CN1483832A - Method for preparing hypoglycosyl anemonic saponin by encyme method hydrolyzing anemonic saponin glycosyl - Google Patents
Method for preparing hypoglycosyl anemonic saponin by encyme method hydrolyzing anemonic saponin glycosyl Download PDFInfo
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- CN1483832A CN1483832A CNA031336396A CN03133639A CN1483832A CN 1483832 A CN1483832 A CN 1483832A CN A031336396 A CNA031336396 A CN A031336396A CN 03133639 A CN03133639 A CN 03133639A CN 1483832 A CN1483832 A CN 1483832A
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- anemoside
- glycosyl
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Abstract
The method for preparing hypoglycosyl anemonic saponin includes the following steps: using the enzyme capable of enzyme hydrolyzing anemonic saponin glycosyl, mixing said enzyme, anemonic saponin and buffer liquor and making them produce reaction for 4-40 hr., its reaction temp. is 4-75 deg.C and pH value is 3-9, so as to extract the invented hypoglycosyl anemonic saponin. Said invention has no pollution, and is high in conversion rate.
Description
Technical field:
The present invention relates to a kind of method for preparing low glycosyl anemoside, especially a kind of enzyme with energy hydrolysis anemoside glycosyl is handled anemoside, the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation.
Background technology:
Root of Chinese Pulsatilla is Ranunculaceae, Pulsatilla plant, belongs to perennial herb, gets its root more and is used as medicine, and has another name called how that grass, powder are careless, Mao aunt flower etc.Mainly be distributed in northeast, ground such as North China and Shaanxi, Anhui, Jiangsu, Hubei, Sichuan.Historical data carry that " Root of Chinese Pulsatilla gives birth to high mountain mountain valley and field, and adopt April.Root of Chinese Pulsatilla is of a great variety, and " Chinese pharmacopoeia is only recorded one kind of Root of Chinese Pulsatilla P.chinensis and used as the certified products Root of Chinese Pulsatilla, both cohosh Root of Chinese Pulsatilla Pulsatilla chinensis (Bge.) Regel.Also have following several in addition: the Root of Chinese Pulsatilla Pulsatilla dahurica of Xingan (Fisch) Spreng, spire Root of Chinese Pulsatilla Pulsatillaturczaninovii Kryl.et Serg. (checking Root of Chinese Pulsatilla), pasque flower Pulsatillakoreana Nakai exMari, Xinjiang Root of Chinese Pulsatilla Pulsatilla ambigua Turcz.exPritz., Altay Root of Chinese Pulsatilla Pulsatilla companella Fisch., palm leaf Root of Chinese Pulsatilla Pulsatillapatens Mill., chrysanthemum Root of Chinese Pulsatilla Pulsatilla sukaczevii Turcz etc. also have some mutation.The main effective constituent of Root of Chinese Pulsatilla is saponin (glycosides), and its glucoside unit is a triterpenes 23-hydroxyl radical white birck acid (anemosapogenin).Its chemical molecular structure is:
R wherein
1And R
2For the side chain of sugar, according to R
1, R
2Difference distinguish, the kind of anemoside can be arranged into down by sequence number:
Saponin 1:R
1=α-L-rha-(1 → 2)-α-L-ara-
R
2＝H-
Saponin 2:R
1=α-L-ara-
R
2＝α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-
Saponin 3:R
1=α-L-rha-(1 → 2)-α-L-ara-
R
2＝α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-
Saponin 4:R
1=α-L-ara-
R
2＝β-D-glc-(1→6)-β-D-glc-
Saponin 5:R
1=α-L-ara-
R
2＝β-D-glc-
Saponin 6:R
1=H-
R
2＝β-D-glc-
Saponin 7:R
1=α-L-ara-
R
2＝H-
Glucoside unit: R
1=H-
R
2＝H-
(glc, glucose; Ara, pectinose; Rha, rhamnosyl)
The higher saponin of content is anemoside 2 and 3 in the Root of Chinese Pulsatilla; With saponin 3 is example, and its formal name used at school is 3-0-[α-L-pyrans rhamnosyl-(1 → 2)-α-L-ratio Arabic glycosyl of muttering]-3 β, 23-dihydroxyl-(alkene) Δ
20 (29)-lupene-28-acid sugar ester [28-0-[α-L-ratio mutter rhamnosyl-(1 → 4)-β-D-Glucopyranose-(1 → 6)-β-D-glucopyranosyl].
