CN101361785A - A kind of preparation method of alfalfa saponin - Google Patents
A kind of preparation method of alfalfa saponin Download PDFInfo
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- CN101361785A CN101361785A CNA2008101199498A CN200810119949A CN101361785A CN 101361785 A CN101361785 A CN 101361785A CN A2008101199498 A CNA2008101199498 A CN A2008101199498A CN 200810119949 A CN200810119949 A CN 200810119949A CN 101361785 A CN101361785 A CN 101361785A
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- Prior art keywords
- alfalfa
- ethanol
- saponins
- saponin
- preparing
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- 150000007949 saponins Chemical class 0.000 title claims abstract description 79
- 229930182490 saponin Natural products 0.000 title claims abstract description 78
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 51
- 239000001397 quillaja saponaria molina bark Substances 0.000 title claims abstract description 49
- 241000219823 Medicago Species 0.000 title claims abstract 17
- 238000002360 preparation method Methods 0.000 title claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 111
- 239000011347 resin Substances 0.000 claims abstract description 57
- 229920005989 resin Polymers 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 21
- 238000001179 sorption measurement Methods 0.000 claims abstract description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000003208 petroleum Substances 0.000 claims abstract description 8
- 235000017709 saponins Nutrition 0.000 claims description 72
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000000284 extract Substances 0.000 claims description 12
- 239000012141 concentrate Substances 0.000 claims description 9
- 239000000469 ethanolic extract Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 4
- 238000009835 boiling Methods 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000010828 elution Methods 0.000 abstract description 19
- 230000000274 adsorptive effect Effects 0.000 abstract description 6
- NQWVSMVXKMHKTF-JKSUJKDBSA-N (-)-Arctigenin Chemical compound C1=C(OC)C(OC)=CC=C1C[C@@H]1[C@@H](CC=2C=C(OC)C(O)=CC=2)C(=O)OC1 NQWVSMVXKMHKTF-JKSUJKDBSA-N 0.000 abstract 1
- YYGRXNOXOVZIKE-UHFFFAOYSA-N Arctigenin Natural products COC1CCC(CC2COC(=O)C2CC3CCC(O)C(C3)OC)CC1OC YYGRXNOXOVZIKE-UHFFFAOYSA-N 0.000 abstract 1
- OIFFJDGSLVHPCW-UHFFFAOYSA-N Guayarol Natural products COc1ccc(CC2C(Cc3ccc(O)c(O)c3)COC2=O)cc1OC OIFFJDGSLVHPCW-UHFFFAOYSA-N 0.000 abstract 1
- NQWVSMVXKMHKTF-UHFFFAOYSA-N L-Arctigenin Natural products C1=C(OC)C(OC)=CC=C1CC1C(CC=2C=C(OC)C(O)=CC=2)C(=O)OC1 NQWVSMVXKMHKTF-UHFFFAOYSA-N 0.000 abstract 1
- 238000005238 degreasing Methods 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- NWFYESYCEQICQP-UHFFFAOYSA-N methylmatairesinol Natural products C1=C(OC)C(OC)=CC=C1CC1C(=O)OCC1CC1=CC=C(O)C(OC)=C1 NWFYESYCEQICQP-UHFFFAOYSA-N 0.000 abstract 1
- 240000004658 Medicago sativa Species 0.000 description 37
- 239000003463 adsorbent Substances 0.000 description 30
- 238000003795 desorption Methods 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 9
- 238000002481 ethanol extraction Methods 0.000 description 9
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 8
- 238000005303 weighing Methods 0.000 description 7
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 description 6
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 6
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 6
- 238000004737 colorimetric analysis Methods 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 229940100243 oleanolic acid Drugs 0.000 description 6
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 6
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 6
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000003480 eluent Substances 0.000 description 5
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- 238000010521 absorption reaction Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- INLFWQCRAJUDCR-IQVMEADQSA-N (1R,2S,4S,5'S,6R,7S,8R,9S,12S,13S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icosane-6,2'-oxane] Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CCCCC4CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@H](C)CO1 INLFWQCRAJUDCR-IQVMEADQSA-N 0.000 description 2
