.7.5 eutectic solvent method Suyue et al uses radix Glycyrrhizae as raw material, adopts ultrasonic-assisted eutectic solvent method, uses choline chloride-isopropanol system with water content of 40% and molar ratio of 1: 3 as extractant, and has extraction temperature of 39 deg.C, material-liquid ratio of 1: 50 (g/mL), ultrasonic time of 30min, ultrasonic power of 250w, and polysaccharide extraction rate of 8.31% (2021, 42, 84-91 for food research and development).
3.7.6 the optimum extraction process of the liquorice residue alkaline-extracted polysaccharide adopted by the alkaline-extracted polysaccharide Zhang Xiaojing is as follows: the concentration of alkali liquor (NaOH) is 5.2%, the extraction time is 3.20 h, the liquid-material ratio (mL/g) is 26.85: 1, and the yield of polysaccharide is 6.44 +/-0.02% (university of Tianjin technology, 2020 academic thesis).
The enzyme method of 3.7.7 selects cellulase and pectinase to extract the liquorice residue polysaccharide respectively, and the polysaccharide yield is 8.43 percent and 10.71 percent respectively (2020, 41, 309-313+319 in the food industry science and technology). Adding water into liquorice for soaking for 1-4 h, filtering to obtain soaked liquorice powder, adding water into the soaked liquorice powder, freezing for 2-3 h at the temperature of- (20-10) DEG C, then unfreezing to 45-55 ℃ at the temperature of 45-55 ℃, adding pectinase accounting for 0.4-1% of the weight of the soaked liquorice powder and papain accounting for 0.1-0.5% of the weight of the liquorice powder, carrying out ultrasonic enzymolysis for 3-9 h at the temperature of 45-55 ℃, heating to 94-98 ℃, keeping the temperature for 8-12 min, inactivating enzyme, adding a leavening agent into the enzymolysis mixture, keeping the temperature of a shaking table at 32-38 ℃, the rotation speed is 120 to 180r/min, the fermentation time is 10 to 20h, the filtration is carried out, the filtrate is heated to 94 to 98 ℃ and is preserved for 8 to 12min for sterilization, the standing is carried out for 2 to 8h, the supernatant after the standing is taken and is decompressed and concentrated to the relative density of 1.055 to 1.075 to obtain the concentrate, 92 to 97 percent of ethanol with the volume fraction of 92 percent to 97 percent is added into the concentrate to ensure that the ethanol with the volume fraction of 70 to 80 percent, the standing is carried out for 4 to 12h, the centrifugation, the filtration and the drying are carried out to obtain the primary extract, the ethanol with the volume fraction of 92 to 97 percent which is 2 to 4 times of the weight of the primary extract is added into the primary extract to be soaked for 2 to 6h, the centrifugation, the filtration and the drying are carried out for 2 to 4 times to obtain the glycyrrhiza polysaccharide (CN 109988795B)
3.7.8 Glycyrrhiza selenoglycoprotein sodium selenite is added under the action of nitric acid catalyst, the Glycyrrhiza selenoglycoprotein and sodium selenite are reacted to generate Glycyrrhiza selenoglycoprotein, the optimal preparation process condition of Glycyrrhiza selenoglycoprotein is as follows: the mass ratio of the glycyrrhiza uralensis glycoprotein to the sodium selenite is 1: 1, the reaction temperature is 70 ℃, the reaction time is 8h, the amount of BaCl2 is 1.0g, the glycyrrhiza uralensis selenoglycoprotein prepared under the conditions has the average selenium content of 4.78mg/g and the average yield of 75.62 percent (Anhui agricultural science, 2018, 46, 17-20). Colophony and the like are prepared by selenizing selenious acid with natural glycyrrhiza polysaccharide as a raw material,the optimal technological conditions for preparing the glycyrrhiza selenoglycosan are that the reaction temperature is 60 ℃, the reaction time is 8 hours, the mass ratio of the glycyrrhiza selenoglycosan to the selenious acid is 1: 1, and the catalyst BaCl is adopted 2 The mass of the polysaccharide is 1.0g, the yield of the glycyrrhiza selenoglycan is 27%, the structural representation of the glycyrrhiza selenoglycan by infrared spectroscopy shows that the glycyrrhiza selenoglycan contains Se-O bonds and Se-C bonds, the selenization of the glycyrrhiza selenoglycan is realized, the mass fraction of selenium in the glycyrrhiza selenoglycan is measured by atomic emission spectroscopy, and the mass fraction of selenium in the glycyrrhiza selenoglycan is 6.59mg/g (chemical engineering, 2009, 37, 63-66). Dissolving glycyrrhetinic acid refined polysaccharide in nitric acid solution, stirring, heating, adding sodium selenite, stirring for reaction, naturally cooling the reaction solution to room temperature, centrifuging, removing precipitate, taking supernatant for later use, adding saturated sodium carbonate solution into the supernatant to adjust the pH value to 5-6, dialyzing with distilled water, concentrating under reduced pressure, precipitating with absolute ethanol, standing at 0-4 ℃ for 8-12 h, centrifuging to remove the supernatant, and drying the precipitate to obtain the glycyrrhetinic acid selenium polysaccharide (CN 106084080A).
3.7.9 supercritical CO 2 Supercritical CO is used for extracting Liqingyu 2 The optimal extraction process conditions are as follows: the temperature is 62.6 ℃, the pressure is 37.7MPa, the time is 82.9min, and the yield of the GPS under the condition is as high as 7.34% (food industry, 2017, 38, 1-5).
3.7.10 the comprehensive method uses water as extraction solvent, and the extraction is carried out for 3h at 80 ℃ and repeated for three times, and the water consumption is 30mL/g raw material each time. The purity of the glycyrrhiza polysaccharide is 40.00 percent, and the yield is 5.40 percent; the crude polysaccharide is subjected to pigment removal by adopting macroporous resin, protein removal by a Sevag method, small molecular impurities removal by deionized water dialysis, and further purification of the glycyrrhiza polysaccharide by adopting diethylamino cellulose (DEAE-32) and Sephadex (Sephadex G-75) column chromatography, wherein the purity of the polysaccharide reaches 96.15%, and the yield is 0.57% (2002 academic paper of northeast forestry university). Mechanically pulverizing Glycyrrhrizae radix, defatting with petroleum ether and anhydrous alcohol, decolorizing, extracting with water to obtain polysaccharide, concentrating the extractive solution, precipitating with ethanol to obtain polysaccharide precipitate, dissolving with distilled water, adding 1/3 volume of mixed solution of chloroform and n-butanol at volume ratio of 4: 1 to remove protein, dialyzing the deproteinized polysaccharide solution, vacuum freeze drying to obtain crude Glycyrrhrizae radix polysaccharide, dissolving with ultrapure water, gradient eluting with ultrapure water and NaCl solution on DEAE-cellulose 52 ion exchange column to obtain neutral polysaccharide and two acidic polysaccharide parts, dialyzing the acidic polysaccharide eluate, concentrating, freeze drying to obtain crude Glycyrrhrizae radix acidic polysaccharide, and further purifying with Sephadex G-100 column chromatography to obtain purified high-purity Glycyrrhrizae radix polysaccharide effective part (CN 112457424B).
3.8 Glycyrrhiza protein
Zhanyanhong and the like establish a technical system suitable for licorice seed protein extraction and SDS-PAGE electrophoresis, a mercaptoethanol method and an acetone precipitation method are adopted as suitable protein extraction methods, 12 strips are obtained by the mercaptoethanol method, 9 protein strips are obtained by the acetone precipitation method, the background color is light, the method is suitable for seed purity detection, the electrophoresis effect is good when 10% separation gel and the material-liquid ratio are 1: 10, and the Glycyrrhiza uralensis and the Glycyrrhiza inflata Linn have the difference of the protein strips (seeds, 2016, 35, 21-24). Niandan and the like, licorice roots are soaked and extracted at normal temperature by using 0.5-2% alkali solvent, 4-6% strong acid is added into filtered filtrate for acidification, the filtrate is filtered, brown black powdery crude licorice protein is obtained after the filtrate is removed, a proper amount of high-concentration ethanol is added for stirring and dissolving, the filtrate is removed, the black brown powdery crude licorice protein is obtained after drying and crushing, water is added, alkali liquor is added at the same time for adjusting the pH to the optimum value for enzymolysis reaction, 0.5-1% amylase is added, 0.5-1% polysaccharase is added, the pH is adjusted to 5.0-6.5, and the black brown powder obtained after drying and crushing is the finished product of high-content licorice protein (CN 114027387A) after the filtrate is removed.
3.9 Glycyrrhiza Lignin
Extracting lignin from Glycyrrhrizae radix residue with ultrasound-assisted general alkaline method to determine the alkali solution concentration of 0.7 mol.L under optimum extraction process conditions -1 The dosage of the alkali liquor is 30mL/g -1 The ultrasonic power is 600w, the ultrasonic time is 60min, and the lignin yield is 22.20% (Zhejiang agricultural science, 2016, 57, 403-405+ 409). The best conditions for separating glycyrrhiza glabra protoplasts by the WangGift principle and the like are as follows: growing 7d aseptic seedling of Glycyrrhiza glabraCotyledon as separation material, 4 ℃, MS culture medium pre-culture for 12h, 1.5% cellulase and 0.5% pectinase as separation protoplast enzyme liquid system, CPW solution containing 0.7mol/L mannitol as penetration protective agent to prepare enzyme liquid, and enzymolysis for 14h under 25 ℃ standing condition (biotechnological communication, 2015, 26, 687-690). The method for extracting lignin from licorice residue as raw material includes 2 methods of alkali method and organic solvent method, and 4 solvents of sodium hydroxide, ammonia water, acetone and glycol, wherein the extraction rates of the 4 solvents are respectively: 17.25%, 5.75%, 11.54%, 12.60%, the content of active groups of lignin extracted by an organic solvent method is higher, and the molecular weight of lignin extracted by acetone is smaller and the distribution is narrower (Jiangsu agricultural science, 2014, 42, 223-225). Zhao thrifto et al, which adopts a general alkaline method to extract lignin from licorice root residue, the optimal extraction process conditions are as follows: the water bath temperature is 40 ℃, the constant temperature time is 2.5h, the alkali liquor concentration is 0.8mol/L, the alkali liquor dosage is 30mL/g, the extraction rate of the lignin is 4.88%, and the total hydroxyl, phenolic hydroxyl and alcoholic hydroxyl contents of the alkali lignin are 5.43%, 3.18% and 2.43% respectively (proceedings of Tarim university, 2011, 23, 42-45). Zhangdao et al add a certain amount of methyl isopropyl ketone (MIPK) -ethanol-water mixed solvent MIPK to ethanol to water = 50: 30: 20 to obtain a turbid solution, the material-liquid ratio is 1: 10g/mL, the turbid solution is treated by ultrasonic waves, after complete precipitation, supernatant B and filter residue C are obtained by centrifugal separation, water is added into the supernatant B to enable insoluble MIPK to generate two-phase layering, the upper layer is separated to be MIPK phase, the MIPK is recovered by vacuum concentration, crystals are obtained, and after filtration, washing and drying, lignin is obtained, the lower layer is hydrated, and the hemicellulose and soluble sugar are obtained by concentration; and drying the filter residue C at the temperature of 80 ℃ for 3-5 h to obtain the cellulose D (CN 101372815B).
3.10 Glycyrrhiza dietary fiber
The Sunyang sheep and the like take licorice root residues as raw materials, and alkali extraction is respectively carried out on soluble dietary fibers and insoluble dietary fibers under the assistance of a water bath and magnetic stirring method, and the optimal extraction process is determined as follows: the feed-liquid ratio is 1: 9 (g/mL), the extraction temperature is 40 ℃, the extraction time is 80min, the yield of insoluble dietary fibers is 81.33 percent at most when the concentration of sodium hydroxide is 6 percent, the feed-liquid ratio is 1: 10 (g/mL), the extraction temperature is 80 ℃, the extraction time is 80min, and the yield of soluble dietary fibers is 8.33 percent at most when the concentration of sodium hydroxide is 7 percent (2019, 40, 96-101 for food research and development). The process for determining the protein feed produced by licorice residue comprises the following steps: 120mg/g of complex enzyme, 5.0 pH value of buffer solution, 52 ℃ hydrolysis temperature, 50r/min of shaking table rotation speed and 23h of hydrolysis time, 100mg/g of yeast is inoculated when the temperature is cooled to room temperature, and the fermentation is carried out for 20h at the temperature of 32 ℃, the protein of the fermentation product is increased by 2616.7%, the lignin is reduced by 24.2%, the cellulose is reduced by 48.43%, and the hemicellulose is reduced by 91.43% (feed industry, 2015, 36, 48-52). Dissolving liquorice waste residue powder in distilled water, performing ultrasonic treatment, filtering to remove dregs to obtain an extracting solution, adjusting the pH value of the extracting solution to 6.0-7.0, adding alpha-amylase, performing hydrolysis reaction for 1-3 h, inactivating enzyme, cooling, adjusting the pH value of the cooled extracting solution to 7.5-8.5, adding protease, reacting for 0.5-1 h at 55-65 ℃, inactivating enzyme, centrifuging, adding 95-100% ethanol into the centrifuged supernatant, performing suction filtration to obtain a precipitate, freeze-drying the obtained precipitate, and micronizing to 80-100 meshes to obtain the liquorice soluble dietary fiber (CN 113595731A).
Disclosure of Invention
At present, one of the developments of liquorice resources in China is to dig the planted liquorice, process and slice the liquorice, and sell the liquorice as a traditional Chinese medicine; one method comprises pulverizing Glycyrrhrizae radix, extracting effective substances with chemical method, and making into extract product for sale. However, these two ways of developing licorice resources do not utilize and develop licorice resources effectively, but rather destroy the licorice resource. The liquorice resources are processed, sliced and sold, the added value is low, a large amount of liquorice is consumed, the residual amount of the liquorice leftover materials is large, and the resources are wasted; effective active substances in the liquorice are extracted by a chemical method to prepare extract products for sale, although the added value is high, the liquorice residue after the extraction of the active ingredients of the liquorice is often treated as solid waste, so that the utilization rate of the liquorice resource is not improved, the environment is polluted and damaged. For a long time, only glycyrrhizin (also known as glycyrrhizic acid) in liquorice is extracted as a main active ingredient in industrial production, and waste residues and residual liquid after the glycyrrhizic acid is extracted are abandoned, so that great environmental pollution and resource waste are caused.
The technical solution of the invention is as follows:
a full-industrial-chain synergic extraction and separation method for effective components of Glycyrrhrizae radix is characterized in that the whole plant of Glycyrrhrizae radix is divided into Glycyrrhrizae radix root and stem; the method comprises the following steps of extracting liquorice leaves and liquorice seeds in a segmented full industrial chain, wherein the method comprises the following steps: (1) Extracting squalene, protein, polysaccharide, oil and other components from the licorice seeds; (2) Extracting volatile components, chlorophyll, tea polyphenols, flavone, polysaccharide, etc. from folium Glycyrrhizae; (3) Extracting radix Glycyrrhizae to obtain polyphenol, glycyrrhizin, licoflavone, liquiritigenin dextran, liquiritigenin, protein, lignin, cellulose, etc.
1. Glycyrrhiza uralensis seed extraction
Extracting squalene, protein, polysaccharide, oil and other components from the licorice seeds, including cleaning, removing impurities, soaking, modifying, coarse crushing, vacuum extruding, puffing, and collecting oil and puffed material;
cleaning and removing impurities, selecting liquorice seeds, and removing impurities such as straws, gravels, metal blocks and the like;
the soaking and tempering are carried out, the cleaned liquorice seeds are soaked in sulfurous acid solution for a period of time, the soaking time is 10-14 hours, the temperature is 30-50 ℃,
the concentration of sulfurous acid in the soak solution is 0.1 to 0.2 percent;
the coarse crushing is carried out, and the soaked liquorice seeds enter a crusher to be crushed;
The vacuum extrusion puffing is carried out, and the vacuum degree is between 0.057 and 0.067 MPa; the water content of the material is 15-20%; the temperature of the sleeve is 87-98 ℃; the rotating speed of the extruder screw is 100-150 rpm;
the aperture of the die hole is 17-22 mm; the extrusion temperature is 120-150 ℃, the extrusion pressure is 10MPa, and the extrusion time is 3min, and the shape of the aperture of the grinding head of the extruder is round or rectangular; the particle size D95 of the puffed material is not more than 125 μm.
Through vacuum extrusion and puffing, oil material tissues are thoroughly destroyed, the puffed material residues are soaked by an organic solvent, the using amount of the solvent is greatly reduced, protein in the raw material is deformed, enzyme is thoroughly inactivated, a grooved wall extrusion and puffing machine is adopted, grease released by material embryos in the extrusion process can be discharged through a gap of a pressing cage, and the material embryos are puffed from a template slotted hole at the tail end of the puffing machine or a conical discharger foundation.
Separating squalene and other oil components from the collected licorice seed oil:
adding petroleum ether (60-90 ℃) into the liquorice seed oil for extraction, wherein the volume ratio of the petroleum ether to the oil is 1: 1-1: 3; extracting for 3 times, combining upper phase extraction liquid, removing a solvent through evaporation and concentration to obtain a brown oily substance, dissolving the brown oily substance by using ethanol, adding 10-20% of sodium hydroxide, and performing saponification reaction for 50min at the reaction temperature of 70 ℃ in a material-liquid ratio of 1: 3;
The concentration is carried out under the conditions of vacuum degree of (0.06-0.08) MPa and temperature of 40-50 ℃;
adding petroleum ether (60-90 ℃) into the saponified solution for extraction, wherein the volume ratio of the petroleum ether to the grease is 1: 1-1: 3; extracting for 3 times, combining the upper phase extraction liquid, evaporating and concentrating to remove the solvent to obtain brown oily matter, dissolving the brown oily matter with ethanol, adding 10-20% sodium hydroxide, and performing saponification reaction at 70 ℃ for 50min at a material-liquid ratio of 1: 3;
the concentration is carried out under the conditions of vacuum degree of (0.06-0.08) MPa and temperature of 40-50 ℃;
adding petroleum ether (60-90 ℃) into the saponification liquid for extraction, wherein the volume ratio of the petroleum ether to the grease is 1: 1-1: 3; extracting for 3 times, mixing the upper phase extractive solutions, evaporating, concentrating to remove solvent to obtain brown oil, preparing with argentized silica gel, loading into column, eluting pigment, gradient eluting with Ag + Removing, decoloring and crystallizing;
the preparation of the silvered silica gel comprises the steps of adding 48-75 mu m of silica gel into a silver nitrate solution containing 8-20% under the condition of keeping out of the sun, fully stirring the mixture into paste, heating and stirring the paste for 20-40 min in a water bath at 90-95 ℃, then cooling the paste to 25-35 ℃, carrying out suction filtration, activating the suction filtration in a vacuum drying oven for 15-25 h to prepare the silvered silica gel, and placing the silvered silica gel in a dark place for standby;
The silver silica gel is filled into a column, is placed into petroleum ether, is stirred to remove bubbles, is kept stand to be fully swelled, is added into a chromatographic column, is wrapped by tinfoil, and is eluted and balanced by the petroleum ether;
dissolving the eluting pigment and brown oily substance with petroleum ether, adding dropwise into the upper end of the sillicon argentea chromatographic column, opening the lower valve of the chromatographic column to allow
Slowly adsorbing the sample liquid on the silver silica gel, and starting gradient elution when the sample is about to be adsorbed;
the gradient elution is carried out by taking petroleum ether and ethyl acetate as eluents with gradient volume ratios of 100: 0, 100: 5, 100: 8, 100: 10, 100: 15, 1 00: 20, 100: 100 and 0: 100 in sequence, TLC and HPLC detection analysis is carried out on each fraction of the eluent respectively, and the same fractions are combined;
the Ag is + Removing, combining the fractions analyzed by TLC and HPLC to obtain eluate mainly containing squalene, rotary evaporating, removing eluent, dissolving with n-hexane, adding saturated sodium chloride solution to remove Ag + Adding anhydrous sodium sulfate for dehydration, and filtering to obtain filtrate;
and (3) decoloring and crystallizing, evaporating filtrate under reduced pressure to dryness, dissolving the filtrate by adding absolute methanol, decoloring the filtrate by adding attapulgite for 30min, performing suction filtration, concentrating the filtrate at the vacuum degree of between- (0.06 and 0.08) MPa and the temperature of between 40 and 50 ℃ to 1/3 to 1/4 of the original volume, standing and crystallizing to obtain squalene.
The structure is as follows:
Figure BSA0000282013940000431
mixing the eluates obtained in the second step, adding attapulgite adsorbent according to the material-liquid ratio of 1: 10-1: 30 (g: mL), refluxing and decolorizing for 30min, filtering, and concentrating the filtrate under reduced pressure to obtain licorice seed oil;
the pressure reduction concentration is carried out, the concentration is carried out under the conditions of vacuum degree of (0.06-0.08) MPa and temperature of 40-50 ℃ until no effluent liquid exists, and the effluent liquid is discharged;
the attapulgite adsorbent is a modified attapulgite adsorbent, and the modification method comprises the steps of treating natural attapulgite by double rollers, adding 5-10 times (mL: g) of purified water, adding 3-8 times (g: g) of magnesium salt or aluminum salt, adding 0.1-1% of chloroacetic acid, fully and uniformly stirring, adding sodium hydroxide to adjust the pH value to 9-11, continuing to react for 5-10 h, carrying out solid-liquid separation after the reaction is finished, calcining the solid product at 200-500 ℃ for 2-6 h, crushing, and sieving by a sieve of 25-75 mu m to obtain the modified attapulgite adsorbent.
