CN101481725B - Method for preparing ginseng saponin F2 by enzymatic hydrolysis of ginseng saponin Rb1 - Google Patents
Method for preparing ginseng saponin F2 by enzymatic hydrolysis of ginseng saponin Rb1 Download PDFInfo
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- CN101481725B CN101481725B CN2009100456390A CN200910045639A CN101481725B CN 101481725 B CN101481725 B CN 101481725B CN 2009100456390 A CN2009100456390 A CN 2009100456390A CN 200910045639 A CN200910045639 A CN 200910045639A CN 101481725 B CN101481725 B CN 101481725B
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- ginsenoside
- enzyme
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Abstract
The present invention discloses a method of using enzymatic hydrolyzed ginsenoside Rb1 to prepare ginsenoside F2, which is characterized in that part of glycosyl in ginsenoside Rb1 molecule is selectively cut by microbial enzyme to generate the target product ginsenoside F2. The enzyme used by the invention is from fusariumspp. CGMCC No.1495 which can be prepared through fermentation and is a catalyst that is cheap and can be obtained easily. The method for preparing the ginsenoside F2 has the advantages of moderate reaction conditions, strong specificity, low cost, high recovery, etc. And the purity of all ginsenoside F2 is above 95 percent. The ginsenoside F2 can be applied to relative pharmaceutical industry.
Description
Technical field
The present invention relates to a kind of enzymically hydrolyse and prepare ginseng saponin F
2Method, specifically, relate to and a kind ofly fall ginsenoside Rb with the hydrolysis of microbial enzyme specificity
1Part glycosyl in the molecule makes it change ginseng saponin F into
2Method.
Technical background
Genseng is as the famous Chinese medicine in the world, and its applicating history is quite remote.Ginsenoside is the main effective constituent of genseng, and up to now, people have isolated more than at least 40 kinds of saponin monomers from genseng.According to the difference of sapogenin structure, ginsenoside can be divided into diol type, triol type and oleanolic acid type three classes at present, and the aglycon structure of each saponins is identical, only be the glycosyl side chain difference that connects, but pharmacologically active has marked difference.Modern study shows that the sugar chain on the saponin(e has material impact to its biological activity, removes specific absorption and active function that the part glycosyl often can change its enteron aisle.How the saponin(e that content in the genseng is higher is converted into the lower saponin(e of content, or the saponin(e that pharmaceutical use is low is converted into the high saponin(e of pharmaceutical use, is the heat subject of genseng research always.
Ginseng saponin F
2Be a kind of highly active diol type saponin(e that contains in the genseng, but its content in genseng is very low, so direct extracting is not favourable industrial production process from plant, and ginsenoside Rb
1Be one of principal monomer in the diol type saponin(e, content reaches 4.3～11.9% in different types of genseng, it and ginseng saponin F
2Difference only be on C-3 position and the C-20 position each multi-link β-D-glucosyl group, so hydrolysis ginsenoside Rb
1Glycosyl be the preparation ginseng saponin F
2An effective way.
Adopt the main saponin(e of hydrolysis diol type to obtain ginseng saponin F at present
2Method mainly contain chemical method and biological process.Chemical method mainly contain heating method (Park.Food Ind.Nutr.2004,9:23-27), acid-hydrolysis method (Feng Zhongyang etc., Chinese Pharmaceutical Journal, 1997,32 (12): 772-774) and alkali hydrolysis method (Im, et al.Kor.J.Ginseng Sci.1995,19:291-294) etc.Though these methods are simple to operate, specificity is poor, and many sapogenins can change by recurring structure in hydrolytic process, are difficult to obtain the certain monomers saponin(e, and the use of simultaneously a large amount of organic solvents and soda acid also can cause environmental pollution.
Microbe fermentation method is to use bioconversion method more widely, is exactly with ginsenoside Rb
1Directly join in the fermented liquid of bacterial strain, utilize the metabolic enzyme of microorganisms to come hydrolysis.Present known Rb
1Most of enteric microorganism (Kobashi, et al.Ginseng Rev.1994,18:10-14) and fungi (Ma Jisheng etc., Acta Pharmaceutica Sinica, 2001,36 (8): 603-605; Dong, et al.Biotechnol.Lett.2003,25:339-344; Cheng, et al.Biotechnol.Lett.2006,28:1121-1127; Chen, et al.Appl.Microbiol.Biotechnol.2008, pathways metabolism 77:1345-1350) is all followed Rb
1-Rd-F
2-C-K-diol, therefore can in meta-bolites, obtain ginseng saponin F
2The microbe fermentation method specificity is stronger, and mild condition is low in the pollution of the environment, but has reaction product complicated component, the loaded down with trivial details shortcoming of separation.
Enzymically hydrolyse is the reaction of a kind of selective hydrolysis, and different types of enzyme can act on the not glycosidic bond of isomorphism type and different sorts sugar, thereby reaches the directionally hydrolyzing purpose.Enzymically hydrolyse method efficient height, specificity is strong, and does not have the back separation problem of microbe fermentation method, is a kind of comparatively ideal saponin glycosyl remodeling method.But the example of enzymic hydrolysis ginsenoside also is not a lot of at present, the main still restriction in enzyme source, therefore pass through screening suitable enzyme and reaction conditions, obtain high-content and active higher secondary saponin and sapogenin and be still the field that is worth exploration, bigger application prospect is also arranged in pharmaceutical industry.
