CN1230553C - Preparation method of novel antitumor ginsenoside - Google Patents

Abstract

The invention relates to enzymatic hydrolysis or microbial fermentation hydrolysis of ginsenosides to prepare low-polarity ginsenosides with free 3-position hydroxyl: 20-O-β-D-xylose (1→6)-β-D-glucose-20(s)- Protopanaxadiol [20-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl-20(S)-protopanaxadiol, referred to as Mx], 20-O-α-L-arabinose (1→ 6)-β-D-glucose-20(S)-protopanaxadiol [20-O-α-L-arabinopyranosyl (1→6)-β-D-glucopyranosyl 20(S)-protopanaxadiol, referred to as C-Y] and 20-O-α-L-arabinose (1→6)-β-D-glucose-20(S)-protopanaxadiol [20-O-α-L-arabinofuranosyl(1→6)-β-D -glucopyranosyl 20 (S)-protopanaxadiol, referred to as the method of Mc], especially relates to the crude helicase hydrolysis preparation method. The helicase used in the present invention has high enzymatic activity, good selectivity, low requirement for ionic strength, strong anti-inactivation ability, convenient source and low price, and the preparation of Mx, C-Y or Mc by using the enzyme is simple and convenient, low in cost and high in recovery rate , The product is of good quality and can meet the needs of the fields of medicine, cosmetics and functional food, and has great application and development value.

Description

Preparation method of novel antitumor ginsenoside

Technical field:

The invention relates to enzymatic hydrolysis or microbial fermentation hydrolysis of ginsenosides to prepare low-polarity ginsenosides with free 3-position hydroxyl: 20-O-β-D-xylose (1→6)-β-D-glucose-20(s)- Protopanaxadiol [20-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl-20(S)-protopanaxadiol, referred to as Mx], 20-O-α-L-arabinose (1→ 6)-β-D-glucose-20(S)-protopanaxadiol [20-O-α-L-arabinopyranosyl (1→6)-β-D-glucopyranosyl 20(S)-protopanaxadiol, referred to as C-Y] and 20-O-α-L-arabinose (1→6)-β-D-glucose-20(S)-protopanaxadiol [20-O-α-L-arabinofuranosyl(1→6)-β-D -glucopyranosyl 20 (S)-protopanaxadiol, referred to as the method of Mc], especially relates to the crude helicase hydrolysis preparation method.

Background technique:

Ginsenosides have a variety of medicinal functions, such as anti-tumor and immune regulation, improving microcirculation, regulating digestive function, calming the nerves, anti-aging, anti-stress, preventing digestive ulcers, improving quality of life, enhancing memory and learning ability, etc. However, natural ginsenosides can hardly be directly absorbed into the blood by the intestinal tract, and have poor pharmacokinetic behavior.

Recently, a variety of metabolites derived from natural ginsenosides through biotransformation have been discovered: low-polarity ginsenosides with free 3-hydroxyl groups, such as CK, CY and Mc. They are respectively proven to be orally absorbed forms of the corresponding natural ginsenosides. Hasegawa et al. found that CK has various biological activities such as inducing tumor cell differentiation, inducing tumor cell apoptosis, inhibiting tumor cell growth, inhibiting tumor-induced angiogenesis, and resisting tumor invasion and metastasis in anti-tumor aspects; in addition, CK has It has good pharmacokinetic behavior and is basically non-toxic or very low toxicity. At present, it has entered phase III clinical research in South Korea. Metabolites CY and Mc derived from natural ginsenosides Rb ₂ and Rc have similar pharmacodynamic activities to CK.

Since natural ginseng or natural ginsenosides are hardly absorbed directly after oral administration, the above three absorbable metabolites are transformed by human intestinal bacteria after oral administration. Because there are individual differences in the metabolism of ginsenoside intestinal bacteria in the body, in theory, direct use of the transformant can avoid absorption disorders, making it easy to master the effective dose in clinical practice.

The glycosidic bond at position 20 of ginsenoside is less stable in acidic media than that at position 3, and the steric hindrance makes the activity of the enzyme hydrolyzing the glycosidic bond at position 20 very low. Therefore, biotransformation is undoubtedly the best method for preparing low-polarity ginsenosides free of the 3-hydroxyl group. Biotransformation includes enzymatic and microbial fermentation methods.

