CN1268759C - Process for preparing low sugar icariin or aglycone by enzymic hydrolysis of icariin glycosylate - Google Patents
Process for preparing low sugar icariin or aglycone by enzymic hydrolysis of icariin glycosylate Download PDFInfo
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- CN1268759C CN1268759C CN 03133635 CN03133635A CN1268759C CN 1268759 C CN1268759 C CN 1268759C CN 03133635 CN03133635 CN 03133635 CN 03133635 A CN03133635 A CN 03133635A CN 1268759 C CN1268759 C CN 1268759C
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- icariine
- icariin
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Abstract
The present invention provides a method for preparing low-carbohydrate icariin or aglycone by hydrolyzing icariin glycosyl with an enzyme method. In the method, icariin glycosyl is hydrolyzed by an enzyme to prepare low-carbohydrate icariin or aglycone. The method comprises the following steps: taking the enzyme which can hydrolyze the icariin glycosyl; mixing the enzyme, the icariin and a buffer solution to react for 1 to 36 hours under the reaction conditions that a temperature is from 4 to 75 DEG C and a pH value is from 3 to 9; extracting the low-carbohydrate icariin or aglycone. The present invention overcomes the disadvantages of large glycoside damage, serious pollution, poor purposefulness, etc. existing in the prior art, and has the advantages of no pollution, strong purposefulness and high conversion rate. Particularly, an enzyme-producing inducer is added during microbial fermentation to greatly improve the yield of the enzyme. Thus, the present invention is helpful to fully hydrolyze the icariin glycosyl, and greatly improves the conversion rate of the low-carbohydrate icariin or aglycone.
Description
Technical field:
The present invention relates to a kind of method for preparing low sugar icariine or glucoside unit, especially a kind ofly prepare the method for low sugar icariine or glucoside unit with enzymic hydrolysis icariine glycosyl.
Background technology:
Herba Epimedii is the Chinese medicine of using always, is Berberridaceae section Epimedium (Epimedium genus) plant.Pharmacopoeia of People's Republic of China (version in 2000) has recorded following five kinds of epimedium herbs, and medicinal material is the exsiccant over-ground part.Be Herba Epimedii (Epimedium brevicornum Maxim), arrow leaf Herba Epimedii (Epimedium sagittatum), pubescence Herba Epimedii (Epimedium pubescens), Epimedium wushanense (Epimedium wushanenes), Herba Epimedii (Epimedium koreanum Nakai) etc.Herba Epimedii mainly contains the flavonoid glycoside (icariine) of isopentene group replacement and the change of ethanoyl replacement contains thing; but its ethanoyl is easy to take off in the process of processing Herba Epimedii, extraction icariine; therefore the glucoside that mainly contains in Herba Epimedii is epimedium flavone glucoside, i.e. icariine.Its main pharmacodynamics is nourishing disney and strengthening bone, strengthens internal secretion, has hormonal action, can promote medullary cell DNA to contain to become, and promotes the growth of osteocyte.The chemical molecular structure of Herba Epimedii glucoside is:
According to R different in the molecular structural formula
2, R
1, R
3, icariine (glycosides) is divided into multiple, can arrange as follows by sequence number:
| R 2 | R 1 | R 3 | |
| Icariine (glycosides) 1: icariine (glycosides) 2: icariine (glycosides) 3: icariine (glycosides) 4: | rha-rha- glc-rha- xyl-rha- rha-rha- | glc- glc- glc- glc- | Me Me Me H |
| Icariine (glycosides) 5: icariine (glycosides) 6: icariine (glycosides) 7: icariine (glycosides) 8: icariine (glycosides) 9: icariine (glycosides) 10: icariine (glycosides) 11: icariine (glycosides) 12: icariine (glycosides) 13: icariine (glycosides) 14: icariine (glycosides) 15: icariine (glycosides) 16: icariine (glycosides) 17: icariine (glycosides) 18:4 '-methoxyl group icariine unit: icariine unit: | glc-rha- xyl-rha- rha-rha- glc-rha- xyl-rha- rha-rha- glc-rha- xyl-rha- rha- rha- rha- H rha- H H H | glc- glc- H H H H H H glc- glc- H glc H glc- H H | H H Me Me Me H H H Me H Me Me H H Me H |
Above-mentioned different icariine all has 3 glycosyls, i.e. polysaccharide base icariine.Polysaccharide base icariine activity is lower, and low glycosyl icariine or icariine unit activity are higher, in order to obtain icariine unit or low glycosyl icariine, once reported peracid alkali hydrolysis method (Sun Pengyue etc., Chinese pharmaceutical chemistry magazine, 1998,30 (4), 281-284), but described method is destructive big to glucoside, seriously polluted, purpose is poor.