The collected swollen licorice seed extract is used for subcritical water extraction of protein and polysaccharide, and the extraction method comprises the following steps:
extracting by using subcritical water, introducing high-purity nitrogen into a subcritical water extraction device, taking deoxidized distilled water as an extracting agent, controlling the material-water ratio to be 1: 10-20 (g: mL), the extraction temperature to be 100-150 ℃, the extraction time to be 30-60 min and the pressure to be 3-10 Mpa, and obtaining extract liquor and filter residue containing glycyrrhiza polysaccharide and protein; by utilizing the subcritical water extraction technology, hydrogen bonds of subcritical water are opened or weakened along with the rise of temperature, so that components with high polarity are extracted, and by controlling the temperature and the pressure of the subcritical water, components with different polarities in the active ingredients of the natural product can be selectively and continuously extracted from water-soluble components to fat-soluble components.
Centrifuging the obtained extract containing glycyrrhiza polysaccharide and protein, concentrating, precipitating with ethanol to obtain crude product, dissolving the crude product with water, adding tert-butanol and ammonium sulfate to form three liquid phase system to obtain upper phase, middle phase and lower phase;
centrifuging at 3000-5000 rpm for 10-20 min;
concentrating by using an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD to 1/3-1/10 of the original volume;
the alcohol precipitation comprises the steps of adding ethanol with volume fraction of 90-95% into crude glycyrrhiza polysaccharide and protein extraction concentrated solution to enable the final volume fraction of the ethanol to be 70-80 wt%, stirring uniformly, standing for precipitation for 5-10 h, performing suction filtration to obtain a precipitate, and washing for 1-3 times by using absolute ethyl alcohol to obtain crude glycyrrhiza polysaccharide and protein;
dissolving Glycyrrhiza polysaccharide and protein crude product with water, adding tert-butanol and ammonium sulfate to form three liquid phase system, and obtaining upper phase, middle phase and lower phase;
dissolving the crude product of the glycyrrhiza polysaccharide and the protein by using water, adding deionized water to the crude product to dissolve the crude product, and preparing a solution with the total concentration of the glycyrrhiza polysaccharide and the protein of 30-50 wt% to obtain a solution containing the polysaccharide and the protein;
the tertiary butanol and ammonium sulfate form a three-liquid phase system, the volume ratio of the solution containing polysaccharide and protein to the tertiary butanol is 1: 1-1: 3, the mass fraction of the ammonium sulfate is 30-50 wt%, the temperature is 35-40 ℃, the time is 30min, and the pH value is 7;
Dialyzing the obtained lower phase to remove inorganic salts, decolorizing in water bath, concentrating, precipitating with ethanol, centrifuging, and drying to obtain Glycyrrhrizae radix seed polysaccharide;
decolorizing in water bath, controlling the temperature of the water bath at 45-50 ℃ for 90min, and taking H for the first 60min 2 O 2 Decolorizing by oxidation method, decolorizing by activated carbon adsorption method for 30 min, vacuum filtering, and collecting filtrate;
centrifuging at 3000-5000 rpm for 10-20 min;
concentrating by using an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD to 1/3-1/10 of the original volume;
the alcohol precipitation is to add the concentrated solution containing the glycyrrhiza polysaccharide into ethanol with the volume fraction of 90-95% to ensure that the volume fraction of the ethanol is 70-80%, to stir evenly, to stand and precipitate for 5-10 h, to perform suction filtration to obtain a precipitate, and to wash the precipitate for 1-3 times by using absolute ethyl alcohol to obtain a crude product;
the centrifugation is carried out for 10-20 min at the rotating speed of 3000-5000 rpm;
and drying for 5-10 h at the temperature of 50-60 ℃ and the vacuum degree of (0.06-0.08) MPa to prepare the liquorice seed polysaccharide.
The fifth step: decolorizing the obtained middle phase in water bath, concentrating, precipitating with ethanol, centrifuging, drying to obtain Glycyrrhrizae radix seed protein, dissolving Glycyrrhrizae radix seed protein with sodium chloride solution, centrifuging, precipitating with ammonium sulfate, and separating to obtain Glycyrrhrizae radix seed total protein;
Decolorizing in water bath, controlling the temperature of the water bath at 45-50 ℃ for 90min, and taking H for the first 60min 2 O 2 Decolorizing with oxidation method, decolorizing with activated carbon adsorption method 30 min later, vacuum filtering, and collecting filtrate;
concentrating the filtrate to 1/2-1/20 of the original volume by using an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD;
precipitating in alcohol, adding absolute ethyl alcohol to make the mass percent concentration of the ethyl alcohol be 75-85%, standing for 5-10 h, centrifuging for 10-15 min at 3000-5000 rp m to obtain a precipitate, and washing the precipitate with the absolute ethyl alcohol for 2-3 times;
dissolving the sodium chloride solution, adding 3-5 times (v/w) of 0.15-0.20 mol/L sodium chloride solution into the licorice seed protein, centrifuging at 3000-5000 rpm for 10-15 min after dissolving, and collecting clear solution;
precipitating by ammonium sulfate, adding the ammonium sulfate into clear liquid to ensure that the saturation degree reaches 70-85%, separating out protein, centrifuging for 10-15 min at 3000-5000 rpm, and collecting precipitate and clear liquid, wherein the precipitate is total protein of licorice seeds;
and drying for 5-10 h at the temperature of 50-60 ℃ and the vacuum degree of- (0.06-0.08) MPa to obtain the total protein of the liquorice seeds.
The invention can form a double-aqueous phase system by different hydrophilic micromolecule organic solvents and inorganic salts, extends liquid-liquid three-phase extraction on the basis of the double-aqueous phase system, has the advantages of high water content of the system, low organic solvent consumption, low price, environmental protection, five pollution and the like, has quick phase separation of the system, strong selectivity and lower viscosity compared with commonly used polymers, and is beneficial to the recovery of the solvent and the separation and purification of target products.
2. Extraction of licorice leaf
Mechanochemistry is used as a new auxiliary extraction means, the auxiliary agent reacts with the effective substances on the fresh plant section, the effective components are modified,
the method has the advantages that the solubility of effective components is changed, the dissolution rate of target components in a specific solvent is improved, the cell wall breaking can be better realized, the cell permeability of the solvent is enhanced, the solid-phase reaction under the action of mechanical force can be utilized to improve the solubility of fat-soluble components, the use of an organic solvent is eliminated or reduced, the extraction efficiency and the extraction selectivity are obviously improved by mechanochemical auxiliary extraction, the use amount of the organic solvent is reduced, and the process is more green and environment-friendly. The plant active ingredients can be solubilized by mechanochemical assistance, the solubility of target ingredients can be improved, and the loss of effective ingredients can be reduced.
Extracting licorice leaves with the assistance of a mechanochemical method, grinding the licorice leaves by using a planetary ball mill, and mixing and ball-milling the licorice leaves and a ball-milling auxiliary agent;
the ball-milling auxiliary agent is sodium carbonate, borax and beta-cyclodextrin, and the mass ratio of the sodium carbonate to the borax is 1: 2; the mass of the ball-milling auxiliary agent is 6.0-7.0 wt% of the mass of the licorice leaves;
the optimal technological parameters are ball milling time of 30min, ball milling rotating speed of 300rpm and filling rate of 26.2 percent, and the material granularity D95 is less than or equal to 37 mu m;
Extracting licorice leaves treated by a mechanochemical method by using ethanol with the volume fraction of 30-50%, adopting a pot group type dynamic countercurrent extraction mode, adopting a three-stage four-pot type countercurrent extraction mode, respectively putting equal parts of the licorice leaves into 4 extraction pots, and circularly extracting for 3 times in each pot in sequence, wherein the stirring frequency of the dynamic extraction is 20-200 rpm;
the extracting solution extracted for the first time by the first extracting tank of the first batch is filtered and then is put into a liquid storage tank for standby, the extracting solution extracted for the second time by the first extracting tank is used as a solvent extracted for the first time by the second extracting tank, the extracting solution is filtered for standby after extraction, the extracting solution extracted for the third time by the first extracting tank is used as a solvent extracted for the second time by the second extracting tank, and the extracting solution obtained by extraction is used as an extracting solvent extracted for the first time by the third extracting tank, namely the solvent added by the N extraction of each extracting tank is the extracting solution of the (N + 1) th time of the previous extracting tank; from the second batch, the first extraction tank extracts the second extraction liquid from the fourth extraction tank for the first time, and the second extraction liquid and the fourth extraction tank are circulated in sequence;
collecting the extracting solution extracted for the first time from each extracting tank;
the first extraction tank of the first batch is added with 5-8 times (mL: g) of 30-50% ethanol solution as an extracting solution for the first extraction, 5-8 times (mL: g) of 30-50% ethanol solution as an extracting solution for the second extraction, and 5-8 times (mL: g) of 30-50% ethanol solution as an extracting solution for the third extraction; the first extraction of the second extraction tank, the third extraction tank and the fourth extraction tank of the first batch is respectively supplemented with 30-50% of ethanol solution on the basis of adding the second extraction solution of the previous extraction tank, and the material-liquid ratio of the extraction is met; from the second batch, the first extraction tank extracts the second extracting solution from the fourth extraction tank for the first time, and the extracting solutions are circulated in sequence;
New solvent is added into the first extraction tank, the second extraction tank, the third extraction tank and the fourth extraction tank for the third extraction;
the extraction time is 60min for the first extraction, 40min for the second extraction and 30min for the third extraction in each tank;
the ratio of the material to the liquid extracted each time is 5-8 times (mL: g).
Mixing the above stored extractive solutions, centrifuging, filtering to remove flocculent precipitate to obtain precipitate-free extractive solution, and distilling under reduced pressure under the following conditions: distilling at 60-80 ℃ and the vacuum degree of- (0.06-0.08) MPa under the condition, recovering ethanol, and concentrating under reduced pressure to obtain extract containing 2 g/mL of crude drug to obtain the licorice leaf extract. Compared with a single-tank reflux extraction process, the tank group countercurrent extraction process greatly saves extraction solvents and reduces energy consumption in a subsequent evaporation concentration process, a plurality of tanks are used for extracting liquid medicine of traditional Chinese medicinal materials, each tank of the traditional Chinese medicinal materials in the group is sequentially and circularly extracted for a plurality of times, the medicinal materials in the tank extracted for the last time are firstly added with the solvents before circular extraction each time, the extracting solution of the medicinal materials is used as the solvent of the medicinal materials in the tank extracted for the next time, the medicinal materials in the tanks extracted for the last time are sequentially and circularly extracted for one time, the dried medicinal materials are added after dregs of the medicinal materials in the tank extracted for the last time are discharged and are used as the last tank of the next tank group for sequential circular extraction, and the extracting solution of each tank of the medicinal materials is obtained after dynamic circular countercurrent extraction for a plurality of times in the extraction process.
Diluting the licorice extract with water to obtain licorice leaf extract diluent, performing steam distillation to obtain distillate which is volatile components of the licorice leaves and water, extracting distillation raffinate with petroleum ether (60-90 ℃) or normal hexane to obtain extract liquor and raffinate, wherein the extract liquor is pigment, the raffinate is fully diluted with water, performing ultrasonic dissolution promotion, centrifuging, and adsorbing the centrifugal liquid with macroporous resin;
diluting the licorice leaf extract with water to obtain diluted licorice leaf extract liquid with relative specific weight of 1.03-1.05;
distilling the diluted solution of the licorice leaf extract with water vapor, heating in oil bath from the bottom of a distillation flask, and distilling after boiling to obtain mixed steam; condensing the obtained mixed steam by a condenser to obtain an oil-water mixture; performing oil-water separation on the oil-water mixture obtained by condensation by an oil-water separator to obtain volatile components of folium eucalypti globueli and liquorice and water; extracting the volatile components of the licorice leaves in an extraction tank, and separating the volatile components of the licorice leaves at the upper layer after layering; performing steam distillation to obtain azeotrope with water, steaming with steam, and collecting volatile oil.
Adding a small amount of sodium chloride into the distillate, standing for layering to obtain a light yellow oily liquid at the upper layer, drying with anhydrous sodium sulfate, performing rotary evaporation by using a rotary evaporation evaporator to purify the product, and collecting the product in a flask;
Diluting residues in a distillation flask with water, extracting with petroleum ether (60-90 ℃) or n-hexane with the same volume, respectively collecting extract liquor and raffinate, and concentrating the extract liquor under reduced pressure to obtain pigment;
the extraction adopts single-section, multi-section or continuous countercurrent extraction;
the extraction conditions are as follows: the extraction temperature is 30-45 ℃; the ratio of the extracting agent to the extracted material is 0.9-1.2: 1;
the single-section and multi-section extraction stirring time is 20-40 rpm;
the countercurrent extraction convection ratio is as follows: the ratio of the extracting agent to the extracted material liquid is = 0.9-1.2: 1, and the flow rate is as follows: the volume of 1 extraction bed is 45-60 min.
The concentration is carried out at the temperature of 50-60 ℃ and the vacuum degree of- (0.06-0.08) MPa.
Heating and volatilizing the raffinate to remove the residual extractant, filling the macroporous resin into a resin column by a wet method, washing and balancing the resin column by water, introducing the raffinate into the resin column, stopping loading the raffinate into the column when the concentration of the flavone in the to-be-detected leakage liquid reaches 5-10% of the initial concentration to obtain a resin column with saturated adsorption, quickly washing the resin column with the saturated adsorption by water with the pH value of 4-6 and the temperature of 10-15 ℃ at 2.0-4.0 BV to obtain polysaccharide eluent, performing gradient elution by using an ethanol aqueous solution, and collecting different eluents;
Performing gradient elution, namely eluting the resin column by using an ethanol water solution with the volume fraction of 20 percent of 2.0-4.0 BV to obtain a component 1; then eluting the resin column by using 2.0-4.0 BV of ethanol water with the volume fraction of 30-50 percent to obtain a component 2; eluting the resin column by using ethanol water solution with the volume fraction of 70-80% of 2.0-4.0 BV to obtain a component 3; eluting the resin column with 85-95% ethanol water solution of 2.0-4.0 BV to obtain component 4, and recovering ethanol from the eluent;
the component 1 is concentrated under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ to obtain the licorice leaf polyphenol;
the component 2 is concentrated under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ until the solid content is 60-70 wt%, thus obtaining the water-soluble flavone of the licorice leaves;
concentrating the component 3 at the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ until the solid content is 60-70 wt%, so as to obtain the isopentenyl flavone from the licorice leaves;
the polysaccharide eluent is passed through a hollow fiber membrane with the molecular weight cutoff of 100,000, the permeate is concentrated through a hollow fiber membrane with the molecular weight cutoff of 10,000 to obtain a polysaccharide concentrated solution, 1.5g of activated carbon and 1.5g of activated clay are added into 100mL of the polysaccharide concentrated solution for decolorization for 30min, and filtration is carried out. Concentrating the filtrate under reduced pressure until the relative specific gravity is 1.05-1.20, adding 1500-2000 mL of ethanol, and standing for 20-40 min at the temperature of 0-4 ℃ to prepare an alcohol precipitation mixture;
The volume fraction of the ethanol is 70-80%;
the alcohol precipitation mixture is moved into a centrifugal tube of a centrifugal machine, centrifugal separation is carried out for 10-20 min under the condition of the rotating speed of 4000-5000 rpm, clear liquid and precipitate are obtained, and the clear liquid is discarded;
drying the precipitate for 5-10 h at the temperature of 50-70 ℃ and the vacuum degree of (0.06-0.08) MPa to obtain licorice leaf polysaccharide;
the macroporous resin is one or a mixture of D-101, HPD722, HPD-100, HPD-300, HPD400, HPD-BJQH, LX-60, ADS-17, A B-8, X-5, HZ-835, ADS-F8, SZ6, SP825, DA201, HP-20 and XDA macroporous resin.
3. Extracting Glycyrrhrizae radix root and stem
Cutting the licorice roots and stems into pieces with the diameter of 20-40 mm to obtain blasting matrixes, adding water according to the mass ratio of the licorice roots, stems and water of 1: 0-1: 4, adjusting the pH value to 9-11 by using ammonia water, then soaking and rehydrating the mixture at normal temperature for 2-8 hours, adding an entrainer into the rehydrated material, uniformly mixing the mixture by using a mixer and maintaining the mixture for 30 minutes, placing the mixture in novel ejection type steam explosion equipment, and taking air and steam as steam explosion media;
the entrainer is a compound of fine-mesh carborundum and ammonium chloride, sodium carbonate or calcium hydroxide powder, the mass ratio of the carborundum to the ammonium chloride, the sodium carbonate or the calcium hydroxide powder is 4: 1-1: 1, the total addition amount of the entrainer accounts for 1.5-3% of the blasting matrix in percentage by mass, the entrainers are added in batches, a mixer is used for uniformly mixing and maintaining for 30min, 1/3-2/5 of the total amount of the entrainer is added in the steam blasting treatment I, and the balance of the entrainer is added in the steam blasting treatment II;
The steam explosion is carried out by a steam explosion treatment I and a steam explosion treatment II, and the interval time of the two-stage steam explosion treatment is 3-6 min;
the conditions of the steam explosion treatment I are as follows: the charging coefficient of the explosion cavity is 0.8-0.9, air is firstly introduced until the pressure in the steam explosion tank is 5-10 kg/cm 2 Then, the steam is quickly introduced into the steam explosion tank until the pressure in the steam explosion tank is 10-18 kg/cm 2 The temperature in the tank reaches 130-250 ℃, steam explosion pressure maintaining treatment is carried out for 5-15 min, and the explosion time is not higher than 0.00875s;
the conditions of the steam explosion treatment II are as follows: blasting in the same cavity of the same blasting equipment, and introducing air until the pressure in the steam explosion tank is 5-10 kg/cm 2 Then the steam is quickly introduced into the steam explosion tank10 to 18kg/cm 2 And (3) leading the temperature in the tank to reach 130-250 ℃, carrying out steam explosion pressure maintaining treatment for 15-45 s, wherein the explosion time is not more than 0.00875s, then quickly relieving pressure, and releasing the material treated in the steam explosion tank into a normal pressure container to obtain the material subjected to steam explosion pretreatment.
The steam explosion technology is a processing method that the raw material is heated to 180-235 ℃ by using steam, the steam is released instantly after the pressure is maintained for several seconds to several minutes, secondary steam is generated when the pressure is reduced instantly, the gas expands rapidly, and the structure of the solid material is destroyed under the action of mechanical force.
Licorice root and stem total extract is prepared by placing Glycyrrhrizae radix and stem and eutectic solvent (DES) in an ultrasonic extractor, ultrasonic extracting for 2-3 times, filtering, mixing filtrates to obtain filtrate I and residue, wherein the filtrate I is the eutectic solvent extract of Glycyrrhrizae radix and stem;
the charging proportion of the raw material of the liquorice root and the liquorice stem to the eutectic solvent is 1: 5-10 (g: mL);
the ultrasonic power is 300-500W, the ultrasonic working frequency is 30-60 KHZ, the extraction temperature is 50-70 ℃, and the extraction time is 0.5-1.5 h;
the eutectic solvent consists of a hydrogen bond acceptor, a hydrogen bond donor and water;
the content of water in the eutectic solvent is 10-30 wt%;
the feed-liquid ratio of the liquorice raw material to the eutectic solvent is 20-100 mg/mL;
the hydrogen bond receptor is selected from one of choline chloride, betaine or methyl trioctyl ammonium chloride;
the hydrogen bond donor is selected from one of lactic acid, urea, citric acid, acetic acid, 1, 4-butanediol, glycerol, malonic acid, ethylene glycol, 1, 3-propanediol or n-propanol;
the molar ratio of the hydrogen bond acceptor to the donor in the eutectic solvent is 1 to (0.5-5).
The eutectic solvent is a substance which is synthesized by quaternary ammonium salt and a hydrogen bond donor and is in a liquid state at room temperature, is a uniform stable system formed by hydrogen bond acceptors and hydrogen bond donors through hydrogen bonds in a certain stoichiometric ratio, is a novel green solvent, is formed by mixing two or more components, has the characteristic of adjusting the structure and the property by selecting different hydrogen donors and hydrogen acceptors, has the advantages of simple preparation process, low price, environmental friendliness and easy degradation, and has the solubility similar to that of the traditional ionic liquid. The atom utilization rate in the synthesis process reaches 100%, the method accords with the concept of green chemistry, and the method is considered to be a good substitute for the traditional volatile organic solvent and the ionic liquid. The method has the advantages of low carbon, sustainability, ecological friendliness and the like, has good development prospect, and is concerned in the extraction and separation and chemical synthesis of the active ingredients of the medicinal plants.