Hasegawa anaerobically cultured natural ginsenosides and intestinal bacteria in vitro, separated C-K, C-Y and Mc from the culture medium, and applied for a US patent (5,919,770). Due to anaerobic culture, the low-polarity ginsenoside with free 3-hydroxyl is not the only product, and the conversion rate of raw materials is low, the separation and purification are cumbersome, the culture conditions are harsh, and the cost is high. Therefore, enzymatic methods have attracted more attention.

At present, the enzyme methods provided in the literature include naringinase and hesperidinase, but the enzyme has the following defects: long hydrolysis time, large amount of enzyme, and difficult source of enzyme, so it is not suitable for industrial preparation.

Our previous research invented the enzymatic preparation method of CK (Chinese patent, application number 01133410.X) and the microbial fermentation method (Chinese patent, application number 02132403.4), which have advantages in both process and cost. Compared with the preparation technology of intestinal bacteria, naringinase and hesperidinase, the two technical inventions have many advantages. Utilize above-mentioned technology simultaneously, with the ginsenoside Rb ₃ that is rich in the Radix Panax notoginseng stem and leaf as raw material, the inventor of the present invention has discovered Mx this new compound that has antitumor activity, Mx has the biological activity similar to CK, and compares Radix Ginseng , Panax notoginseng and its stems and leaves have more obvious resource advantages in China, and its structural formula is as follows:

Technical content of the invention:

In order to prepare Mx, CY and Mc simply, conveniently, at low cost and in large quantities, the present invention provides a method for preparing Mx, CY or Mc from ginsenoside by enzymolysis or microbial fermentation hydrolyzing ginsenoside, including hydrolysis, collection and The enzyme recycling step is characterized in that glycosidase is used to selectively hydrolyze ginsenoside Rb ₃ or notoginsin, Rb ₂ , and Rc in a buffer solution, and the weight ratio of saponin to glycosidase is 1:1 to 10:1 , the temperature is 30-45°C, the pH is 3.0-5.5, and the hydrolysis time is 2-72 hours.

In the method for preparing Mx, C-Y or Mc by enzymatically hydrolyzing ginsenosides provided by the present invention, the glycosidase is required to have good selectivity for the hydrolysis of glycosidic bonds, that is, only the 3-position glycosidic bonds are hydrolyzed without affecting the 20-position xylosyl and glucose base. Glycosidase can be derived from commercial enzymes such as helicase, hesperidinase, naringinase, etc., and can also be screened and isolated from nature for microorganisms with selective glycosidase activity, such as Helicobacter, Campylobacter, and Flexibacter , Pseudomonas, Xanthomonas, Phyllobacter, Methylococcus, Neibacterium, Flavobacterium, Bacteroides, Actinobacillus, Bordetella, Paracoccus , Enterobacter spp., Proteus spp., Photobacterium spp., Ciliobacterium spp., Succinovibrio spp., Lueromonas spp., Micrococcus spp., Stomatobacter spp., Enterococcus spp., Lactococcus spp., Anaerobic Streptococcus spp. , Leuconostoc, Pediococcus, Peptococcus, Faecococcus, Bacillus, Lactobacillus, Clostridium, Lactobacillus, Listeria, Corynebacterium, Arthrobacter, Short Bacillus, Caseobacter, Microbacterium, Fibrobacterium, Eubacterium, Actinomyces, Mycobacterium, Nocardia, Saccharopolyspora, Promicromonospora, Candida Genus, Penicillium, Aspergillus, Mucor, Cryptococcus, Candida, etc., biological products with selective glycosidase activity of the above microorganisms can also be used.

In the method for preparing Mx, C-Y or Mc by enzymatically hydrolyzing ginsenoside provided by the present invention, the enzymatic process is simple, and product separation and purification are easy, which is superior to microbial fermentation hydrolysis.

In the method for preparing Mx, CY or Mc by enzymatically hydrolyzing ginsenoside provided by the present invention, the raw materials for preparing Mx, CY and Mc are ginsenoside Rb ₃ , Rb ₂ , Rc, or plants containing Rb ₃ , Rb ₂ , Rc, Plant extract or plant culture fluid.

