CN115403589A - Separation and purification method of homoharringtonine - Google Patents
Separation and purification method of homoharringtonine Download PDFInfo
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Abstract
A separation and purification method of homoharringtonine comprises the following steps: (1) vacuum blasting treatment; the method comprises the following steps of (2) extracting by using a eutectic solvent, (3) separating by resin adsorption, (4) separating and purifying by using ionic liquid and aqueous two phases, (5) separating and purifying homoharringtonine, wherein cephalotaxus grandis bark is treated by vacuum explosion, then the total alkaloids are extracted by using the eutectic solvent, the total alkaloids are adsorbed by using macroporous resin, the total alkaloids are separated, the eutectic solvent is regenerated, the macroporous resin is subjected to gradient elution to obtain high-content alkaloid, and the high-content alkaloid is further purified by ionic liquid and aqueous two phase extraction, and finally the homoharringtonine with the content of more than 99% is obtained by pH zone countercurrent chromatographic separation or medium pressure column chromatography.
Description
Technical Field
A method for separating and purifying homoharringtonine is provided.
Background
Cephalotaxus fortunei (RamuluexFolium Cephalotaxus) is a unique tree species in China, originates in the commingled forest of coniferous trees and broadleaf trees, and is produced in China, anhui, zhejiang, shaanxi, gansu, sichuan, yunnan, guizhou and the like. Cephalotaxus sinensis (red. Et wils.) Li), also known as Cephalotaxus sinensis, eastern Cephalotaxus sinensis and Cephalotaxus sinensis, is a Cephalotaxus plant of cephalotaxaceae, is a special tree species in China, is widely distributed, is naturally distributed in the Yangtze river basin and in the southern areas, is produced in south of Jiangsu, southern Anhui, southern Shaanxi, southern Gansu, zhejiang, fujian, jiangxi, henan, hunan, hubei, sichuan, southeast of Yunnan, southeast of Guizhou, guangxi and southwest, and is mostly produced in granite, sandstone and limestone mountain land with the altitude of 600-2200 meters. The torreya grandis is the most valuable natural anticancer medicinal plant, is the domestic tree species containing the most kinds and the highest content of tumor-inhibiting alkaloids, and is confirmed to be the most potential natural anticancer medicine source and the last line of defense of advanced cancers.
1. Chemical composition
Chinese materia medica records that branches and leaves of cephalotaxus contain various alkaloids, wherein the alkaloids belonging to cephalotaxus comprise: cephalotaxine (cephalotaxine), epicephalotaxine (epicephalotaxine), levorotatory and dextrorotatory acetylcephalotaxine (acetylcephalotaxine), cephalotaxine (harringtonine), homocephalotaxine (homoharringtonine), isocephalotarine (isoharringtonine), deoxycephalotaxine (deoxyharringtonine), 11-hydroxytryptophane (11-hydroxycephalotaxine), demethylcephalotaxine (desmethylcephalotaxine), desmethylcephalotaxine (4-hydroxycephalotaxine), cephalotaxine (cephalotaxine), isocephalanaline (isocephalanaline), 4-hydroxytryptophane (4-hydroxycephalotaxine), cephalotaxine (isocephalotanine), isocephalotanine (isocephalotanine, etc.; the alkaloid belonging to the high erythrina alkaloid class comprises: taiwan cephalotaxine (wilsonine), epi-taiwan cephalotaxine (epiwilsonine), fujian cephalotaxine (caalofortuneine), cephalotaxine (fortuneine), epi-fujian cephalotaxine (epicalofortunei-ne), 3-epi-kawasaki (3-epideschelherminic), 3-epi-methyishihem (3-epimethyishihalmerine B), and the like. Further comprises cephalotaxus hainanensis lactone (hainanolide), cephalotaxus hainanensis lactone alcohol (hainanolidol) and apigenin (api-gen), chrysoenol, sequoyitol, D-1-O-methyl mucoinositol (D-1-O-methyl mucoinositol), pinitol (pinitol), 12, 16-dihydroxy abietane-6, 8, 11, 13-tetraen-3-one separated from cephalotaxus fortunei stems and leaves, junenol, cephalotaxol, margoclin, cephalotodoidB, torreyanyunin, cephalotaxus gondii diterpene D, 6 alpha-hydroxy shanda pinic acid, cephalotaxus hainanensis lactone and cephalotaxus hainanensis lactone (Chinese herbal medicine, 2021, 52, 3174-3179). Are obtained by separating from Cephalotaxus fortunei and are shonanin, arctigenin, alpha-hemerin, matairesinol, nortrachelogenin, epi-nortrachelogenin, (7 'S) -hydroxymatairesinol, (7' R) -hydroxymatairesinol, (7 'S) -hydroxynarcotinin, secoisolariciresinol, 4' -di-O-methylephamfortin A, 5- (3 ',4' -dimethoxyphenyl) -3-hydroxy-3- (4 '-hydroxy-3' -methoxybenzyl) -4-hydroxymethyl dihydrofuran-2-one, cephafortin B, dihydrodehydro-guaiacyl alcohol, 7R,8S-4,7,9 '-tetrahydroxy-3, 3' -dimethoxy-8-O-4 '-neolignan, 7R,8R-4,7, 9' -tetrahydroxy-3, 3 '-dimethoxy-8-O-4' -neolignan and threo-1, 2-bis (4-hydroxy-3-methoxyphenyl) -1, 3-propanediol (Chinese herbal medicine, 2020, 51, 36-42). 