CN105648021A - Preparation method for rare ginsenoside C-K and F1 and four kinds of isomer ginsengenin - Google Patents
Preparation method for rare ginsenoside C-K and F1 and four kinds of isomer ginsengenin Download PDFInfo
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Abstract
The invention discloses a preparation method for rare ginsenoside C-K and F1 and four kinds of isomer panoxadiol saponin and panaxatriol sapogenin. Ginseng is used as a raw material, panoxadiol saponin (mixture), panaxatriol sapogenin (mixture) and a ginseng saponin enzyme compound are extracted. Panoxadiol saponin and an organic solvent are prepared into a substrate solution, panaxatriol sapogenin and a buffer solution are prepared into a substrate solution, the two substrate solutions are fermented with aspergillus microorganisms to obtain crude enzyme liquid for a reaction, there is almost no other byproduct saponin, and rare ginsenoside C-K or rare ginsenoside F1is obtained; after the reaction, the enzyme can be recovered and used repeatedly. The obtained C-K or F1 saponin reacts with the ginseng saponin enzyme compound (a proper amount of organic acid is added), and then four kinds of isomer panoxadiol saponin and panaxatriol sapogenin are obtained. Operation is easy, yield is high, cost is low, and the method is suitable for mass production; the obtained products can be used for medicine development, ginseng products, health care products and cosmetics.
Description
Technical field
The preparation method that the present invention relates to the rare saponin of a kind of Radix Ginseng and four kinds of isomer ginsengenins, especially a kind of simple to operate, cost is low, yield and purity high, be suitable for the preparation method of Radix Ginseng rare saponin C-K, F1 and the four kinds of isomer ginsengenins produced in enormous quantities.
Background technology
The Radix Ginseng that China's current production rate is high is mainly Radix Ginseng (PanaxginsengC.A.Mayer), Radix Panacis Quinquefolii (P.quinguefolusL.) and Radix Notoginseng (P.notoginsengburk). The main saponin of Radix Ginseng is Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1; The main saponin of Radix Panacis Quinquefolii is based on Rb1 and Re, also containing Rb2, Rc, Rd, Rg1 saponin; The main saponin of Radix Notoginseng is based on Rb1 and Rg1, also containing Rd, Re, R1 saponin; Other saponin contents are relatively low.
Radix Ginseng main protopanoxadiol class (PPD) saponin structure is as follows:
Note: Glc, ��-D-glucopyranosyl; Arap, ��-L-arabopyranose base; Araf, ��-L-arabinofuranosyl; Xyl, ��-D-xylopyranosyl.
Radix Ginseng Rubra exists Panaxadiol saponin unit and protopanaxatriol ginsenoside unit (document: the LarsP.Christensened.Ginsenosides:Chemistry of four kinds of isomers, Biosynthesis, Analysis, andPotentialHealthEffects.AdvancesinFoodandNutritionRese arch. (2009) Volume55, p.1-99.ElsevierInc.).
The Panaxadiol saponin unit of the four kinds of isomers existed in Radix Ginseng Rubra is 20 (S)-Panaxadiol saponin unit [20 (S)-PPDol], 20 (R)-Panaxadiol saponin unit [20 (R)-PPDol], 20-hydroxyl de-20 (21), 24-diene Panaxadiol saponin unit [PPDol (-H2O)-20 (21), 24-diene], the 20 (22) of 20-hydroxyls dehydrate, 24-diene Panaxadiol saponin unit [PPDol (-H2O)-20 (22), 24-diene];
The Panaxadiol saponin meta structure of Radix Ginseng rare saponin C-K and four kinds of isomers is as follows:
Radix Ginseng main protopanaxatriol (PPT) saponin structure is as follows:
Note: Glc, ��-D-glucopyranosyl;Rha, ��-L-rhamnopyranosyl; Xyl, ��-D-xylopyranosyl.
The protopanaxatriol ginsenoside unit of the four kinds of isomers existed in Radix Ginseng Rubra is 20 (S)-protopanaxatriol ginsenoside unit [20 (S)-PPTol], 20 (R)-protopanaxatriol ginsenoside unit [20 (R)-PPTol], 20-hydroxyls dehydrate protopanaxatriol ginsenoside unit-20 (21), 24-diene [PPTol (-H2O)-20 (21), 24-diene], 20-hydroxyls dehydrate protopanaxatriol ginsenoside unit-20 (22), 24-diene [PPTol (-H2O)-20 (22), 24-diene]. Above-mentioned ginsengenin isomer, in Radix Ginseng Rubra, extremely trace exists.
The protopanaxatriol ginsenoside meta structure of Radix Ginseng rare saponin F1 and four kinds of isomers is as follows:
The physiologically active of the Panaxadiol saponin unit of four kinds of isomers and the protopanaxatriol ginsenoside unit of four kinds of isomers is far above ginsenoside.
After Radix Ginseng is oral, under the effect of intestinal, Radix Ginseng glycols saponin (Rb1, Rb2, Rb3, Rc, Rd) is converted into C-K, and panaxatriol's saponins (Re, Rg1) is converted into F1, is then absorbed, plays drug effect; Therefore, ginseng saponin C-K and F1 are the rare saponin of Radix Ginseng (document: MKanaoke, TOkao, KKobashi.JTradMed.199411,241-245) that absorbance is high, physiologically active is high.
Therefore, prepare in a large number high activity Radix Ginseng rare saponin C-K and F1, four kinds of isomers Panaxadiol saponin unit and protopanaxatriol ginsenoside unit, ginseng product, dietetic food and cosmetics and drug development meaning are very big.