Soaking the treated resin in the filtrate I, stirring by a glass rod at intervals, adsorbing for 2-3 times, and filtering to obtain macroporous resin and filtrate II;
the macroporous resin is one or the mixture of two of D-101, HPD722, HPD-100, HPD-300, HPD400, HPD-BJQH, LX-60, ADS-17, AB-8, X-5, HZ-835, ADS-F8, SZ6, SP825, DA201, HP-20 and XDA macroporous resin;
adding monopotassium phosphate into the filtrate II, fully and uniformly mixing, completely dissolving the monopotassium phosphate, standing for 60-100 min, centrifuging the solution for 10-2 min at the centrifugal rotating speed of 3000-5000 rpm to form a double-aqueous-phase extraction system;
the dosage of the monopotassium phosphate is 30-60% (g/mL) of the filtrate II;
in the DES/alcohol double-aqueous phase system, small molecular alcohol is enriched in an upper phase, two components of an eutectic solvent are enriched in a lower phase, two phases are formed due to the difference of the abilities of the eutectic solvent and alcohol to combine with water molecules, and related to the hydrophobicity of the eutectic solvent and the alcohol, the larger the difference of the hydrophobicity of the eutectic solvent and the small molecular alcohol is, the stronger the phase forming ability of the constructed double-aqueous phase system is, the temperature influences the hydrogen bond acting force in the system, the raising temperature weakens the ability of the eutectic solvent to form the double-aqueous phase, the increase of the alcohol content in the double-aqueous phase forming process can ensure that the eutectic solvent and the alcohol are more thoroughly separated in the two phases, and the gradient of the two phases is correspondingly increased. The double aqueous phases refer to a system in which two water-soluble substances are incompatible to cause a mixed solution of the two water-soluble substances to separate into two phases, and the two phases are extracted and purified through the difference of distribution coefficients of solutes between the two phases or directly used as an extracting agent, so that the system has the advantages of rich raw materials, low price, small solvent viscosity, high mass transfer speed, easy recovery of reagents and the like, and has attracted wide attention.
The upper phase after the double aqueous phase extraction contains licorice protein, the protein is extracted by reverse micelles, the reverse micelle system is added into the double aqueous phase extraction system formed by the filtrate II in the former extraction, so that the protein is transferred into the reverse micelle system from the DES, a buffer solution is added into the former extraction solution, so that the protein is transferred into the aqueous phase from the reverse micelle to separate the protein, the back extraction of the protein is realized, and the aqueous solution of licorice root and stem protein is obtained by centrifugal separation;
the reverse micelle system consists of a surfactant-cosolvent-inorganic salt buffer solution;
w is o Represents the molar concentration ratio of water to surfactant, W, in the reverse micelle solution o ＝[H 2 O]/[ surfactant ]]；
The surfactant is one of Sodium Dodecyl Sulfate (SDS), dioctadecyl dimethyl ammonium chloride, dodecyl trimethyl ammonium chloride or dioctyl sodium sulfosuccinate (AOT);
the cosolvent is one of glycerol, isooctane or n-decanol;
the addition amount of the cosolvent is 0-10% of the total amount of the reverse micelle solution;
the inorganic salt is sodium chloride or potassium chloride;
the pre-extraction has been performedThe process: adjusting the pH value of DES liquid containing liquiritin to 4.0-6.0, the concentration of surfactant to 0.05-0.08 g/m L, the ratio of material to liquid to 1: 25, the concentration of salt to 0.07mol/L, and W 0 =27, mixing the extract with a reverse micelle solution, extracting for 10-35 min, centrifuging at the temperature of 40 ℃ and the rpm of 1500-5000, layering, and taking an upper organic phase;
the post-extraction process comprises the following steps: mixing the obtained upper layer organic phase and potassium chloride-phosphate buffer solution according to the volume ratio of 1: 1-2, performing back extraction, mixing for 5-40 min, performing centrifugal layering at 1500-5000 rpm, separating to obtain a protein-containing water phase, performing protein separation on the protein-containing water phase, and performing freeze drying to obtain the licorice protein.
Pre-extracting the primary extract with reverse micelle extract to separate out upper organic phase, back-extracting the separated upper organic phase with back-extracted water phase to separate out lower water phase, hydrolyzing the separated lower water phase with acid solution to obtain acidolysis solution, and purifying the obtained acidolysis solution.
Diluting the lower phase after aqueous two-phase extraction with water, passing the water solution through Sephadex LH20 gel column chromatography, washing with water to obtain glycyrrhiza polysaccharide eluate, dialyzing the eluate, collecting polysaccharide solution, concentrating, decolorizing, precipitating with ethanol, collecting precipitate, drying to obtain crude glycyrrhiza polysaccharide, and purifying to obtain refined glycyrrhiza polysaccharide;
dialyzing, namely dialyzing the used dialysis bag with molecular interception of 8000-12000 Da in sequence in running water and deionized water, and collecting liquid in the dialysis bag;
Concentrating under the vacuum degree of- (0.06-0.08) MPa and at the temperature of 50-60 ℃ to obtain concentrated solution with the relative ratio of 1.05-1.10;
the decolorization is carried out by adding 3-5% (w/w) of H 2 O 2 Heating at 60 deg.C for 30min;
precipitating the ethanol, adding ethanol with the volume fraction of 85-95% to ensure that the volume fraction of the ethanol reaches 65-70%, and standing the mixture for 2-40 min at the temperature of 0-4 ℃ to prepare an ethanol precipitate;
filtering, transferring into a centrifuge tube of a centrifuge, and centrifuging for 10-20 min under the condition that the rotating speed is 4000-5000 rpm;
the drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and under the vacuum degree of between 0.06 and 0.08 MPa;
the crude product of glycyrrhiza polysaccharide is purified by S-8 resin, and the technological parameters are as follows: the glycyrrhiza polysaccharide sample concentration is 5.23mg/mL, the initial PH value is 5, and the flow rate is 2.0BV/H.
Eluting the Sephadex LH20 gel column eluted with the glycyrrhiza polysaccharide by using a 10-20% sodium chloride solution to obtain glycyrrhiza glucan, decoloring, filtering, concentrating the filtrate to be dry, dissolving the filtrate in purified water, filtering, collecting the filtrate, concentrating the filtrate, adding ethanol for precipitation, collecting the precipitate, and drying to obtain the glycyrrhiza glucan;
the decolorization is carried out by adding 3-5% (w/w) of H 2 O 2 Heating at 60 deg.C for 30min;
The concentration is carried out at the vacuum degree of between 0.06 and 0.08 MPa and the temperature of between 50 and 60 ℃ to obtain concentrated solution with the relative ratio of between 1.05 and 1.10;
precipitating the ethanol, adding ethanol with the volume fraction of 85-95% to ensure that the volume fraction of the ethanol reaches 65-70%, and standing the mixture for 2-40 min at the temperature of 0-4 ℃ to prepare an ethanol precipitate;
filtering, transferring into a centrifuge tube of a centrifuge, and centrifuging for 10-20 min under the condition that the rotating speed is 4000-5000 rpm;
the drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and the vacuum degree of between 0.06 and 0.08 MPa;
adding 1-2 times (w/w) of sodium carbonate or sodium bicarbonate solution with the pH value of 9-10 into the filter residue I after DES extraction, heating and boiling for 30-80 min, carrying out suction filtration to obtain clear liquid and filter residue II, washing the filter cake with water until the effluent liquid is clear, and combining the washing liquid and the filtrate to obtain insoluble dietary fiber extracting solution;
concentrating the lignin extract under reduced pressure until the relative specific gravity is 1.10-1.20, adjusting the pH value to 1-3 by using one of hydrochloric acid, sulfuric acid or phosphoric acid, precipitating, performing filter pressing, and drying a filter cake to obtain insoluble dietary fiber;
adding 4-8 times (w/w) of calcium hydroxide solution with the pH value of 11-13 into the filter residue H, adding 3-10% (w/v) of hydrogen peroxide or sodium hypochlorite solution while stirring, adjusting the pH value to 11-12 by using one of hydrochloric acid, sulfuric acid or phosphoric acid, performing filter pressing, washing with water until the effluent is neutral, and drying to obtain the soluble dietary fiber.
Adding activated carbon into the eutectic solvent which is effectively and fully extracted by the regeneration of the eutectic solvent, heating to 40-70 ℃, preserving the heat for 30min, filtering, concentrating the filtrate at the temperature of 60-70 ℃ and under the vacuum degree of 72.6-83.8 KPa to obtain the regenerated eutectic solvent.
Extracting the obtained macroporous resin with ethanol, concentrating the extractive solution under reduced pressure, removing ethanol, adding purified water, ultrasonic diluting, centrifuging, adsorbing the centrifugate with polyamide resin, washing with water, and collecting the water washing solution; eluting polyamide resin with gradient ethanol, and collecting eluate;
putting the macroporous resin in an ultrasonic extractor, and desorbing by using ethanol as an desorbing agent under the action of ultrasonic waves;
extracting the macroporous resin with 80-90% ethanol by volume for 30min at 40-50 ℃, repeating the extraction for 2-3 times until the extract is clear, and combining the ethanol extracts;
concentrating the ethanol extract under reduced pressure to obtain a concentrated solution with the solid content of 50-60 wt%, and adding water for ultrasonic dissolution promotion;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
adding water for ultrasonic dilution, adding purified water into the concentrated solution, and fully stirring uniformly under ultrasonic to form a diluted solution with a considerable proportion of 1.02-1.05;
the ultrasonic frequency is 25kHz, the ultrasonic power is 100-300W, the ultrasonic extraction temperature is 30-55 ℃, the ultrasonic extraction time is 10-30 min,
The centrifugation step, the diluent is moved into a centrifuge tube of a centrifuge, and the centrifugal separation is carried out for 10-20 min under the condition that the rotating speed is 4000-5000 rpm, so as to obtain a centrifugate;
adsorbing the centrifugate with polyamide resin, washing with water, and collecting water washing solution which is glycyrrhizin;
the height ratio of the column diameter of the resin is 1: 10-1: 20, the sample loading amount is 10-30 BV, the adsorption flow rate is 2-10 BV/h, and after the adsorption is finished, 2-8 BV of deionized water is used for eluting to obtain glycyrrhizin washing liquid;
eluting the polyamide resin after water washing by gradient ethanol, and eluting the macroporous resin adsorption column by 20 to 30 weight percent ethanol solution for 2.0 to 4.0BV to obtain a component A which is a polyphenol compound; then eluting the macroporous resin adsorption column by using 70 to 80 weight percent ethanol solution for 2.0 to 5.0BV to obtain a component B which is a flavonoid compound;
concentrating the component 1 under reduced pressure to obtain the licorice polyphenol with the solid content of 60-70 wt%, and drying in vacuum;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
the vacuum drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and under the vacuum degree of between 0.06 and 0.08 MPa.
The glycyrrhizin washing liquid is used as initial feed liquid, and a first-stage defoaming liquid is obtained through two-stage foam separation;
The first stage foam separation is carried out at the initial concentration of feed liquid of 2.0 mg.mL -1 Liquid loading amount of 300mL, pH 4.0, concentration of 2.0 mg/mL -1 Nitrogen flow rate 200mL.min -1 Electrolyte sodium chloride concentration 0.20 mol.L -1 The foam separation temperature is 60 ℃;
the second stage of foam separation is carried out at the nitrogen flow rate of 300 mL-min -1 The concentration of sodium chloride in the electrolyte is 0.20 mol.L -1 The foam separation temperature is 50 ℃, the liquid loading amount is 270mL, and the pH value is 4.5;
the second-stage foam separation is to take residual liquid left in the first-stage foam separation tower as feed liquid of the second-stage foam separation to enter a second-stage foam separation tower;
and mixing the second-stage defoaming solution and the glycyrrhizin water washing solution, adding the mixture into the feed of the first-stage foam separation tower, and performing next-batch foam separation.
The flotation separation and enrichment technology is an effective method for separating and enriching trace substances, and is a method for separating a target substance in a solution from a mother solution by using the adsorption effect of bubbles, wherein the target substance in the solution to be separated is gathered at a gas-liquid interface, and is easily adsorbed on the surface of micro bubbles due to the small interfacial tension (or strong hydrophobicity) of the component to be separated with surface activity (or hydrophobicity), and is brought to the top of a flotation column along with the rising of the bubbles, and after the bubbles are broken, the target substance to be separated and an organic phase at the top of the flotation column have certain effect (directly dissolve or dissolve after undergoing a complexing reaction with a complexing agent in the organic phase). The method has the advantages of simple operation, high separation efficiency, high enrichment coefficient, mild mass transfer, low organic solvent consumption, simple equipment, normal-temperature operation, small occupied area, low cost and the like, is easy to operate and grasp, has mild separation process and the like, and has good application prospect. The solution contains surface active ingredients which are one of the necessary conditions for flotation separation, the saponin in the liquorice has the characteristics of a surfactant, stable foam can be generated during strong stirring or boiling, the necessary conditions for foam separation are met, and the flotation separation enrichment technology is widely applied to the extraction and separation of natural active substances.
Adding warm water with the temperature of 30-45 ℃ into the obtained first-stage defoaming solution, uniformly dispersing the first-stage defoaming solution into liquid with the concentration of 50-100 mg/mL, adsorbing the liquid by macroporous resin at the sampling speed of 0.5mL/min, statically adsorbing the liquid for 30-50 min, performing gradient elution, collecting eluent and concentrating the eluent to obtain concentrated solution;
the gradient elution is carried out by adopting purified water with the pH value of 2.0-4.0 BV of 4-6 and the temperature of 15-25 ℃ to wash, and impurities are removed until the eluent is clear;
eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 5-10 percent to obtain a component (1);
eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 50-70 percent to obtain a component (2);
finally eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 80-90 percent to obtain a component (3);
the macroporous resin, one of AB-8, D101, D140, HPD100, LX-38, LSA-12S, LX-68, S-8, FL-2, DM130, X-5, D201, HZ806, LSA-5B and NAK-12;
concentrating the component (2) under reduced pressure, and vacuum drying to obtain Glycyrrhrizae radix and stem saponin;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
the vacuum drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and under the vacuum degree of between 0.06 and 0.08 MPa.
Concentrating the obtained component B under reduced pressure to obtain an extract, adding warm water at 40-60 ℃, fully stirring, standing at room temperature, extracting with ethyl acetate and n-butyl alcohol respectively, collecting ethyl acetate extract and n-butyl alcohol extract respectively, and concentrating under reduced pressure to obtain ethyl acetate part extract and n-butyl alcohol part extract respectively;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
the solid content of the extract is 70-80 wt%;
dissolving part of the ethyl acetate extract by using methanol, filtering, adding 1-2 times of 48-75 mu m silica gel into the filtrate, uniformly stirring, volatilizing the solvent, drying in the air, crushing, sieving by using a 75 mu m sieve, adding 48-75 mu m column chromatography silica gel which is 15-20 times of the weight of the dried substance II into a silica gel chromatographic column, and carrying out chromatographic separation;
the silica gel chromatographic column has the diameter-height ratio of 1: 10-1: 20, the chloroform-methanol volume ratio is sequentially 100: 0, 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 for gradient elution, eluent is identified by TLC, and 7 parts Fr.1-Fr.7 are obtained by classification and combination;
the Fr.1 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by gradient elution is subjected to thin-layer chromatography identification, classification and combination, and glabridin are obtained by separation through centrifugal partition chromatography;
The centrifugal partition chromatography is used for obtaining a satisfactory separation effect by carrying out systematic optimization experiments on parameters such as a two-phase solvent system, a rotor rotating speed, a mobile phase flow rate and the like, wherein the optimal two-phase solvent system is n-hexane-ethyl acetate-methanol-water (1: 5: 1: 5, v/v), an upper phase organic phase is a stationary phase, a lower phase aqueous phase is a mobile phase, the rotor rotating speed is 1500rpm, the flow rate is 3.0mL/min, and the ultraviolet detection wavelength is 287nm.
Figure BSA0000282013940000531
The Fr.2 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, the eluent is identified by TLC, isoliquiritigenin is obtained at the position where the volume ratio of the petroleum ether to the ethyl acetate is 100: 20, and liquiritigenin is obtained at the position where the volume ratio of the petroleum ether to the ethyl acetate is 100: 30;
Figure BSA0000282013940000532
performing thin layer chromatography, performing silica gel column chromatography on the eluent with the volume ratio of petroleum ether to ethyl acetate of 100: 50, performing gradient elution with the volume ratio of petroleum ether to ethyl acetate of 100: 10, 100: 20, 100: 30, 100: 50 and 100: 100 in sequence, and performing thin layer chromatography on the eluent to obtain the liquiritigenin at the position with the volume ratio of petroleum ether to ethyl acetate of 100: 20; the glabrene is obtained at the volume ratio of petroleum ether to ethyl acetate of 100: 20.
Figure BSA0000282013940000533
And the Fr.3 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 10, 100: 15, 100: 20, 100: 30, 100: 40, 100: 100 and 0: 100 in sequence, the eluent obtained by the gradient elution is subjected to thin layer chromatography identification, classification and combination, the eluent is subjected to Sephadex LH-20 hydroxypropyl Sephadex column chromatography, elution is carried out by using chloroform and methanol as eluent according to the volume ratio of 6: 4, then the same components are combined through thin layer tracking detection, and the licoflavonol A are obtained through separation.
Figure BSA0000282013940000541
The Fr.4 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by the gradient elution is subjected to thin-layer chromatography identification, classification and combination, and is subjected to supercritical fluid chromatography separation to obtain liquiritin P, liquiritin N, liquiritin G, liquiritin O, liquiritin I and liquiritin M;
the supercritical fluid chromatographic separation conditions are as follows: the supercritical fluid chromatographic separation conditions are as follows: polar chromatographic column ZorBaxSB-CN (9.4 mm. Times.250mm, 5 μm) as stationary phase and CO 2 Methanol (containing 0.2% of trifluoroacetic acid and 0.1% of ethanolamine) (90: 10, v/v) as a mobile phase, an entrainer content of 13.64wt%, an entrainer (methanol-0.2 wt% of phosphoric acid), an ultraviolet detection wavelength of 280nm, a UV detection wavelength of CO 2 The flow rate is 10-20 g.min -1 Column pressure is 12-14 MPa, sample injection amount is 0.05mL, flow rate is 2mL/min, column temperature: 40 ℃, column pressure program: the initial pressure is 8MPa, the pressure is increased to 12-14 MPa at the speed of 300kPa/min, and 2 mu L of sample is injected.
The supercritical fluid chromatography as a novel chromatographic separation technology combines the characteristics of gas chromatography and liquid chromatography, and because the density of the supercritical fluid is similar to that of liquid, the viscosity is similar to that of gas, the self-diffusion coefficient is far greater than that of liquid, mass transfer can be carried out more quickly, the equilibrium time is short, and CO is low 2 The method has the advantages of high efficiency, high speed, high separation efficiency, short separation time, small organic solvent consumption in the separation process, simple post-treatment, high product quality, less organic solvent residue in the product, environmental protection and the like. Thus, supercritical CO 2 The fluid becomes the first choice of the mobile phase, is called as the 'green environmental protection technology', is more and more widely applied in the fields of chiral drug resolution, separation and purification of traditional Chinese medicines and natural products and the like, is widely concerned, and has wide application prospect.
Figure BSA0000282013940000542
Figure BSA0000282013940000551
The Fr.5 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by the gradient elution is identified by thin-layer chromatography, classified and combined, and is separated by coordination chromatography to obtain licoisoflavone A, licoflavone A, licoisoflavan A, glycyrrhiza isoflavone A, licoisoflavone B, licoricone and licoisoflavanone;
performing coordination chromatographic separation, namely preparing a coordination chromatographic column with the central ion content of 7-10% by taking metal ions as central ions, loading a sample on the coordination chromatographic column, sequentially identifying eluent by thin-layer chromatography according to the gradient volume ratio of chloroform to methanol of 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100, and separating out licoisoflavone A, licoflavone A, licoisoflavone B, licoricone and licoisoflavanone;
the metal ion is Se 4+ 、Cu 2+ 、Mg 2+ 、Ni 2+ 、Al 3+ Or Zn 2+ One of them.
Figure BSA0000282013940000552
Figure BSA0000282013940000561
According to the theory of multi-element coordination chromatography, the mixed components flow out on a chromatographic column in sequence according to the stability difference of the complexes formed by the mixed components and central ions to achieve separation, and the main factors influencing the separation efficiency of the coordination chromatography comprise the selection of the central ions and the change of a mobile phase. The central ion is the basis of separation, the space arm has an auxiliary effect on the separation, and the loss performance of the central ion is a main factor influencing the separation repeatability; the change of the mobile phase can improve the separation efficiency, shorten the separation time and reduce the consumption of the solvent. The natural product and the central ion have common functions, and compared with the traditional chromatographic separation, the coordination chromatography has high separation degree, high yield, less solvent consumption and better protection on unstable compounds, so the coordination chromatography is beneficial to improving the separation efficiency, reducing the cost, being suitable for industrial scale-up production, having less solvent pollution and being easier to meet the requirement of environmental protection.
The Fr.6 part is subjected to silica gel column chromatography, the gradient volume ratio of chloroform to methanol is 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, and eluent is subjected to thin layer chromatography identification to finally obtain a mixture containing licochalcones A-E;
separating and purifying the mixture of licochalcone A-E by high-speed counter-current chromatography to obtain licochalcone A-E;
the high-speed countercurrent chromatography adopts n-hexane, ethyl acetate, methanol and water (1.0: 2.5: 1.5: 0.1, v/v/v/v) as a two-phase solvent system, the lower phase as a stationary phase and the upper phase as a mobile phase, the reverse rotation is carried out, the rotating speed is 850r/min, the temperature of a constant-temperature water bath is 25 ℃, and the licochalcone A-E5 components are obtained by separation under the conditions that the flow rate of the mobile phase is 1.5mL/min and the sample injection concentration is 20 mg/mL;
Figure BSA0000282013940000562
concentrating the Fr.7 part into extract, dissolving in water, adsorbing with macroporous adsorption resin, eluting with 70-80 vol% methanol as eluent, concentrating the eluent into extract, subjecting the obtained extract to silica gel column chromatography, gradient elution with chloroform and isopropanol at volume ratio of 20: 1, 10: 1, 5: 1, 1: 1, 0: 1, detecting the eluent by thin layer chromatography, classifying, mixing, and separating by microemulsion electric capillary chromatography to obtain isoliquiritin, liquiritin, neoliquiritin, neoisoliquiritin;
According to the microemulsion electric capillary chromatography, when a microemulsion system is 50mmol/L boric acid buffer solution (pH 95), 10% (volume fraction) n-butanol, 80mmol/L n-heptane, 120mmol/L sodium dodecyl sulfate and 5mmol/L sulfonated beta-cyclodextrin, the separation voltage is 20kV, and the column temperature is 35 ℃, the microemulsion electric capillary chromatography for separating isoliquiritin, liquiritin, neoliquiritin and neoisoliquiritin is established, and the separation of the 4 components in a sample solution is realized within 30 min.