6 compounds were isolated from Cephalotaxus fortunei and identified as 20-hydroxyecdysone, arhat's sterone, 7, 8-dihydro-20 hydroxyecdysone, 5,4' -dihydroxy-7-methoxy-8-O-beta-D-glucoflavonol glycoside, 5-O-beta-D-glucoapigenin, and 3-O-beta-L-rhamnose quercetin (Shenyang pharmaceutical science university report, 2018, 35, 89-91+ 97), respectively. Sugiol, junipediol A, (+) -catechin, (-) -epicatechin, (+) -isolarch pinoresinol-9' -O-beta-D-glucoside, seco-dehydrodiformyl alcohol-4-O-beta-D-glucopyranoside and icariside E3 are separated from branches and leaves of cephalotaxus olivaceus (Chinese medicinal materials, 2017, 40, 2594-2597). Isolating Cephalotaxus fortunei sesquiterpene A, cephalotaxus fortunei sesquiterpene B, cephalotaxus fortunei C9-hydroxy-4, 7-medimagadien-3-one, corochool C9, 10-dihydroxy-4, 7-medine-3-one 5, 12-epoxy-9-hydroxy-7-medine 5, 12-epoxy-6, 9-hydroxy-7-medine-3-one, lilolide, (3S, 5R,8S-5, 8-4-epoxy-6-medine-3, 9-hydroxy-7-medine-3-one, 2015, 46, 320-324) from Cephalotaxus fortunei branches and leaves, isolating and purifying to obtain sitosterol, beta-cephalothin, acetylcephalothin, oxycedrine, trialkamine, trialkanol, 7-voxaline, jasminol, 4' -trihydroxyflavone, cryptomerin, cephalotaxine, succinic acid, cephalotaxine, taiwan cephaloflavanone-B, acetyl cephalotaxine, cephalotaxus hainanensis lactone, kayaflavanone, daucosterol, apigenin, chrysin, cephalotaxine and desmoxigenin (Zhou Mei, guizhou university, 2009 Master paper.) isolation of desmoxigenin, cephalotaxine, epi-cephalotaxine, lijiang cephalotaxine and acetyl cephalotaxine (Jiang, zhejiang university, 2009 university.) isolation of cephalotaxus hainanensis from cephalotaxus hainanensis to cephalotaxine, 2, 3-epi-2-hydroxyxytaxine, cephalotaxine, deoxycephalotaxine, isotrichtotone, cephalotaxine, (+) -cephalotaxine ester, cephalotaxine-R) -cephalotaxine, (R) -cephalotaxine, (S) -Lijiang cephalotaxine, cephalomannine G, (2R, 3S,4S, 5S) -2-, 3-dihydroxy cephalotaxine, iso-3-epilicamitine, cephalomannine H, iso-cephalotaxine, epierythtation, desmoxidotrine, 1, 4-N-diisopropylaminoanthraquinone, indazole, drummondol, benzoic acid (2-ethyl) -N-hexyl ester, 3-methoxy-4-hydroxyphenylethanol, coniferal, p-hydroxybenzaldehyde, 3, 4-dihydroxybenzaldehyde, isovanillin, vanillic acid, hydroxypyropimiacaiacone, β -sitosterol, stigmasterol and di (α -ethylhexyl) phthalate (in Cantonella 562815656281, university of Wood 2019 academic papers). Cephalotaxine, iso-cephalotaxine, cephalotaxine G, (2R, 3S,4S, 5S) -2, 3-dihydroxy cephalotaxine, acetyl cephalotaxine, deoxycephalotaxine, (R) -Lijiang cephalotaxine, (S) -Lijiang cephalotaxine, 3-epi-hydrastine isomer, 1, 4-N-diisopropylamine anthraquinone, benzoic acid (2-ethyl) -N-hexyl ester and beta-sitosterol (Chinese herbal medicine, 2019, 50, 1541-1545) are separated from cephalotaxine total alkali. 8-oxoharringtonine, 8-oxodeoxyl 8-H8-arrringtonine, cephaforutine A, 8-oxophalotaxine, deoxy-9-harringtonine, acetylcephaotaxine, cephaotaxine, epichaotaxine and cephaotaxine (Chinese journal of Chinese medicine, 2022, 47, 2994-2999) are separated from Chinese torreya. <xnotran> 6-c-methyl-7,4',4"' -tri-o-methylamento-flavoneapigenin-6-C-methyl-o- β -D-glucopyranosideamentoflavone-7,7",4',4"' -tetramethylether, 7,4',7" -tri-O-methyl amentoflavone, taiwanhomoflavoneAginkgetin2,3-dihydro-6-methylginkgetin2,3-dihydro-6-methylsequoiaflavoneapigenin5-o- α -L-rhamnopyranosyl- (1 → 3) β -D-glucopyranoside, ladanetin-6-O- β -D-glucoside, isoscutellarein7- β -D-glucopyronoside, apigeninnaringenin6-C-methylnaringenin, cephalancin A, cephalotaxineP-2, 11-epoxy-1,2-dihydro-8-oxo- (2 α,11 α) -9 (N), cephalancetine B, drupacine, 11-hydroxyhainanolidol, fortunolideA, rel- (7 α,8 β -3-methoxy-4',7-epoxy-8,3'-12-oxyne-olignan-4,9,9' -triol [3,3',4,4' -tetrahydro-6,6'-dimethoxy-3,3' -bi-2H-benzopyran ] -4,4'-diol, (+) -7' (S) -hydroxyarctigenin, 5- (3 ",4" -dimethoxyphenyl) -3-hydorxy-3- (4 '-14-Hydroxy-3' -methoxybenzyl) -4- (hydaoxymethyl) -dihydrofuran-2-onedehydrovomifoliol, </xnotran> (6R, 7E, 9S) -9-hydroxy-4, 7-megastimadien-3-one, (6R, 9R) -9-hydroxy-4-megastimein-3-one, seiricaldenone A, eremopetasinone A, methyl 17-hydroxydocosanoate-3 beta-glucoside-6' -o-palmate, beta-sitosterol, daucosterol, p-hydroxybenzazole 3-methoxy-4-hydroxybenzazole, cemananin A, 19-cemanaxin B, cefadrain A, and cephamarin C, cephamarin D, cephamidine adoxyharringtonine, drupacine, cephamidine A, cephamazine alpha-N-oxide, cephamazine beta-N-oxide, 3-epischemhommerine, taiwanoflavone A, ginkgetin, 2, 3-dihydro-methylgeketin, apigenin, fortunolide A, 3 beta-hydroxy-5, 6-dehydrosugol, (5 alpha, 20R) -pregn-2-en-20-ol, (3. Beta., 5. Alpha., 20S) -20-methylpregnane-3, 21-diol, (22S) -5. Alpha. -ergosta-3. Alpha., 22-diol,. Beta. -sitosterol, glucosterol, ferulyl aldehyde, [8] -bishydroshodiol, p-hydroxybenzalode and methyl 6-hydroxytetracarbonate (Zhang Yunmei, university of Yunnan Master, 2014-year school bit). Cephalotaxus sinensis bark is divided into cephalotaxus hainanensis lactone, iso-cephalotaxus fortunei ester alkali, homoharringtonine, hydroxyl cephalotaxus yedoensis, cephalotaxus fortunei alkali, oxo cephalotaxus fortunei alkali, epi-phenoline Alkaloid 6 and Taiwan cephalotaxus fortunei alkali (Chinese herbal medicines, 1981, 12, 1-4). Deoxyharringtonine, isotrichodermine, harringtonine, homoharringtonine, cephalotaxine, desmethyl cephalotaxine, 3-epi-methamphetamine and epi-cephalotaxine were isolated from cephalotaxus hainanensis bark (journal of chemistry 1976, 04, 283-293). 4,4 '-diphenylmethane methyl dicarbamate, arctigenin, shonanin, (7R, 8S) -dihydrodehydrodiconiferyl alcohol 9' -O-beta-D-glucopyranoside (Shenyang pharmaceutical department university report, 2018, 35, 89-91+ 97) are separated and obtained from cephalotaxus fortunei. Apigenin, beta-sitosterol, acetyl cephalotaxine, chrysin, desmoxigenin, norhendecanol, 7,3',4' -trihydroxyflavone, cryptomerin, cephalotaxine, taiwan cephalotaxine and cephalotaxus hainanensis lactone (proceedings of Chinese pharmaceutical university, 2009, 40, 209-212) are separated from cephalotaxus hainanensis produced from Qian.