In order to obtain high activity, easily absorb the rare saponin of Radix Ginseng, International Patent Application PCT/CN00/00744(China ZL0082112.9, Europe EP1354944, U.S. patent Nos US7,759,101B2, Japan JP-4953547; Document 2) etc., disclose the high Rb1 of content from Radix Ginseng, Rh2, Rg3, C-K, Rg2 and Rh1 etc. are prepared in Rb2, Rc, Rd, Re and Rg1 enzymatic conversion; Disclosed ginsenosidases is from antibacterial, mycete, yeast, Fructus Hordei Germinatus, panax ginseng plant, animal liver, different according to hydrolyzing saponin glycosyl position, it is divided into four kinds of ginsenosidases: ginsenosidases I type, 3-O-(the 3rd carbon of Panaxadiol saponin Rb1, Rb2, Rb3, Rc etc. can be hydrolyzed) glycosyl and 20-O-glycosyl, progressively obtain Rd �� F2 �� C-K(or Rh2) etc. saponin; Ginsenosidases II type, can be hydrolyzed the 20-O-glycosyls such as Panaxadiol saponin Rb1, Rb2, Rb3, Rc, progressively generates Rd and Rg3; Ginsenosidases type III, can be hydrolyzed the 3-O-glycosyl of ginsenoside Rd, generates C-K; Ginsenosidases IV type, can be hydrolyzed the 6-O-glycosyl of ginsenoside Re and Rg2, generates Rg1 and Rh1.
Available ginsenosidases type III conversion Rd monomer saponin prepares C-K as mentioned above, but Rd monomer and ginsenosidases III pure enzyme cost are too high, and industrialization difficulty is big; Other diol saponin enzyme reactions, although produce C-K saponin, but reactant has a lot of other saponin by-product, not only C-K low conversion rate, but also add the step separating C-K in product, troublesome poeration.
Paper (J.Microbiol.Biotechnol.2011,21 (10), 1057 1063) report, ginsenosidases IV from aspergillus oryzae is hydrolyzed the 6-O-rhamnose of Re and R1 saponin and xylosyl becomes Rg1, the 6-O-glucosyl group being hydrolyzed Rg1 further becomes F1 saponin, and Rg2 is converted into Rh1 and 20 (S)-Panaxoside A aglycone Aj.
Paper (J.Microbiol.Biotechnol.2012; 22,343 ~ 351;Paper PLOSONE, 2014, (9), 1-16, e96914 (PosOne:June2014/Volume9/Issue6/e96914, document 5) report, the ginsenosidases that the enzyme of a lot of newfound microorganisms, transgene clone obtain, can be hydrolyzed ginsenoside Rb1, Rb2, Rc, Rd, the generation rare saponin F2 of corresponding low glycosyl of Re and Rg1, C-K, F1,20 (S)-Rg2,20 (S)-Rg3,20 (S)-Rh1,20 (S)-Rh2; But these enzyme reactions produce a lot of by-products.
(document: MKanaoke, TOkao, the KKobashi.JTradMed.199411 such as Japan's foot bridge, 241-245) report, after Radix Ginseng is oral, under the effect of intestinal, Radix Ginseng glycols saponin Rb1 etc. gradates as Rd, F2, C-K, 20 (S)-diol saponins units; Panaxatriol saponins Re etc. gradates and is absorbed for Rg1, F1,20 (S)-saponin triol units, but its conversion is faint.
Therefore, enzyme transforming process hydrolysis panoxadiol saponins Rb1, Rb2, Rb3, Rc, Rd disclosed in prior art prepares C-K saponin or enzyme transforming process hydrolysis panaxatriol saponins Re, R1, Rg1 prepare F1 saponin, all there is by-product many, the shortcomings such as rare saponin C-K or F1 low conversion rate, and needing separating-purifying from numerous by-products, troublesome poeration, difficulty are big; And above-mentioned enzyme reaction can only generate 20 (S)-Panaxadiol saponin units and 20 (S)-protopanaxatriol ginsenoside unit in trace ground, it is impossible to produce the sapogenin of four kinds of isomers.
Summary of the invention
The present invention is to solve the above-mentioned technical problem existing for prior art, it is provided that a kind of simple to operate, cost is low, yield and purity high, be suitable for the preparation method of Radix Ginseng rare saponin C-K, F1 and the four kinds of isomer ginsengenins produced in enormous quantities.
The technical solution of the present invention is: the preparation method of a kind of Radix Ginseng rare saponin C-K, F1 and four kinds of isomer ginsengenins, it is characterised in that carry out in accordance with the following steps:
A. with Radix Ginseng for raw material, panoxadiol's saponins mixture and panaxatriol's saponins mixture are extracted;
Specifically can be as follows:
Radix Ginseng, Radix Panacis Quinquefolii or Radix Notoginseng are shredded, extract 6��24 hours room temperature or 30��50 DEG C with 8��10 times of (weight/volume) methanol or 70��80% ethanol, repeat to extract 3 times, united extraction liquid, it is evaporated to Baume degrees 20��24(and reclaims organic solvent), obtain concentrated solution; By the concentrated solution petroleum ether degreasing 2��3 times of 30��50% volumes, after defat, Radix Ginseng extractive solution adds 3��dilution of the water of 5 times of volumes, macroporous resin column 2 times of volumes of Radix Ginseng weight (column volume be) adsorbs saponin repeatedly, with the deionized water eluting of 5��8 times of volumes except impurity such as sugar; Then with alcohol-water gradient elution resin column: ethanol concentration gradient is 30%��84%, eluant cumulative volume is 7��10 times of column volume. Detect saponin by TLC method, collect Protopanaxatriol's saponins eluent part and protopanoxadiol saponins eluent part respectively; Distinguish concentrating under reduced pressure (recovery ethanol) again, dry, respectively obtain protopanaxatriol (PPT) saponin and protopanoxadiol class (PPD) saponin.