The capillary electrophoresis is a novel separation and analysis technology which takes an electric field as a driving force and a capillary as a separation channel, and has the characteristics of multiple separation modes, high separation efficiency, high analysis speed, less reagent and sample consumption, low cost, wide application range and the like.
Figure BSA0000282013940000571
Mixing the n-butanol part extract with silica gel with the particle size of 48-75 μm, loading the mixture on a dry column for silica gel column chromatography, performing gradient elution by using chloroform-methanol as an eluent, wherein the gradient volume ratio of chloroform to methanol is 30: 1, 20: 1, 10: 1, 5: 1, 3: 1, 1: 1 and 0: 1 in sequence, and performing thin-layer chromatography identification and combination on corresponding eluents to obtain 4 parts Fr.I-Fr.IV;
subjecting the Fr.I part to silica gel column chromatography, sequentially detecting the eluate with ethyl acetate-methanol volume ratio of 100: 0, 100: 5, 100: 10, 100: 20, 100: 50, 100: 100 and 0: 100 by thin layer chromatography, subjecting the eluate to silica gel column chromatography again, sequentially detecting the eluate with chloroform-methanol volume ratio of 100: 5, 100: 10, 100: 20, 100: 30, 100: 100 and 0: 100 by thin layer chromatography, and subjecting the eluate to apiosyisoliquiritin at gradient volume ratio of chloroform-methanol of 100: 10;
Figure BSA0000282013940000572
And performing silica gel column chromatography with chloroform-methanol at gradient volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 40, 100: 100 and 0: 100, subjecting the eluate to thin layer chromatography, subjecting the eluate to Sephadex LH20 column gradient elution with methanol of 10%, 15% and 20% volume fraction, and subjecting the eluate to thin layer chromatography to obtain isoliquiritigenin-4' -O-apiose (1 → 2) glucoside.
Figure BSA0000282013940000581
And (3) performing gradient elution on the Fr.III part by silica gel column chromatography with the volume ratio of chloroform to methanol of 100: 5, 100: 10, 100: 15, 100: 20, 100: 25, 100: 100 and 0: 100 in sequence, detecting the eluent by using thin layer chromatography, performing isocratic elution by using chloroform-methanol as an eluent, wherein the volume ratio of chloroform to methanol is 100: 10, and obtaining the apiosyl liquiritin.
Figure BSA0000282013940000582
The fr.iv fraction need not be isolated.
Drawings
The process flow chart is shown in the figures 1 to 10.
FIG. 1 extraction of Glycyrrhiza uralensis seeds and separation of oil and fat components
FIG. 2 Glycyrrhiza seed protein and polysaccharide separation
FIG. 3 Licorice leaf extraction and pigment and volatile component separation
FIG. 4 separation of Glycyrrhiza glabra leaf polysaccharide and prenylflavonoid
FIG. 5 extraction of licorice root and stem and separation of protein and polysaccharide
FIG. 6 separation of soluble and insoluble dietary fibers from the root and stem of Glycyrrhiza glabra
FIG. 7 initial separation of Glycyrrhiza glabra root, stem Polyphenol and flavonoid
FIG. 8 separation of Licorice root and Stem saponins
FIG. 9 separation of flavonoid glycosides from radix Glycyrrhizae
FIG. 10 separation of flavonoids from licorice root and stem
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The method not specifically mentioned in the following examples is selected in accordance with conventional methods and conditions, or in accordance with the commercial instructions. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
In the following examples, room temperature means 15 to 25 ℃.
Materials and instruments: choline chloride, lactic acid, 1, 4-butanediol, glycerin, urea, malonic acid, ethylene glycol, glucose, 1, 3-propanediol, n-propanol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide, anhydrous sodium sulfate, ethanol, t-butanol, ammonium sulfate, diethyl ether, petroleum ether (60-90 ℃), isopropyl alcohol, copper sulfate, hydrochloric acid, methanol, sulfurous acid, ethyl acetate, calcium hydroxide, silver nitrate, n-hexane, sodium chloride, sodium carbonate, copper chloride, aluminum trichloride hexahydrate, zinc chloride, nickel chloride, aqueous ammonia, sodium dioctyl sulfosuccinate, glycerin, potassium chloride, aluminum nitrate, hydrogen peroxide, borax, β -cyclodextrin, hydrochloric acid, sulfuric acid, phosphoric acid, n-butanol, obtained from alatin, berlingwei, aldrich, tianjinke michelsen chemical agents ltd and national chemical agents ltd; waters high performance liquid chromatography, uv-260 Uv-visible spectrophotometer (Shimadzu corporation), 800 centrifugal precipitator (Shanghai easy instruments, ten factories), water bath (Shanghai-Hengsheng scientific instruments Co., ltd.), rotary evaporator (Shanghai Shensheng scientific Co., ltd.), vacuum drying oven (Nanjing Wao Ring science and technology Co., ltd.), dialysis bag (Shanghai Living packaging materials Co., ltd.), D-101, HPD722, HPD-100, HPD-300, HPD400, HPD-BJQH, LX-60, ADS-17, AB-8, X-5, HZ-835, HZ-F8, SZ6, SP825, DA201, HP-20, XDA macroporous resin (Senah blue West-Chun science and technology Co., ltd.), sephadex LH20 gel, polyamide resin, sephadex resin, and silica gel fine carborundum, a pharmacopoeia sieve (Dahan vibration machinery Co., ltd., xinxiang city), 48-75 μm silica gel (Qingdao ocean chemical industry Co., ltd.), a silica gel thin-layer plate (Qingdao ocean chemical industry Co., ltd.), a sample application capillary tube (Waxi medical university apparatus factory), attapulgite (Lingshu county Xuanguang mineral product processing factory), activated carbon (Li Yang Zijin activated carbon Co., ltd.), a vacuum water pump SHB-IIIG (Zhengzhou Changcheng apparatus Co., ltd.), a French GILSON centrifugal distribution chromatograph, a waters supercritical fluid chromatograph, a vacuum extrusion swelling machine, a separating funnel, a stirrer (Shanghai Shensheng science and technology Co., ltd.), tin paper, a chromatographic column, a TTC-24 subcritical water extraction instrument (Hangzhou Hui apparatus), ultrafiltration membrane, active carbon, buchner flask, planetary ball mill (Changsha Tianchuang powder technology limited), multifunctional extraction tank, continuous countercurrent extraction instrument (Qingdao Jinghong environmental protection technology), hollow fiber membrane, foam separator, TBE-300A type preparative countercurrent chromatograph (Shanghai Tongtian biotechnology limited), instantaneous ejection type steam explosion machine (Henan Zhengdao Qibao environmental protection technology limited), ultrasonic extraction instrument (Qingdao Juchuang environmental protection group limited), TCLX200 ultrasonic traditional Chinese medicine extraction instrument (Ningbo Sha national pharmaceutical equipment manufacturing limited).
1. Licorice seed
100kg of liquorice seeds are taken, impurities such as straws, gravels, metal blocks and the like are removed, the cleaned liquorice seeds are soaked in a sulfurous acid solution for a period of time, the soaking time is 10-14 h, the temperature is 30-50 ℃, the concentration of the sulfurous acid in a soaking solution is 0.1-0.2%, the water content is 15-20%, the soaked liquorice seeds enter a crusher to be crushed, and the crushed liquorice seeds enter a vacuum extrusion bulking machine. Vacuum degree of-0.07 MPa, sleeve temperature of 87 ℃, extruder screw rotation speed of 100rpm, die hole diameter of 17mm, feeding amount of 3.2kg/min, extrusion temperature of 120-150 ℃, extrusion pressure of 10MPa, extrusion time of 3min, round or rectangular shape of the aperture of the grinding head of the extruder, 48.5kg of licorice seed oil obtained after extruding the bulking machine, and granularity D95 of bulking material of not more than 125 μm.
100g of liquorice seed oil is taken and added with petroleum ether (60-90 ℃) for extraction, and the volume ratio of the petroleum ether to the oil is 1: 1-1: 13; extracting for 3 times, combining the upper phase extraction liquid, evaporating and concentrating under the conditions of vacuum degree- (0.06-0.08) MPa and temperature 40-50 ℃ to remove the solvent, and obtaining brown oily matter; dissolving the brown oily substance in ethanol, adding 10-20% sodium hydroxide, and performing saponification reaction for 50min at the temperature of 70 ℃ and the material-liquid ratio of 1: 3; adding petroleum ether (60-90 ℃) into the saponified solution for extraction, wherein the volume ratio of the petroleum ether to the grease is 1: 1-1: 3; extracting for 3 times, combining the upper phase extraction liquid, and removing the solvent by evaporation and concentration under the conditions of vacuum degree of (0.06-0.08) MPa and temperature of 40-50 ℃ to obtain brown oily matter; dissolving the brown oily substance in ethanol, adding 10-20% sodium hydroxide, and performing saponification reaction at 70 ℃ for 50min at a material-liquid ratio of 1: 3; adding petroleum ether (60-90 ℃) into the saponification liquid for extraction, wherein the volume ratio of the petroleum ether to the grease is 1: 1-1: 3; extracting for 3 times, mixing the upper phase extractive solutions, evaporating, concentrating to remove solvent to obtain brown oil, preparing with argentized silica gel, loading into column, eluting pigment, gradient eluting with Ag + Removing, decoloring and crystallizing. Preparing silvered silica gel, adding 48-75 μm silica gel into silver nitrate solution containing 8-20% under the condition of keeping out of the sun, fully stirring into paste, heating and stirring for 20-40 min under the condition of 90-95 ℃ water bath, then cooling to 25-35 ℃, carrying out suction filtration, activating the suction filtration in a vacuum drying oven for 15-25 h, and preparing the silvered silica gel which is placed in the shade for standby. Packing the silver silica gel into a column, placing the column into petroleum ether, stirring to remove bubbles, standing to fully swell the column, adding the column into a chromatographic column, wrapping the chromatographic column with tinfoil, and eluting and balancing the column with the petroleum ether. Dissolving the brown oily substance with petroleum ether, dropwise adding into the upper end of a silver silica chromatographic column, opening a valve at the lower end of the chromatographic column to allow the sample liquid to be slowly adsorbed on the silver silica gel, and starting gradient elution when the sample is about to be adsorbed; the gradient elution is carried out by taking the gradient volume ratio of petroleum ether to ethyl acetate as eluent of 100: 0, 100: 5, 100: 8, 100: 10, 100: 15, 100: 20, 100: 100 and 0: 100 in sequence, detecting and analyzing each flow part of the eluent by TLC and HPLC respectively, and combining the same flow parts. Ag + Removing, combining the fractions analyzed by TLC and HPLC to contain squalene, collecting eluate, rotary evaporating, and removing eluate Removing agent, dissolving with n-hexane, adding saturated sodium chloride solution to remove Ag + Adding anhydrous sodium sulfate for dehydration, and filtering to obtain filtrate; evaporating the filtrate under reduced pressure, adding anhydrous methanol for dissolving, adding attapulgite for decoloring for 30min, performing suction filtration, concentrating the filtrate at the temperature of 40-50 ℃ under the vacuum degree of- (0.06-0.08) MPa to the original volume of 1/3-1/4, standing, and crystallizing to obtain 0.72g of squalene with the content of 98.6%. A detection method for measuring squalene content by high performance liquid chromatography adopts an Xbridge RP C18 chromatographic column (250 mm multiplied by 4.6mm,3.5 mu m), a mobile phase acetonitrile-methanol (60: 40, v/v), a flow rate of 1.0mL/min, a detector of a photodiode array detector, a detection wavelength of 210nm and a column temperature of 30 ℃.
The method separates the substances according to the column chromatography separation principle, namely, the substances are separated according to different adsorption forces of the substances on a stationary phase, and the squalene is separated and extracted by utilizing the complex formed by silver ions in the silvered silica gel chromatographic column and carbon-carbon double bonds of unsaturated fatty acid.
Treating natural attapulgite by double rollers, adding purified water of which the volume is 5-10 times (mL: g), adding magnesium salt or aluminum salt of which the volume is 3-8 times (g: g), adding chloroacetic acid of which the concentration is 0.1-1%, fully and uniformly stirring, adding sodium hydroxide to adjust the pH value to 9-11, continuously reacting for 5-10 hours, carrying out solid-liquid separation after the reaction is finished, calcining the solid product at 200-500 ℃ for 2-6 hours, crushing, and sieving by a sieve of 25-75 mu m to obtain the modified attapulgite adsorbent. Mixing the eluates from which squalene is separated, adding attapulgite adsorbent according to the material-liquid ratio of 1: 10-1: 30 (g: mL), refluxing and decolorizing for 30min, filtering, concentrating the filtrate under reduced pressure, and discharging when no effluent is available under the conditions of vacuum degree of- (0.06-0.08) MPa and temperature of 40-50 ℃ to obtain licorice seed oil 74.2 g.
Putting 100g of a liquorice puffed product into a subcritical water extraction device, introducing high-purity nitrogen, taking deoxidized distilled water as an extracting agent, adding deionized water at a material-water ratio of 1: 1-20 (g: mL), extracting at 100-150 ℃ for 10-120 min, uniformly stirring, screwing down an extraction kettle cover to seal the extraction kettle, controlling the pressure of the extraction kettle at 3-10 Mpa, then placing the extraction kettle on a temperature heating controller, controlling the preheating temperature of the temperature heating controller at 100-200 ℃, heating the extraction kettle to 140 ℃ by the temperature heating controller, keeping the temperature of materials in the extraction kettle at 140 ℃ for 10min, taking out the extraction kettle, cooling to room temperature by cold water within 5min, opening a valve at the bottom of the extraction kettle, allowing an extract liquid to flow into a stirring tank, separating liquid residues in the materials, and coarsely filtering to obtain liquid containing glycyrrhiza polysaccharide and protein and filter residues.
Centrifuging the filtrate in the stirring tank at 3000-5000 rpm for 10-20 min, and concentrating the supernatant with ultrafiltration membrane with cut-off molecular weight of 10-50 kD to 1/3-1/10 of the original volume to obtain crude concentrated solution containing glycyrrhiza polysaccharide and protein; adding the crude extract concentrated solution containing the glycyrrhiza polysaccharide and the protein into ethanol with the volume fraction of 90-95% to ensure that the final volume fraction of the ethanol is 70-80%, uniformly stirring, standing and precipitating for 5-10 h, carrying out suction filtration to obtain a precipitate, washing the precipitate for 1-3 times by using absolute ethanol, and carrying out vacuum drying to obtain a crude product containing the glycyrrhiza polysaccharide and the protein. Dissolving the glycyrrhiza polysaccharide and the crude protein product in deionized water to prepare a solution containing glycyrrhiza polysaccharide and protein with the concentration of 30-50 wt%, obtaining a solution containing sugar and protein, adding tert-butyl alcohol and ammonium sulfate to form a three-liquid-phase system, wherein the volume ratio of the solution containing sugar and protein to the tert-butyl alcohol is 1: 1-1: 3, the mass fraction of the ammonium sulfate is 30-50 wt%, the temperature is 35-40 ℃, the time is 30min, the pH value is 7, separating into three phases, the upper phase is pigment, the middle phase is protein, the lower phase is polysaccharide, and collecting the lower phase polysaccharide and the middle phase protein respectively. Decolorizing the middle phase protein in water bath at 45-50 deg.c for 90min and H for the first 60min 2 O 2 Decolorizing by an oxidation method, decolorizing by an activated carbon adsorption method after 30min, performing suction filtration, concentrating the filtrate to 1/2-1/20 of the original volume by an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD, adding absolute ethyl alcohol into the concentrated solution to ensure that the volume fraction of the ethyl alcohol is 75-85%, standing for 5-10 h, centrifuging for 10-15 min at 3 000-5000 rpm to obtain a precipitate, and washing the precipitate for 2-3 times by the absolute ethyl alcohol to obtain a licorice seed protein semi-finished product; adding 3-5 times (v/w) of 0.15-0.20 mol/L sodium chloride solution into the licorice seed protein, dissolving, centrifuging for 10-15 min at 3000-5000 r/min,collecting clear liquid; adding ammonium sulfate into the clear liquid to ensure that the saturation degree reaches 70-85%, separating out protein, centrifuging for 10-15 min at 3000-5000 rp m, and collecting precipitate and clear liquid, wherein the precipitate is total protein of licorice seeds; drying for 5-10 h at 50-60 ℃ and vacuum degree- (0.06-0.08) MPa to obtain 38.5g of total protein of the liquorice seeds.
Dialyzing the lower phase polysaccharide to remove inorganic salt, decolorizing the polysaccharide solution in water bath at 45-50 deg.C for 90min, and adding H for the first 6 min 2 O 2 Decolorizing by an oxidation method, decolorizing by an activated carbon adsorption method after 30min, performing suction filtration, concentrating the filtrate to 1/2-1/20 of the original volume by an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD, adding absolute ethanol into the concentrated solution to ensure that the volume fraction of the ethanol is 75-85%, standing for 5-10 h, and then centrifuging at 3000-5000 rpm for 10-15 min to obtain a precipitate, wherein the precipitate is a glycyrrhiza polysaccharide semi-finished product; and washing the glycyrrhiza polysaccharide semi-finished product with absolute ethyl alcohol for 2-3 times, taking a precipitate, and drying to obtain 29.6g of glycyrrhiza polysaccharide.
2. Extraction of licorice leaves
Extracting licorice leaves with the assistance of a mechanochemical method, grinding the licorice leaves by using a planetary ball mill, and mixing 200g of the licorice leaves with a ball milling auxiliary agent for ball milling; the ball milling auxiliary agent is Na 2 CO 3 And Na 2 B 4 O 7 With beta-cyclodextrin, na 2 CO 3 And Na 2 B 4 O 7 The mass ratio of (A) to (B) is 1: 2; the mass of the ball-milling auxiliary agent is 6.0-7.0 wt% of the mass of the licorice leaves; the optimal technological parameters are that the ball milling time is 30min, the ball milling rotating speed is 300rpm, the filling rate is 26.2 percent, and the material granularity D95 is less than or equal to 37 mu m; adopting a three-stage four-pot type countercurrent extraction mode, wherein the pot bodies are numbered A, B, C and D, 100kg of licorice leaves treated by a mechanochemical method are added into each pot, 600L of 40% ethanol solution is firstly added into the pot A, the mixture is stirred and extracted for 60min at the temperature of 75 ℃, solid-liquid separation and extraction liquid filtration are carried out to obtain extraction liquid A1, and the extraction liquid A1 is collected and stored;
adding 600mL of 40% ethanol solution into the tank A, stirring and leaching for 40min at the temperature of 75 ℃ to obtain a leaching solution A2, pumping the leaching solution A2 into the tank B, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 60min at the temperature of 75 ℃ to obtain an extracting solution B1, and collecting and storing the B1;
adding 600mL of 40% ethanol solution into a tank A, stirring and leaching for 20min at the temperature of 75 ℃, obtaining an extracting solution A3 and material residues after leaching is finished, removing the material residues from the tank A, adding 100g of liquorice leaves treated by a mechanochemical method into the tank A, adding the extracting solution A3 into a tank B, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 40min at the temperature of 75 ℃ to obtain an extracting solution B2, adding the B2 into a tank C, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 60min at the temperature of 75 ℃ to obtain an extracting solution C1, and collecting and storing the C1;
Adding 600mL of 40% ethanol solution into a tank B for leaching, stirring and leaching for 30min at the temperature of 75 ℃ to obtain an extracting solution B3 and material residues, removing the material residues in the tank B, adding 100g of licorice leaves treated by a mechanochemical method into the tank B, adding a filtrate B3 into a tank C, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 40min at the temperature of 75 ℃ to obtain an extracting solution C2, adding the C2 into a tank D, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 60min at the temperature of 75 ℃ to obtain an extracting solution D1, and storing the D1;
adding 600mL of 40% ethanol solution into a C tank, stirring and leaching for 30min at the temperature of 75 ℃ to obtain leaching liquor C3 and material residues, removing the material residues in the C tank, adding 100g of licorice leaves treated by a mechanochemical method into the C tank, adding a C3 extracting solution into a D tank, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 40min at the temperature of 75 ℃ to obtain extracting solution D2, adding the D2 into a A tank, supplementing 40% ethanol solution according to the extracted material-liquid ratio, stirring and leaching for 60min at the temperature of 75 ℃ to obtain a second batch of extracting solution A1 in the A tank, and storing the A1;
Adding 600mL of 40% ethanol solution into D tank, stirring and leaching at 75 deg.C for 30min to obtain leaching solution D3 and residue, removing residue in D tank, adding 100g of mechanically and chemically treated folium Glycyrrhizae into D tank, and circularly extracting according to the above method;
mixing the above stored extractive solutions, centrifuging, filtering to remove flocculent precipitate to obtain extractive solution without precipitate, and distilling the extractive solution under reduced pressure under the following conditions: distilling at 60-80 deg.c and vacuum degree of- (0.06-0.08) MPa, recovering ethanol, and vacuum concentrating to obtain extractum containing crude drug 2 g/mL to obtain licorice leaf extract. Diluting the licorice extract with water to obtain diluted licorice leaf extract, steam distilling to obtain volatile licorice leaf component and water as distillate, extracting the distilled residual liquid with petroleum ether at 60-90 deg.c or n-hexane to obtain extracted liquid and extracted residual liquid, diluting the extracted liquid with water, ultrasonic dissolving, centrifuging and adsorbing the centrifuged liquid with macroporous resin.