The anemoside germ resistance is extensive, and it can suppress the growth of streptococcus aureus, Pseudomonas aeruginosa, dysentery bacterium, Bacillus subtilus, Corynebacterium diphtheriae, salmonella etc., kills trichomonas vaginitis; Also the tuberculosis of cervical lymph nodes, lung's squama cancer and melanoma etc. there is certain curative effect.But the higher saponin of content in the Root of Chinese Pulsatilla, containing glycosyl quantity is 4-5, the hemolytic height, antibacterial effect is low.And low glycosyl anemoside or glucoside unit activity are higher, and hemolytic is low.The method of removing low glycosyl anemoside of glycosyl preparation or glucoside unit from anemoside mainly was the acid and alkali hydrolysis method in the past.As use ammonia treatment, the sugar ester key on can hydrolysis 28 sugar, from anemoside 4 obtain saponin 2 (Wu Zhenjie etc., China Medicine University's journal, 1991,22 (5), 265-269); Acidic treatment can obtain glucoside unit (leaf literary talent etc., China Medicine University's journal, 1990,21 (5), 264-266).Above-mentioned acid and alkali hydrolysis method, the purpose of glycosyl hydrolase is poor, the whole hydrolysis sugar ester bonds of basic hydrolysis, acid hydrolysis is seriously polluted, destructive big to glucoside unit.
Summary of the invention:
The present invention is in order to solve existing technical problem in the prior art, to provide a kind of enzymatic hydrolysis content higher anemoside glycosyl, and the method for the low glycosyl anemoside of preparation is pollution-free, purpose is strong, transformation efficiency is high.
Technical solution of the present invention is: a kind of enzymatic hydrolysis anemoside glycosyl prepares the method for low sugar basis soap glucoside, is with enzymic hydrolysis anemoside glycosyl, the low glycosyl anemoside of preparation.
Comprise the steps:
A. get the enzyme of energy hydrolysis anemoside glycosyl;
B. with enzyme, anemoside and damping fluid hybrid reaction 4-40 hour, reaction conditions was temperature 4-75 ℃, pH value 3-9.
C. extract low glycosyl anemoside.
Described a step can be prepared by microbial fermentation.
Described microbial fermentation preparation is to obtain containing the enzyme mixed solution with liquid state fermentation or solid state fermentation, adds ammonium sulfate or extraction using alcohol enzyme in containing the enzyme mixed solution.
Described microorganism is bacterium, streptomycete, mould, yeast, basidiomycetes.
Described microorganism is Aspergillus (Aspergillus genus) mould.
Described microbial fermentation participates in fermentation for the enzyme induction of producing thing is arranged.
The anemoside concentration of described b step is the 0.02-15% of total reactant volume.
Described damping fluid is acetic acid or phosphoric acid.
Described c step is for extracting or use n-butanol extraction with ethanol sedimentation.
The present invention compares with prior art, and maximum characteristics are with enzymic hydrolysis anemoside glycosyl, low glycosyl anemoside of preparation or glucoside unit.Overcome shortcomings such as existing in prior technology is big, seriously polluted to the glucoside destructiveness, purpose difference, had advantage pollution-free, that purpose is strong, transformation efficiency is high.Not only improve antibacterial effect, also can eliminate hemolytic simultaneously, medicine, protective foods are developed highly significant.Especially in microbial fermentation, add product enzyme induction thing, can improve its yield of enzyme greatly, helped abundant hydrolysis anemoside glycosyl, improved the transformation efficiency of low glycosyl anemoside more.