Homoharringtonine has the following structural formula:
2. pharmacological action
Homoharringtonine (Homoharringtonine) is an alkaloid obtained from Cephalotaxus fortunei of the family Cephalotaxus or its congeneric plants. The effect of homoharringtonine in combination with azacitidine and Venetocclax on the treatment of refractory/relapsed acute myeloid leukemia is positive, and the patient tolerance is good (proceedings of the New county medical college, 2021, 38, 580-584). The effect of homoharringtonine on proliferation of liver cancer cell PLC5 and its possible mechanism, homoharringtonine can induce PLC5 cell to generate DNA damage, activate DNA damage repair signal pathway, block cell cycle, thereby inhibiting cell proliferation (Chinese pharmacological report, 2021, 37, 380-385). The combined use of homoharringtonine with cladribine, decitabine and cidanil, respectively, has a synergistic effect on the proliferation inhibition of Acute Myeloid Leukemia (AML) cells and has important guiding significance for clinical treatment of AML (journal of western medicine, 2021, 36, 35-38). Effect of homoharringtonine on human melanoma A375 proliferation and possible mechanism of action, HHT blocks A375 cells from G 2 In the/M phase, the proliferation of A375 cells is inhibited, HHT can activate a mitochondrial apoptosis signaling pathway and induce the apoptosis of melanoma A375 cells (Chinese pharmacological Notification, 2020, 36, 1100-1105). The High Harringtonine (HHT) can regulate the proliferation and apoptosis of HT29 human colorectal tumor cells by blocking an mTOR signaling pathway, so that the HT29 human colorectal tumor cells can be inhibited from proliferation and induced to be apoptotic (journal of cellular and molecular immunology, 2018, 34, 346-353). The homoharringtonine can inhibit the activation of PI3K/Akt pathway by reducing the expression of IRS4 protein so as to block cell cycle at G 0 /G 1 And thereby inhibiting proliferation of melanoma resistant cells (proceedings of the Chinese biochemistry and molecular biology 2020, 36, 970-976). For the middle and high risk group of the oldMyelodysplastic syndrome patients are treated by chemotherapy with a small dose of high harringtonine or a scheme containing a small dose of high harringtonine, and can achieve certain curative effect, have small adverse reaction and be easily tolerated by patients (Chinese medical guidelines, 2008, 03, 71-72). The curative effect and the safety of treating myelodysplastic syndrome by combining homoharringtonine with cytarabine are obviously better than the effect of treating myelodysplastic syndrome by using cytarabine alone, the effect of treating myelodysplastic syndrome by combining homoharringtonine with cytarabine is capable of effectively improving the quality of life level of patients, the safety is higher, and the combination is possibly related to the reduction of IL-6 and TNF-alpha levels of serum, the increase of IL-10 level and the reduction of inflammatory response (the modern biomedical progress is 2020, 20, 2762-2765+ 2794). Modern clinical research is used for treating malignant lymphoma, leukemia, gynecological cancer, breast cancer, polycythemia vera and the like, is used for treating acute myelocytic leukemia, can improve the curative effect when being used together with VCR, ara-C and prednisone, has certain curative effect when being used for treating acute monocytic leukemia and malignant lymphoma, and can also be used for treating polycythemia vera, chronic myelocytic leukemia, promyelocytic leukemia and the like.