The protopanoxadiol saponins extracted from Radix Ginseng detects through HPLC method, and mainly containing Rb1, Rb2, Rc, Rd, on a small quantity containing Rb3, Protopanaxatriol's saponins is mainly containing Re and Rg1; From the protopanoxadiol saponins of Radix Panacis Quinquefolii extraction mainly containing Rb1, Rb2, Rc, Rd, Protopanaxatriol's saponins is mainly containing Re and Rg1;From the protopanoxadiol saponins of Radix Notoginseng extraction mainly containing Rb1, Rd, Protopanaxatriol's saponins is mainly containing Rg1, Re, R1.
B. respectively with panoxadiol's saponins mixture or panaxatriol's saponins mixture for raw material, become substrate solution with organic solvent and buffer, then the crude enzyme liquid that substrate solution and aspergillosis fermentable obtain is reacted;
C. extract saponin in reactant liquor respectively, obtain Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng.
In described substrate solution, the pH of buffer is 4.5��6.0, and concentration is 0.01��0.05M, and the mass concentration of organic solvent is 5��40%, and the mass concentration of panoxadiol's saponins mixture or panaxatriol's saponins mixture is 0.5��8%; Described buffer is acetate buffer solution, citrate buffer solution or phosphate buffer, and described organic solvent is propanol, acetone, ethanol or methanol.
Described crude enzyme liquid is to adopt Aspergillus niger or aspergillus oryzae liquid fermentation or solid fermentation, and the product enzyme induction thing of enzyme fermentation culture medium is Radix Ginseng powder, Radix Ginseng total saponins or Flos Sophorae powder; To the centrifugal slagging-off of fermentation enzyme liquid or solid fermentation zyme extract, collect pheron precipitation, pheron is precipitated and dissolved in buffer.
Specifically can be as follows: the enzyme induction thing dosage that produces of liquid culture medium is the 0.005��0.1% of culture medium quality; The enzyme induction thing dosage that produces of solid medium is the 1��25% of culture medium quality; Fermentation culture temperature 27��35 DEG C, 3��9 days cultivation and fermentation time. To the centrifugal slagging-off of fermentation enzyme liquid or solid fermentation zyme extract, supernatant ammonium sulfate, methanol or ethanol precipitation pheron, collect precipitation, its pheron be precipitated and dissolved in 0.005��0.1 mole, pH be 4.5��6.5 acetic acid, citric acid or phosphate buffer; The buffer consumption of lyase albumen precipitation: 1/5��1/20 volume of liquid fermentation fermentation liquid; During solid fermentation fermentation, the bulking value of 0.5��2 times of solid material, it is available crude enzyme liquid.
It is mixed with crude enzyme liquid by substrate solution that described substrate solution reacts with crude enzyme liquid, reacts 16��48 hours at 35��60 DEG C; The volume ratio of described substrate solution and crude enzyme liquid is 1:0.1 ~ 1.
It is as follows that described step c extracts saponin in reactant liquor: adds the organic solvent deposit pheron of 2��4 times of volumes of reactant liquor, obtain supernatant, concentrated solution is obtained through decolouring macroporous resin column decolouring, concentrating under reduced pressure and recovery organic solvent, again gained concentrated solution is adsorbed saponin repeatedly through macroporous resin column, with the deionized water eluting impurity of 5��8 times of column volumes, again with the organic solvent eluting saponin that the mass concentration of 4��6 times of column volumes is 80��96%, concentration, crystallization and dry eluent, obtain Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng. The pheron precipitated, is dissolved in the buffer of 70��80% original enzyme liquid volumes, is recycled enzyme liquid, reusable; That is, enzyme is without immobilization, reuses.
Obtained Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng can be reacted with Radix Ginseng self the saponin multienzyme complex extracted from Radix Ginseng, react 1.5��10 hours at 60��85 DEG C, extract saponin in reactant liquor respectively, obtain by 20 (S)-Panaxadiol saponin unit, 20 (R)-Panaxadiol saponin unit, its 20-hydroxyls dehydrate 20 (21), 24-diene Panaxadiol saponin unit and 20 (22), the Panaxadiol saponin unit of four kinds of isomer compositions of 24-diene Panaxadiol saponin unit;Or obtain 20 (S)-protopanaxatriol ginsenoside unit, 20 (R)-protopanaxatriol ginsenoside unit, its 20-hydroxyls dehydrate 20 (21), 24-diene protopanaxatriol ginsenoside unit and 20 (22), the protopanaxatriol ginsenoside unit of four kinds of isomer compositions of 24-diene protopanaxatriol ginsenoside unit.
Described Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng is 1:0.1��10 with the mass ratio of Radix Ginseng self the saponin multienzyme complex of extraction from Radix Ginseng, added with the formic acid of reaction volume 0.1��50%, acetic acid or citric acid in described reaction system, personal rare saponin C-K or Radix Ginseng rare saponin F1 reaction mass concentration 0.1��10%.