Summary of the invention
The invention provides a kind of with enzymatic specificity hydrolysis ginsenoside Rb
1The preparation ginseng saponin F
2Method, it is characterized in that, adopt microbial enzyme hydrolysis ginsenoside Rb in damping fluid
1, make it change ginseng saponin F into
2Wherein the add-on of enzyme is 2～50U/mg substrate, and the add-on of substrate ginsenoside is 0.5～30g/L, and the pH of buffer value is 3.0～7.0, and temperature of reaction is 30～70 ℃, and the reaction times is 2～120 hours.
Enzymically hydrolyse provided by the present invention prepares ginseng saponin F
2Method in employed enzyme source in Fusarium (Fusarium proliferatum) ECU2042, preserving number is CGMCC No.1495, is one and is easy to get relatively and cheap catalyzer.
Enzymically hydrolyse provided by the invention prepares ginseng saponin F
2Method in, catalyzer adopts crude zyme preparation and immobilized enzyme dual mode, wherein, immobilized enzyme makes through chitosan absorption and glutaraldehyde cross-linking by crude enzyme liquid, and cross-linking method is that the concentration of crude enzyme liquid is 0.04～0.24g albumen/L, and the add-on of chitosan is 50～100g/L, immobilized pH value of reaction system is 6.0～7.0, adsorption time is 0.5～15 hour, and the adding weight/volume of linking agent glutaraldehyde is 0.1%～1% (w/v), and crosslinking time is 2～20 hours.
It is 4.5～6.0 acetic acid or phosphoric acid saline solution that described damping fluid is recommended as the pH value.The recommendation response temperature is 45～55 ℃.
The invention provides a kind of method of utilizing microbial enzyme selectivity catalytic hydrolysis high abundance ginsenoside preparation under mild conditions to have more the rare saponin(e of application potential.Compare with other method, present method not only have reaction conditions gentleness, specificity strong, be convenient to plurality of advantages such as directed control, and be that the technology cost in enzyme source is lower with the microorganism, help large batch of preparation.Prepare ginseng saponin F with this method
2, the purity of gained saponin(e can reach more than 95%.
Embodiment
To be further elaborated technology contents of the present invention by embodiment below, and its objective is to be better understanding content of the present invention.Therefore, the cited case does not limit protection scope of the present invention.
Embodiment 1
Adopt Cha Shi culture medium culturing Fusarium ECU2042, substratum is formed: sucrose 30g/l, NaNO
33.0g/l, K
2HPO
41.0g/l, KCl 0.5g/l, MgSO
47H
2O 0.5g/l, FeSO
47H
2O 0.01g/l.
Seed liquor is cultivated after 18 hours in 30 ℃, 180rpm shaking table, and the inoculum size by 4% is linked into 500ml and shakes (liquid amount 100ml) in the bottle, cultivates under similarity condition after 72 hours, filters and collects thalline.
Adopt the abrasive method smudge cells, cytoclasis liquid high speed freezing centrifuge centrifugal (12,000rpm, 30min), collect supernatant liquor, slowly add isopyknic freezing acetone under the condition of ice bath, continue to stir and use high speed freezing centrifuge centrifugal after 30 minutes, precipitation is the Fusarium crude zyme preparation through vacuum lyophilization.
Embodiment 2
In 100ml ground triangular flask, add prepared thick enzyme among the 600mg embodiment 1 successively, 60mg ginsenoside Rb
1, adding 30ml acetate buffer (pH 5.0 for NaAc/HAc, 0.2M) again, the jam-pack grinding port plug seals with raw material band, and after 72 hours, sampling is carried out HPLC and is analyzed (detection: UV 203nm in reaction on 50 ℃ of rotary shaking tables of following 200rpm; Moving phase: methanol=85/15, v/v), transformation efficiency about 30.5%.
Embodiment 3
In 100ml ground triangular flask, add prepared thick enzyme among the 300mg embodiment 1 successively, 60mg ginsenoside Rb
1, add 30ml acetate buffer (NaAc/HAc, 0.2M again, pH 5.0), the jam-pack grinding port plug seals with raw material band, after reacting 72 hours on 50 ℃ of rotary shaking tables of following 200rpm, add the thick enzyme of 300mg, continue reaction 48 hours, add propyl carbinol 30ml extraction 4 times respectively, collect the propyl carbinol phase, after rotary evaporation concentrates, separate, obtain the 16mg ginseng saponin F through preparative chromatography post (Venusil XBP C18)
2, transformation efficiency is about 43.2%, and yield is 87%, preparation gained ginseng saponin F
2Purity 〉=95%.