3. Extraction and separation process
The NRA resin is selected to be feasible for alkaloid purification, and the macroporous resin can achieve the optimal purification effect under certain factors (Guizhou science, 2009, 27, 67-69+ 85). Cephalotaxine is separated by utilizing a pH zone countercurrent chromatography, trifluoroacetic acid is added into an upper phase (stationary phase) of an ethyl acetate-petroleum ether-water (3: 1: 4) solvent system to serve as a retention agent, and a lower phase (mobile phase) containing 10% ammonia water, neutral and trifluoroacetic acid is sequentially subjected to gradient elution, so that a good separation effect is obtained, and the cephalotaxine and acetyl cephalotaxine have the purities of 95.4% and 98.9% respectively (J. of pharmaceutical analysis, 2009, 29, 188-191). Separating the mixture of harringtonine and homoharringtonine by improved countercurrent distribution method, using chloroform saturated with pH5 buffer solution as mobile phase, disodium hydrogen phosphate-citric acid saturated with chloroform as stationary phase, ethyl acetate and acetone (6: 2.5) as developing agent, and alkaline silica gel plate as thin layer plate, separating 5 batches of mixture to obtain harringtonine and homoharringtonineWhite crystals (journal of pharmacy in China, 1998, 18, 28-29). By adopting 89-A type high-speed countercurrent chromatography, the three main components of the anticancer drug, namely the iso-harringtonine, homoharringtonine and harringtonine, can be completely separated by taking chloroform/0.07 mol/L phosphorus jun sodium-0.04 mol/L citric acid buffer solution (pH 5.0) as the upper phase as the stationary phase and taking the lower phase as the mobile phase (journal of Chinese medicine industry, 1991, 22, 72-73). The patent CN201210096947.8 adopts acidic ethanol for extraction, the extract is obtained by concentrating, tartaric acid is used for dissolving the extract, ethyl acetate is used for extraction and degreasing, and saturated Na is used for a water layer 2 CO 3 Adjusting pH to about 9, extracting with equal volume of chloroform for 3 times, recovering chloroform to obtain cephalotaxin with total alkali yield of 0.298%, and extracting with Al 2 O 3 As adsorbent, dissolving cephalotaxine with dichloromethane, loading onto column, eluting with diethyl ether, collecting eluate, and obtaining pure cephalotaxine with purity of 96.4%. CN200710099906.3 takes cephalotaxus extract, adjusts pH value to 1-7, filters, passes through a cation exchange resin column, firstly washes to remove impurities, then soaks the resin column with 0.5-20% saline solution, then elutes with 0.5-20% saline solution containing acid or alcohol, collects eluant, desalts, and obtains cephalotaxus fortunei total alkaloids by concentration and drying. Patent CN02150323.0 dilute ammonia alkalization → supercritical CO 2 Fluid extraction → separation and recovery of cephalotaxus fortunei total alkaloids → adsorption resin continuous column chromatography → recovery of cephalotaxus fortunei mixed ester alkali → preparation of high-speed countercurrent chromatography and ultraviolet detection to obtain cephalotaxus fortunei and homoharringtonine monomers and a small amount of mixture of the cephalotaxus fortunei and homoharringtonine → respective recrystallization and purification to obtain pure products for preparation. Patent CN201410762622.8 uses mobile phase to dissolve homoharringtonine crude product, uses silica gel as stationary phase and uses ethyl acetate/n-hexane/DIEA mixed solution as mobile phase to perform column chromatography, collects chromatographic solution containing homoharringtonine, concentrates into solid, dissolves the solid with ethyl acetate, then adds n-hexane or n-heptane to mix uniformly, stands for crystallization, performs suction filtration, and obtains homoharringtonine solid after filter cake drying. Patent CN201910348404.2 methanol-chloroform mixed solution is extracted at room temperature, concentrated to obtain extract, chloroform is added into the extract, the extract is settled and filtered, the filtrate is concentrated to obtain crude homoharringtonine product, and the homoharringtonine product is obtained by separating homoharringtonine from homoharringtonineDissolving the crude product of harringtonine with acetonitrile, filtering, adding water into the filtrate, loading into a mixed resin column for separation and purification, collecting and combining eluates containing homoharringtonine, concentrating, adding chloroform for extraction, collecting a chloroform layer, concentrating and draining to obtain a semi-finished product of homoharringtonine, adding chloroform into the semi-finished product of homoharringtonine for dissolution, loading into an acidic alumina chromatographic column for separation and purification after the semi-finished product is dissolved, eluting with a chloroform-methanol mixed solution, monitoring the eluates with a TLC plate, collecting and combining eluates containing homoharringtonine, concentrating and draining, adding chloroform for dissolution, adding n-pentane for recrystallization. The method comprises the steps of pretreatment of raw materials, crushing, methanol extraction, primary reduced pressure concentration, acid precipitation, extraction decolorization, ester alkali extraction, secondary reduced pressure concentration, column chromatography elution, tertiary reduced pressure concentration, distribution chromatography elution, four-time reduced pressure concentration, dissolution, evaporation to dryness, crystallization and the like in patent CN 202110868850.3. Extracting with methanol in CN202110828420.9 for multiple times to obtain an extracting solution, concentrating the extracting solution to obtain an extract, adding 1 percent of HCl aqueous solution into the obtained extract, fully stirring and dissolving, standing the acid aqueous solution after the pH is adjusted, centrifuging to obtain an acid precipitation supernatant and a precipitate, adding petroleum ether into the obtained acid precipitation supernatant, stirring and extracting, standing, layering, extracting an upper ether layer, extracting for multiple times to obtain an acid aqueous solution, adjusting the pH of the obtained acid aqueous solution 2 to 4-6 with 10 percent ammonia water, adding chloroform, stirring and extracting, standing, layering, discharging a lower chloroform layer, repeatedly extracting in the way to obtain a chloroform solution and an acid aqueous solution, drying the obtained chloroform solution with anhydrous sodium sulfate, concentrating under reduced pressure to obtain an extract, adding chloroform into the obtained extract for dissolving, and obtaining a sample; the optimal extraction process parameters of the main alkaloids in cephalotaxus hainanensis are as follows: the solvent is H 2 SO 4 (pH 1), the particle diameter of the raw material is 10 meshes, the ratio of material to liquid is 1: 70 (g: mL), the extraction time is 60min, the extraction temperature is 50 ℃, the ultrasonic power is 480W, and the extraction times are 4 times, so that the optimal enrichment technological parameters of the cephalotaxine are obtained: the reaction time is 10min, the reaction temperature is room temperature (28 ℃), the alkali used in the reaction is KOH, the concentration of KOH solution is 0.1mol/L, a method for separating and purifying cephalotaxin by preparative high performance liquid chromatography is established, and cephalotaxin is successfully separated and purified by the methodAlkali monomer (Shenyun, university of northwest, master's paper 2020).
Disclosure of Invention
The raw materials are 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 torreya grandis bark is damaged under the action of mechanical force. The method for separating homoharringtonine by adopting the ionic liquid-aqueous two-phase system has the advantages of stronger selectivity, high enrichment degree, low price, high efficiency, mild and nontoxic extraction, small activity loss on the extract and the like, and is a green process. The pH zone countercurrent chromatography is particularly suitable for separating ionic alkaloid, and the homoharringtonine is separated and purified by adopting the pH zone countercurrent chromatography and medium-pressure column chromatography.
A separation and purification method of homoharringtonine comprises the following steps: (1) vacuum blasting treatment; the method comprises the following steps of (2) eutectic solvent extraction, (3) resin adsorption separation, (4) ionic liquid-aqueous two-phase separation, and (5) separation and purification of homoharringtonine.
The steam explosion treatment in the step (1) comprises the steps of cutting off cephalotaxus sinensis barks to 30-40 mm to be used as an explosion matrix, adding water according to the mass ratio of the dry cephalotaxus sinensis barks to the water of 1: 0-1: 4, then soaking at normal temperature for 2-8 h, adding an entrainer into the rehydrated materials, uniformly mixing by using a mixer and maintaining for 30min, placing in a novel ejection type steam explosion device, and using air and steam as steam explosion media;
the entrainer is fine-mesh carborundum and NH 4 Cl、Na 2 CO 3 Or Ca (OH) 2 Composites of powders, corundum and NH 4 Cl、Na 2 CO 3 Or Ca (OH) 2 The mass ratio of the 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 tank 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.