Described Radix Ginseng self saponin multienzyme complex is prepared as follows: extract in the ginseng residue after panoxadiol's saponins mixture and panaxatriol's saponins mixture in described a step, add 5��6 times of water of ginseng residue's dry weight, stir 2��3 hours at 55��75 DEG C, centrifugal collection supernatant, below 65 DEG C, fine vacuum is concentrated into Baume degrees 20��25, is Radix Ginseng self saponin multienzyme complex.
The present invention is that the thick enzyme obtained with aspergillosis fermentable reacts with panoxadiol's saponins (mixture of Rb1, Rb2, Rb3, Rc, Rd saponin) or panaxatriol's saponins (mixture of Re, Rg1, R1 saponin), reaction add organic solvent and controls reaction temperature, can efficiently prepare Radix Ginseng rare saponin C-K and F1; Prepared rare saponin C-K or F1 of Radix Ginseng reacts with Radix Ginseng self saponin multienzyme complex, can prepare Panaxadiol saponin unit and the protopanaxatriol ginsenoside unit of four kinds of isomers respectively. Efficiency of pcr product of the present invention and purity high (equal more than 90%), almost without by-product saponin, it is not necessary to separate further, simple to operate, cost is low, it is suitable for producing in enormous quantities; Products obtained therefrom can be used for drug development, ginseng product, health product and cosmetics.
Accompanying drawing explanation
Fig. 1 is the Panaxadiol saponin unit isomer HPLC figure of the embodiment of the present invention 3 preparation.
Fig. 2 is the protopanaxatriol ginsenoside unit isomer HPLC figure of the embodiment of the present invention 4 preparation.
Fig. 3 is that the embodiment of the present invention 5 is separated the Panaxadiol saponin unit HPLC figure obtained.
Fig. 4 is that the embodiment of the present invention 6 is separated the protopanaxatriol ginsenoside unit HPLC figure obtained.
Detailed description of the invention
Embodiment 1:
A. with Radix Ginseng for raw material, panoxadiol's saponins mixture and panaxatriol's saponins mixture are extracted:
Radix Ginseng (band fibrous root) 1 kilogram, cuts into slices (2��4 millimeters thick), adds 8 liters of methanol and soaks, at 35��40 DEG C, and stirring extraction 6��12 hours, extraction fluid, in triplicate; Filtration, united extraction liquid, concentrating under reduced pressure (recovery methanol), to proportion Baume degrees 20, adds 500 milliliters of petroleum ether, stirring defat 1 hour, separates oil layer, repeat 2 times. Radix Ginseng extractive solution after defat is added 3��dilution of the water of 5 times of volumes, in macroporous resin (AB-8 resin or the HP-20 resin) post of 1.5 liters of volumes, repeatedly adsorb saponin, with the deionized water eluting post of 7.5��10 liters, except the impurity such as sugared; The macroporous resin column of its absorption saponin, use alcohol-water gradient elution: ethanol concentration gradient is 30%��84%, eluant cumulative volume 11 liters, every 300 milliliters of fraction collections: with TLC method detection saponin, containing Protopanaxatriol's saponins in its eluent forward part, containing protopanoxadiol saponins in eluent rear section; Its protopanaxatriol saponin fraction, protopanoxadiol saponins part, merge respectively, distinguish concentrating under reduced pressure (recovery ethanol), dry, obtain 20.3 grams of panaxatriol's class (PPT) saponin, 31.7 grams of panoxadiol's class (PPD) saponin.
Same method, methanol or 70��80% soak with ethanol Radix Panacis Quinquefolii or Radix Notoginseng, separable extraction protopanoxadiol saponins and Protopanaxatriol's saponins.
Protopanoxadiol saponins and Protopanaxatriol's saponins of 4��8% can be extracted from Radix Ginseng or Radix Panacis Quinquefolii root; 8��11% protopanoxadiol saponins and Protopanaxatriol's saponins can be extracted from Radix Notoginseng root.
B. with panoxadiol's saponins mixture for raw material, become substrate solution with organic solvent and buffer, then the crude enzyme liquid that substrate solution and aspergillosis fermentable obtain reacted:
Described crude enzyme liquid is prepared according to following conventional method:
By Aspergillus niger Aspergillusnigerg.848 bacterium, (from Daqu (massive raw stater for alcholic liquor), separation obtains; Document, Liu Chunying etc. JGinsengResearch2015,39,221-229. Culture presevation institute of Dalian Polytechnic University, preserving number: Aspergillusnigerg.848 bacterium) it is inoculated in 5% malt extract medium of the Radix Ginseng total saponins containing 0.03%, 28��33 DEG C of cultivations, stirring fermentations 4��6 days of ventilating, centrifugal slagging-off; Supernatant adds the ammonium sulfate powder of 75% saturation, at 4 DEG C overnight, precipitates pheron, collect pheron precipitation; Dialyse with the acetate buffer solution of 0.01 mole, pH5.0, add 0.02 mole, the fermentating liquid volume of the acetate buffer solution to 1/10th of pH5.0, crude enzyme liquid.
Above-mentioned prepared ginseng raw glycols saponin 30 grams adds 90 ml methanol and the acetate buffer solution of 210 milliliters 0.02 mole, pH5.0, stirring and dissolving, adds the crude enzyme liquid of 300 milliliters of above-mentioned preparations, 45 DEG C of stirring reactions 30 hours. Detecting by TLC method, do not have other saponin in reactant liquor, more than 90% is C-K saponin.