- VCNKUCWWHVTTBY-UHFFFAOYSA-N 18alpha-Oleanane Natural products C1CCC(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C)(C)CC5C4CCC3C21C VCNKUCWWHVTTBY-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- IDGXIXSKISLYAC-WNTKNEGGSA-N Medicagenic acid Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C IDGXIXSKISLYAC-WNTKNEGGSA-N 0.000 description 2
- 235000010624 Medicago sativa Nutrition 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- SIOMFBXUIJKTMF-UHFFFAOYSA-N hypoglauterpenic acid Natural products C1CC(O)C(C)(C)C2=CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C SIOMFBXUIJKTMF-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- FLDQIFMZAMYFJR-UHFFFAOYSA-N medicagenic acid Natural products CC1(C)CCC2(CCC3(C)C(=CCC4(C)C5(C)CC(O)C(O)C(C)(C5CCC34C)C(=O)O)C2C1)C(=O)O FLDQIFMZAMYFJR-UHFFFAOYSA-N 0.000 description 2
- BPAWXSVOAOLSRP-UHFFFAOYSA-N oleanane Natural products CCCCCCCCCCCCCCCC(=O)OC1CCC2(C)C(CCC3(C)C2CC=C4C5CC(C)(C)CCC5(C)C(O)CC34C)C1(C)C BPAWXSVOAOLSRP-UHFFFAOYSA-N 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
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- 238000002791 soaking Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- RWNHLTKFBKYDOJ-UHFFFAOYSA-N (-)-arjunolic acid Natural products C1C(O)C(O)C(C)(CO)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C RWNHLTKFBKYDOJ-UHFFFAOYSA-N 0.000 description 1
- RWNHLTKFBKYDOJ-JEERONPWSA-N (2beta,3beta,4alpha)-2,3,23-trihydroxyolean-12-en-28-oic acid Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(CO)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C RWNHLTKFBKYDOJ-JEERONPWSA-N 0.000 description 1
- NTWLPZMPTFQYQI-UHFFFAOYSA-N (3alpha)-olean-12-ene-3,23-diol Natural products C1CC(O)C(C)(CO)C2CCC3(C)C4(C)CCC5(C)CCC(C)(C)CC5C4=CCC3C21C NTWLPZMPTFQYQI-UHFFFAOYSA-N 0.000 description 1
- YOQAQNKGFOLRGT-UXXABWCISA-N (3beta,22beta)-olean-12-ene-3,22,24-triol Chemical compound C1C[C@H](O)[C@](C)(CO)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C)[C@H](O)CC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C YOQAQNKGFOLRGT-UXXABWCISA-N 0.000 description 1
- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 description 1
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
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- 241000220485 Fabaceae Species 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
- GCGBHJLBFAPRDB-UHFFFAOYSA-N Hederagenin Natural products CC1(C)CCC2(CCC3(C)C4CCC5C(C)(CO)C(O)CCC5(C)C4CC=C3C2C1)C(=O)O GCGBHJLBFAPRDB-UHFFFAOYSA-N 0.000 description 1
- URRZRRQMNMZIAP-UHFFFAOYSA-N Kudzusapogenol C Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)C(O)CC3(C)CCC21C URRZRRQMNMZIAP-UHFFFAOYSA-N 0.000 description 1
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- GCGBHJLBFAPRDB-KCVAUKQGSA-N Scutellaric acid Natural products CC1(C)CC[C@@]2(CC[C@@]3(C)[C@@H]4CC[C@H]5[C@@](C)(CO)[C@H](O)CC[C@]5(C)[C@H]4CC=C3[C@@H]2C1)C(=O)O GCGBHJLBFAPRDB-KCVAUKQGSA-N 0.000 description 1
- FTPPTNQVRLPEAE-UHFFFAOYSA-N Soyasapogenol-E Natural products CC1(C)CC(=O)C2(C)CCC3C(=CCC4C3(C)CCC5C(C)(CO)C(O)CCC45C)C2(C)C1 FTPPTNQVRLPEAE-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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- PLERLWUIYWAWRU-UHFFFAOYSA-N Zanhic acid Natural products C1C(O)C(O)C(C)(C(O)=O)C2CCC3(C)C4(C)CC(O)C5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C PLERLWUIYWAWRU-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 229930013930 alkaloid Natural products 0.000 description 1
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- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
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- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
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Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a method for purifying the saponin of alfalfa. The saponin is prepared by carrying out the steps of: extracting from the upper part of the alfalfa by using 80 percent of ethanol, degreasing with petroleum ether and purifying with AB-8 macroporous adsorptive resins. The invention confirms the ratio between total quantity of saponin of alfalfa and Arctigenin quantity is 2.03 for the first time, simultaneously confirms the maximum adsorption volume and the elution movement of the AB-8 macroporous adsorptive resins; in the total saponin of alfalfa prepared, the content of saponin is not less than 40 percent and can be used in the industrial fields of medicine, food and the like.