Chinese patent application number is that 01803603.1 application for a patent for invention discloses a kind of " extracting method of flavonoid ".This method may further comprise the steps: " in the enzymatic mode flavonoid glycosides or its joiner are changed into the flavonoid aglycon; Regulate pH so that the flavonoid aglycon is solvable and remove insoluble part; With regulate pH so that the flavonoid aglycon becomes relative insoluble and form the extract that contains them." because the flavonoid glycosides is two kinds of absolutely different products of configuration with icariine, disclosed enzyme at all can not the hydrolysis icariine in the literary composition.For this reason, up to now, people also prolong with acid and alkali hydrolysis method processing icariine always, still exist the problems referred to above.
Summary of the invention:
The present invention is in order to solve the existing in prior technology technical problem, a kind of enzymatic hydrolysis icariine glycosyl to be provided, prepare the method for low sugar icariine or glucoside unit, and is pollution-free, purpose is strong, transformation efficiency is high.
Technical solution of the present invention is:
Comprise the steps: that a kind of enzymatic hydrolysis icariine glycosyl prepares the method for low sugar icariine or glucoside unit, is characterized in that comprising the steps:
A. be bacterium, mould, saccharomycetes to make fermentation product enzyme induction thing with the Herba Epimedii, obtain containing the enzyme mixed solution behind liquid state fermentation or the solid state fermentation, in containing the enzyme mixed solution, add ammonium sulfate or extraction using alcohol enzyme;
B. with the enzyme, icariine and the acetic acid that are extracted or phosphoric acid hybrid reaction 1-36 hour, reaction conditions was temperature 4-75 ℃, pH value 3-9;
C. extract low sugar icariine or glucoside unit.
The icariine concentration of described b step is the 0.1-10% of total reactant volume.
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 to be that bacterium, mould, saccharomycetes to make fermentation produce the enzymic hydrolysis icariine glycosyl that the enzyme induction thing extracted and prepare low sugar icariine or glucoside unit in order to Herba Epimedii.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.
Embodiment:
Embodiment 1:
A. produce the enzyme induction thing with high-temperature aerobic bacterium Clostridium thermocopriea (document 1) containing 3% Semen Maydis powder, 1%---in Herba Epimedii extract (dry) substratum, be under 60 ℃ the condition in temperature, shaking culture 30-60 hour, bactofugation; Toward the middle ammonium sulfate precipitation zymoprotein that adds 65% saturation ratio of supernatant liquor (containing the enzyme mixed solution), with 0.02M, the dialysis of pH5 acetate buffer solution, centrifugal slagging-off, freeze-drying obtains the glucoside enzyme.
B. with the above-mentioned glucoside enzyme of 0.2 gram, the 3 gram mixtures of icariines 1,2,3 or monomer, 150 milliliters of acetate buffer solution (0.2M, pH5.0) mix, make the mixture of icariine 1,2,3 account for the 0.1-10% of total reaction volume, stirring reaction is 1 hour under the condition of 75 ℃ of temperature.
C. react Hou and add 300 milliliters of ethanol sedimentation zymoproteins, remove by filter the zymoprotein precipitation, the filtrate decompression evaporate to dryness obtains 2.1 gram crude products.
Change into monose icariine 16, glucoside unit with the glucoside of high performance liquid chromatography (document 2) detected result more than 85%.Separate (document 3) through silicagel column, dissolving in hot methanol, cold methanol precipitation obtain secondary glucoside 13, the 0.05 gram monose icariine 15 of pure 0.1 gram Herba Epimedii and 16 and 0.02 gram, 4 '-methoxyl group icariine unit.