C. extract saponin in reactant liquor, obtain the rare saponin C-K of Radix Ginseng:
Adding the methanol mixed of 2000 milliliters in reactant liquor, left undisturbed overnight makes pheron precipitate, and centrifugation pheron precipitates, and adds 210 milliliters of buffer, recovery enzyme, reuse, the enzyme response rate is up to more than 70��90%.
The supernatant that pheron precipitate and separate obtains, the D-280 macropore decolorizing resin post decolouring of 700 ml volumes through anticipating, again with the saponin in the methanol solution eluting resin column of 1500 milliliters of mass concentrations 80%, its destaining solution concentrating under reduced pressure (recovery methanol) dilutes with 300 milliliters of water afterwards, macroporous resin column (AB-8 or HP-20 resin) then through 700 ml volumes adsorbs saponin repeatedly, with impurity such as the saccharides in the deionized water eluting post of 4200 milliliters, then with mass concentration 85��90% methanol-eluted fractions saponin of 3500 milliliters, concentrating under reduced pressure, crystallization, dry, obtain 16 grams of rare saponin C-K of product Radix Ginseng, HPLC detects, its purity more than 90%.
In above-mentioned reaction: utilize the buffer (acetate buffer solution, citrate buffer solution or phosphate buffer) containing organic solvent (propanol, acetone, ethanol or methanol) from panoxadiol's saponins (PPD) of Radix Ginseng or Radix Panacis Quinquefolii or the extraction of Radix Notoginseng root, be configured to the substrate solution of panoxadiol's saponins; The pH of buffer of substrate solution is 4.5��6.0, and concentration is 0.01��0.05M, and organic solvent mass concentration is 5��40%; Panoxadiol's saponins substrate mass concentration is 0.5��8%; Substrate solution is mixed according to volume ratio 1:0.1 ~ 1 with crude enzyme liquid, reacts 16��40 hours at 35��60 DEG C; Products therefrom is nearly all C-K saponin, the organic solvent (propanol, acetone, ethanol or methanol) adding 2��4 times of volumes of reactant liquor precipitates pheron, centrifugal collection pheron, it is dissolved in the acetate buffer solution of the pH5 of original enzyme liquid mass concentration 70��80%, citrate buffer solution or phosphate buffer, is crude enzyme liquid reusable.
The supernatant of centrifugation pheron, through decolouring macroporous resin column (column volume, 20 times of volumes of PPD saponin raw material weight) decolouring, concentrating under reduced pressure, recovery organic solvent, its concentrated solution is through macroporous resin column (column volume, 20 times of volumes of PPD saponin raw material weight) repeatedly adsorb saponin, with impurity such as the deionized water eluting of 5��8 times of column volumes sugar; Again with organic solvent (propanol, acetone, ethanol or methanol) the eluting saponin of the mass concentration 80��96% of 4��6 times of column volumes, concentration, crystallization, dry, be similarly obtained the rare saponin C-K of Radix Ginseng of purity more than 90%.
Embodiment 2:
A. with Radix Ginseng for raw material, panaxatriol's saponins mixture is extracted with embodiment 1;
B. with panaxatriol's saponins mixture for raw material, become substrate solution with organic solvent and buffer, then the crude enzyme liquid that substrate solution and aspergillosis fermentable obtain reacted:
Described crude enzyme liquid is prepared according to following conventional method:
Aspergillus oryzae Aspergillusoryzaesp.39g(separates from Daqu (massive raw stater for alcholic liquor) and obtains; Document, Wang Dongming, the red sudden strain of a muscle of fish etc. ProcessBiochemistry2012,47,133-138. Culture presevation institute of Dalian Polytechnic University, preserving number: Aspergillusoryzaesp.39g) be inoculated in the moisture 50% equipped with sterilizing 1 kilogram of Testa Tritici bent casket in (containing 50 grams of Radix Ginseng powder), 28��32 DEG C, 4��7 days (every day stirs 4��6 times) of cultivation, then leach enzyme with the acetate buffer solution of 0.02 mole of 5 liters, pH5.0; Enzyme liquid is centrifuged, and its supernatant ammonium sulfate solids precipitates pheron; Centrifugal collecting precipitation, is dissolved in 0.02 mole of 500 milliliters, the acetate buffer solution of pH5.0; It is crude enzyme liquid.
Protopanaxatriol's saponins (mainly containing Rg1, Re, R1 saponin) that prepared by Example 1 20 grams extract from Radix Notoginseng adds the citrate buffer solution of 30 milliliters of ethanol and 270 milliliters 0.02 mole, pH5.0, stirring and dissolving, add the crude enzyme liquid 300 milliliters of above-mentioned preparation, 45 DEG C of stirring reactions 48 hours, detect by TLC method, reactant liquor is mainly F1 saponin;
C. extract saponin in reactant liquor, obtain the rare saponin F1 of Radix Ginseng:
In reactant liquor add 1800 milliliters ethanol mixing, left undisturbed overnight makes pheron precipitate, centrifugation pheron precipitate, add 210 milliliters of buffer, recovery enzyme, reusable as mentioned above.
Separate enzyme and precipitate the enzyme reaction supernatant obtained, the D-280 decolouring macroporous resin decolouring of 700 ml volumes through anticipating, again with the saponin in the ethanol eluting post of 1500 milliliters of mass concentrations 75%, its destaining solution concentrating under reduced pressure (recovery ethanol), dilute with 300 milliliters of water, saponin is repeatedly adsorbed through macroporous resin column (AB-8 or HP-20 resin), with impurity such as the saccharides in the deionized water eluting post of 4200 milliliters, then with the mass concentration 85% ethanol elution saponin of 3500 milliliters, concentrating under reduced pressure, crystallization, dry, obtain 11 grams of product F1, HPLC detects, its purity more than 90%.