Embodiment 4
In 100ml ground triangular flask, add prepared thick enzyme among the 500mg embodiment 1 successively, 50mg ginsenoside Rb
1Add 50ml acetate buffer (pH 5.0 for NaAc/HAc, 0.2M) again, the jam-pack grinding port plug, seal with raw material band, after 48 hours, add propyl carbinol 25ml extraction 4 times respectively in reaction on 50 ℃ of rotary shaking tables of following 200rpm, collect the propyl carbinol phase, rotary evaporation concentrates, and separates through the preparative chromatography post, obtains the 15.2mg ginseng saponin F
2, transformation efficiency is near 50%, and yield is 86%, preparation gained ginseng saponin F
2Purity 〉=95%.
Embodiment 5
Get the 8g chitosan, join that prepared thick enzyme aqueous solution (contains 0.2g albumen/L among the 100ml embodiment 1, dipotassium hydrogen phosphate-potassium phosphate buffer, 0.2M pH 6.0) in, add the glutaraldehyde of 0.4% (w/v) again, 25 ℃, 100rpm concussion 18 hours, with damping fluid washing three times, vacuum filters collecting precipitation, is immobilized enzyme.Immobilization result is as follows: enzyme activity is 0.37U/mg, and carrying capacity is the 0.84mg/g carrier on the albumen, and activity recovery is 66.8%.
At 2ml substrate ginsenoside Rb
1Concentration is in the reaction system of 0.5g/L, adds immobilized enzyme 120mg, is reaction 24 hours under 50 ℃, 200rpm condition in temperature, centrifugal and sucking-off reaction solution, with damping fluid washing immobilized enzyme three times, join and begin reaction in the fresh reaction system once more, so repetitive operation is 10 times.When the 10th hydrolysis reaction, the vigor of immobilized enzyme has descended 22%, and the transformation efficiency of reaction still can maintain about 70%, the product ginseng saponin F
2Through being accumulate to about 6mg behind 10 secondary responses.
Claims (6)
1. enzymically hydrolyse ginsenoside Rb
1The preparation ginseng saponin F
2Method, it is characterized in that, adopt narrow spectrum microbial enzyme specificity hydrolysis ginsenoside Rb in damping fluid
1, make it change ginseng saponin F into
2Wherein, the add-on of enzyme is 2～50U/mg substrate, and the add-on of substrate ginsenoside is 0.5～30g/L, and the pH of buffer value is 3.0～7.0, and temperature of reaction is 30～70 ℃, and the reaction times is 2～120 hours;
Described microorganism is Fusarium (Fusarium proliferatum) ECU2042, and preserving number is CGMCC No.1495.
2. method according to claim 1 is characterized in that, wherein said microbial enzyme is crude zyme preparation and two kinds of catalysis forms of immobilized enzyme.
3. method according to claim 2 is characterized in that, described immobilized enzyme is the goods of microorganism crude enzyme liquid through chitosan absorption and glutaraldehyde cross-linking gained.
4. method according to claim 3, it is characterized in that, described microorganism crude enzyme liquid is that the concentration of described crude enzyme liquid is 0.16～0.24g albumen/L through chitosan absorption and glutaraldehyde cross-linking method, the add-on of chitosan is 70～100g/L, pH value of reaction system is 6.0～7.0, adsorption time is 0.5～2 hour, and the adding weight/volume of linking agent glutaraldehyde is 0.3%～0.5% (w/v), and crosslinking time is 16～20 hours.
5. method according to claim 1 is characterized in that, described damping fluid is that the pH value is 4.5～6.0 acetic acid or phosphoric acid saline solution.
6. method according to claim 1 is characterized in that, temperature of reaction is 45～55 ℃.
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| KR101621356B1 (en) * | 2014-09-05 | 2016-05-17 | 한국과학기술원 | Use of ginsenoside F2 for prophylaxis and treatment of liver disease |
| CN107312720B (en) * | 2017-03-01 | 2020-02-04 | 东北林业大学 | Cochinchinensis endophytic fungus for efficiently converting ginsenoside Rb1 into Rd and application thereof |
| CN111617120B (en) * | 2020-06-03 | 2021-08-13 | 广州鹰远生物科技有限公司 | Preparation method of ginseng cordyceps sinensis fermentation extract |
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| CN1793320A (en) * | 2005-12-05 | 2006-06-28 | 华东理工大学 | Strain of sickle mycete and process for preparing ginseng saponin Rh2 thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1793320A (en) * | 2005-12-05 | 2006-06-28 | 华东理工大学 | Strain of sickle mycete and process for preparing ginseng saponin Rh2 thereof |
Non-Patent Citations (3)
| Title |
|---|
| 刘颖等.壳聚糖- 戊二醛交联吸附法固定β - 葡萄糖苷酶的研究.食品科学.2008,第29卷(第5期),315-318. |
| 刘颖等.壳聚糖- 戊二醛交联吸附法固定β- 葡萄糖苷酶的研究.食品科学.2008,第29卷(第5期),315-318. * |
| 马吉胜等.真菌对人参皂苷Rb1及人参二醇系皂苷的代谢作用.药学学报.2001,第36卷(第8期),第603页摘要,第604页表1. * |
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