Embodiment 2:
A. containing 4% Semen Maydis powder, 0.2% product enzyme induction thing with high-temperature aerobic bacterium Clostridium thermocopriea (document 1)---in Herba Epimedii extract (dry) substratum, be under 60 ℃ the condition in temperature, shaking culture 30-60 hour, bactofugation, in supernatant liquor (containing the enzyme mixed solution), add the ethanol sedimentation zymoprotein, freeze-drying obtains the glucoside enzyme.
B. the above-mentioned glucoside enzyme of 0.3 gram, 2 is restrained mixture, 120 milliliters of acetate buffer solution (0.2M of icariines 1,2,3, pH5.0) mix, making the mixture of icariine 1,2,3 account for the 0.1-10% of total reaction volume, is stirring reaction 36 hours under 5 ℃ the condition in temperature.
C. react Hou and add 300 milliliters of ethanol sedimentation zymoproteins, remove by filter the zymoprotein precipitation, the filtrate decompression evaporate to dryness obtains 1.7 gram crude products.
Change into monose icariine 14,17 and 18 with the glucoside of high performance liquid chromatography (document 2) detected result more than 90%.Separate (document 3) through silicagel column, dissolving in hot methanol, cold methanol precipitation obtain pure 0.3 gram icariine, 14,0.1 gram icariine 17 and 0.1 gram icariine 18.
Embodiment 3:
A. containing 3% Semen Maydis powder, 1% product enzyme induction thing with high-temperature aerobic bacterium Clostridium thermocopriea (document 1)---in genseng extract (dry) substratum, be under 60 ℃ the condition in temperature, shaking culture 30-60 hour, bactofugation, in supernatant liquor (containing the enzyme mixed solution), add the ethanol sedimentation zymoprotein, collect albumen, freeze-drying obtains the glucoside enzyme.
B. the above-mentioned glucoside enzyme of 0.4 gram, 2 is restrained mixture, 120 milliliters of phosphoric acid buffer (0.2M of icariines 4,5,6, pH5.0) mix, make the mixture of icariine 4,5,6 account for the 0.1-10% of total reaction volume, under 60 ℃ of conditions of temperature, stirring reaction 18 hours.
C. react Hou and add 300 milliliters of ethanol sedimentation zymoproteins, remove by filter albumen precipitation, the filtrate decompression evaporate to dryness obtains low glycosyl icariine 1.5 gram crude products.
Separate (document 3) through silicagel column, dissolving in hot methanol, cold methanol precipitation obtain pure 0.3 gram icariine, 14,0.1 gram icariine 17 and 0.06 gram icariine unit.
Embodiment 4:
A. with black-koji mould (Aspegillus niger) in containing the substratum that 4% Fructus Hordei Germinatus extract, 0.5% produces enzyme induction thing-Herba Epimedii extract (dry), stir cultivation 50-100 hour of ventilating at 28-30 ℃, 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, ammonium sulfate is removed in dialysis, centrifugal slagging-off is enzyme liquid.
B. with the 3 gram mixtures of icariines 4,5,6 or monomer whose (accounting for the 0.1-10% of total reaction volume), 100 milliliters of acetate buffer solutions (0.02M, pH5.0) and 20 milliliters of above-mentioned enzyme liquid mix, make 20-55 ℃ of reaction 1-4 hour.
C. the n-butanol extraction saponin three times that adds 1/3 total reaction volume, evaporated under reduced pressure obtains secondary saponin crude product 2 grams of low glycosyl.
Obtain the pure icariine 17,18 and the 0.15 icariine unit of 0.1 gram with silicagel column separation method (document 3).
Embodiment 5:
A. with embodiment 4;
B. mixture or monomer whose (accounting for the 0.1-10% of total reaction volume), 50 milliliters of acetate buffer solutions (0.02M, pH 5.0) and the 20 milliliters of enzyme liquid with 1 gram icariine 7,8,9 mixed, 20-55 ℃ of reaction 1-4 hour.
C. the n-butanol extraction saponin three times that adds 1/3 total reaction volume, evaporated under reduced pressure obtains secondary saponin crude product 2 grams of low glycosyl.