In above-mentioned reaction, panaxatriol's saponins from Radix Ginseng or American ginseng root or the extraction of Radix Notoginseng root, utilize the buffer (acetate buffer solution, citrate buffer solution or phosphate buffer) containing organic solvent (propanol, acetone, ethanol or methanol), be configured to the substrate solution of panaxatriol's saponins (PPT); In substrate solution, pH of buffer is 4.5��6.0, and concentration is 0.01��0.5M, and organic solvent content is mass concentration 5��40%, and panaxatriol's saponins (PPT) substrate content is mass concentration 0.5��8%.Its substrate solution crude enzyme liquid mixes according to volume ratio 1:0.1 ~ 10, reacts 16��48 hours at 35��60 DEG C, and product is nearly all F1 saponin. Add the organic solvent (propanol, acetone, ethanol or methanol) of 2��4 times of volumes of reactant liquor, precipitate pheron, centrifugal collection pheron equally, being dissolved in the buffer (acetate buffer solution, citrate buffer solution or phosphate buffer) of the pH5 of original enzyme liquid mass concentration 70��80%, enzyme is reusable.
Separate the centrifugal supernatant of pheron, through decolouring macroporous resin column (column volume, 20 times of volumes of PPT saponin raw material weight) decolouring, concentrating under reduced pressure, recovery organic solvent, its concentrated solution is through macroporous resin column (column volume, 20 times of volumes of PPT saponin raw material weight) repeatedly adsorb saponin, with impurity such as the deionized water eluting of 5��8 times of column volumes sugar; Again with organic solvent (propanol, acetone, ethanol or methanol) the eluting saponin of the mass concentration 80��94% of 4��6 times of column volumes, concentration, crystallization, dry, be similarly obtained the rare saponin of F1 of purity more than 90%.
Embodiment 3:
Prepared by Radix Ginseng self saponin multienzyme complex: take the deionized water adding 6 liters in the slag after Radix Ginseng described in 1 kilogram of embodiment 1 extracts saponin, stir 2��3 hours at 55��65 DEG C, centrifugal collection supernatant, fine vacuum concentration (product temperature less than 65 DEG C) is to about about 500 milliliters of Baume degrees 25(), it is Radix Ginseng self saponin multienzyme complex.
Obtained Radix Ginseng self saponin multienzyme complex is reacted with the obtained C-K of embodiment 1, reaction system adds the formic acid of reaction volume 0.1��50%, acetic acid or citric acid and reduces pH reaction, the Panaxadiol saponin unit of four kinds of isomers can be prepared.
Specific as follows: take the product C-K saponin of 20 grams of embodiments 1,50 grams Radix Ginseng self saponin multienzyme complex, deionized water 350 milliliters, citric acid 25 grams mixing, 70��75 DEG C react 2 hours. Detect by TLC method, after reaction thoroughly, add 400 milliliters of water-saturated n-butanols and extract saponin reactant, in triplicate, merge n-butanol layer, with the deionized water wash n-butyl alcohol 3��4 times of 600 milliliters, concentrating under reduced pressure (recovery n-butyl alcohol), dry, obtain 12��14 grams of Panaxadiol saponin units. Detecting through HPLC, its aglycon is four kinds of isomer compositions, specifically as shown in Fig. 1 and table 1:
The HPLC data of 1 four kinds of isomer Panaxadiol saponin units of table
As can see from Figure 1, prepared Panaxadiol saponin unit is made up of four kinds of isomers, through nmr for the determination structure, four kinds of isomers be 20 (S)-Panaxadiol saponin unit [20 (S)-PPDol], 20 (R)-Panaxadiol saponin unit [20 (R)-PPDol], its 20-hydroxyls dehydrate 20 (21), 24-diene Panaxadiol saponin unit [PPDol (-H2O-20 (21), 24-diene] and 20 (22), 24-diene Panaxadiol saponin unit [PPDol (-H2O-20 (22), 24-diene]; Each sapogenin content in total sapogenins is than 32.4:16.7:23.3:27.6, and in product, the mass content of total sapogenins is more than 90%.
The above results, the ratio (weight) of ginseng saponin C-K aqueous solution and Radix Ginseng self multienzyme complex enzyme (calculating of Radix Ginseng dry product raw material) is 1:0.1��10 times; The dosage of formic acid, acetic acid or citric acid is the 0.1��50% of reaction volume; In reactant liquor, ginseng saponin C-K mass concentration is 0.1 ~ 10%, in 60��85 DEG C are reacted 1.5��10 hours, obtains same result; Product detects through HPLC, four kinds of isomer Panaxadiol saponin unit content be total aglycones to more than the 90% of quality, wherein 20 (S)-Panaxadiol saponin units and the first content ratio at total sapogenins of 20 (R)-Panaxadiol saponin are between quality 20 ~ 80%.
Embodiment 4:
Radix Ginseng self saponin multienzyme complex is prepared with embodiment 3.
Obtained Radix Ginseng self saponin multienzyme complex is reacted with the obtained F1 of embodiment 2, reaction system adds the formic acid of reaction volume 0.1��50%, acetic acid or citric acid and reduces pH reaction, the Panaxoside A aglycone Aj of four kinds of isomers can be prepared.