Description
Technical field
The present invention relates to a kind of preparation method of alfalfa total saponins, belong to pharmaceutical field.
Background technology
Alfalfa (Medicago sativa Linn.) is a kind of pulse family (Leguminosae) Papillionoideae (Faboideae) clover.Except that as the good herbage, alfalfa has important application equally at drought resisting, anti-blown sand, aspect such as improve the soil, prevent erosion, preserve the ecological environment.Alfalfa contains multiple secondary metabolites such as flavone, triterpene saponin, alkaloid, coumarin, antithrombotic, analgesia, antiinflammatory, antioxidation, antibiotic, pest-resistant, change aspect such as sense and have wide biological activity.Three terpene components are mainly oleanane (Oleanane) type pentacyclic triterpene and glycocide thereof in the alfalfa.The Herba Medicaginis triterpenoid can be subdivided into medicagenic acid (Medicagenic acid) type, helexin (Hederagenin) type, Bayogenine (Bayogenin) type, soybean soapstock aglycon B (Soyasapogenol B) type, soybean soapstock aglycon E (Soyasapogenol E) type, Zanhic acid type according to the aglycon type.
Macroporous adsorbent resin (Macroporous adsorption resin) is the molecular material that adsorptivity and molecular sieve principle combine, and has good absorption and separating property, has been widely used in pharmacy and active skull cap components and has extracted.Utilize alternately eluting of water alcohol, can well the target product in the natural extract aqueous dispersions be extracted.
The present invention adopts 80% ethanol 80 ℃ of following reflux, extract,, concentrates the back and obtains ethanol extraction (extractum), behind defat with petroleum ether, obtains total saponins with the AB-8 purification with macroreticular resin.The purpose of this invention is to provide a kind of total saponin of alfalfa preparation method that can satisfy the large-scale production needs, the total saponins impurity that adopts this method to obtain is few, the purity height.
Summary of the invention
The present invention relates to extraction, purification and the hydrolysis of alfalfa saponin, following technical method is provided.
1, the preparation of Herba Medicaginis ethanol extraction
Adopt 80% ethanol at 80 ℃ of following reflux, extract, Herba Medicaginis materials, concentrate the back and obtain ethanol extraction, yield is 19.03% (g/g dry weight).
2, the all-hydrolytic of alfalfa saponin and hydrolysis coefficient
Total saponin of alfalfa is carried out all-hydrolytic under acid condition, obtain aglycon, the ratio of total saponins amount and aglycon amount (being the hydrolysis coefficient) is 2.03.
3, the mensuration of alfalfa saponin relative amount
With the oleanolic acid is standard substance, by chemical reaction, adopts spectrophotography, and the linear equation (Y=10.572X-0.0073) of setting up content analysis draws the amount of sapogenin, can draw the relative amount of alfalfa saponin by the ratio of total saponins amount and aglycon amount.
4, the AB-8 macroporous adsorbent resin is to the static adsorption rate and the desorption efficiency of alfalfa saponin
The AB-8 macroporous adsorbent resin is respectively 11.38mg/gdw and 50.61% to the static adsorption rate and the desorption efficiency of alfalfa saponin.