Distilling the diluted solution of the licorice leaf extract with water vapor, heating in oil bath from the bottom of a distillation flask, and distilling after boiling to obtain mixed steam; condensing the obtained mixed steam by a condenser to obtain an oil-water mixture; performing oil-water separation on the oil-water mixture obtained by condensation by an oil-water separator to obtain volatile components of folium eucalypti globueli and liquorice and water; extracting the volatile components of the eucalyptus leaves and the liquorice leaves in an extraction tank, and separating the upper layer which is the volatile components of the liquorice leaves after layering; adding a small amount of NaCl into the distillate, standing for layering to obtain upper layer yellowish oily liquid, anhydrous Na 2 SO 4 Drying, purifying the product by rotary evaporation through a rotary evaporation evaporator, and collecting the product in a flask.
Diluting the residue in the distillation flask with water, transferring to a continuous countercurrent extraction instrument, adding 1-2 parts by weight of hydrophobic organic solvent, and performing single-stage, multi-stage or continuous countercurrent extraction; the extraction temperature is 30-45 ℃; the ratio of the extracting agent to the extracted material is 0.9-1.2: 1; the single-section and multi-section extraction stirring time is 20-40 rpm; the countercurrent extraction convection ratio is as follows: the ratio of the extracting agent to the extracted material liquid is = 0.9-1.2: 1, and the flow rate is as follows: 1 extraction bed is 4-60 min, dark green solvent layer and brown water solution layer containing oil soluble substance of Glycyrrhrizae radix leaf are obtained, and the extract is concentrated under reduced pressure to obtain pigment 26.3g.
Soaking AB-8 resin in 90-95% or anhydrous alcohol, and stirring with glass rod to make resin and alcohol contact fully and swell fully, the resin soaking time is 18-24 h; vertically fixing a glass chromatographic column on an experimental frame, padding a layer of absorbent cotton on a sand core, compacting, opening a bottom column piston, adding a certain amount of distilled water into the column, then slowly adding resin with water into the chromatographic column while stirring, keeping the water level in the chromatographic column higher than the resin layer by 3-5 cm, and discharging excessive water from the bottom column piston; filling the soaked resin into a column, and then washing with absolute ethyl alcohol until the effluent liquid is white and turbid when equal amount of distilled water is added; washing the resin with a large amount of distilled water until the effluent liquid has no alcohol smell, and detecting the alcoholic strength of the effluent liquid by an alcohol meter to be more than or equal to 3.0% vol. Heating and volatilizing the obtained raffinate to remove residual extractant, filling the treated AB-8 macroporous resin into a resin column by a wet method, after water washing and balancing, introducing the raffinate into the resin column, stopping loading the raffinate into the column when the concentration of the flavone in the to-be-detected effluent reaches 5-10% of the initial concentration to obtain a resin column with saturated adsorption, quickly washing the resin column with saturated adsorption by using water with the pH value of 2.0-4.0 BV of 4-6 and the temperature of 10-15 ℃ to obtain polysaccharide eluent, performing gradient elution by using an ethanol aqueous solution, and collecting different eluents. Eluting the resin column by using 2.0-4.0 BV of ethanol water solution with volume fraction of 20 percent to obtain a component 1; then eluting the resin column by using 2.0-4.0 BV of ethanol water solution with the volume fraction of 30-50% to obtain a component 2; eluting the resin column by using ethanol water with the volume fraction of 70-80% of 2.0-4.0 BV to obtain a component 3; eluting the resin column with 2.0-4.0 BV of 85-95 vol% ethanol water solution to obtain component 4, and recovering ethanol from the eluent. Concentrating the component 1 at the temperature of 50-60 ℃ under the vacuum degree of (0.06-0.08) MPa to obtain the polyphenol of the licorice leaves; concentrating the component 2 at the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ until the solid content is 60-70 wt%, and obtaining 2.6g of water-soluble flavone of licorice leaves; and concentrating the component 3 at the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ until the solid content is 60-70 wt%, thus obtaining 1.8g of the prenylflavonoid of the liquorice leaves. The types of flavones in licorice are many, and the flavones mainly include water-soluble flavones with high polarity and isoamylene flavonoids with low polarity according to polarity.
The obtained polysaccharide eluent passes through a hollow fiber membrane with the molecular weight cutoff of 100,000, the permeate is concentrated by a hollow fiber membrane with the molecular weight cutoff of 10,000 to obtain polysaccharide concentrated solution, 1.5g of activated carbon and 1.5g of activated clay are added into 100mL of the polysaccharide concentrated solution for decolorization for 30min, and filtration is carried out. Concentrating the filtrate under reduced pressure until the relative specific gravity is 1.05-1.20, adding 1500-2000 mL of ethanol to ensure that the volume fraction of the ethanol is 70-80%; standing the mixture for 20 to 40min at the temperature of between 0 and 4 ℃ to prepare an alcohol precipitation mixture; transferring the obtained alcohol precipitation mixture into a centrifugal tube of a centrifugal machine, performing centrifugal separation for 10-20 min under the condition of the rotation speed of 4000-5000 rpm to obtain clear liquid and precipitate, and discarding the clear liquid; drying the obtained precipitate for 5-10 h at the temperature of 50-70 ℃ and the vacuum degree of (0.06-0.08) MPa to obtain the licorice leaf polysaccharide 28.3g of the total weight of the powder.
Resin regeneration: soaking the macroporous resin in 80-95% ethanol water solution for 12-15 h, swelling fully, leaching with absolute ethanol until no turbidity exists, soaking the macroporous resin in 15-20 wt% sodium hydroxide water solution for at least 8h, taking out the macroporous resin, washing with deionized water until the discharged washing liquid is neutral, soaking the macroporous resin in 15-20 wt% hydrochloric acid solution for at least 8h, taking out the macroporous resin, washing with deionized water until the discharged washing liquid is neutral, and pouring the macroporous resin into a chromatographic column.
3. Total extraction of licorice root and stem
And (3) steam explosion treatment: cutting 500g of liquorice roots and stems into pieces with the diameter of 20-40 mm to be used as a blasting matrix, adding water according to the mass ratio of the liquorice roots and stems to the water of 1: 0-1: 4, adjusting the pH value to 9-11 by using ammonia water, then soaking and rehydrating at normal temperature for 2-8 h, adding an entrainer into the rehydrated materials, uniformly mixing the materials by using a mixer and maintaining the mixture for 30min, placing the mixture in novel ejection type steam explosion equipment, and taking air and steam as steam explosion media; the entrainer is a compound of fine-mesh carborundum and ammonium chloride, sodium carbonate or calcium hydroxide powder, the mass ratio of the carborundum to the ammonium chloride, the sodium carbonate or the calcium hydroxide powder is 4: 1-1: 1, the total addition amount of the entrainer accounts for 1.5-3% of the blasting matrix in percentage by mass, the entrainer is added in batches, the mixture is uniformly mixed by a mixer and is maintained for 30min, and 1/3-2 & ltANG & gt of the total addition amount of the steam blasting treatment I is added5, adding the rest entrainer into the steam explosion treatment II; the steam explosion is carried out by a steam explosion treatment I and a steam explosion treatment II, and the interval time of the two-stage steam explosion treatment is 3-6 min; the conditions of the steam explosion treatment I are as follows: the charging coefficient of the explosion cavity is 0.8-0.9, air is firstly introduced until the pressure in the steam explosion tank is 5-10 kg/cm 2 Then, the steam is quickly introduced into the steam explosion tank until the pressure in the steam explosion tank is 10-18 kg/cm 2 The temperature in the tank reaches 130-250 ℃, steam explosion pressure maintaining treatment is carried out for 5-15 min, and the explosion time is not higher than 0.00875s; the conditions of the steam explosion treatment II are as follows: blasting in the same cavity of the same blasting equipment, and introducing air until the pressure in the steam explosion tank is 5-10 kg/cm 2 Then, the steam is quickly introduced until the pressure in the steam explosion tank is 10-18 kg/cm 2 And (3) leading the temperature in the tank to reach 130-250 ℃, carrying out steam explosion pressure maintaining treatment for 15-45 s, wherein the explosion time is not higher than 0.00875s, then rapidly relieving pressure, and releasing the material treated in the steam explosion tank into a normal pressure container to obtain the material subjected to steam explosion pretreatment.
Preparation of eutectic solvent: mixing choline chloride and one of lactic acid, urea, citric acid, acetic acid, 1, 4-butanediol, glycerol, malonic acid, ethylene glycol, 1, 3-propanediol or N-propanol at a molar ratio of 1: 2 in a reactor with a drying tube, placing the mixture in a drying vessel for mixing, heating the mixture in a water bath kettle at constant temperature and heating the mixture to 80 ℃ under mechanical stirring, and heating the mixture in a N-phase reactor 2 Heating and stirring the mixture to react in an atmosphere and under an oil bath condition, obtaining a colorless and transparent eutectic solvent DES after the reaction is finished, stirring the mixture for 2 hours until a transparent and uniform solvent is formed, adding deionized water into the prepared eutectic solvent to prepare an extraction solvent with the water content of 10-30%, and storing the extraction solvent in a room temperature environment for later use.
Putting 100g of liquorice root, stem and a eutectic solvent (DES) into an ultrasonic extractor, carrying out ultrasonic extraction for 2-3 times, filtering, and combining filtrate to obtain filtrate and filter residue. According to the mass of the raw material powder: the volume ratio of DES is 1 g: 5-15 mL, the raw material powder and DES are placed in an ultrasonic extractor, the raw material powder and DES are stirred and mixed uniformly, then the first ultrasonic extraction is carried out for 0.5-1.5 h under the conditions that the ultrasonic power is 300-500W, the ultrasonic working frequency is 30-60 KHZ and the temperature is 50-70 ℃, the extracting solution is centrifuged for the first time, the first filtrate and filter residue are respectively collected, the first collected filter residue is placed in the ultrasonic extractor again, and then the mass of the filter residue is calculated as follows: the volume ratio of DES is 1 g: 5-1 5mL, then the first collected filter residue and DES are placed in an ultrasonic extractor, the mixture is stirred and mixed evenly, then the second ultrasonic extraction is carried out for 0.5-1.5 h under the conditions that the ultrasonic power is 300-500W, the ultrasonic working frequency is 30-60 KHZ and the temperature is 50-70 ℃, then the second centrifugation is carried out on the extracting solution, and then the filtrate I and the filter residue are respectively collected. Soaking the treated resin in the filtrate I, stirring with a glass rod at intervals, adsorbing for 2-3 times, and filtering to obtain macroporous resin and filtrate II; the macroporous resin is one or the mixture of two of D-101, HPD722, HPD-100, HPD-300, HPD400, HPD-BJQH, LX-60, ADS-17, AB-8, X-5, HZ-835, ADS-F8, SZ6, SP825, DA201, H P-20 and XDA macroporous resin. Dissolving a certain amount of potassium dihydrogen phosphate in distilled water to prepare a potassium dihydrogen phosphate solution, wherein the using amount of the potassium dihydrogen phosphate is 30-60% (g/mL) of the filtrate II, adding the potassium dihydrogen phosphate solution into the filtrate II, fully and uniformly mixing, uniformly shaking, keeping the external temperature constant, observing whether the filtrate II becomes clear two phases, successfully constructing the two phases after cleaning the two phases, flushing and preparing a DES and potassium dihydrogen phosphate solution if the two phases are not clear, centrifuging the solution for 10-20 min at the centrifugal rotation speed of 3000-5000 rpm until the two phases are clear, retaining the clear DES and potassium dihydrogen phosphate solutions of the two phases, and standing for 60-100 min to form a double-water-phase extraction system.
And the upper phase after the double aqueous phase extraction contains the glycyrrhiza uralensis protein, the protein is extracted by reverse micelles, the reverse micelle system is added into the double aqueous phase extraction system formed by the filtrate II in the former extraction, so that the protein is transferred into the reverse micelle system from the DES, a buffer solution is added into the former extraction solution, so that the protein is transferred into the aqueous phase from the reverse micelles, the protein is separated, the back extraction of the protein is realized, and the aqueous solution of the glycyrrhiza uralensis and the stem protein is obtained by centrifugal separation.
Preparing a reverse micelle solution: adding surfactant dioctyl sodium sulfosuccinate and cosolvent glycerol into organic solvent, stirring to obtain a mixtureThe surfactant is completely dissolved to be uniformly distributed in an organic phase, wherein the concentration of sodium dioctyl sulfosuccinate in the organic phase is 0.05-0.08 g/mL, potassium chloride-phosphate buffer solution is added after complete dissolution, the salt concentration reaches 0.07mol/L, and water is added after uniform mixing to ensure that the system W is o Value =27, resulting in a clear, transparent, stable reverse-micellar solution. The pre-extraction process comprises the following steps: adjusting the pH value of DES liquid containing liquiritin to 4.0-6.0, the concentration of surfactant to 0.05-0.08 g/mL, the ratio of material to liquid to 1: 25, the concentration of salt to 0.07mol/L, and W 0 And =27, mixing and extracting the mixture with a reverse micelle solution for 10-35 min at 40 ℃ and 1500-5000 rpm, centrifuging and layering the mixture, and taking an upper organic phase. The post-extraction process comprises the following steps: mixing the obtained upper layer organic phase and potassium chloride-phosphate buffer solution according to the volume ratio of 1: 1-2, performing back extraction, mixing for 5-40 min, performing centrifugal layering at 1500-5000 rpm, separating to obtain a protein-containing water phase, performing protein separation on the protein-containing water phase, and performing freeze drying to obtain 32.5g of licorice root and stem protein.
Diluting the lower phase after the two-aqueous-phase extraction with water, passing the water solution through Sephadex LH20 gel column chromatography, washing with water to obtain glycyrrhiza polysaccharide eluent, dialyzing the eluent, collecting polysaccharide solution, concentrating, decolorizing, precipitating with ethanol, collecting precipitate, drying to obtain crude glycyrrhiza polysaccharide, and purifying to obtain refined glycyrrhiza polysaccharide 27.8g. The molecular interception of the dialysis bag is 8000-12000 Da, dialyzing in running water and deionized water in turn, collecting the liquid in the dialysis bag, concentrating under the vacuum degree of- (0.06-0.08) MPa and the temperature of 50-60 ℃ to obtain the concentrated solution of 1.05-1.10 in the relative ratio, then adding 3-5% (w/w) H 2 O 2 Heating at 60 ℃ for 30min, adding 85-95% by volume of ethanol to make the volume fraction of the ethanol reach 65-70%, standing at 0-4 ℃ for 20-40 min to obtain an alcohol precipitate, transferring the alcohol precipitate into a centrifuge tube of a centrifuge, centrifuging at 4000-5000 rpm for 10-20 min to obtain a precipitate, and drying the precipitate for 5-10 h at 50-60 ℃ under the vacuum degree of- (0.06-0.08) MPa; the crude product of glycyrrhiza polysaccharide is purified by S-8 resin, and the technological parameters are as follows: the concentration of the glycyrrhiza polysaccharide sample is 5.23mg/mL, and the initial pH value is 5, the flow rate is 2.0BV/h. Eluting the SephadexLH20 gel column eluted with the glycyrrhiza polysaccharide by using a 10-20% sodium chloride solution to obtain glycyrrhiza glucan, decoloring, filtering, concentrating the filtrate to be dry, adding purified water for dissolving, filtering, collecting the filtrate, concentrating the filtrate, adding ethanol for precipitation, collecting the precipitate, and drying to obtain 15.2g of glycyrrhiza glucan. The decolorization is carried out by adding 3-5% (w/w) of H 2 O 2 Heating at 60 deg.C for 30min; the concentration is carried out at the vacuum degree of between 0.06 and 0.08 MPa and the temperature of between 50 and 60 ℃ to obtain concentrated solution with the relative ratio of between 1.05 and 1.10; precipitating the ethanol, adding ethanol with the volume fraction of 85-95% to ensure that the volume fraction of the ethanol reaches 65-70%, and standing the mixture for 20-40 min at the temperature of 0-4 ℃ to prepare an ethanol precipitate; filtering, transferring into a centrifuge tube of a centrifuge, and centrifuging for 10-20 min under the condition that the rotating speed is 4000-5000 rpm; the drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and the vacuum degree of between 0.06 and 0.08 MPa.
Extracting macroporous resin adsorbing effective components in DES with ethanol, concentrating the extract under reduced pressure at 50-60 deg.C under vacuum degree of (0.06-0.08) MPa to remove ethanol, adding purified water into the concentrated solution, stirring with ultrasound to obtain diluted solution with a certain proportion of 1.02-1.05, transferring the diluted solution into a centrifuge tube of a centrifuge, centrifuging at 4000-5000 rpm for 10-20 min to obtain centrifugate, adsorbing the centrifugate with polyamide resin, washing with water, and collecting water washing solution as glycyrrhizin; the polyamide resin is eluted by gradient ethanol, and the eluent is collected. The polyamide resin is placed in an ultrasonic extractor and desorbed by using ethanol as an analytical agent under the action of ultrasonic waves, the ultrasonic frequency is 25 kHz, the ultrasonic power is 100-300W, the ultrasonic extraction temperature is 30-55 ℃, the ultrasonic extraction time is 10-30 min, the polyamide resin is extracted for 30min at 40-50 ℃ by using 80-90% of ethanol by volume fraction, the steps are repeated for 2-3 times until the extract is clear, the ethanol extract is combined, the ethanol extract is decompressed and concentrated into concentrated solution with the solid content of 50-60 wt%, and water is added for ultrasonic dilution. The height ratio of the column diameter of the resin is 1: 10-1: 20, the sample loading amount is 10-30 BV, the adsorption flow rate is 2-10 BV/h, and after the adsorption is finished, 2-8 BV of deionized water is used for eluting to obtain glycyrrhizin washing liquid; eluting the polyamide resin after water washing by gradient ethanol, and eluting the macroporous resin adsorption column by 20 to 30 weight percent ethanol solution for 2.0 to 4.0BV to obtain a component A which is a polyphenol compound; eluting the macroporous resin adsorption column by using 70 to 80 weight percent ethanol solution for 2.0 to 5.0BV to obtain a component B which is a flavonoid compound; the component A is decompressed and concentrated to have the solid content of 60 to 70 weight percent under the conditions of the vacuum degree of (0.06 to 0.08) MPa and the temperature of 50 to 60 ℃, and is dried for 5 to 10 hours under the conditions of the temperature of 50 to 60 ℃ and the vacuum degree of (0.06 to 0.08) MPa to obtain 10.5g of the liquorice polyphenol.
The glycyrrhizin washing liquid is used as initial feed liquid, and a first-stage defoaming liquid is obtained through two-stage foam separation; the first stage foam separation is carried out at the initial concentration of feed liquid of 2.0 mg/mL -1 Liquid loading amount of 300mL, pH 4.0, concentration of 2.0 mg/mL -1 Nitrogen flow rate of 200 mL/min -1 Electrolyte sodium chloride concentration 0.20 mol. L -1 The foam separation temperature is 60 ℃; second stage foam separation at nitrogen flow rate 300mL min -1 The concentration of sodium chloride in the electrolyte is 0.20 mol.L -1 The foam separation temperature is 50 ℃, the liquid loading amount is 270mL, and the pH is 4.5; the second stage foam separation is to take residual liquid left in the first stage foam separation tower as feed liquid of the second stage foam separation to enter a second stage foam separation tower; mixing the second stage defoaming solution and glycyrrhizin water washing solution, adding into the feed of the first stage foam separation tower, and performing next foam separation. And adding warm water at 30-45 ℃ into the obtained first-stage defoaming solution, uniformly dispersing into liquid of 50-100 mg/mL, adsorbing by using D101 macroporous resin at a sampling speed of 0.5mL/min, statically adsorbing for 30-50 min, performing gradient elution, collecting eluent, and concentrating to obtain concentrated solution. The gradient elution is carried out by adopting purified water with the pH value of 4-6 of 2.0-4.0 BV and the temperature of 15-25 ℃ to wash, and impurities are removed until the eluent is clear; eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 5-10 percent to obtain a component (1); eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 50-70 percent to obtain a component (2); finally eluting the macroporous resin adsorption column by using ethanol solution with the volume fraction of 80-90 percent for 2.0-4.0 BV to obtain a component (3); concentrating the component (2) under reduced pressure at the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃, concentrating at the temperature of 50-60 ℃, Drying for 5-10 h under the vacuum degree of- (0.06-0.08) MPa to obtain 9.6g of liquoric root and stem saponin;
concentrating the component B into an extract under reduced pressure, adding warm water at 40-60 ℃, fully stirring, standing at room temperature, extracting with ethyl acetate and n-butanol respectively, collecting ethyl acetate extract and n-butanol extract respectively, and concentrating under reduced pressure to obtain an ethyl acetate part extract and an n-butanol part extract respectively. Dissolving 10g of the ethyl acetate part of the extract with methanol, filtering, adding 1-2 times of 48-75 mu m silica gel into the filtrate, uniformly stirring, volatilizing the solvent, drying in the air, crushing, sieving with a 75 mu m sieve, adding 48-75 mu m column chromatography silica gel which is 15-20 times of the weight of the dried substance II into a silica gel chromatographic column, and carrying out chromatographic separation; the silica gel chromatographic column has height ratio of 1: 10-1: 20, gradient elution with chloroform-methanol in the volume ratio of 100: 0, 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100, TLC detection of the eluted liquid, classification and merging to obtain 7 parts Fr.1-Fr.7.