Extracting the torreya grandis subjected to vacuum blasting by using the eutectic solvent to obtain a torreya grandis extracting solution;
the eutectic solvent is choline chloride, a hydrogen bond donor and water;
the content of water in the eutectic solvent is 10-30 wt%;
the feed-liquid ratio of the cephalotaxus sinensis to the eutectic solvent is 20-100 mg/mL;
the hydrogen bond donor is selected from one of lactic acid, 1, 4-butanediol, glycerol or malonic acid;
the mol ratio of choline chloride to a hydrogen bond donor in the eutectic solvent is 1: 0.5-5;
the extraction temperature is 60-70 ℃, and the extraction time is 1-15 min.
Washing the cephalotaxus sinensis extract obtained in the step (3) with water through a macroporous adsorption resin column to obtain a eutectic solvent, and performing gradient elution to obtain alkaline elution liquid containing homoharringtonin;
the step of washing, which is to wash the effluent liquid by purified water with the pH value of 6-7 until the effluent liquid is clear, and concentrate the water wash liquid under reduced pressure and dry the water wash liquid to obtain a regenerated eutectic solvent for the next use;
the macroporous adsorbent resin is specifically one of D101, D140, HPD100, LX-38 or HPD 850;
the gradient elution is to elute impurities with larger polarity absorbed by the resin column by using 2.0 to 4.0BV of 10 to 20 volume percent of ethanol aqueous solution to obtain a component 1, discard the eluent, elute impurities with medium polarity absorbed by the resin column by using 2.0 to 4.0BV of 20 to 40 volume percent of ethanol aqueous solution to obtain a component 2, elute alkaloid absorbed by the resin column by using 2.0 to 4.0BV of 50 to 70 volume percent of ethanol aqueous solution to obtain a component 3, and collect the component; eluting the impurities with weak polarity adsorbed in the resin by using an ethanol water solution with the volume solubility of 80-90% of 2.0-4.0 BV, discarding, recovering ethanol, washing by using purified water, and regenerating the resin;
and carrying out reduced pressure concentration on the alkaline eluent containing homoharringtonine to obtain a homoharringtonine concentrate.
The preparation method of claim 1, wherein the ionic liquid-aqueous two-phase separation in step (4) is performed, the homoharringtonine concentrate obtained in step (3) is extracted by taking the ionic liquid as an extractant, then inorganic salt is added, the mixture is uniformly mixed to form a suspension, ultrasonic extraction is performed on the suspension, the suspension is kept stand to obtain an aqueous two-phase system, the extractant phase is collected, and the extractant phase is subjected to reverse extraction and extractant regeneration;
the aqueous two-phase system consists of ionic liquid and inorganic salt, and the imidazole hydrophobic ionic liquid consists of cation M + And the anion N - Two-part, in which the cation M + Including but not limited to one of a substituted imidazolium cation, pyridinium cation, amine cation, or substituted pyrroline cation, and an anion including but not limited to N - Is one of hexafluorophosphate radical, tetrafluoroborate radical, bis (trifluoromethanesulfonyl) imide radical or nonafluorobutyl sulfonate radical;
the substituent is one of C- (1) -C- (8) alkyl, C- (1) -C- (3) alkenyl or C- (6) -C- (15) aryl;
saidSalt solution phases include, but are not limited to, naH 2 PO 4 、(NH 4 ) 2 SO 4 、Na 2 HPO 4 、KH 2 PO 4 、K 2 HPO 4 、K 2 CO 3 、Na 2 CO 3 、K 3 PO 4 Or Na 3 PO 4 ；
The mass concentration of the ionic liquid is 400-600 g/L, and the pH value is 6.8;
the ratio of the homoharringtonine concentrate to the ionic liquid is 0.5-1.0 g/mL, the extraction temperature is 50-60 ℃, and the extraction time is 10-20 min;
the ultrasonic conditions of the ionic liquid aqueous two-phase system are as follows: the ultrasonic frequency is 40kHz, the power is 190W, the temperature is 45 ℃, the time is 1-1.5 h, and the degassing condition is as follows: operating pressure intensity of 0.05-0.07 MPa, degassing time of 6-10 min, adjusting pH value to 3.8-4.0 by using 0.6mol/L phosphoric acid, standing for 8-10 h at 8-10 ℃, and taking an upper liquid ionic phase;
the mass percentage of salt in the ionic liquid aqueous two-phase system is 10-30%.
Adding an organic solvent with the volume 1-5 times (v/v) of that of the ionic liquid phase obtained in the step (4) for extraction for 1-3 times, wherein the organic phase obtained by separation is a cephalotaxus sinensis total alkaloid extract phase, the raffinate obtained by extraction is an ionic liquid and can be recycled, acidifying the total alkaloid extract phase to remove the organic phase, collecting the water phase, alkalifying the water phase, filtering, dissolving the precipitate obtained by filtering in the organic solvent, filtering, and recrystallizing to obtain cephalotaxus sinensis total alkaloid;
the organic solvent is one of dichloromethane, 1-dichloroethane, 1, 2-dichloroethane and chloroform.
Separating and purifying homoharringtonine in the step (5), and adopting one of pH zone countercurrent chromatography separation or medium pressure preparative chromatography technologies;
the solvent system for the pH zone countercurrent chromatographic separation is as follows: one of petroleum ether-ethyl acetate-methanol-water (5: 4: 6, v/v), petroleum ether-ethyl acetate-methanol-water (3: 7: 1: 9, v/v) or petroleum ether-ethyl acetate-methanol-water (1: 1, v/v);
the acidic substance is one of trifluoroacetic acid, hydrochloric acid or acetic acid;
the alkaline substance is one of ammonia water or triethylamine.