Change into monose icariine 17, glucoside unit with the glucoside of high performance liquid chromatography (document 2) detected result more than 85%.Obtain the pure icariine 17 and the 0.05 gram icariine unit of 0.1 gram with silicagel column separation method (document 3).
Embodiment 6:
A. aspergillus oryzae (Aspegillus oryzae) is containing 5% wheat bran extract, 0.2% product enzyme induction thing---in the substratum of the extract of icariine or rutin or 1% flavonoid source plant, be to stir under 28-30 ℃ the condition in temperature, ventilate and cultivated 50-100 hour, bactofugation must contain the enzyme mixed solution, add the ethanol sedimentation zymoprotein, collect albumen, add the acetate buffer solution (0.02M of 1/5 fermentating liquid volume, pH 5.0), be enzyme liquid.
B. above-mentioned enzyme liquid is pressed b, the c step process icariine 1,2,3 of embodiment 4, obtained monose icariine 13,15,16 and 4 '-methoxyl group glucoside unit; Handle various icariines 4,5,6, obtain monose icariine 14,17,18 and glucoside unit.
Embodiment 7:
A. with embodiment 6;
B. 10 gram Herba Epimedii extracts (salidroside content is more than 60%), 50 milliliters, 400 milliliters acetate buffer solutions of enzyme liquid (0.02M, pH 5.0) are blended in 20-55 ℃ of reaction 1-4 hour.
C. the liquor-saturated extraction saponin of positive fourth three times that adds 1/3 total reaction volume, evaporated under reduced pressure obtains secondary glucoside crude product 5 grams of low glycosyl.
With high performance liquid chromatography (document 2) detected result, mainly contain monose icariine 15,16,17,18, a spot of icariine 13,14 and 4 '-methoxyl group icariine unit and glucoside unit.
Embodiment 8:
A. candiyeast is containing upward inoculation of the wheat bran substratum of Herba Epimedii powder 10% (dry 1000 grams), be divided in the eggplant bottle 30 ℃ of solid culture 3～6 days, collect culture, the physiological saline that in the substratum dry, adds 6000 milliliters, soaked one hour, centrifuging and taking supernatant (containing the enzyme mixed solution), the ammonium sulfate that adds saturation ratio 60～75%, make the zymoprotein precipitation, collecting precipitation, with 800 milliliters pH5, the dissolving of 0.02M sodium-acetate buffer, ammonium sulfate is removed in dialysis, centrifugal slagging-off promptly gets the enzyme liquid about 1000 milliliters.
B. above-mentioned enzyme liquid is handled icariine 1,2,3,4,5,6 by the method for embodiment 4 and mixture obtains single, double sugared icariine or glucoside unit.
Document described in the embodiment is:
1.Fengxie Jin et al (Jin Feng is mediate etc.): J.Int.Syst.Bact, 1988,38,279-281.
2. Ma Zhuo etc., traditional Chinese medicine magazine, 2002,22 (11), 697.
3. Sun Peng pleases etc., Chinese pharmaceutical chemistry magazine, 1998,30 (4), 281-284.
Claims (3)
1. an enzymatic hydrolysis icariine glycosyl prepares the method for low sugar icariine or glucoside unit, it is characterized in that comprising the steps:
A. be bacterium, mould, saccharomycetic enzymatic production inductor with the Herba Epimedii, obtain containing the enzyme mixed solution behind liquid state fermentation or the solid state fermentation, in containing the enzyme mixed solution, add ammonium sulfate or extraction using alcohol enzyme;
B. with the enzyme, icariine and the acetic acid that are extracted or phosphoric acid hybrid reaction 1-36 hour, reaction conditions was temperature 4-75 ℃, pH value 3-9;
C. extract low sugar icariine or glucoside unit.
2. enzymatic hydrolysis icariine glycosyl according to claim 1 prepares the method for low sugar icariine or glucoside unit, it is characterized in that: the icariine concentration of described b step is the 0.1-10% of total reactant volume.
3. enzymatic hydrolysis icariine glycosyl according to claim 1 and 2 prepares the method for low sugar icariine or glucoside unit, it is characterized in that: described c step is for extracting or use n-butanol extraction with ethanol sedimentation.
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