The Fr.1 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by gradient elution is subjected to thin-layer chromatography identification, classification and combination, and is separated by centrifugal partition chromatography to obtain 0.082g of glabridin and 0.122g of glabridin, wherein the content is more than 98%; a satisfactory separation effect is obtained by carrying out systematic optimization experiments on parameters such as a two-phase solvent system, a rotor rotating speed, a mobile phase flow rate and the like, wherein the optimal two-phase solvent system is n-hexane-ethyl acetate-methanol-water (1: 5: 1: 5, v/v), an upper phase organic phase is a stationary phase, a lower phase aqueous phase is a mobile phase, the rotor rotating speed is 1500rpm, the flow rate is 3.0mL/min, and the ultraviolet detection wavelength is 280nm. The extraction cavity of the centrifugal distribution chromatograph is provided with an inlet and an outlet of a solvent, the solvent serving as a stationary phase flows into the extraction cavity firstly, after the extraction cavity starts to rotate, a liquid pump starts to input a mobile phase from the inlet of the extraction cavity, the flowing mobile phase solvent forms tiny droplets called atomization according to Stokes' law, the droplets start to settle through the stationary phase, a huge interaction area is generated between the droplets and the stationary phase, the droplets are extracted, and when the droplets reach the tail end of the extraction cavity, the droplets are gathered together again under the action of surface tension to generate settlement. When a sample mixture is fed into the mobile phase, different substances in the mixture are eluted in sequence according to the distribution coefficients of the different substances, the centrifugal partition chromatography only needs a two-phase solvent, the solvent ratio can be adjusted according to the distribution coefficients of the different substances, and a high-selectivity separation result is ensured.
The Fr.2 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, the eluent is identified by TLC, isoliquiritigenin eluent is obtained at the position where the volume ratio of the petroleum ether to the ethyl acetate is 100: 20, and liquiritigenin-containing eluent is obtained at the position where the volume ratio of the petroleum ether to the ethyl acetate is 100: 30; performing thin layer chromatography, subjecting the eluate with the volume ratio of petroleum ether to ethyl acetate of 100: 50 to silica gel column chromatography, performing gradient elution with the volume ratio of petroleum ether to ethyl acetate of 100: 10, 100: 20, 100: 30, 100: 50 and 100: 100, subjecting the eluate to thin layer chromatography to obtain liquiritigenin 0.063g at the position of petroleum ether to ethyl acetate with the volume ratio of 100: 20, and obtaining liquiritin 0.054g at the position of petroleum ether to ethyl acetate with the volume ratio of 100: 50, wherein the content is greater than 98%.
And the Fr.3 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 10, 100: 15, 100: 20, 100: 30, 100: 40, 100: 100 and 0: 100 in sequence, eluent obtained by the gradient elution is subjected to thin layer chromatography identification, classification and combination, sephadex LH-20 hydroxypropyl Sephadex column chromatography is carried out on the eluent, chloroform and methanol are used as eluent according to the volume ratio of 6: 4 for elution, then the same components are combined through thin layer tracking detection, and 0.132g of licoflavonol and 0.042g of licoflavonol A are obtained through separation, wherein the content is more than 98%.
The Fr.4 fraction is subjected to silica gel column chromatography, and gradient chromatography with chloroform-methanol volume ratio of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 50, 100: 100, and 0: 100Eluting, identifying the eluates obtained by gradient elution by thin-layer chromatography, classifying and mixing, and separating by supercritical fluid chromatography to obtain liquiritin P0.032G, liquiritin N0.021G, liquiritin G0.324G, liquiritin O0.225G, liquiritin I0.182G and liquiritin M0.178G, wherein the content is more than 98%. The supercritical fluid chromatographic separation conditions are as follows: the supercritical fluid chromatographic separation conditions are as follows: polar chromatographic column ZorBaxSB-CN (9.4 mm. Times.250mm, 5 μm) as stationary phase and CO 2 Methanol (containing 0.2% of trifluoroacetic acid and 0.1% of ethanolamine) (90: 10, v/v) is used as a mobile phase, the content of an entrainer is 13.64wt%, the entrainer (methanol-0.2 wt% of phosphoric acid) and the ultraviolet detection wavelength are 280nm 2 The flow rate is 10-20 g.min -1 Column pressure is 12-14 MPa, sample injection amount is 0.05mL, flow rate is 2mL/min, column temperature: 40 ℃, column pressure program: the initial pressure is 8MPa, the pressure is increased to 12-14 MPa at the speed of 300kPa/min, and the sample injection is 2 mu L; the supercritical fluid chromatograph is composed of a sample introduction system, a high-pressure pump, a chromatographic column, a current limiter, a detector and the like, and the whole system is basically in a high-pressure airtight state. (1) The sample system supercritical fluid chromatograph generally adopts HPLC manual or automatic sample valve, adopts Rheodyne type six-way sample valve with sample tube for packed column, adopts dynamic flow-dividing similar to gas chromatograph and timing flow-dividing sample introduction of opening time of sample valve controlled by microcomputer for capillary column, and can also be connected with SFE on-line to use column head sample introduction. (2) The high-pressure pumps commonly used for the high-pressure pump supercritical chromatograph mainly comprise two types, one type is a spiral injection pump, and the other type is a reciprocating plunger pump. (3) The chromatographic column mainly comprises a capillary column or an open tubular column, a capillary packed column and a packed column. The open tubular column is a quartz thick-wall capillary tube with the inner diameter of 50-100 mu m, the thickness of the fixed phase liquid film is 0.25-several micrometers, the thickness of one wall is more than or equal to 200 mu m, the open tubular column can bear the high pressure of (400-600) multiplied by 105Pa, and the length of the open tubular column is 10-20 m. The packed capillary column is a thick-wall capillary with the inner diameter of 250-530 microns, the packing particle size is 3-10 microns, the length is 20-100 cm, the packed column packing particle size is similar to that of HPLC, and the column inner diameter is 2-4.6 mm, and the length is 10-20 cm. (4) A flow restrictor, i.e. a damper, placed before or after the detector, respectively, is a hydrogen Flame Ionization Detector (FID), the inlet end of the flow restrictor is a chromatographic column and the outlet end is a detector, which functions to maintain the separation system on the one hand The mobile phase is in a supercritical state, and the detector works in a normal-pressure gas state; another effect is that the chromatography column effluent, including the mobile phase and sample components, undergoes rapid phase changes and transfer through the flow restrictor. (5) Detectors various GC and HPLC detectors can be used for the supercritical fluid chromatograph. Most used are FIDs, with the optimal distance of the flow restrictor to the FID nozzle being 5-7 mm. When the mobile phase contains an organic modifier, it is not suitable for FID, and therefore an Evaporative Light Scattering Detector (ELSD) is used as a general purpose detector.
And the Fr.5 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by the gradient elution is identified by thin-layer chromatography, classified and combined, and is separated by coordination chromatography to obtain 0.083g of licoisoflavone A, 0.045g of licoflavone A, 0.035g of licoisoflavan A, 0.064g of glycyrrhiza isoflavone A, 0.046g of licoisoflavone B, 0.024g of licoricone and 0.063g of licoisoflavanone. Performing coordination chromatographic separation, namely preparing a coordination chromatographic column with the central ion content of 7-10% by taking metal ions as central ions, loading a sample on the coordination chromatographic column, sequentially identifying eluent by thin-layer chromatography according to the gradient volume ratio of chloroform to methanol of 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100, and separating eluent of licoisoflavone A, licoflavone A, licoisoflavone B, licoricone and licoisoflavanone; the metal ion is Se 4+ 、Cu 2+ 、Mg 2+ 、Ni 2+ 、Al 3+ Or Zn 2+ One of them. Preparation of a coordination chromatography column: with metal ions Cu 2+ Taking the filler and the coordination metal ion compound copper chloride as central ions, putting the filler and the coordination metal ion compound copper chloride into a sample crusher, grinding to obtain powder with the particle size of 5-15 mu m, and preparing the central ion Cu 2+ Adding ethyl acetate into a coordination chromatographic column with the content of 7-10%, uniformly mixing, filling into the chromatographic column, standing for 1d to ensure that coordination ions are fully coordinated and combined with the filler, and thus obtaining the coordination chromatographic column. The coordination chromatographic column is balanced and washed by ethyl acetate-methanol-concentrated ammonia test solution until no coordination existsUntil the ions react. After the sample is loaded on a coordination chromatographic column, gradient volume ratios of chloroform to methanol are 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, eluent is identified by thin-layer chromatography, and eluent of licoisoflavone A, licoflavone A, licoisoflavone A, glycyrrhetin A, licoisoflavone B, licoricone and licoisoflavanone is separated.
And the Fr.6 part is subjected to silica gel column chromatography, the gradient volume ratio of chloroform to methanol is 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, the eluent is subjected to thin-layer chromatography identification, the eluent containing the mixture of licochalcones A-E is finally obtained, and the licochalcones A-E are obtained through high-speed counter-current chromatography separation and purification. Designing a separation solvent according to factors such as polarity, viscosity, specific gravity and solubility of various solvents, then preparing 20mL of the separation solvent according to the proportion of the separation solvent, respectively sucking 1mL of upper and lower phases of the separation solvent, adding 1mg of a sample to be separated, fully shaking, standing for layering, respectively taking upper and lower layer solutions, and measuring the concentrations of licochalcone A-E contained in the upper and lower layer solutions by using a high performance liquid chromatography to obtain the distribution coefficient K of the licochalcone A-E in the separation solvent, wherein the K = Cs/Cm, wherein Cs represents the mass concentration of a solute in the upper phase, and Cm represents the mass concentration of the solute in the lower phase; selecting a separation solvent with a K value within the range of 0.5-1.5 as a separation preparation system; adopting n-hexane, ethyl acetate, methanol and water (1.0: 2.5: 1.5: 0.1, v/v/v/v) as a two-phase solvent system, placing 2L of the prepared separation solvent in a separating funnel, fully shaking, standing for 12h, layering, taking an upper phase as a stationary phase, and taking a lower phase as a mobile phase; adopt the TBE-300A type preparative countercurrent chromatograph who produces of Shanghai Hotan biotechnology limited company, countercurrent chromatograph comprises plunger pump, injection valve, ultraviolet detector, record appearance and chromatographic separation post, and the chromatographic separation post is the spiral tube column that forms by polytetrafluoroethylene pipe multilayer winding, and its capacity is 230 mL, and the connection order is: a plunger pump, a sample injection valve, a chromatographic separation column, an ultraviolet detector and a recorder; firstly, a sample injection valve is in a sample injection state, a stationary phase is filled in a chromatographic separation column of a semi-preparative countercurrent chromatograph by a pump at the flow rate of 30mL/min, a speed controller is started, the elution mode of the chromatograph is adjusted to be in reverse connection and forward rotation, when the rotation speed reaches 850r/min, the rotation speed is stable, the flow rate of a mobile phase is set to be 1.5mL/min, the temperature of a constant-temperature water bath is 25 ℃, the mobile phase is started to be pumped into a spiral tube, when the mobile phase flows through the chromatographic separation column, the mobile phase is continuously pumped into the balance for 30min, a certain amount of sample is weighed and put into a mixed solution of an upper phase and a lower phase for dissolving, the sample is injected into a liquid storage tube of the sample injection valve of the countercurrent chromatograph by using an injector, the sample injection valve is in a connection column state, timing is started, each separated peak is collected according to an ultraviolet spectrogram of a detector, the purity is measured by using high-efficiency liquid chromatography, and the content of collected light licochalcones A-E is all more than 98%.
And the Fr.7 part is concentrated into an extract, dissolved by adding water, adsorbed by macroporous adsorption resin, eluted by using methanol with volume fraction of 70-80% as an eluent, the eluent is concentrated into the extract, the obtained extract is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to isopropanol of 20: 1, 10: 1, 5: 1, 1: 1 and 0: 1 in sequence, the eluent is identified by thin-layer chromatography, classified and combined, and is separated by microemulsion electric capillary chromatography to obtain 0.183g of isoliquiritin, 0.257g of liquiritin, 0.054g of neoliquiritin and 0.026g of neoisoliquiritin. According to the microemulsion electrokinetic capillary chromatography, when a microemulsion system is 50mmol/L boric acid buffer solution (pH 95), 10% (volume fraction) n-butanol, 80mmol/L n-heptane, 120mmol/L sodium dodecyl sulfate and 5mmol/L sulfonated beta-cyclodextrin, the separation voltage is 20kV, and the column temperature is 35 ℃, the microemulsion electrokinetic capillary chromatography for separating isoliquiritin, liquiritin, neoliquiritin and neoisoliquiritin is established, and the separation of the 4 components in a sample solution is realized within 30 min.
Taking 5g of n-butanol part extract, dissolving, mixing with silica gel with the particle size of 48-75 mu m, loading the mixture on a column by a dry method for silica gel column chromatography, performing gradient elution by using chloroform-methanol as an eluent, wherein the gradient volume ratio of chloroform to methanol is 30: 1, 20: 1, 10: 1, 5: 1, 3: 1, 1: 1 and 0: 1 in sequence, and performing thin-layer chromatography identification and combination on corresponding eluents to obtain 6 parts Fr.I-Fr.VI;
Part Fr.ISeparating, passing through silica gel column chromatography, sequentially detecting the eluent with ethyl acetate-methanol volume ratio of 100: 0, 100: 5, 100: 10, 100: 20, 100: 50, 100: 100 and 0: 100 by thin layer chromatography, performing silica gel column chromatography again, sequentially detecting the eluent with chloroform-methanol volume ratio of 1 00: 5, 100: 10, 100: 20, 100: 30, 100: 100 and 0: 100 by thin layer chromatography, and obtaining 0.034g of apigenin-glycoside at gradient volume ratio of chloroform to methanol of 100: 10, wherein the content is more than 98%. Using ACQUITY UPLC BEH C 18 Chromatography column (2.1 mm. Times.100mm, 1.7 μm) with acetonitrile (A) -0.05% phosphoric acid aqueous solution (B) as mobile phase, gradient elution was carried out at a flow rate of 0.3 mL. Min -1 The detection wavelength is 360nm and the column temperature is 40 ℃. And performing silica gel column chromatography on the Fr.II part by using chloroform-methanol, wherein gradient volume ratios of chloroform to methanol are 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 40, 100: 100 and 0: 10 in sequence, detecting eluent by using thin layer chromatography, performing gradient elution by using Sephadex LH20 and methanol with volume fractions of 10%, 15% and 20% in sequence, and detecting the eluent by using the thin layer chromatography to obtain 0.029g of isoliquiritigenin-4' -O-apiose (1 → 2) glucoside, wherein the content is more than 9%. And (3) performing gradient elution on the Fr.III part by silica gel column chromatography with the volume ratio of chloroform to methanol of 100: 5, 100: 10, 100: 15, 100: 20, 1 00: 25, 100: 100 and 0: 100 in sequence, performing thin layer chromatography on the eluent, performing isocratic elution by using chloroform-methanol as an eluent, wherein the volume ratio of chloroform to methanol is 100: 10, and obtaining 0.056g of apiosyl liquiritin with the content of more than 98%. Using ACQUITY UPLC BEH C 18 Chromatography column (2.1 mm. Times.100mm, 1.7 μm) with acetonitrile (A) -0.05% phosphoric acid aqueous solution (B) as mobile phase, gradient elution was carried out at a flow rate of 0.3 mL/min -1 The detection wavelength is 276nm and the column temperature is 40 ℃. The fr.iv fraction need not be isolated.
Taking 100g of filter residue I after DES extraction, adding 1-2 times (w/w) of sodium carbonate or sodium bicarbonate solution with the pH value of 9-10, heating and boiling for 30-80 min, carrying out suction filtration to obtain clear liquid and filter residue II, washing a filter cake with water until effluent liquid is clear, and combining the washing liquid and filtrate to obtain insoluble dietary fiber extracting solution; concentrating the lignin extract under reduced pressure until the relative specific gravity is 1.10-1.20, adjusting the pH value to 1-3 by using one of hydrochloric acid, sulfuric acid or phosphoric acid, precipitating, performing filter pressing, and drying a filter cake to obtain 43.6g of insoluble dietary fiber; adding 4-8 times (w/w) of calcium hydroxide solution with the pH value of 11-13 into the filter residue II, adding 3-10% (w/v) of hydrogen peroxide or sodium hypochlorite solution while stirring, adjusting the pH value to 11-12 by using one of hydrochloric acid, sulfuric acid or phosphoric acid, performing filter pressing, washing with water until the effluent is neutral, and drying to obtain 35.2g of soluble dietary fiber. Adding activated carbon into the eutectic solvent after the regeneration extraction of the eutectic solvent, heating to 40-70 ℃, preserving the temperature for 30min, filtering, concentrating the filtrate at the vacuum degree of 72.6-83.8 KPa and the temperature of 60-70 ℃ to obtain the regenerated eutectic solvent.

Claims (10)

1. A full-industrial-chain synergic clean extraction and separation method for effective components of Glycyrrhrizae radix is characterized in that the whole plant of Glycyrrhrizae radix is divided into Glycyrrhrizae radix root and stem; the method comprises the steps of extracting liquorice leaves and liquorice seeds in a segmented whole industrial chain, and comprises the following steps: (1) Extracting squalene, protein, polysaccharide, oil and other components from Glycyrrhrizae radix seed; (2) Extracting volatile components, chlorophyll, tea polyphenols, flavone, polysaccharide, etc. from folium Glycyrrhizae; (3) Extracting radix Glycyrrhizae to obtain polyphenol, glycyrrhizin, licoflavone, liquiritigenin dextran, liquiritigenin, protein, lignin, cellulose, etc.
2. A full-industrial-chain synergic cleaning extraction and separation method for effective components of liquorice is characterized in that in the step (1) in claim 1, the liquorice seeds are used for extracting squalene, protein, polysaccharide, oil and other components, and the method is characterized by comprising the steps of cleaning, impurity removal, soaking, tempering, coarse crushing, vacuum extrusion and puffing, and collecting oil and puffed substances;
cleaning and removing impurities, selecting liquorice seeds, and removing impurities such as straws, gravels, metal blocks and the like;
soaking and tempering, namely soaking the cleaned liquorice seeds in a sulfurous acid solution for a period of time, wherein the soaking time is 10-14 h, the temperature is 30-50 ℃, and the concentration of sulfurous acid in a soaking solution is 0.1-0.2%;
The coarse crushing is carried out, and the soaked liquorice seeds enter a crusher to be crushed;
the vacuum extrusion puffing is characterized in that the vacuum degree is (0.057-0.067) MPa; the water content of the material is 15-20%; the temperature of the sleeve is 87-98 ℃; the rotating speed of the extruder screw is 100-150 rpm; the aperture of the die hole is 17-22 mm; the extrusion temperature is 120-150 ℃, the extrusion pressure is 10MPa, and the extrusion time is 3min; the shape of the aperture of the grinding head of the extruding machine is circular or rectangular; the particle size D95 of the puffed material is not more than 125 μm.
3. A full-industrial-chain synergic cleaning extraction and separation method for effective components of Glycyrrhrizae radix is characterized in that in step (2) of claim 1, folium Glycyrrhizae is extracted to obtain chlorophyll, phenolic acid, flavone, volatile components, polysaccharide, etc.;
the first step is as follows: extracting licorice leaves with the assistance of a mechanochemical method, grinding the licorice leaves by using a planetary ball mill, and mixing and ball-milling the licorice leaves and a ball-milling auxiliary agent;
the ball-milling auxiliary agent is sodium carbonate, borax and beta-cyclodextrin, and the mass ratio of the sodium carbonate to the borax is 1: 2; the mass of the ball-milling auxiliary agent is 6.0-7.0 wt% of the mass of the licorice leaves;
the optimal process parameters are ball milling time of 30min, ball milling rotating speed of 300rpm and filling rate of 26.2 percent, and the material granularity D95 is less than or equal to 37 mu m;
The second step is that: extracting licorice leaves treated by a mechanochemical method by using ethanol with the volume fraction of 30-50%, adopting a pot group type dynamic countercurrent extraction mode, adopting a three-stage four-pot type countercurrent extraction mode, respectively putting equal parts of the licorice leaves into 4 extraction pots, and circularly extracting for 3 times in each pot in sequence, wherein the stirring frequency of the dynamic extraction is 20-200 rpm;
the extracting solution extracted for the first time by the first extracting tank of the first batch is filtered and then is put into a liquid storage tank for standby, the extracting solution extracted for the second time by the first extracting tank is used as a solvent extracted for the first time by the second extracting tank, the extracting solution is filtered for standby after extraction, the extracting solution extracted for the third time by the first extracting tank is used as a solvent extracted for the second time by the second extracting tank, and the extracting solution obtained by extraction is used as an extracting solvent extracted for the first time by the third extracting tank, namely the solvent added by the N extraction of each extracting tank is the extracting solution of the (N + 1) th time of the previous extracting tank; from the second batch, the first extraction tank extracts the second extracting solution from the fourth extraction tank for the first time, and the extracting solutions are circulated in sequence;
collecting the extracting solution extracted for the first time in each extracting tank;
the first extraction tank of the first batch is added with 5-8 times (mL: g) of 30-50% ethanol solution as an extracting solution for the first extraction, 5-8 times (mL: g) of 30-50% ethanol solution as an extracting solution for the second extraction, and 5-8 times (mL: g) of 30-50% ethanol solution as an extracting solution for the third extraction; the first extraction of the second extraction tank, the third extraction tank and the fourth extraction tank of the first batch is respectively supplemented with 30-50% of ethanol solution on the basis of adding the second extraction solution of the previous extraction tank, and the material-liquid ratio of the extraction is met; from the second batch, the first extraction tank extracts the second extraction liquid from the fourth extraction tank for the first time, and the second extraction liquid and the fourth extraction tank are circulated in sequence;
Adding new solvent into the first extraction tank, the second extraction tank, the third extraction tank and the fourth extraction tank for the third extraction;
the extraction time is 60min for the first extraction, 40min for the second extraction and 30min for the third extraction in each tank;
the ratio of the material to the liquid extracted each time is 5-8 times (mL: g);
the third step: mixing the above stored extractive solutions, centrifuging, filtering to remove flocculent precipitate to obtain precipitate-free extractive solution, and distilling under reduced pressure under the following conditions: distilling at 60-80 deg.c and vacuum degree of- (0.06-0.08) MPa, recovering ethanol, and vacuum concentrating to obtain extractum containing crude drug 2g/mL to obtain licorice leaf extract.