Separating and purifying homoharringtonine in the step (5), adopting a medium-pressure preparative chromatography technology, performing medium-pressure chromatographic column equilibrium activation, preparing and loading a sample, performing gradient elution by an elution solvent, detecting and collecting fractions: concentrating and crystallizing the eluent to separate and purify homoharringtonine;
the medium-pressure chromatographic column is activated in a balanced manner, and after the mobile phase washes the column for 30-40 min at a higher flow rate, the mobile phase with different polarities at a lower flow rate activates the chromatographic column for 1-2 h;
preparing and loading a sample, dissolving a concentrate by using 1-5 times (v/v) of mobile phase, centrifuging at 12000r/min, pumping a supernatant into a chromatographic column, or uniformly mixing the supernatant with a chromatographic filler, and loading the dried chromatographic filler into a column head of the chromatographic column;
and (3) gradient elution of the elution solvent, namely, linear gradient elution is performed by taking water (A) -methanol (B) as a solvent system respectively, the gradient elution is performed for 110min, namely, the flow rate is 10-50 mL/min, the volume percentage of the methanol in the solvent system is B%, and the gradient of each time period and the solvent system is: 0-20 min B% is 15-30%, 20-50 min B% is 30-45%, 50-65 min B% is 45-78%, 65-95 min B% is 78-85%, 95-100 min B% is 85-100%, 100-110 min B% is 100%; gradient elution is carried out for 110min, and the flow rate is 10-50 mL/min;
detecting and collecting fractions, monitoring at 220nm wavelength, and collecting eluate fractions of corresponding absorption peaks by an automatic fractionation collector according to peak emergence time;
and (3) concentrating and crystallizing the eluent, concentrating the eluent of each component under reduced pressure at 50 ℃ under the vacuum degree of 90-150 mbar, removing methanol, and cooling the concentrated sample to room temperature.
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.
In the following examples, room temperature means 15 to 25 ℃.
Instruments and materials: cephalotaxus sinensis barks are produced in Shanxi Shandong, instantaneous ejection type steam explosion machine (Henan Zhengdao Qibao environmental protection science and technology Co., ltd.), ultrasonic extraction instrument (Qingdao Juchuang environmental protection group Co., ltd.), high-speed countercurrent chromatograph (AECS Co., england), HPD850 macroporous resin (Xian Langxi), ionic liquid (Minnean chemical technology (Shanghai)) ammonium sulfate, chloroform, ethyl acetate, ammonia water, triethylamine, trifluoroacetic acid, hydrochloric acid, acetic acid, dichloromethane, carborundum, na 2 CO 3 Choline chloride, 1, 4-butanediol, ethanol were purchased from ACROS, carbofuran, aldrich, tianjin, kemikou chemical reagents ltd and national drug group chemical reagents ltd.
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.
50kg of Qinling cephalotaxus sinensis barks are cut into 30-40 mm and used as blasting substrates, 150kg of water is added, the materials are soaked for 6 hours at room temperature, and fine carborundum and Na are added into the materials after rehydration 2 CO 3 Adding entrainer in batches, mixing with mixer uniformly for 30min, steam blasting I adding fine emery 0.75kg, adding Na 0.25kg 2 CO 3 Uniformly mixing the components by using a mixer, maintaining the mixture for 30min, placing the mixture in novel ejection type steam explosion equipment, and taking air and steam as steam explosion media; steam explosion treatment II, fine emery powder 0.30kg, na 0.20kg was added 2 CO 3 The interval time of the two-stage steam explosion treatment is 5min; 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 6kg/cm 2 Then, the steam is rapidly introduced until the pressure in the steam explosion tank is 18kg/cm 2 The temperature in the tank reaches 210 ℃, and the steam explosion protection is carried outPressing for 10min, wherein the blasting time is not more 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 10kg/cm 2 Then, the steam is rapidly introduced until the pressure in the steam explosion tank is 16kg/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 quickly relieving pressure, and releasing the material treated in the steam explosion tank into a normal pressure container to obtain the cephalotaxus sinensis material subjected to steam explosion pretreatment.
The eutectic solvent consists of choline chloride, a hydrogen bond donor and choline chloride hydrate, wherein the hydrogen bond donor is 1, 4-butanediol, the molar ratio of the choline chloride to the 1, 4-butanediol is 1: 3, the water content is 10-50% (v/v), the mixture is uniformly mixed, the mixture is heated and stirred at the temperature of 60-110 ℃ until transparent liquid is formed, the transparent liquid is stored at the temperature of 45-50 ℃ overnight after being formed, whether solid is separated out from the liquid is observed, the liquid is stable and does not have solid separated out, and the deep eutectic solvent for extraction is obtained. Adding 500-1000L of prepared eutectic solvent into the cephalotaxus sinensis material after steam explosion pretreatment, uniformly mixing, and extracting for 1-15 min at 60-70 ℃. Centrifuging to obtain supernatant after extraction; and adsorbing the obtained eutectic solvent extracting solution by macroporous resin, then carrying out gradient elution by pure water-ethanol, respectively collecting eluates, and combining the same components. Adopting a chromatographic column with the column diameter-height ratio of 1: 4, taking the treated HPD850 macroporous resin as a filler, soaking the HPD850 macroporous resin for 24 hours by using absolute ethyl alcohol, fully swelling, then leaching by using the absolute ethyl alcohol until no turbidity exists, finally washing by using deionized water until no alcohol smell exists, washing with purified water for balance, introducing an extracting solution into the resin column, stopping loading the column when effluent to be detected contains alkaloid to obtain a resin column with saturated adsorption, and quickly washing the resin column with the water with the pH value of 4-6 of 2.0-4.0 BV and the temperature of 10-15 ℃ to remove impurities; gradient elution: eluting impurities with larger polarity adsorbed by the resin column by using an ethanol aqueous solution with the solubility of 10-20% by volume of 2.0-4.0 BV to obtain a component 1, discarding the eluent, eluting impurities with the solubility of 20-40% by volume of 2.0-4.0 BV to obtain a component 2, eluting alkaloid adsorbed by the resin column by using an ethanol aqueous solution with the solubility of 50-70% by volume of 2.0-4.0 BV to obtain a component 3, and collecting the component; eluting the impurities with weak polarity adsorbed in the resin by using an ethanol water solution with the volume solubility of 80-90% of 2.0-4.0 BV, discarding, recovering ethanol, washing by using purified water, and regenerating the resin. And carrying out reduced pressure concentration on the homoharringtonine-containing alkaline washing liquid to obtain a homoharringtonine concentrate.