In the method for preparing Mx, CY or Mc by enzymatically hydrolyzing ginsenoside provided by the present invention, snail enzyme hydrolyzes raw material ginsenoside with high enzymatic activity, good selectivity, strong anti-inactivation ability, convenient source and low price, and is especially suitable for Mx, CY or Mc. Production preparation of CY and Mc. The optimum temperature for crude snail enzyme hydrolysis raw material ginsenoside is 40°C, the optimum pH is 4.5, the lower the ionic strength, the better, and the type of buffer has little effect on the enzyme activity; the present invention selects pH4.5, and the ionic strength is 0.001 mol·L ^-1 , of phosphate-citrate buffer solution; the ratio of raw material to enzyme is 6:1; according to this condition, the production of Mx will not change after the hydrolysis time exceeds 8 hours, therefore, the hydrolysis time is 8 hours is appropriate.

In the method for preparing Mx, C-Y or Mc by enzymatically hydrolyzing ginsenoside provided by the present invention, the enzymatic reaction liquid can be recycled after removing sediment and small molecule impurities.

In the method for preparing Mx, C-Y or Mc by enzymatically hydrolyzing ginsenoside provided by the present invention, when screening and separating microorganisms with selective glycosidase activity, enzyme induction method and antibiotic resistance induction method are used to increase the content of microbial glycosidase or enhance The hydrolysis ability of microorganisms and the antibiotics added at the same time can also prevent the contamination of bacteria.

In the method for preparing Mx, CY or Mc by enzymatically hydrolyzing ginsenosides provided by the present invention, the collection process is: (1) enzymatically collect the enzyme reaction precipitate to make a suspension; extract three times with ethyl acetate, and combine the ethyl acetate liquid , after recovery of ethyl acetate under reduced pressure, the collected residue is Mx, CY or Mc; if the raw material is a plant, plant extract or plant culture solution containing Rb ₃ , Rb ₂ and Rc, the obtained crude product needs to be processed Adsorption resin purification. (2) The microbial fermentation method extracts the culture medium three times with water-saturated n-butanol, combines the n-butanol extracts, decolorizes and deodorizes with activated carbon, concentrates under reduced pressure to recover n-butanol, dissolves the residue in ethyl acetate and uses silica gel column chromatography Mx, CY or Mc are obtained after purification by reverse phase chromatography.

In the method for preparing Mx, C-Y or Mc by enzymatically hydrolyzing ginsenoside provided by the present invention, a certain amount of organic solvent is added to help dissolve the intermediate product, which is beneficial to the normal progress of the enzymatic hydrolysis reaction. The type and amount of the organic solvent should not affect the enzyme activity. After screening, 5-15% ethanol is the best.

The invention provides an enzyme with convenient source and low price for preparing ginsenoside Mx, C-Y or Mc by enzymatic hydrolysis of ginseng diol saponin, and the preparation process of Mx, C-Y and Mc by using the enzyme is simple and convenient, with low cost and high recovery rate. The contents of Mx, C-Y and Mc prepared by the method are more than 95%, and the yield of aglycon is more than 91%.

Detailed ways:

Example 1

Ginsenoside Rb ₃ (500mg) and helicase (84mg) were dissolved in 32mL phosphate-citrate buffer (pH 4.5, ionic strength 0.001mol·L ^-1 , containing 10% ethanol), in a water bath at 40°C Hydrolyze for 1 day; centrifuge the reaction solution, collect the precipitate, make the precipitate into a suspension with water, extract the suspension with ethyl acetate (10mL×5), combine the ethyl acetate solution, and remove the ethyl acetate under reduced pressure to obtain Mx (106 mg).

Example 2

Ginsenoside Rb ₃ (50 mg) and helicase (8.4 mg) were dissolved in 3.2 mL of phosphate-citrate buffer solution (ionic strength 0.001 mol·L ^-1 , containing 10% ethanol) with different pH, and water bath at 40 °C hydrolyzed for 1 day. Centrifuge the reaction solution and collect the precipitate. The precipitate was made into a suspension with water, extracted with ethyl acetate (1mL×5), the suspension was combined, the ethyl acetate was combined, ethyl acetate was removed under reduced pressure, the collected residue was dissolved in 5.00mL of methanol, and analyzed by HPLC Detection of Mx content. The results showed that the optimal pH of helicase was 4.5.