4. A full-industrial-chain synergic cleaning extraction and separation method for effective components of Glycyrrhrizae radix is characterized in that in step (3) of claim 1, the Glycyrrhrizae radix and stem extract polyphenol, alkaloid, glycyrrhizin, licoflavone, liquiritin dextran, glycyrrhiza polysaccharide, protein, lignin, cellulose, etc.;
the first step is as follows: cutting the licorice roots and stems into pieces with the diameter of 20-40 mm to obtain blasting matrixes, adding water according to the mass ratio of the licorice roots, stems and water of 1: 0-1: 4, adjusting the pH value to 9-11 by using ammonia water, then soaking and rehydrating the mixture at normal temperature for 2-8 hours, adding an entrainer into the rehydrated material, uniformly mixing the mixture by using a mixer and maintaining the mixture for 30 minutes, placing the mixture in novel ejection type steam explosion equipment, and taking air and steam as steam explosion media;
The entrainer is a compound of fine-mesh carborundum and ammonium chloride, sodium carbonate or calcium hydroxide powder, the mass ratio of the carborundum to the ammonium chloride, the sodium carbonate or the calcium hydroxide powder is 4: 1-1: 1, the total addition amount of the entrainer accounts for 1.5-3% of the blasting matrix in percentage by mass, the entrainers are added in batches, a mixer is used for uniformly mixing and maintaining for 30min, 1/3-2/5 of the total amount of the entrainer is added in the steam blasting treatment I, and the balance of the entrainer is added in the steam blasting treatment II;
the steam explosion is carried out by a steam explosion treatment I and a steam explosion treatment II, and the interval time of the two-stage steam explosion treatment is 3-6 min;
the conditions of the steam explosion treatment I are as follows: the charging coefficient of the explosion cavity is 0.8-0.9, air is firstly introduced until the pressure in the steam explosion tank is 5-10 kg/cm 2 Then, the steam is quickly introduced until the pressure in the steam explosion tank is 10-18 kg/cm 2 The temperature in the tank reaches 130-250 ℃, steam explosion pressure maintaining treatment is carried out for 5-15 min, and the explosion time is not higher than 0.00875s;
the conditions of the steam explosion treatment II are as follows: blasting in the same cavity of the same blasting equipment, and introducing air until the pressure in the steam explosion tank is 5-10 kg/cm 2 Then, the steam is quickly introduced into the steam explosion tank until the pressure in the steam explosion tank is 10-18 kg/cm 2 The temperature in the tank reaches 130-250 ℃, steam explosion pressure maintaining treatment is carried out for 15-45 s, the explosion time is not higher than 0.00875s, then the pressure is quickly relieved, and the material treated in the steam explosion tank is released into a normal pressure container, thus obtaining the material pretreated by steam explosion;
The second step: preparing a liquorice root and stem total extract: putting the liquorice root, the liquorice stem and a eutectic solvent (DES) into an ultrasonic extractor, carrying out ultrasonic extraction for 2-3 times, filtering, and combining filtrate to obtain filtrate I and filter residue, wherein the filtrate I is the eutectic solvent extracting solution of the liquorice root and the liquorice stem;
the feeding proportion of the raw materials of the liquorice roots and the stems to the eutectic solvent is 1: 5-10 (g: mL);
the ultrasonic power is 300-500W, the ultrasonic working frequency is 30-60 KHZ, the extraction temperature is 50-70 ℃, and the extraction time is 0.5-1.5 h;
the eutectic solvent consists of a hydrogen bond acceptor, a hydrogen bond donor and water;
the content of water in the eutectic solvent is 10-30 wt%;
the feed-liquid ratio of the licorice raw material to the eutectic solvent is 20-100 mg/mL;
the hydrogen bond receptor is selected from one of choline chloride, betaine or methyl trioctyl ammonium chloride;
the hydrogen bond donor is selected from one of lactic acid, urea, citric acid, acetic acid, 1, 4-butanediol, glycerol, malonic acid, ethylene glycol, 1, 3-propanediol or n-propanol;
the molar ratio of the hydrogen bond acceptor to the donor in the eutectic solvent is 1 to (0.5-5);
the third step: soaking the treated resin in the filtrate I, stirring by a glass rod at intervals, adsorbing for 2-3 times, and filtering to obtain macroporous resin and filtrate II;
The macroporous resin is one or a mixture of D-101, HPD722, HPD-100, HPD-300, HPD400, HPD-BJQH, LX-60, ADS-17, AB-8, X-5, HZ-835, ADS-F8, SZ6, SP825, DA201, HP-20 and XDA macroporous resin;
the fourth step: adding monopotassium phosphate into the filtrate II, fully and uniformly mixing, completely dissolving the monopotassium phosphate, standing for 60-100 min, centrifuging the solution for 10-20 min at the centrifugal rotating speed of 3000-5000 rpm to form a double-aqueous-phase extraction system;
the dosage of the monopotassium phosphate is 30-60% (g/mL) of the filtrate II;
the fifth step: the upper phase after the two-aqueous phase extraction in the fourth step contains licorice protein, the protein is extracted by reverse micelle, the reverse micelle system is added into the two-aqueous phase extraction system formed by the filtrate II in the former extraction, so that the protein is transferred from DES to the reverse micelle system, a buffer solution is added into the former extraction, so that the protein is transferred from the reverse micelle to the aqueous phase, so that the protein is separated, the back extraction of the protein is realized, and the aqueous solution of licorice root and stem protein is obtained by centrifugal separation;
the reverse micelle system consists of a surfactant, a cosolvent and an inorganic salt buffer solution;
the W is o Represents the molar concentration ratio of water to surfactant, W, in the reverse micelle solution o ＝[H 2 O]/[ surfactant ]]；
The surfactant is one of Sodium Dodecyl Sulfate (SDS), dioctadecyl dimethyl ammonium chloride, dodecyl trimethyl ammonium chloride or dioctyl sodium sulfosuccinate (AOT);
the cosolvent is one of glycerol, isooctane or n-decanol;
the addition amount of the cosolvent is 0 to 10 percent of the total amount of the reverse micelle solution,
the inorganic salt is sodium chloride or potassium chloride;
the pre-extraction process comprises the following steps: adjusting the pH value of DES liquid containing liquiritin to 4.0-6.0, the concentration of surfactant to 0.05-0.08 g/mL, the ratio of material to liquid to 1: 25, the concentration of salt to 0.07mol/L, and W 0 =27, mixing the extract with a reverse micelle solution, extracting for 10-35 min, centrifuging at the temperature of 40 ℃ and the rpm of 1500-5000 for layering, and taking an upper organic phase;
the post-extraction process comprises the following steps: mixing the obtained upper layer organic phase and potassium chloride-phosphate buffer solution according to the volume ratio of 1: 1-2, performing back extraction, mixing for 5-40 min, performing centrifugal layering at 1500-5000 rpm, separating to obtain a protein-containing water phase, performing protein separation on the protein-containing water phase, and performing freeze drying to obtain licorice root and stem protein;
and a sixth step: diluting the lower phase after the two-aqueous-phase extraction in the fourth step with water, passing the water solution through Sephadex LH20 gel column chromatography, washing with water to obtain glycyrrhiza polysaccharide eluent, dialyzing the eluent, collecting polysaccharide solution, concentrating, decolorizing, precipitating with ethanol, collecting precipitate, drying to obtain crude glycyrrhiza polysaccharide, and purifying to obtain fine glycyrrhiza polysaccharide;
Dialyzing, namely dialyzing the used dialysis bag with molecular interception of 8000-12000 Da in sequence in running water and deionized water, collecting liquid in the dialysis bag and concentrating;
concentrating under the vacuum degree of- (0.06-0.08) MPa and at the temperature of 50-60 ℃ to obtain concentrated solution with the relative specific gravity of 1.05-1.10;
the decolorization is carried out by adding 3-5% (w/w) of H 2 O 2 Heating at 60 deg.C for 30min;
precipitating the ethanol, adding ethanol with the volume fraction of 85-95% to ensure that the volume fraction of the ethanol reaches 65-70%, and standing the mixture for 20-40 min at the temperature of 0-4 ℃ to prepare an ethanol precipitate;
filtering, transferring into a centrifuge tube of a centrifuge, and centrifuging for 10-20 min under the condition that the rotating speed is 4000-5000 rpm;
the drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and the vacuum degree of between 0.06 and 0.08 MPa;
the crude product of glycyrrhiza polysaccharide is purified by S-8 resin, and the technological parameters are as follows: the concentration of the glycyrrhiza polysaccharide sample is 5.23mg/mL, the initial pH value is 5, and the flow rate is 2.0BV/h;
the seventh step: eluting the Sephadex LH20 gel column eluted with the glycyrrhiza polysaccharide by using a 10-20% sodium chloride solution to obtain glycyrrhiza glucan, decoloring, filtering, concentrating the filtrate to be dry, adding purified water for dissolving, filtering, collecting the filtrate, concentrating the filtrate, adding ethanol for precipitation, collecting the precipitate, and drying to obtain the glycyrrhiza glucan;
The decolorization is carried out by adding 3-5% (w/w) of H 2 O 2 Heating at 60 deg.C for 30min;
concentrating under the vacuum degree of- (0.06-0.08) MPa and at the temperature of 50-60 ℃ to obtain concentrated solution with the relative specific gravity of 1.05-1.10;
precipitating the ethanol, adding ethanol with the volume fraction of 85-95% to ensure that the volume fraction of the ethanol reaches 65-70%, and standing the mixture for 20-40 min at the temperature of 0-4 ℃ to prepare an ethanol precipitate;
filtering, transferring into a centrifuge tube of a centrifuge, and centrifuging for 10-20 min under the condition that the rotating speed is 4000-5000 rpm;
the drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and the vacuum degree of between 0.06 and 0.08 MPa;
eighth step: adding 1-2 times (w/w) of sodium carbonate or sodium bicarbonate solution with the pH value of 9-10 into the filter residue in the second step, heating and boiling for 30-80 min, carrying out suction filtration to obtain clear liquid and filter residue, washing the filter cake with water until the effluent liquid is clear, and combining the washing liquid and the filtrate to obtain insoluble dietary fiber extracting solution;
concentrating the insoluble dietary fiber extracting solution under reduced pressure until the relative specific gravity is 1.10-1.20, adjusting the pH value to 1-3 by using one of hydrochloric acid, sulfuric acid or phosphoric acid, precipitating, performing filter pressing, and drying a filter cake to obtain insoluble dietary fiber;
the ninth step: adding 4-8 times (w/w) of calcium hydroxide solution with the pH value of 11-13 into the filter residue obtained in the eighth step, adding 3-10% (w/v) of hydrogen peroxide or sodium hypochlorite solution while stirring, adjusting the pH value to 11-12 by using one of hydrochloric acid, sulfuric acid or phosphoric acid, performing filter pressing, washing with water until the effluent liquid is neutral, and drying to obtain soluble dietary fiber;
The tenth step: adding activated carbon into the eutectic solvent which is effectively and fully extracted after the regeneration of the eutectic solvent, heating to 40-70 ℃, preserving the temperature for 30min, filtering, and concentrating the filtrate at the temperature of 60-70 ℃ and under the vacuum degree of- (0.06-0.08) MPa to obtain the regenerated eutectic solvent.
5. A full-industrial-chain synergic clean extraction and separation method for effective components of liquorice is characterized in that liquorice seed oil collected in the step 2 is separated into squalene and other oil components;
the first step is as follows: adding petroleum ether (60-90 ℃) into the liquorice seed oil for extraction, wherein the volume ratio of the petroleum ether to the oil is 1: 1-1: 3; extracting for 3 times, combining the upper phase extraction liquid, evaporating and concentrating to remove the solvent to obtain brown oily matter, dissolving the brown oily matter with ethanol, adding 10-20% sodium hydroxide, and performing saponification reaction for 50min at the reaction temperature of 70 ℃ in a material-liquid ratio of 1: 3;
the concentration is carried out under the conditions of vacuum degree of (0.06-0.08) MPa and temperature of 40-50 ℃;
the second step is that: adding petroleum ether (60-90 ℃) into the saponified solution for extraction, wherein the volume ratio of the petroleum ether to the grease is 1: 1-1: 3; extracting for 3 times, mixing the upper phase extractive solutions, evaporating, concentrating to remove solvent to obtain brown oil, and purifying with silver Preparing silica gel, loading in column, eluting pigment, gradient eluting, and Ag + Removing, decoloring and crystallizing;
preparing the silvered silica gel, namely adding 48-75 mu m of silica gel into a silver nitrate solution containing 8-20% under the condition of keeping out of the sun, fully stirring the mixture into paste, heating and stirring the paste in a water bath at 90-95 ℃ for 20-40 min, then cooling the paste to 25-35 ℃, carrying out suction filtration, activating the filtrate in a vacuum drying oven for 15-25 h to prepare the silvered silica gel, and placing the silvered silica gel in the shade for later use;
filling the silver silica gel into a column, placing the column into petroleum ether, stirring to remove bubbles, standing to fully swell the column, adding the column into a chromatographic column, wrapping the chromatographic column with tinfoil, and eluting and balancing the column with the petroleum ether;
dissolving the brown oily matter by using petroleum ether, dropwise adding the dissolved brown oily matter into the upper end of a silconium silicagel chromatographic column, opening a valve at the lower end of the chromatographic column to slowly adsorb sample liquid on the silconium silicagel, and starting gradient elution when the sample is about to be adsorbed;
the gradient elution is carried out by taking the gradient volume ratio of petroleum ether to ethyl acetate as eluent of 100: 0, 100: 5, 100: 8, 100: 10, 100: 15, 100: 20, 100: 100 and 0: 100 in sequence, detecting and analyzing each flow part of the eluent by TLC and HPLC respectively, and merging the same flow parts;
The Ag is + Removing, combining the fractions analyzed by TLC and HPLC to obtain eluate mainly containing squalene, rotary evaporating, removing eluent, dissolving with n-hexane, adding saturated sodium chloride solution to remove Ag + Adding anhydrous sodium sulfate for dehydration, and filtering to obtain filtrate;
the decoloring crystallization, the filtrate is decompressed and evaporated to dryness, absolute methanol is added for dissolution, attapulgite is added for decoloring for 30min, the filtration is carried out, the filtrate is concentrated under the vacuum degree of- (0.06-0.08) MPa and the temperature of 40-50 ℃ until the original volume is 1/3-1/4, and the squalene is obtained after standing and crystallization;
the third step: mixing the eluates obtained in the second step, adding attapulgite adsorbent according to the material-liquid ratio of 1: 10-1: 30 (g: mL), refluxing and decolorizing for 30min, filtering, and concentrating the filtrate under reduced pressure to obtain licorice seed oil;
the vacuum concentration is carried out, the vacuum degree is between 0.06 and 0.08 MPa, and the concentration is carried out at the temperature of between 40 and 50 ℃ until no effluent liquid exists;
the attapulgite adsorbent is a modified attapulgite adsorbent, and the modification method comprises the steps of carrying out double-roller treatment on natural attapulgite, adding 5-10 times (mL: g) of purified water, adding 3-8 times (g: g) of magnesium salt or aluminum salt, adding 0.1-1% of chloroacetic acid, fully and uniformly stirring, adding sodium hydroxide to adjust the pH value to 9-11, continuously reacting for 5-10 h, carrying out solid-liquid separation after the reaction is finished, calcining the solid product at 200-500 ℃ for 2-6 h, crushing, and sieving by using a sieve of 25-75 mu m to obtain the modified attapulgite adsorbent.
6. A full-industrial-chain synergic cleaning extraction and separation method for effective components of liquorice is characterized in that the liquorice seed puffed material collected in claim 2 is subjected to subcritical water extraction of protein and polysaccharide, and the extraction method comprises the following steps:
the first step is as follows: extracting by using subcritical water, introducing high-purity nitrogen into a subcritical water extraction device, taking deoxidized distilled water as an extracting agent, controlling the material-water ratio to be 1: 10-20 (g: mL), the extraction temperature to be 100-150 ℃, the extraction time to be 30-60 min and the pressure to be 3-10 Mpa, and obtaining extract liquor and filter residue containing glycyrrhiza polysaccharide and protein;
the second step is that: centrifuging the obtained extract containing glycyrrhiza polysaccharide and protein, concentrating, precipitating with ethanol to obtain glycyrrhiza polysaccharide and protein crude product, and dissolving glycyrrhiza polysaccharide and protein crude product with water;
the centrifugation is carried out for 10-20 min at the rotating speed of 3000-5000 rpm;
the concentration is carried out, and an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD is used for concentrating to 1/3-1/10 of the original volume;
the alcohol precipitation comprises the steps of adding ethanol with volume fraction of 90-95% into crude glycyrrhiza polysaccharide and protein extraction concentrated solution to enable the final volume fraction of the ethanol to be 70-80 wt%, stirring uniformly, standing for precipitation for 5-10 h, performing suction filtration to obtain a precipitate, and washing for 1-3 times by using absolute ethyl alcohol to obtain crude glycyrrhiza polysaccharide and protein;
Dissolving the crude product of the glycyrrhiza polysaccharide and the protein by using water, adding deionized water to the crude product to dissolve the crude product, and preparing a solution with the total concentration of the glycyrrhiza polysaccharide and the protein of 30-50 wt% to obtain a solution containing the polysaccharide and the protein;
the third step: dissolving Glycyrrhiza polysaccharide and protein crude product with water, adding tert-butanol and ammonium sulfate to form three liquid phase system, and obtaining upper phase, middle phase and lower phase;
the tertiary butanol and ammonium sulfate form a three-liquid phase system, the volume ratio of the solution containing polysaccharide and protein to the tertiary butanol is 1: 1-1: 3, the mass fraction of the ammonium sulfate is 30-50 wt%, the temperature is 35-40 ℃, the time is 30min, and the pH value is 7;
the fourth step: dialyzing the obtained lower phase to remove inorganic salts, decolorizing in water bath, concentrating, precipitating with ethanol, centrifuging, and drying to obtain Glycyrrhrizae radix seed polysaccharide;
decolorizing in water bath, controlling the temperature of the water bath at 45-50 ℃ for 90min, and taking H for the first 60min 2 O 2 Decolorizing with oxidation method, decolorizing with activated carbon adsorption method 30min later, vacuum filtering, and collecting filtrate;
the centrifugation is carried out for 10-20 min at the rotating speed of 3000-5000 rpm;
the concentration is carried out, and an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD is used for concentrating to 1/3-1/10 of the original volume;
the alcohol precipitation is to add the concentrated solution containing the glycyrrhiza polysaccharide into ethanol with the volume fraction of 90-95% to ensure that the volume fraction of the ethanol is 70-80%, to stir evenly, to stand and precipitate for 5-10 h, to perform suction filtration to obtain a precipitate, and to wash the precipitate for 1-3 times by using absolute ethyl alcohol to obtain a crude product;
Centrifuging at 3000-5000 rpm for 10-20 min;
drying for 5-10 h at 50-60 ℃ and (0.06-0.08) MPa to obtain liquorice seed polysaccharide;
the fifth step: decolorizing the obtained middle phase in water bath, concentrating, precipitating with ethanol, centrifuging, drying to obtain Glycyrrhrizae radix seed protein, dissolving Glycyrrhrizae radix seed protein with sodium chloride solution, centrifuging, precipitating with ammonium sulfate, and separating to obtain Glycyrrhrizae radix seed total protein;
decolorizing in water bath, controlling the temperature of the water bath at 45-50 ℃ for 90min, and taking H for the first 60min 2 O 2 Decolorizing with oxidation method, decolorizing with activated carbon adsorption method 30min later, vacuum filtering, and collecting filtrate;
concentrating the filtrate to 1/2-1/20 of the original volume by using an ultrafiltration membrane with the molecular weight cut-off of 10-50 kD;
precipitating in alcohol, adding absolute ethyl alcohol to make the mass percent concentration of the ethyl alcohol be 75-85%, standing for 5-10 h, centrifuging for 10-15 min at 3000-5000 rpm to obtain a precipitate, and washing the precipitate for 2-3 times by using the absolute ethyl alcohol;
dissolving the sodium chloride solution, adding 3-5 times (v/w) of 0.15-0.20 mol/L sodium chloride solution into the licorice seed protein, centrifuging at 3000-5000 rpm for 10-15 min after dissolving, and collecting clear solution;
Precipitating by ammonium sulfate, adding the ammonium sulfate into clear liquid to ensure that the saturation degree reaches 70-85%, separating out protein, centrifuging for 10-15 min at 3000-5000 rpm, and collecting precipitate and clear liquid, wherein the precipitate is total protein of licorice seeds;
and drying for 5-10 h at the temperature of 50-60 ℃ and the vacuum degree of- (0.06-0.08) MPa to obtain the total protein of the liquorice seeds.