Preparing mixed liquor by taking imidazole salt ionic liquid and an organic solvent, wherein the mass concentration of the ionic liquid is 400-600 g/L, and the pH value is 6.8; then adding inorganic salt, wherein the mass percent of the salt is 10-30%. Mixing uniformly to form a suspension, performing ultrasonic extraction on the suspension, and standing to obtain an aqueous two-phase system. Placing the obtained homoharringtonine concentrate in a closed extraction tank, vacuumizing until the vacuum degree in the extraction tank reaches-100 kPa, injecting distilled water into the extraction tank, adding ionic liquid-aqueous two-phase for extraction, wherein the ratio of the homoharringtonine concentrate to the ionic liquid is 0.5-1.0 g/mL, the extraction temperature is 50-60 ℃, and extracting for 10-20 min; the extraction temperature is controlled by an automatic controller, the heat source is an ultrasonic generating device arranged at the bottom of the extraction tank, a glass window is arranged at the bottom of the extraction tank, the ultrasonic penetrates through the extraction tank, the ultrasonic frequency is 40kHz, the power is 190W, the temperature is 45 ℃, the time is 1-1.5 h, and the degassing condition is as follows: operating pressure intensity of 0.05-0.07 MPa, degassing time of 6-10 min, adjusting pH value to 3.8-4.0 by using 0.6mol/L phosphoric acid, standing for 8-10 h at 8-10 ℃, and taking an upper liquid ionic phase; and after extraction is finished, adding dichloromethane with the volume 1-5 times (v/v) of that of the ionic liquid phase for extraction for 1-3 times, separating to obtain an organic phase which is a cephalotaxus sinensis total alkaloid extract phase, taking raffinate which is the ionic liquid, concentrating to remove most of water, adding activated carbon for refluxing, filtering, concentrating filtrate, keeping the temperature in a vacuum drying oven at 30-60 ℃, drying for 30-60 min, and recovering to obtain the ionic liquid which can be recycled. Acidifying the extract phase of total alkaloids to remove organic phase, collecting water phase, alkalifying the water phase, filtering, dissolving the precipitate obtained by filtering in organic solvent, filtering, and recrystallizing to obtain Cephalotaxus sinensis total alkaloids.
Weighing 25-65 g of ammonium sulfate, adding 45-135 mL of water for dissolving, adding 65-126 mL of 70-90% ethanol, fully mixing, establishing an ethanol/ammonium sulfate aqueous two-phase system, adding 10mL of homoharringtonine concentrated solution into 50-200 mL of ethanol/ammonium sulfate aqueous two-phase system to ensure that the mass concentration of ethanol is 30-40%, oscillating, shaking uniformly, mixing, standing for phase separation or centrifugal phase separation to obtain an upper phase and a lower phase, collecting the upper phase, concentrating the upper phase under reduced pressure, and taking a concentrated paste. Adding solvent systems consisting of chloroform-ethyl acetate-methanol-water (3: 1: 3, v/v) into a separating funnel, fully shaking, standing for layering, taking the upper phase, adding inorganic acid with the final concentration of 10-20 mmol/L as a stationary phase, and adding alkali with the final concentration of 10-20 mmol/L as a mobile phase. Dissolving the ethanol concentrated paste of homoharringtonine with a stationary phase, wherein the addition amount of the stationary phase is 25-60 mL/g of ethanol concentrate of homoharringtonine, and preparing a sample solution after dissolving; filling a separation column of a counter-current chromatograph with the stationary phase, then injecting a sample solution, starting a speed controller after sample injection is finished, enabling the separation column to rotate forwards, continuously injecting a mobile phase at the flow rate of 1.5-2.5 mL/min of volume flow at the rotation speed of 750-850 r/min, detecting by using an ultraviolet detector with the wavelength of 190-400 nm, collecting eluent by using an automatic part collector, tracking and detecting by using thin layer chromatography until the eluent only contains homoharringtonine components, collecting the eluent, concentrating and crystallizing to obtain homoharringtonine with the purity of more than 99%.
Claims (8)
1. A separation and purification method of homoharringtonine comprises the following steps: (1) vacuum blasting treatment; (2) extraction by eutectic solvent, (3) resin adsorption separation, (4) ionic liquid-aqueous two-phase separation, and (5) separation and purification of homoharringtonine.
2. The preparation method according to claim 1, wherein the steam explosion treatment in the step (1) comprises the steps of cutting off cephalotaxus sinensis barks to 30-40 mm to obtain an explosion matrix, adding water according to the mass ratio of the dry cephalotaxus sinensis barks to the water of 1: 0-1: 4, soaking at normal temperature for 2-8 h, adding an entrainer into the rehydrated material, uniformly mixing the materials by using a mixer and maintaining for 30min, placing the mixture in a novel ejection type steam explosion device, and taking air and steam as steam explosion media;
the entrainer is fine-mesh carborundum and NH 4 Cl、Na 2 CO 3 Or Ca (OH) 2 Composites of powders, corundum and NH 4 Cl、Na 2 CO 3 Or Ca (OH) 2 The mass ratio of the 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 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.
3. The preparation method according to claim 1, wherein the extraction of the eutectic solvent in the step (2) is performed by extracting the vacuum explosion treated cephalotaxus sinensis with the eutectic solvent to obtain a cephalotaxus sinensis extract;
the eutectic solvent is choline chloride, a hydrogen bond donor and water;
the content of water in the eutectic solvent is 10-30% (v/v);
the feed-liquid ratio of the cephalotaxus sinensis to the eutectic solvent is 20-100 mg/mL;
the hydrogen bond donor is selected from one of lactic acid, 1, 4-butanediol, glycerol or malonic acid;
the mol ratio of choline chloride to a hydrogen bond donor in the eutectic solvent is 1: 0.5-5;
the extraction temperature is 60-70 ℃, and the extraction time is 1-15 min.