Example 3

Dissolve ginsenoside Rb ₃ and helicase in different proportions in 3.2 mL of phosphate-citrate buffer (pH 4.5, ionic strength 0.001 mol·L ^-1 , containing 10% ethanol), and hydrolyze 1 days; centrifuge the reaction solution and collect the precipitate. Make the precipitate into a suspension with water, extract the suspension with ethyl acetate (1mL×5), combine the ethyl acetate solution, remove the ethyl acetate under reduced pressure, and dissolve the collected residue in 5.00mL methanol, HPLC Analysis and detection of Mx content. The results showed that the optimum ratio of raw material/enzyme was 6/1.

Example 4

Ginsenoside Rb ₃ (50 mg) and helicase (8.4 mg) were dissolved in 3.2 mL of phosphate-citrate buffer (pH 4.5, ionic strength of 0.001 mol·L ^-1 , containing 10% ethanol), at different temperatures Hydrolyze in a water bath for 1 day; centrifuge the reaction solution and collect the precipitate. Make the precipitate into a suspension with water, extract the suspension with ethyl acetate (1 mL×5), combine the ethyl acetate solution, remove the ethyl acetate under reduced pressure, and dissolve the collected residue in 5.00 mL of methanol, HPLC analysis detects Mx content. The result shows: the optimum temperature is 45℃.

Example 5

Ginsenoside Rb ₃ (50mg) and helicase (8.4mg) were dissolved in 3.2mL phosphate-citrate buffer (pH 4.5, containing 10% ethanol), and the ionic strength was changed, and hydrolyzed in a water bath at 40°C for 1 day; Centrifuge the reaction solution and collect the precipitate. Make the precipitate into a suspension with water, extract the suspension with ethyl acetate (1mL×5), combine the ethyl acetate solution, remove the ethyl acetate under reduced pressure, and dissolve the collected residue in 5.00mL methanol, HPLC Analysis and detection of Mx content. The results show that the lower the ionic strength, the better.

Example 6

Ginsenoside Rb ₃ (50 mg) and helicinase (8.4 mg) were dissolved in 3.2 mL pH4.5 ionic strength of 0.001 mol L ^-1 containing 10% ethanol in different buffers (including 1 for glycine-HCl, 2 for o Potassium hydrogen phthalate-HCl, 3 is Na ₂ HPO ₄ -citric acid, 4 is citric acid-sodium citrate, 5 is acetic acid-sodium acetate), hydrolyzed in water bath at 40°C for 8 hours; the reaction solution was collected by centrifugation Precipitation; the precipitate was made into a suspension with water; extracted with ethyl acetate (1mL×5), the ethyl acetate solution was combined, and after the ethyl acetate was recovered under reduced pressure, the collected residue Mx was dissolved in 5.00mL of methanol, and HPLC Analysis of Mx content. The results showed that the type of buffer had little effect on the activity of helicase.

Example 7

Ginsenoside Rb ₃ (50 mg) and helicase (8.4 mg) were dissolved in 3.2 mL of phosphate-citrate buffer (pH 4.5, ionic strength 0.001 mol·L ^-1 , containing 10% ethanol), at 40°C In the water bath, change the hydrolysis time. Centrifuge the reaction solution, collect the precipitate, and make the precipitate into a suspension with water; extract the suspension with ethyl acetate (1mL×5), combine the ethyl acetate solution, remove the ethyl acetate under reduced pressure, and collect the residue Dissolved in 5.00mL methanol, HPLC analysis and detection of Mx content. Result explanation: after reacting 10 hours, the increment of Mx is not big.

Example 8

The Aspergillus niger v.Tiegh (Aspergillus niger v.Tiegh) YLY bacterial strain solution obtained by the co-cultivation and domestication of tetracyclines obtained through ginsenoside Rb ₃ co-cultivation is cultivated overnight in Chapei's liquid medium (100mL) before use, and the colonies are picked out. Introduce into Chapei's liquid medium (100 mL) containing ginsenoside Rb ₃ (3%), and culture continuously for 3 days on a shaker at 37 degrees. The culture solution was extracted three times with saturated n-butanol (10mL×5), the n-butanol extract was combined, decolorized and deodorized by activated carbon, n-butanol was removed under reduced pressure, the residue was dissolved in ethyl acetate and subjected to silica gel column chromatography ( The eluent is chloroform/ethyl acetate/ethanol=60/30/10, lower layer 500mL) and reverse phase chromatography purification (mobile phase is 50% ethanol 5L), and Mx (515mg) is obtained, and the content after inspection is ≥95% .