5, in the AB-8 macroporous adsorptive resins 30% ethanol and 90% ethanol to the desorption efficiency of alfalfa saponin
(30%) ethanol of low concentration in the AB-8 macroporous adsorptive resins and high concentration (90%) ethanol are respectively 25.98mg/gdw and 95.11mg/gdw to the desorption efficiency of alfalfa saponin.
6, AB-8 macroporous adsorbent resin maximal absorptive capacity
Along with the increase of applied sample amount, the adsorbance of AB-8 macroporous adsorbent resin is also increasing, and when applied sample amount: during amount of resin (dw)=1.0:1.1 (g/g), adsorbance reaches maximum.
7, the excessive upward sample analysis of AB-8 macroporous adsorbent resin
After applied sample amount reaches AB-8 macroporous adsorbent resin maximal absorptive capacity, increase applied sample amount again, promptly begin to occur leaking, so the water layer eluate of leaking is carried out going up sample the second time, observe the relation between applied sample amount and the adsorbance.Twice total 90% ethanol elution thing is 0.0925 with the meansigma methods of total applied sample amount ratio, and promptly the thick saponin amount of Herba Medicaginis (adopting gravimetric method to try to achieve) accounts for 9.25% of total extractum.Adopt colorimetric method for determining, the purity of thick total saponins is 40%, and saponin content is 3.7% in total extractum.
8, AB-8 macroporous adsorbent resin eluting is dynamic
Measure eluate and saponin amount under the different gradient respectively by gravimetric method and colorimetry, the result of two kinds of method mensuration is similar.15%-35% ethanol elution amount is lower, and when carrying out eluting with 45% ethanol, the eluate amount obviously increases, and under 55%-65% ethanol elution gradient, it is maximum that elution amount reaches, and elution amount reduces gradually subsequently.
Advantage of the present invention: the present invention with the Herba Medicaginis ethanol extraction water suspendible that obtains after, through defat with petroleum ether, directly cross macroporous adsorbent resin, with comparing after macroporous adsorbent resin behind the n-butanol extraction, reduced technological process with traditional, saved cost.The ratio relation of the present invention is clear and definite first alfalfa saponin and alfalfa saponin unit is 2.03, and it is dynamic to the absorption of alfalfa saponin to have studied the AB-8 macroporous adsorbent resin simultaneously, and in the thick total saponins of the Herba Medicaginis of preparation, saponin content reaches more than 40%.Purpose is to make full use of the alfalfa plant resource, obtains highly purified saponin active component on a large scale, for the development and utilization of alfalfa saponin active component provides foundation.
In order to understand the present invention better, further specify essentiality content of the present invention below in conjunction with embodiments of the invention, but content of the present invention is not limited thereto.
The specific embodiment
Embodiment 1: the preparation of Herba Medicaginis ethanol extraction
Take by weighing 500g alfalfa ground top dry powder, place the 5000mL round-bottomed flask, add 80% ethanol 4000mL by solid-liquid ratio 1:8 (g:mL) and extract, first soaked overnight is then 80 ℃ of following reflux, extract, 3h (repeating 3 times).Extracting solution is merged, and being concentrated into small size does not have the alcohol flavor simultaneously, adds an amount of distilled water and is settled to 500mL, adds isopyknic petroleum ether (boiling range is 60-90 ℃) again and extracts re-extract 3 times.Respectively petroleum ether layer and water layer extract are concentrated evaporate to dryness, petroleum ether layer is 19.55g, and yield is 3.91%; Water layer 75.61g, yield is 15.12%; The ethanol extraction yield is 19.03%.