Embodiment:
Embodiment 1:
A. containing 3% Semen Maydis powder, 0.2% product enzyme induction thing with high-temperature aerobic bacterium Bacillus sp.JF bacterium (document 1)---in Root of Chinese Pulsatilla extract (dry), the 0.01%MgSO4 substratum, under 60 ℃ of conditions of temperature, ventilate and cultivated 30-40 hour, bactofugation must contain the enzyme mixed solution, with 50-80% ethanol sedimentation zymoprotein, freeze-drying obtains the saponin enzyme.
B. with the above-mentioned saponin enzyme of 10 grams, 30 gram anemosides, 1500 milliliters of acetate buffer solutions (0.2M, pH5.0) and 150 milliliters of ethanol mix, making reactant concn is 0.01-15%, stirring reaction is 4 hours under 75 ℃ of conditions of temperature.
C. react Hou and add 6000 milliliters of ethanol, remove by filter albumen precipitation, the filtrate decompression evaporate to dryness obtains 20 gram low sugar basis soap glucoside crude products.
Change into the low secondary saponin of glycosyl with the anemoside of thin layer chromatography (document 2) detected result more than 80%.Obtain four kinds of anemoside enzyme reaction product monomers with silicagel column separation method (document 3), through hot methanol dissolving, the refining monomer of crystallisation by cooling method.Determine structure through magnetic nuclear resonance method (document 4,5): the monomer whose structure is anemoside 5,6,7 and glucoside unit, and the amount of obtaining is followed successively by 4.1 grams, 3.5 grams, 1.6 grams, 0.2 gram.
Above-mentioned enzyme liquid DEAE-Cellulose ion exchange resin column method is separated purification anemoside zymoprotein with BioRed protein Preparation chromatographic instrument (document 6), and with SDS electrophoresis method determining molecular weight (document 7), the molecular weight of enzyme is 5.0 ten thousand.
Embodiment 2:
Other is with embodiment 1, and the product enzyme induction thing in the substratum is 0.05% soybean isoflavones.
Embodiment 3:
A. black-koji mould (Aspegillus niger) is containing 5% wheat bran extract, 1% product enzyme induction thing---in the substratum of Root of Chinese Pulsatilla extract (dry), stirring under temperature 28-30 ℃ condition ventilates cultivated 50-100 hour, bactofugation must contain enzyme mixed solution, the ammonium sulfate precipitation zymoprotein with the 60-75% saturation ratio, collection albumen, be dissolved in the acetate buffer solution (0.02M of 1/10 fermentating liquid volume, pH5.0) in, ammonium sulfate is removed in dialysis, and centrifugal slagging-off is enzyme liquid.
B. with 4 gram anemosides, 100 milliliters of acetate buffer solutions (0.02M, pH5.0) and 50 milliliters of above-mentioned enzyme liquid mix, making reactant concn is 0.01-15%, is under 4 ℃ of conditions in temperature, reacts 40 hours.
C. the n-butanol extraction saponin three times that adds 1/3 volume again, evaporated under reduced pressure obtains the low secondary saponin crude product of glycosyl.
Obtain the secondary saponin of pure low glycosyl of 0.5-1.4 gram with silicagel column separation method (document 3).Determine structure (document 4,5) through magnetic nuclear resonance method, its structure is an anemoside 6.
Embodiment 4:
Other is with embodiment 3, with 1 gram anemoside, 100 milliliters of acetate buffer solutions (0.02M, pH5.0) and 50 milliliters of above-mentioned enzyme liquid mix, obtain two kinds of the secondary saponins of the 0.4 pure low glycosyl of gram and 0.1 and restrain glucoside unit.Determine structure (document 2,3) through magnetic nuclear resonance method, its structure is anemoside 7 and 8.
Above-mentioned enzyme liquid DEAE-Cellulose ion exchange resin column method is separated purification anemoside zymoprotein with BioRed protein Preparation chromatographic instrument (document 6), and with SDS electrophoresis method (document 7) determining molecular weight, the molecular weight of enzyme is 5.3 ten thousand.