4. The preparation method according to claim 1, wherein the cephalotaxus sinensis extract in the step (3) is subjected to macroporous adsorption resin column and water washing to obtain a eutectic solvent, and then gradient elution is carried out to obtain alkaline elution liquid containing homoharringtonine;
the step of washing, which is to wash the effluent liquid by purified water with the pH value of 6-7 until the effluent liquid is clear, and concentrate the water wash liquid under reduced pressure and dry the water wash liquid to obtain a regenerated eutectic solvent for the next use;
the macroporous adsorption resin is specifically one of D101, D140, HPD100, LX-38 or HPD 850;
the gradient elution is to elute impurities with larger polarity adsorbed by the resin column by using 10 to 20 volume percent of ethanol aqueous solution with 2.0 to 4.0BV to obtain a component 1, the eluent is discarded, then elute impurities with medium polarity adsorbed by the resin column by using 20 to 40 volume percent of ethanol aqueous solution with 2.0 to 4.0BV to obtain a component 2, elute alkaloid adsorbed by the resin column by using 50 to 70 volume percent of ethanol aqueous solution with 2.0 to 4.0BV to obtain a component 3, and collect the component; eluting the impurities with weak polarity adsorbed in the resin by using an ethanol aqueous solution with the volume solubility of 80-90% of 2.0-4.0 BV, discarding, recovering ethanol, washing by using purified water, and regenerating the resin;
and carrying out reduced pressure concentration on the alkaline eluent containing homoharringtonine to obtain a homoharringtonine concentrate.
5. The preparation method of claim 1, wherein the ionic liquid-aqueous two-phase separation in step (4) is performed, the homoharringtonine concentrate obtained in step (3) is extracted by taking the ionic liquid as an extractant, then inorganic salt is added, the mixture is uniformly mixed to form a suspension, ultrasonic extraction is performed on the suspension, the suspension is kept stand to obtain an aqueous two-phase system, the extractant phase is collected, and the extractant phase is subjected to reverse extraction and extractant regeneration;
the aqueous two-phase system consists of ionic liquid and inorganic salt, and the imidazole hydrophobic ionic liquid consists of cation M + And the anion N - Two-part, in which the cation M + Including but not limited to one of a substituted imidazolium cation, pyridinium cation, amine cation, or substituted pyrroline cation, and an anion including but not limited to N - Is one of hexafluorophosphate radical, tetrafluoroborate radical, bis (trifluoromethanesulfonyl) imide radical or nonafluorobutyl sulfonate radical;
the substituent is one of C- (1) -C- (8) alkyl, C- (1) -C- (3) alkenyl or C- (6) -C- (15) aryl;
the salt solution phase includes, but is not limited to, naH 2 PO 4 、(NH 4 ) 2 SO 4 、Na 2 HPO 4 、KH 2 PO 4 、K 2 HPO 4 、K 2 CO 3 、Na 2 CO 3 、K 3 PO 4 Or Na 3 PO 4 ；
The mass concentration of the ionic liquid is 400-600 g/L, and the pH value is 6.8;
the ratio of the homoharringtonine concentrate to the ionic liquid is 0.5-1.0 g/mL, the extraction temperature is 50-60 ℃, and the extraction time is 10-20 min;
the ultrasonic conditions of the ionic liquid aqueous two-phase system are as follows: the ultrasonic frequency is 40kHz, the power is 190W, the time is 1-1.5 h at 40-50 ℃, and the degassing condition is as follows: operating pressure of 0.05-0.07 MPa, degassing for 6-10 min, adjusting pH value to 3.8-4.0 with 0.6mol/L phosphoric acid, standing for 8-10 h at 8-10 ℃, and taking upper liquid ionic phase;
the mass percentage of salt in the ionic liquid aqueous two-phase system is 10-30%.
6. The preparation method according to claim 1, wherein the ionic liquid phase obtained in step (4) is extracted by adding an organic solvent with a volume 1-5 times (v/v) of the ionic liquid phase for 1-3 times, the organic phase obtained by separation is a cephalotaxus sinensis total alkaloid extract phase, the raffinate is an ionic liquid which can be recycled, the cephalotaxus sinensis total alkaloid extract phase is acidified to remove the organic phase, the aqueous phase is collected, the aqueous phase is alkalized and filtered, and the precipitate obtained by filtering is dissolved in the organic solvent, filtered and recrystallized to obtain cephalotaxus sinensis total alkaloid;
the organic solvent is one of dichloromethane, 1-dichloroethane, 1, 2-dichloroethane and chloroform.
7. The process according to claim 1, wherein the separation and purification of homoharringtonine in step (5) is carried out by one of pH-band countercurrent chromatography or medium-pressure preparative chromatography;
the solvent system for the pH zone countercurrent chromatographic separation is as follows: one of petroleum ether-ethyl acetate-methanol-water (5: 4: 6, v/v), petroleum ether-ethyl acetate-methanol-water (3: 7: 1: 9, v/v) or petroleum ether-ethyl acetate-methanol-water (1: 1, v/v);
the acidic substance is one of trifluoroacetic acid, hydrochloric acid or acetic acid;
the alkaline substance is one of ammonia water or triethylamine.
8. The method of claim 1, wherein the separation and purification of homoharringtonine in step (5) is performed by medium pressure preparative chromatography, medium pressure chromatographic column equilibrium activation, sample loading, gradient elution with elution solvent, detection and collection of fractions: concentrating and crystallizing the eluent to separate and purify homoharringtonine;
the medium-pressure chromatographic column is activated in a balanced way, and after the mobile phase washes the column for 30-40 min at a higher flow rate, the chromatographic column is activated for 1-2 h at a lower flow rate by the mobile phase with different polarities;
preparing and loading the sample, dissolving the concentrate by using 1-5 times (v/v) of mobile phase, centrifuging at 12000r/min, pumping the supernatant into a chromatographic column, or uniformly mixing the supernatant with chromatographic packing, and loading the dried chromatographic packing into the column head of the chromatographic column;
and (3) gradient elution of the elution solvent, namely, linear gradient elution is performed by taking water (A) -methanol (B) as a solvent system respectively, the gradient elution is performed for 110min, namely, the flow rate is 10-50 mL/min, the volume percentage of the methanol in the solvent system is B%, and the gradient of each time period and the solvent system is: 0-20 min B% is 15-30%, 20-50 min B% is 30-45%, 50-65 min B% is 45-78%, 65-95 min B% is 78-85%, 95-100 min B% is 85-100%, 100-110 min B% is 100%; gradient elution is carried out for 110min, and the flow rate is 10-50 mL/min;
detecting and collecting fractions, monitoring at a wavelength of 220nm, and collecting eluate fractions of corresponding absorption peaks by an automatic fractionation collector according to peak emergence time;
and (3) concentrating and crystallizing the eluent, namely concentrating the eluent of each component under reduced pressure at 50 ℃ under the vacuum degree of 90-150 mbar, removing methanol, and cooling the concentrated sample to room temperature.
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