Embodiment 2: the acquisition of the all-hydrolytic of alfalfa saponin and hydrolysis coefficient
Get the about 450mg-500mg of a certain amount of pure total saponin of alfalfa (content 95%) (repeating 3 times), add 10% hydrochloric acid solution of 100mL with the preparation of 50% ethanol, behind 80 ℃ of following hydrolysis 4h, in ice bath, cool off, use the 100mL chloroform extraction, repeat combined chloroform extraction part, washing chloroform layer (each 300mL distilled water 3 times, repeat 3 times), 40 ℃ of following concentrating under reduced pressure chloroform parts, weigh, the results are shown in Table 1, hydrolysate detects with thin layer chromatography (TLC), show that hydrolysis is thorough, main lucerne unit has 3 in the hydrolyzate, and Rf value is respectively 0.537,0.426 and 0.370.The thin layer testing conditions is as follows: purification on normal-phase silica gel (Qingdao Marine Chemical Co., Ltd.); Development system is: chloroform: methanol=20:1 (v/v); Developer is: methanol: acetic anhydride: concentrated sulphuric acid=50:5:5 (v/v).The ratio of total saponins amount and total sapogenins amount is 2.03 (w/w).
The ratio of table 1 alfalfa saponin amount and total sapogenins amount
Embodiment 3: the assay of total saponin of alfalfa
Precision takes by weighing 5.01mg oleanolic acid (Oleanolic acid) standard substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute), is dissolved in the volumetrical volumetric flask of 5.0mL, adds dissolve with methanol and is settled to scale.Draw respectively: 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, 100 μ L and 120 μ L place 10mL tool plug test tube, water-bath (40 ℃) volatilizes methanol, the amount of oleanolic acid is respectively in the test tube: 0.04008mg, 0.05010mg, 0.06012mg, 0.07014mg, 0.08016mg, 0.09018mg, 0.1002mg and 0.12024mg, add 5% vanillin-glacial acetic acid solution 0.2mL after cooling respectively, add 0.8mL perchloric acid respectively after the jolting dissolving, shake up to be placed in 70 ℃ of water-baths and heat 15min, then with ice cooling rapidly, respectively add glacial acetic acid 5mL again, shake well is measured absorbance in the 545nm place behind the static 10min.Get regression equation Y=10.572X-0.0073 (coefficient R=0.99201; Y is the absorbance value at 545nm place; X is the amount of the oleanolic acid in every pipe, and unit is mg/tube).
In order to detect the content of total saponins in the Herba Medicaginis sample (extract, thick total saponins), get a certain amount of sample Amg, be dissolved in the volumetrical volumetric flask of 5.0mL, getting certain volume B μ L (repeats 3 times) in 10mL tool plug test tube, operating procedure is same as the assay of above-mentioned oleanolic acid, absorbance value Y substitution equation Y=10.572X-0.0073 with the 545nm place, can obtain X (is the sapogenin amount in every pipe, mg/tube), the total saponins amount is 2.03Xmg in every pipe thereby draw, and the total saponin content computing formula is as follows in the Herba Medicaginis sample:
Embodiment 4: macroporous adsorbent resin and pretreatment thereof
The AB-8 macroporous adsorbent resin that adopts is Tianjin sea light chemical industry company limited production, is styrene type low pole copolymer, is milky white granules, and particle size range is 0.3-1.25mm (a 60-16 order), and specific surface area is 480-520m
2/ g, the aperture is 13.0-14.0nm.
Take by weighing the AB-8 macroporous adsorbent resin of 20g fresh weight (being converted to dry weight 12g), be soaked in 95% ethanol, again it is joined the glass column that internal diameter is 1.6cm (the high 13cm of post, blade diameter length ratio is 1:8) in, make the ethanol liquid level be higher than the about 2cm of resin, soak 24h, fully swelling.Use the flow velocity drip washing resin of 2BV/h then, till not occurring white opacity after effluent adds a small amount of distilled water.Use drip washing behind the 5%HCl solution soaking 2-4h of 2BV then, drip washing behind the 2%NaOH solution soaking 2-4h of reuse 2BV is neutral with distilled water drip washing to pH at last.