Embodiment 5:
A. aspergillus oryzae (Aspegillus oryzae) is containing 5% wheat bran extract, 0.2% product enzyme induction thing---soybean isoflavones or rutin, perhaps in the substratum of the extract of 0.6% its source plant, be 28-30 ℃ in temperature and stir cultivation 50-100 hour of ventilating, bactofugation must contain the enzyme mixed solution.With the ammonium sulfate precipitation zymoprotein of 60-75% saturation ratio, collect albumen, be dissolved in 1/10 fermentating liquid volume acetate buffer solution (0.02M, pH5.0) in, dialysis remove sulfuric acid by, centrifugal slagging-off is enzyme liquid.
B. above-mentioned enzyme liquid is pressed the method for embodiment 1 and handled anemoside.
Purify and the result of measurement of enzymatic reaction products: the transformation efficiency that anemoside changes into the low secondary saponin of glycosyl is more than 60%, and product mainly is anemoside 7 and 8, in addition saponin 6 and a spot of glucoside unit.
(separate purification anemoside zymoprotein, with SDS electrophoresis method determining molecular weight, the molecular weight of enzyme is 6.0 ten thousand to above-mentioned enzyme liquid with BioRed protein Preparation chromatographic instrument through DEAE-Cellulose ion exchange resin column method.
Embodiment 6:
A. candiyeast is containing upward inoculation of the wheat bran substratum of Root of Chinese Pulsatilla powder 10% (dry 1000 grams), be divided in the eggplant bottle, temperature is 30 ℃, cultivated 3～6 days, collect the physiological saline that culture adds 6000 milliliters, soaked one hour, centrifuging and taking supernatant (containing the enzyme mixed solution), the adding saturation ratio is 60～75% ammonium sulfate, make the zymoprotein precipitation, collecting precipitation with 800 milliliters pH5, the dissolving of 0.02M sodium-acetate buffer, gives ammonium sulfate, centrifugal slagging-off promptly gets the enzyme liquid about 1000 milliliters.
B. above-mentioned enzyme liquid is pressed the method for embodiment 1 and handled anemoside.
Purify and the result of measurement of enzymatic reaction products: the transformation efficiency that anemoside changes into the low secondary saponin of glycosyl is more than 60%, and product mainly is anemoside 7 and 8, in addition saponin 6 and a spot of glucoside unit.The molecular weight of enzyme is 4.3 ten thousand.
Embodiment 7: basidiomycetes
Mushroom bacteria (Agaricus campestris) is cultivated on Root of Chinese Pulsatilla powder and each wheat bran substratum of 10% of rice skin, was cultivated 6-8 days under 25 ℃ of conditions of temperature, presses the method for embodiment 6 and extracts enzyme liquid, presses the method for embodiment 3 and handles anemoside.
Separate and purify and measurement of enzymatic reaction products, 50% anemoside changes into low sugar basis soap glucoside, and product has anemoside 5,6,7 and a small amount of glucoside unit.
Document described in the embodiment is:
1.Fengxie Jin, et al (Jin Feng is mediate etc.): J.Gen Appl.Bact., 1990,36,415-424.
2.Hongshan Yu, et al (fish Red Hill etc.): J.Ginseng Res., 1999,23 (10,50-54.
3. Xu Rensheng edits, natural goods chemistry, Science Press, 1997, p17-20.
4. Wu Zhen is clean etc.: China Medicine University's journal, 1991,22 (5), 265-269.
5. leaf literary talent etc.: China Medicine University's journal, 1990,21 (5), 264-266).
6.Hongshan Yu, et al (fish Red Hill etc.): Chem.Pharm.Bull., 50 (2), 175-178.
7. open Long Xiang etc.: distillation experimental technique and technology, Higher Education Publishing House, 1997, p100-111.
Claims (10)
1. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation is characterized in that: with enzymic hydrolysis anemoside glycosyl, prepare low glycosyl anemoside.
2. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 1 is characterized in that comprising the steps:
A. get the enzyme of energy hydrolysis anemoside glycosyl;
B. with enzyme, anemoside and damping fluid hybrid reaction 4-40 hour, reaction conditions was temperature 4-75 ℃, pH value 3-9.