Example 9

Dissolve ginsenoside Rb ₂ (500mg) and helicase (84mg) in 32mL phosphate-citrate buffer (pH 4.5, ionic strength 0.001mol·L ^-1 , containing 10% ethanol), in a water bath at 40°C Hydrolyze for 1 day; centrifuge the reaction solution, collect the precipitate, make the precipitate into a suspension with water, extract the suspension with ethyl acetate (10mL×5), combine the ethyl acetate solution, and remove the ethyl acetate under reduced pressure to obtain CY (103 mg).

Example 10

Dissolve ginsenoside Rc (500mg) and helicase (84mg) in 32mL phosphate-citrate buffer solution (pH 4.5, ionic strength 0.001mol·L ^-1 , containing 10% ethanol), and hydrolyze in a water bath at 40°C 1 day; centrifuge the reaction solution, collect the precipitate, make the precipitate into a suspension with water, extract the suspension with ethyl acetate (10mL×5), combine the ethyl acetate solution, and remove the ethyl acetate under reduced pressure to obtain Mc( 105mg).

Claims (4)

1, a kind of with the low polarity ginsenoside of 3 hydroxyl free of enzymic hydrolysis panoxadiol type saponin(e preparation: 20-O-β-D-wood sugar (1 → 6)-β-D-glucose-20 (s)-protopanoxadiol [20-O-β-D-xylopyranosyl (1 → 6)-β-D-glucopyranosyl-20 (S)-protopanaxadiol, be called for short Mx], 20-O-α-L-arabinose (1 → 6)-β-D-glucose-20 (S)-protopanoxadiol [20-O-α-L-arabinopyranosyl (1 → 6)-β-D-glucopyranosyl 20 (S)-protopanaxadiol, be called for short C-Y] or 20-O-α-L-arabinose (1 → 6)-β-D-glucose-20 (S)-protopanoxadiol [20-O-α-L-arabinofuranosyl (1 → 6)-β-D-glucopyranosyl 20 (S)-protopanaxadiol, be called for short Mc] method, comprise hydrolysis, product collection and enzyme are recycled step, with helicase difference selective hydrolysis ginsenoside Rb in damping fluid ₃Preparation Mx, ginsenoside Rb ₂Preparation C-Y or Ginsenoside Rc prepare Mc, and it is characterized in that: the weight ratio of saponin(e and helicase is 1: 1～10: 1, and temperature is 30～45 ℃, and pH 3.0～5.5, hydrolysis time 2 hours～3 days, and the product collection method is enzyme process or microbe fermentation method:

A) enzyme process is collected the enzyme reaction throw out, makes suspension liquid; Ethyl acetate extraction three times, combined ethyl acetate liquid, after the reclaim under reduced pressure ethyl acetate, the resistates of collection is Mx, C-Y or Mc; If raw material is for containing Rb ₃, Rb ₂With plant, plant milk extract or the plant culturing liquid of Rc, institute's crude product that obtains also needs through purification on adsorbent resins;

B) microbe fermentation method is with water saturated n-butanol extraction substratum three times, merge n-butanol extracting liquid, the gac deodorizing of discoloring, concentrating under reduced pressure reclaims propyl carbinol, and resistates is dissolved in ethyl acetate and with obtaining Mx, C-Y or Mc behind silica gel column chromatography and the reversed phase chromatography purifying.

2, prepare the method for Mx, C-Y or Mc according to claim 1 is described with enzymic hydrolysis, it is characterized in that: the weight ratio of used saponin(e and helicase is 6: 1, and pH is 4.5, and hydrolysis temperature is 40 ℃, and hydrolysis time is 8 hours.

3, prepare the method for Mx, C-Y or Mc according to claim 1 is described with enzymic hydrolysis, it is characterized in that: describedly removed sedimentary enzyme reaction solution, after small molecular weight impurity is removed in ultrafiltration, reclaimed and use as dhdps enzyme.

4, prepare the method for Mx, C-Y or Mc according to claim 1 is described with enzymic hydrolysis, it is characterized in that: contain 5～15% ethanol in the used damping fluid.