Embodiment 5:AB-8 macroporous adsorbent resin is to the static adsorption rate and the desorption efficiency of the thick saponin of Herba Medicaginis
Take by weighing pretreated AB-8 macroporous adsorbent resin 2.0g fresh weight (conversion is the 1.20g dry weight) and in 50mL volume triangular flask, add 20mL Herba Medicaginis ethanol extract (extract concentrations is 19.6mg/mL).After sealing triangular flask is placed constant temperature (25 ℃) agitator, vibration 24h, rotating speed is 150 rev/mins.Fully after the absorption, filter, filtrate concentrates, and weighs, and the adsorption rate of AB-8 resin sees Table 2.In adsorbing saturated resin, add 40mL95% ethanol, after sealing triangular flask is placed constant temperature (25 ℃) agitator, vibration 24h, rotating speed is 150 rev/mins.Fully after the desorbing, filter, filtrate concentrates, and weighs, and the desorption efficiency of AB-8 resin sees Table 2.Adsorption rate and desorption efficiency computing formula are as follows:
Table 2 AB-8 macroporous adsorbent resin is to the static adsorption rate and the desorption efficiency of alfalfa saponin
Embodiment 6: 30% ethanol and 90% ethanol are to the desorption efficiency of alfalfa saponin in the AB-8 macroporous adsorptive resins
Take by weighing Herba Medicaginis and extract extractum 10g, be dissolved in (concentration is 100mg/mL) in the 100mL water, add in the AB-8 macroporous adsorptive resins, resin fresh weight 20g (being converted to dry weight 12g), the high 13cm of post, column internal diameter 1.6cm with the flow velocity of 2BV/h, carries out eluting with 5BV water, 5BV30% ethanol, 5BV90% ethanol one by one.Abandon water elution liquid, collect 30% ethanol elution, 90% ethanol elution respectively, concentrate, take by weighing dry weight, calculate the desorption efficiency of 30% ethanol and 90% ethanol elution respectively, the results are shown in Table 3.The AB-8 macroporous adsorbent resin is as follows to the desorption efficiency computing formula of alfalfa saponin in adsorption resin column:
The desorption efficiency of table 3 AB-8 macroporous adsorbent resin in adsorption resin column
Embodiment 7:AB-8 macroporous adsorbent resin maximal absorptive capacity
Get some parts of pretreated AB-8 macroporous resin 20.0g (being converted to the 12g dry weight), adorn post respectively.Use the Herba Medicaginis ethanol extraction upper prop of variable concentrations then successively, it is dynamic to investigate AB-8 absorption with macroporous adsorbent resin amount.Behind preliminary definite maximal absorptive capacity, sample on the 50%-80% Herba Medicaginis extracting solution of selection maximal absorptive capacity, use 5BV water, 5BV30% ethanol, 5BV90% ethanol elution more successively, collect eluent, weigh after concentrating, and the concentrate weight of different eluents compared, thereby investigate AB-8 macroporous adsorbent resin maximal absorptive capacity, the results are shown in Table 4.
Increase along with applied sample amount, the adsorbance of AB-8 macroporous adsorbent resin is also increasing, when applied sample amount: during amount of resin (dw)=1.0:1.1 (g/g), adsorbance reaches maximum, this moment, 90% alcohol desorption rate was 108.375mg/g dw, along with applied sample amount increases, adsorbance no longer obviously increases, and illustrates that at this moment resin reaches capacity afterwards.Applied sample amount before saturated is carried out trend analysis, and the linear relationship of applied sample amount and 90% alcohol desorption rate (mg/gdw) is: Y=0.0077X-0.2446, R=0.9918; Y is 90% alcohol desorption rate (mg/gdw), and X is each applied sample amount (mg), and R is a correlation coefficient.Because applied sample amount and 90% alcohol layer desorption efficiency have the better linearity relation, thereby can predict under different applied sample amount situations 90% alcohol desorption rate.
Table 4 AB-8 macroporous adsorbent resin maximal absorptive capacity
Annotate: 90% alcohol desorption rate (mg/g dw) is the adsorbance of macroporous adsorbent resin to total saponin of alfalfa.
The excessive upward sample analysis of embodiment 8:AB-8 macroporous adsorbent resin
Get 4 parts of pretreated AB-8 macroporous resin 20.0g (being converted to the 12g dry weight), adorn post respectively.After applied sample amount (Herba Medicaginis ethanol extraction) reaches the AB-8 maximal absorptive capacity, increase applied sample amount again, promptly begin to occur leaking, so the water layer eluate of leaking is carried out going up sample the second time, observe the relation between applied sample amount and the adsorbance.Twice total 90% ethanol elution thing is 0.0925 with the meansigma methods of total applied sample amount ratio, and promptly the thick saponin amount of Herba Medicaginis accounts for 9.25% (annotate: genuine soap glycosides content is 3.7% in total extractum) of total extractum, the results are shown in Table 5.Adopt colorimetry, the saponin concentration of measuring in the thick total saponins is 40%.