Concrete grammar can be as follows:
The F1 saponin of 10 grams, Radix Ginseng self the saponin multienzyme complex of 50 grams, deionized water 120 milliliters, acetic acid (acetic acid) 30 milliliters mixing, react 2 hours at 70��75 DEG C; Detect by TLC method, after reaction thoroughly, add 200 milliliters of water-saturated n-butanols and extract saponin reactant, in triplicate, merge n-butanol layer, with the deionized water wash n-butyl alcohol 3��4 times of 300 milliliters, concentrating under reduced pressure (recovery n-butyl alcohol), dry, obtain 6 grams of protopanaxatriol ginsenoside units. Detecting through HPLC, its aglycon is four kinds of isomer compositions: as shown in Fig. 2 and table 2:
The HPLC data of 2 four kinds of isomer protopanaxatriol ginsenoside units of table
From Fig. 2 and table 2 it can be seen that, prepared protopanaxatriol ginsenoside unit is made up of four kinds of isomers, through nmr for the determination structure, four kinds of isomers are 20 (S)-protopanaxatriol ginsenoside unit [20 (S)-PPTol], 20 (R)-protopanaxatriol ginsenoside unit [20 (R)-PPTol], the 20 (21) of its 20-hydroxyls dehydrate, 24-diene protopanaxatriol ginsenoside unit [PPTol (-H2O-20 (21), 24-diene] and 20 (22), 24-diene protopanaxatriol ginsenoside unit [PPTol (-H2O-20 (22), 24-diene], each sapogenin content in total sapogenins compares 26.5:22.4:14.4:36.7, in product, the mass content of total sapogenins is more than 90%.
The above results, the ratio (weight) of GF1 aqueous solution and Radix Ginseng self multienzyme complex (calculating of Radix Ginseng dry product raw material) is 1:0.1��10 times; The dosage of formic acid, acetic acid or citric acid is the 0.1��50% of reaction volume; In reactant liquor, GF1 mass concentration is 0.1 ~ 10%, in 60��85 DEG C are reacted 1.5��10 hours, obtains same result; Both in product, Panaxoside A aglycone Aj's mass content of four kinds of isomers was more than 90%, and wherein 20 (S)-protopanaxatriol ginsenoside units and 20 (R)-protopanaxatriol ginsenoside unit are between 20 ~ 80% in the content ratio of total sapogenins.
Embodiment 5:
Each sapogenin monomer separation of four kinds of isomer Panaxadiol saponin units prepared by embodiment 3: adopt the high performance liquid preparative chromatography instrument (any product is all available) of conventional city's pin, separate with C18 preparative hplc post, acetonitrile-water gradient.
Take 10 grams of embodiment 3 gained Panaxadiol saponin units, separate with C18 preparative hplc post, acetonitrile-water gradient, respectively obtain 20 (S)-Panaxadiol saponin units of 2.5 grams successively, 20 (R)-Panaxadiol saponin units of 1.2 grams, the 20 (21) of the 20-hydroxyls dehydrate of 1.6 grams, 24-diene Panaxadiol saponin unit, the 20 (22) of the 20-hydroxyls dehydrate of 1.8 grams, 24-diene Panaxadiol saponin unit; Detecting through HPLC, its four kinds of sapogenin purity are more than 90%.
As shown in Figure 3: 1,20 (S)-Panaxadiol saponin unit; 2,20 (R)-Panaxadiol saponin units; The 20 (21) of 3,20-hydroxyls dehydrate, 24-diene Panaxadiol saponin unit; 4, the 20 (22) of 20-hydroxyls dehydrate, 24-diene Panaxadiol saponin unit.
Embodiment 6:
Each sapogenin monomer separation of four kinds of isomer protopanaxatriol ginsenoside units prepared by embodiment 4: adopt the high performance liquid preparative chromatography instrument (any product is all available) of conventional city's pin, separate with C18 preparative hplc post, acetonitrile-water gradient.
Take the protopanaxatriol ginsenoside unit of embodiment 4 preparation of 10 grams, separate with C18 preparative hplc post, acetonitrile-water gradient, respectively obtain 20 (S)-protopanaxatriol ginsenoside units of 1.4 grams successively, 20 (R)-protopanaxatriol ginsenoside units of 1.2 grams, the 20 (21) of the 20-hydroxyls dehydrate of 0.9 gram, 24-diene protopanaxatriol ginsenoside unit, the 20 (22) of 2.2 grams, 24-diene protopanaxatriol ginsenoside unit; Detecting through HPLC, the purity of each sapogenin is more than 90%.
As shown in Figure 4:
1,20 (S)-protopanaxatriol ginsenoside unit; 2,20 (R)-protopanaxatriol ginsenoside units; The 20 (21) of 3,20-hydroxyls dehydrate, 24-diene protopanaxatriol ginsenoside unit; 4, the 20 (22) of 20-hydroxyls dehydrate, 24-diene protopanaxatriol ginsenoside unit.