7. A liquorice active ingredient full-industrial-chain collaborative cleaning extraction separation method is characterized in that liquorice extract obtained in claim 3 is diluted by water to obtain liquorice leaf extract diluent, steam distillation is carried out, distillate is liquorice leaf volatile ingredients and water, distillation raffinate is extracted by petroleum ether (60-90 ℃) or normal hexane to obtain extract liquor and raffinate, the extract liquor is pigment, the raffinate is fully diluted by water, ultrasonic dissolution promotion is carried out, centrifugation is carried out, and centrifugal liquid is adsorbed by macroporous resin;
the first step is as follows: diluting the licorice leaf extract with water to obtain diluted licorice leaf extract liquid with the relative specific gravity of 1.03-1.05;
the second step: distilling the diluted solution of the licorice leaf extract with water vapor, heating in oil bath from the bottom of a distillation flask, and distilling after boiling to obtain mixed steam; condensing the obtained mixed steam by a condenser to obtain an oil-water mixture; performing oil-water separation on the oil-water mixture obtained by condensation by an oil-water separator to obtain volatile components of folium eucalypti globueli and liquorice and water; extracting the obtained volatile components of the licorice leaves in an extraction tank, and separating the volatile components of the licorice leaves at the upper layer after layering;
Adding a small amount of sodium chloride into the distillate, standing for layering to obtain an upper layer of yellowish oily liquid, drying with anhydrous sodium sulfate, purifying the product by rotary evaporation with a rotary evaporation evaporator, and collecting the product in a flask;
the third step: diluting residues in a distillation flask with water, extracting with petroleum ether (60-90 ℃) or n-hexane with the same volume, respectively collecting extract liquor and raffinate, and concentrating the extract liquor under reduced pressure to obtain pigment;
the extraction adopts single-section, multi-section or continuous countercurrent extraction;
the extraction conditions are as follows: the extraction temperature is 30-45 ℃; the ratio of the extracting agent to the extracted material liquid is 0.9-1.2: 1;
the single-section and multi-section extraction stirring time is 20-40 rpm;
the countercurrent extraction convection ratio is as follows: the ratio of the extracting agent to the extracted material liquid is = 0.9-1.2: 1, and the flow rate is as follows: the volume of 1 extraction bed is 45-60 min.
The concentration is carried out at the temperature of 50-60 ℃ and the vacuum degree of- (0.06-0.08) MPa;
the fourth step: heating the raffinate to volatilize and remove the residual extractant, filling the macroporous resin into a resin column by a wet method, washing with water for balancing, introducing the raffinate into the resin column, stopping loading the column when the concentration of the flavone in the to-be-detected leakage liquid reaches 5-10% of the initial concentration to obtain a resin column with saturated adsorption, quickly washing the resin column with saturated adsorption by using water with the pH value of 4-6 and the temperature of 10-15 ℃ in a 2.0-4.0 BV range to obtain polysaccharide eluent, performing gradient elution by using an ethanol aqueous solution, and collecting different eluents;
Performing gradient elution, namely eluting the resin column by using an ethanol water solution with the volume fraction of 20% of 2.0-4.0 BV to obtain a component 1; then eluting the resin column by using 2.0-4.0 BV of ethanol water solution with the volume fraction of 30-50% to obtain a component 2; eluting the resin column by using ethanol water solution with the volume fraction of 70-80% of 2.0-4.0 BV to obtain a component 3; eluting the resin column by using 2.0-4.0 BV of ethanol aqueous solution with the volume fraction of 85-95% to obtain a component 4, and recovering the ethanol from the eluent;
the component 1 is concentrated under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ to obtain the licorice leaf polyphenol;
the component 2 is concentrated under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ until the solid content is 60-70 wt%, thus obtaining the water-soluble flavone of the licorice leaves;
concentrating the component 3 at the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃ until the solid content is 60-70 wt%, so as to obtain the isopentenyl flavone from the licorice leaves;
the polysaccharide eluent is passed through a hollow fiber membrane with the molecular weight cutoff of 100,000, the permeate is concentrated through a hollow fiber membrane with the molecular weight cutoff of 10,000 to obtain a polysaccharide concentrated solution, 1.5g of activated carbon and 1.5g of activated clay are added into 100mL of the polysaccharide concentrated solution for decolorization for 30min, and filtration is carried out. Concentrating the filtrate under reduced pressure until the relative specific gravity is 1.05-1.20, adding 1500-2000 mL of ethanol, and standing for 20-40 min at the temperature of 0-4 ℃ to prepare an alcohol precipitation mixture;
The volume fraction of the ethanol is 70-80%;
transferring the alcohol precipitation mixture into a centrifugal tube of a centrifugal machine, performing centrifugal separation for 10-20 min under the condition of the rotation speed of 4000-5000 rpm to obtain clear liquid and precipitate, and discarding the clear liquid;
drying the precipitate for 5-10 h at the temperature of 50-70 ℃ and the vacuum degree of (0.06-0.08) MPa to obtain licorice leaf polysaccharide;
the macroporous resin is one or the mixture of two of D-101, HPD722, HPD-100, HPD-300, HPD400, HPD-BJQH, LX-60, ADS-17, AB-8, X-5, HZ-835, ADS-F8, SZ6, SP825, DA201, HP-20 and XDA macroporous resin.
8. A full-industrial-chain synergic cleaning extraction separation method for effective components of Glycyrrhrizae radix is characterized in that macroporous resin obtained in the third step in claim 4 is extracted with ethanol, the extract is concentrated under reduced pressure, ethanol is removed, purified water is added for ultrasonic dilution, centrifugation is carried out, the centrifugate is adsorbed by polyamide resin, water washing is carried out, and water washing liquid is collected; eluting polyamide resin with gradient ethanol, and collecting eluate;
the first step is as follows: putting the macroporous resin in an ultrasonic extractor, and desorbing by using ethanol as an desorbing agent under the action of ultrasonic waves;
extracting the macroporous resin with 80-90% ethanol by volume for 30min at 40-50 ℃, repeating the extraction for 2-3 times until the extract is clear, and combining the ethanol extracts;
The second step is that: concentrating the ethanol extract under reduced pressure to obtain a concentrated solution with the solid content of 50-60 wt%, and adding water for ultrasonic dissolution promotion;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
adding water for ultrasonic dilution, adding purified water into the concentrated solution, and fully stirring the mixture uniformly under ultrasonic to form a diluted solution with a certain proportion of 1.02-1.05;
the ultrasonic frequency is 25kHz, the ultrasonic power is 100-300W, the ultrasonic extraction temperature is 30-55 ℃, the ultrasonic extraction time is 10-30 min,
the centrifugation step, the diluent is moved into a centrifuge tube of a centrifuge, and the centrifugal separation is carried out for 10-20 min under the condition that the rotating speed is 4000-5000 rpm, so as to obtain a centrifugate;
the third step: adsorbing the centrifugate with polyamide resin, washing with water, and collecting water washing solution which is glycyrrhizin;
the height ratio of the resin column diameter is 1: 10-1: 20, the sample loading amount is 10-30 BV, the adsorption flow rate is 2-10 BV/h, and after adsorption is finished, 2-8 BV deionized water is used for elution to obtain glycyrrhizin water washing liquid;
the fourth step: eluting the polyamide resin after water washing by gradient ethanol, and eluting the macroporous resin adsorption column by 20 to 30 weight percent ethanol solution for 2.0 to 4.0BV to obtain a component A which is a polyphenol compound; eluting the macroporous resin adsorption column by using 70 to 80 weight percent ethanol solution for 2.0 to 5.0BV to obtain a component B which is a flavonoid compound;
The fifth step: concentrating the component A under reduced pressure to obtain the component A with the solid content of 60-70 wt%, and drying in vacuum to obtain licorice polyphenol;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
the vacuum drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and under the vacuum degree of between 0.06 and 0.08 MPa.
9. A full-industrial-chain synergic clean extraction and separation method for effective components of Glycyrrhrizae radix is characterized in that glycyrrhizin water washing liquid obtained in claim 8 is used as initial material liquid, and is subjected to two-stage foam separation to obtain first-stage defoaming solution;
the first step is as follows: the first-stage foam separation is carried out at the initial concentration of feed liquid of 2.0 mg.mL -1 Liquid loading amount of 300mL, pH 4.0, concentration of 2.0 mg/mL -1 Nitrogen flow rate of 200 mL/min -1 Electrolyte sodium chloride concentration 0.20 mol.L -1 The foam separation temperature is 60 ℃;
the second stage of foam separation was carried out at a nitrogen flow rate of 300mL min -1 The concentration of sodium chloride in the electrolyte is 0.20 mol.L -1 The foam separation temperature is 50 ℃, the liquid loading amount is 270mL, and the pH is 4.5;
the second-stage foam separation is to take residual liquid left in the first-stage foam separation tower as feed liquid of the second-stage foam separation to enter a second-stage foam separation tower;
mixing the second-stage defoaming solution and glycyrrhizin washing solution, adding the mixture into the feed of the first-stage foam separation tower, and performing next-batch foam separation;
The second step is that: adding warm water with the temperature of 30-45 ℃ into the obtained first-stage defoaming solution, uniformly dispersing the first-stage defoaming solution into liquid with the concentration of 50-100 mg/mL, adsorbing the liquid by macroporous resin at the sampling speed of 0.5mL/min, statically adsorbing the liquid for 30-50 min, performing gradient elution, collecting eluent and concentrating the eluent to obtain concentrated solution;
the gradient elution is carried out by adopting purified water with the pH value of 4-6 at 2.0-4.0 BV and the temperature of 15-25 ℃ to wash, and impurities are removed until the eluent is clear;
eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 5-10 percent to obtain a component (1);
eluting the macroporous resin adsorption column by using 2.0-4.0 BV of ethanol solution with the volume fraction of 50-70 percent to obtain a component (2);
finally eluting the macroporous resin adsorption column by using ethanol solution with the volume fraction of 80-90 percent for 2.0-4.0 BV to obtain a component (3);
the macroporous resin, one of AB-8, D101, D140, HPD100, LX-38, LSA-12S, LX-68, S-8, FL-2, DM130, X-5, D201, HZ806, LSA-5B, and NAK-12;
the third step: concentrating the component (2) under reduced pressure, and vacuum drying to obtain glycyrrhizin;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
the vacuum drying is carried out for 5 to 10 hours at the temperature of between 50 and 60 ℃ and under the vacuum degree of between 0.06 and 0.08 MPa.
10. A full-industrial-chain synergic cleaning extraction separation method for active ingredients of liquorice is characterized in that a component B obtained in the third step in claim 8 is concentrated into an extract under reduced pressure, warm water at 40-60 ℃ is added to be fully stirred, the mixture is placed at room temperature and is respectively extracted by ethyl acetate and n-butyl alcohol, ethyl acetate extract and n-butyl alcohol extract are respectively collected and are concentrated under reduced pressure to respectively obtain ethyl acetate part extract and n-butyl alcohol part extract;
the vacuum concentration is carried out, and the concentration is carried out under the vacuum degree of (0.06-0.08) MPa and the temperature of 50-60 ℃;
the solid content of the extract is 70-80 wt%;
the first step is as follows: dissolving part of the ethyl acetate extract by using methanol, filtering, adding 1-2 times of silica gel with the particle size of 48-75 mu m into the filtrate, uniformly stirring, volatilizing the solvent, drying in the air, crushing, sieving by using a 75 mu m sieve, adding column chromatography silica gel with the particle size of 48-75 mu m into a silica gel chromatographic column, wherein the weight of the dried substance II is 15-20 times that of the dried substance II, and carrying out chromatographic separation;
the silica gel chromatographic column diameter height ratio is 1: 10-1: 20, gradient elution is carried out according to the chloroform-methanol volume ratio of 100: 0, 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, eluent is identified by TLC, and 7 parts Fr.1-Fr.7 are obtained through classification and combination;
The Fr.1 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by gradient elution is subjected to thin-layer chromatography identification, classification and combination, and glabridin are obtained by separation through centrifugal partition chromatography;
the centrifugal distribution chromatography is used for carrying out systematic optimization experiments on parameters such as a two-phase solvent system, the rotating speed of a rotor, the flow velocity of a mobile phase and the like to obtain a satisfactory separation effect, wherein the optimal two-phase solvent system is n-hexane-ethyl acetate-methanol-water (1: 5: 1: 5, v/v), an upper phase organic phase is a stationary phase, a lower phase aqueous phase is a mobile phase, the rotating speed of the rotor is 1500rpm, the flow velocity is 3.0mL/min, and the ultraviolet detection wavelength is 280nm;
the Fr.2 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 3, 100: 5, 100: 10, 100: 15, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, the eluent is identified by TLC, isoliquiritigenin eluent is obtained at the position where the volume ratio of the petroleum ether to the ethyl acetate is 100: 20, and liquiritigenin is obtained at the position where the volume ratio of the petroleum ether to the ethyl acetate is 100: 30;
Performing thin layer chromatography, subjecting the eluate with the volume ratio of petroleum ether to ethyl acetate of 100: 50 to silica gel column chromatography, performing gradient elution with the volume ratio of petroleum ether to ethyl acetate of 100: 10, 100: 20, 100: 30, 100: 50 and 100: 100 in sequence, subjecting the eluate to thin layer chromatography, and obtaining an eluate containing Glycyrrhrizae radix Ciceris at the volume ratio of petroleum ether to ethyl acetate of 100: 20; obtaining the glabrene at the volume ratio of petroleum ether to ethyl acetate of 100: 50;
the Fr.3 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 100: 10, 100: 15, 100: 20, 100: 30, 100: 40, 100: 100 and 0: 100 in sequence, the eluent obtained by the gradient elution is subjected to thin layer chromatography identification, classification and combination, the eluent is subjected to SephadexLH-20 hydroxypropyl sephadex gel column chromatography, chloroform and methanol are used as eluent according to the volume ratio of 6: 4, then the same components are combined through thin layer tracking detection, and the licoflavonol A are obtained through separation;
the Fr.4 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 50, 100: 100 and 0: 100 in sequence, the eluent obtained by the gradient elution is subjected to thin-layer chromatography identification, classification and combination, and supercritical fluid chromatography separation is carried out to obtain liquiritin P, liquiritin N, liquiritin G, liquiritin O, liquiritin I and liquiritin M;
The supercritical fluid chromatographic separation conditions are as follows: polar chromatographic column ZorBaxSB-CN (9.4 mm. Times.250mm, 5 μm) as stationary phase, with CO 2 Methanol (containing 0.2% of trifluoroacetic acid and 0.1% of ethanolamine) (90: 10, v/v) as a mobile phase, an entrainer content of 13.64wt%, an entrainer (methanol-0.2 wt% of phosphoric acid), an ultraviolet detection wavelength of 280nm, a UV detection wavelength of CO 2 The flow rate is 10-20 g.min -1 Column pressure is 12-14 MPa, sample injection amount is 0.05mL, flow rate is 2mL/min, column temperature: 40 ℃, column pressure program: the initial pressure is 8MPa, the pressure is increased to 12-14 MPa at the speed of 300kPa/min, and the sample injection is 2 mu L;
the Fr.5 part is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 50, 100: 100 and 0: 100 in sequence, eluent obtained by the gradient elution is identified by thin-layer chromatography, classified and combined, and is separated by coordination chromatography to obtain licoisoflavone A, licoflavone A, licoisoflavan A, glycyrrhiza isoflavone A, licoisoflavone B, licoricone and licoisoflavanone;
performing coordination chromatographic separation, namely preparing a coordination chromatographic column with the central ion content of 7-10% by taking metal ions as central ions, loading a sample on the coordination chromatographic column, sequentially identifying eluent by thin-layer chromatography according to the gradient volume ratio of chloroform to methanol of 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100, and separating eluent of licoisoflavone A, licoflavone A, licoisoflavone A, glycyrrhiza isoflavone B, licoricone and glycyrrhiza isoflavone;
The metal ion is Se 4+ 、Cu 2+ 、Mg 2+ 、Ni 2+ 、Al 3+ Or Zn 2+ One of (1) and (b);
the Fr.6 part is subjected to silica gel column chromatography, the gradient volume ratio of chloroform to methanol is 100: 5, 100: 10, 100: 20, 100: 30, 100: 50, 100: 100 and 0: 100 in sequence, and eluent is subjected to thin layer chromatography identification to obtain licochalcone A-E mixture finally;
separating and purifying the mixture of licochalcone A-E by high-speed counter-current chromatography to obtain licochalcone A-E;
the high-speed countercurrent chromatography adopts n-hexane, ethyl acetate, methanol and water (1.0: 2.5: 1.5: 0.1, v/v/v/v) as a two-phase solvent system, the lower phase as a stationary phase and the upper phase as a mobile phase, the reverse rotation is carried out, the rotating speed is 850r/min, the temperature of a constant-temperature water bath is 25 ℃, and the licochalcone A-E5 components are obtained by separation under the conditions that the flow rate of the mobile phase is 1.5mL/min and the sample injection concentration is 20 mg/mL;
the Fr.7 part is concentrated into an extract, dissolved by adding water, adsorbed by macroporous adsorption resin, eluted by using methanol with volume fraction of 70-80% as an eluent, the eluent is concentrated into the extract, the obtained extract is subjected to silica gel column chromatography, gradient elution is carried out according to the volume ratio of chloroform to isopropanol of 20: 1, 10: 1, 5: 1, 1: 1 and 0: 1 in sequence, the eluent is identified by thin-layer chromatography, classified and combined, and isoliquiritin, liquiritin, neoliquiritin and neoisoliquiritin are obtained by separation through microemulsion electric capillary chromatography;
According to the microemulsion electrokinetic capillary chromatography, when a microemulsion system is 50mmol/L boric acid buffer solution (pH 9), 10% (volume fraction) n-butanol, 80mmol/L n-heptane, 120mmol/L sodium dodecyl sulfate and 5mmol/L sulfonated beta-cyclodextrin, the separation voltage is 20kV, and the column temperature is 35 ℃, the microemulsion electrokinetic capillary chromatography for separating isoliquiritin, liquiritin, neoliquiritin and neoisoliquiritin is established, and the separation of the 4 components in a sample solution is realized within 30 min;
the second step is that: mixing the n-butanol part extract with silica gel with the particle size of 48-75 μm, loading the mixture on a dry column for silica gel column chromatography, performing gradient elution by using chloroform-methanol as an eluent, wherein the gradient volume ratio of chloroform to methanol is 30: 1, 20: 1, 10: 1, 5: 1, 3: 1, 1: 1 and 0: 1 in sequence, and performing thin-layer chromatography identification and combination on corresponding eluents to obtain 6 parts Fr.I-Fr.IV;
subjecting the Fr.I part to silica gel column chromatography, sequentially detecting eluates at ethyl acetate-methanol volume ratio of 100: 0, 100: 5, 100: 10, 100: 20, 100: 50, 100: 100 and 0: 100 by thin layer chromatography, subjecting the eluates to silica gel column chromatography again, sequentially detecting eluates at chloroform-methanol volume ratio of 100: 5, 100: 10, 100: 20, 100: 30, 100: 100 and 0: 100 by thin layer chromatography, and obtaining apiosyisoliquiritin at gradient volume ratio of chloroform to methanol of 100: 10;
Performing silica gel column chromatography with chloroform-methanol at gradient volume ratio of chloroform to methanol of 100: 0, 100: 5, 100: 10, 100: 15, 100: 20, 100: 40, 100: 100, and 0: 100, subjecting the eluate to thin layer chromatography, subjecting the eluate to Sephadex LH20 column gradient elution with methanol of 10%, 15%, and 20% volume fraction, and subjecting the eluate to thin layer chromatography to obtain isoliquiritigenin-4' -O-apiose (1 → 2) glucoside;
performing gradient elution on the Fr.III part by silica gel column chromatography with chloroform-methanol volume ratio of 100: 5, 100: 10, 100: 15, 100: 20, 100: 25, 100: 100 and 0: 100 in sequence, detecting the eluate by thin layer chromatography, performing silica gel column chromatography, and performing isocratic elution with chloroform-methanol as eluent, wherein the chloroform-methanol volume ratio is 100: 10 to obtain apiosyl liquiritin;
the fr.iv fraction need not be isolated.
CN202211015511.1A 2022-08-12 2022-08-12 Method for extracting and separating effective components of liquorice root in full-industrial-chain cooperation manner Pending CN115721671A (en)

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CN116370378A (en) * 2023-03-31 2023-07-04 广东诗漾医疗科技有限公司 Licorice root extract skin care product composition and preparation method thereof
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* Cited by examiner, † Cited by third party
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CN116350675A (en) * 2023-03-13 2023-06-30 武汉市中西医结合医院（武汉市第一医院） Preparation method of licorice extract, licorice extract and application
CN116370378A (en) * 2023-03-31 2023-07-04 广东诗漾医疗科技有限公司 Licorice root extract skin care product composition and preparation method thereof
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CN116574668B (en) * 2023-06-13 2023-12-05 浙江觅得优生物科技有限公司 Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium
CN116807031A (en) * 2023-08-28 2023-09-29 云南邦纳科技有限公司 Preparation process of truffle extract for truffle wine
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