The excessive upward sample analysis of table 5 AB-8 macroporous adsorbent resin
Annotate: applied sample amount is the corresponding water elution part first time for the second time.
Embodiment 9:AB-8 macroporous adsorbent resin eluting is dynamic
Eluting to the AB-8 macroporous adsorbent resin is investigated dynamically, can effectively determine the concentration of alcohol and the eluting scope of eluent, obtains highly purified alfalfa saponin.Get pretreated AB-8 macroporous resin 20.0g (being converted to the 12g dry weight) dress post, adopt the thick saponin of 2.9654g (i.e. 90% ethanol elution part, saponin content is 40%) excessive dress post, behind the excessive eluting of distilled water, use 5BV15% ethanol, 5BV25% ethanol, 5BV35% ethanol, 5BV45% ethanol, 5BV55% ethanol, 5BV65% ethanol, 5BV75% ethanol, 5BV85% ethanol and 5BV95% ethanol elution respectively, collect eluent, concentrate the title dry weight.Adopt gravimetric method different gradient alfalfa saponin content to be compared respectively, the results are shown in Table 6 with colorimetry.
Measure eluate and saponin content under the different gradient respectively by gravimetric method and colorimetry, the measurement result unanimity of two kinds of methods.Adopt to concentrate the back weighing method, lower in 15%-35% ethanol elution thing amount as can be seen, when carrying out eluting with 45% ethanol, the eluate amount obviously increases, and under 55%-65% ethanol elution gradient, it is maximum that elution amount reaches, and elution amount is reducing gradually subsequently.Adopt alfalfa saponin content under the different gradient of colorimetric method for determining, find the highest at 55% alcohol layer alfalfa saponin content, more relatively at 55%-75% alcohol layer alfalfa saponin content.
In sum, adopt the ratio of ethanol extraction applied sample amount and resin dry weight to be lower than 1.0:1.1, use earlier the excessive water eluting, use 5BV 30% ethanol (being the described low-concentration ethanol of this patent) eluting then, reuse 5BV90% ethanol (being the described high concentration ethanol of this patent) eluting is also collected eluent, can obtain purity and be 40% total saponin of alfalfa.
The eluting of table 6 AB-8 macroporous adsorbent resin is dynamic
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912436A (en) * | 2010-08-31 | 2010-12-15 | 扬州大学 | Ultrasonic extraction method of alfalfa saponin |
| CN104606280A (en) * | 2015-01-09 | 2015-05-13 | 河南工业大学 | Method for leaching alfalfa saponins with ethanol |
| CN106377568A (en) * | 2016-11-15 | 2017-02-08 | 上海诺德生物实业有限公司 | Method for extracting alfalfa total saponin |
| CN109402164A (en) * | 2018-11-21 | 2019-03-01 | 中国农业科学院北京畜牧兽医研究所 | A kind of alfalfa is overexpressed the method and application of squalene epoxidase enzyme gene |
-
2008
- 2008-09-12 CN CNA2008101199498A patent/CN101361785A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912436A (en) * | 2010-08-31 | 2010-12-15 | 扬州大学 | Ultrasonic extraction method of alfalfa saponin |
| CN104606280A (en) * | 2015-01-09 | 2015-05-13 | 河南工业大学 | Method for leaching alfalfa saponins with ethanol |
| CN106377568A (en) * | 2016-11-15 | 2017-02-08 | 上海诺德生物实业有限公司 | Method for extracting alfalfa total saponin |
| CN109402164A (en) * | 2018-11-21 | 2019-03-01 | 中国农业科学院北京畜牧兽医研究所 | A kind of alfalfa is overexpressed the method and application of squalene epoxidase enzyme gene |
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