C. extract low glycosyl anemoside.
3. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 2, it is characterized in that: described a step can be prepared by microbial fermentation.
4. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 3, it is characterized in that: described microbial fermentation preparation is to obtain containing the enzyme mixed solution with liquid state fermentation or solid state fermentation, adds ammonium sulfate or extraction using alcohol enzyme in containing the enzyme mixed solution.
5. according to the method for claim 3 or the low glycosyl anemoside of 4 described enzymatic hydrolysis anemoside glycosyl preparations, it is characterized in that: described microorganism is bacterium, streptomycete, mould, yeast, basidiomycetes.
6. according to the method for claim 3 or the low glycosyl anemoside of 4 described enzymatic hydrolysis anemoside glycosyl preparations, it is characterized in that: described microorganism is Aspergillus (Aspergillus genus) mould.
7. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 3 is characterized in that: described microbial fermentation participates in fermentation for the enzyme induction of producing thing is arranged.
8. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 2, it is characterized in that: the anemoside concentration of described b step is the 0.02-15% of total reactant volume.
9. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 2, it is characterized in that: described damping fluid is acetic acid or phosphoric acid.
10. the method for the low glycosyl anemoside of enzymatic hydrolysis anemoside glycosyl preparation according to claim 2, it is characterized in that: described c step is for extracting or use n-butanol extraction with ethanol sedimentation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100337635C (en) * | 2004-09-28 | 2007-09-19 | 徐东铭 | Medicine containing general pasqueflower saponin composition and its preparation method and uses |
| CN101130802A (en) * | 2007-07-30 | 2008-02-27 | 金凤燮 | Method for preparing astragaloside iv by enzymic hydrolysis for astragalus saponin glycosyl |
| CN102805750A (en) * | 2011-05-31 | 2012-12-05 | 天津药物研究院 | Composition of aescine derivative and salt thereof, preparation method and medical uses thereof |
| CN103860668A (en) * | 2014-03-18 | 2014-06-18 | 于法周 | Medicinal composition for treating hemolytic anemia and application thereof |
-
2003
- 2003-08-01 CN CN 03133639 patent/CN1268763C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100337635C (en) * | 2004-09-28 | 2007-09-19 | 徐东铭 | Medicine containing general pasqueflower saponin composition and its preparation method and uses |
| CN101130802A (en) * | 2007-07-30 | 2008-02-27 | 金凤燮 | Method for preparing astragaloside iv by enzymic hydrolysis for astragalus saponin glycosyl |
| CN102805750A (en) * | 2011-05-31 | 2012-12-05 | 天津药物研究院 | Composition of aescine derivative and salt thereof, preparation method and medical uses thereof |
| CN103860668A (en) * | 2014-03-18 | 2014-06-18 | 于法周 | Medicinal composition for treating hemolytic anemia and application thereof |
| CN103860668B (en) * | 2014-03-18 | 2015-11-18 | 陈金杰 | A kind of pharmaceutical composition and application thereof for the treatment of hemolytic anemia |
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| CN1268763C (en) | 2006-08-09 |
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Effective date of registration: 20180510 Address after: 300000 Tianjin Binhai high tech Zone, Binhai science and Technology Park, 19 Kangtai Avenue, 19. Patentee after: TIANJIN TIANSU GUANGHUA HEALTH TECHNOLOGY CO.,LTD. Address before: 116034 College of biology and food engineering, Dalian Light Industry Institute, No. 1 Light Industrial Park, Ganjingzi District, Dalian, Liaoning Patentee before: Jin Fengxie |
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Address after: 300457 Building 19, 59 Kangtai Avenue, Binhai Science Park, Binhai New District, Tianjin Patentee after: Sunflower Pharmaceutical Group (Tianjin) Pharmaceutical Research Institute Co.,Ltd. Address before: 300000 Tianjin Binhai high tech Zone, Binhai science and Technology Park, 19 Kangtai Avenue, 19. Patentee before: TIANJIN TIANSU GUANGHUA HEALTH TECHNOLOGY CO.,LTD. |
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