It is ginseng saponin C-K and four kinds of isomers of Panaxadiol saponin unit respectively to confirm the product prepared by embodiment, four kinds of isomers of GF1 and protopanaxatriol ginsenoside unit, by Switzerland's BrukeAVANCE600 NMR spectrometer with superconducting magnet, popping one's head in as 5mmBBO, solvent is Pyridine-D5(deuterated pyridine), determines four kinds of isomer monomers of above-mentioned prepared C-K and Panaxadiol saponin unit, four kinds of isomer monomer structures of GF1 and protopanaxatriol ginsenoside unit: its13C-NMR data, as shown in table 3:
The four kinds of isomers of table 3. ginseng saponin C-K and Panaxadiol saponin unit, four kinds of isomers 13C-NMR data (ppm) of GF1 and protopanaxatriol ginsenoside unit
The four kinds of isomers of the ginseng saponin C-K of table 3 and Panaxadiol saponin unit, the determination data of four kinds of isomers of GF1 and protopanaxatriol ginsenoside unit and list of references:
1) InTanaka, O.andKasai, R.:Saponinsofginsengandrelatedplants, Progressinthechemistryoforganicnaturalproducts.1984, Volume46,1-76, theIV.ModernTechniquesUsedinStructureDetermination, p22-36. are (2.13C-NMRSpectroscopyofDammaraneTypeTriterpenes,p23-26;3.GlycosylationShiftsin13C-NMRSpectra,p26-35)
2) Li Yaping (chief editor). ginsenoside's NMR standard diagram. Chemical Industry Press, Beijing, 2012.
Through comparing, consistent with list of references. Prove that prepared product is the four kinds of isomers of ginseng saponin C-K and Panaxadiol saponin unit:
20 (S)-Panaxadiol saponin unit [20 (S)-PPDol],
20 (R)-Panaxadiol saponin unit [20 (R)-PPDol],
The 20 (21) of 20-hydroxyls dehydrate, 24-diene Panaxadiol saponin unit [PPDol (-H2O-20(21),24-diene],
20 (22), 24-diene Panaxadiol saponin unit [PPDol (-H2O-20 (22), 24-diene];
Its structure is consistent with described in background technology.
Prove that prepared product is the four kinds of isomers of GF1 and protopanaxatriol ginsenoside unit and is simultaneously:
20 (S)-protopanaxatriol ginsenoside unit [20 (S)-PPTol];
20 (R)-protopanaxatriol ginsenoside unit [20 (R)-PPTol];
The 20 (21) of 20-hydroxyls dehydrate, 24-diene protopanaxatriol ginsenoside unit [PPTol (-H2O-20 (21), 24-diene],
The 20 (22) of 20-hydroxyls dehydrate, 24-diene protopanaxatriol ginsenoside unit [PPTol (-H2O-20 (22), 24-diene];
Its structure is consistent with described in background technology.
The ginsenoside's assay method adopted in the embodiment of the present invention:
1) thin layer chromatography (TLC) measures ginsenoside:
Radix Ginseng PPD saponin and PPT saponin measure: chromatoplate, silica gel plate SilicaGel60F254(Germany Merck product); Developing solvent, n-butyl alcohol: ethyl acetate: water=4:1:2. After the rare saponin of Radix Ginseng launches on the tlc plate, dry up, spray 10% sulphuric acid, 110 DEG C of colour developings;
Enzyme reaction generates the mensuration of C-K, F1: chromatoplate, silica gel plate SilicaGel60F254(Germany Merck product); Developing solvent, chloroform: methanol: water=7.5:2.5:0.5. After the rare saponin of Radix Ginseng launches on the tlc plate, dry up, spray 10% sulphuric acid, 110 DEG C of colour developings;
C-K, F1 convert and obtain the mensuration of panoxadiol's aglycon and Panaxoside A aglycone Aj: chromatoplate, silica gel plate SilicaGel60F254(Germany Merck product); Developing solvent, chloroform: methanol: water=7.5:2.5:0.5. After the rare saponin of Radix Ginseng launches on the tlc plate, dry up, spray 10% sulphuric acid, 110 DEG C of colour developings.
2) high performance liquid chromatography (HPLC) method measures ginsenoside:
Ginsenoside, C-K and F1 mensuration: chromatograph, U.S. Waters2695 efficient liquid phase chromatographic analysis instrument, Waters2996 photodiode array (PDA) detector and Empower chromatographic work station; Chromatographic column: KnauerC18 post (5 ��m, �� 3mm �� 250mm); Mobile phase: acetonitrile (A)-water (B): 0-20min, 20%A etc. spend; 20-31min, 20%A-32%A linear gradient, 31-40min, 32%A-43%A linear gradient; 40-70min, 43%A-100%A linear gradient; Sample size: 10 �� L; Column temperature: 35 DEG C; Volume ratio flow velocity: 0.6mL/min; Detection wavelength: 203nm.
The mensuration of four kinds of isomer Panaxadiol saponin units and protopanaxatriol ginsenoside unit: chromatographic condition: chromatograph, Waters2695 efficient liquid phase chromatographic analysis instrument, Waters2996 photodiode array (PDA) detector and Empower chromatographic work station; Chromatographic column: UnitaryC18 post (5 ��m, �� 4.6mm �� 250mm); Mobile phase: acetonitrile (A)-water (B): 0-5min, 20%A-40%A etc. spend; 5-10min, 40%A-60%A linear gradient, 10-70min, 60%A-100%A linear gradient; Sample size: 10 �� L; Column temperature: 35 DEG C; Volume ratio flow velocity: 0.6mL/min; Detection wavelength: 203nm.
3) nuclear magnetic resonance method (NMR) measures the structure of Radix Ginseng rare saponin C-K and the F1 that enzyme reaction obtains, each sapogenin:
Instrument is Switzerland BrukeAVANCE600 superconduction nuclear magnetic resonance, NMR POP instrument; Probe, 5mmBBO; Solvent, Pyridine-D5 (deuterated pyridine). With nmr for the determination: the rare saponin of monomer Radix Ginseng that enzymatic conversion obtains one-dimensional1HNMR��13CNMR, DEPT collection of illustrative plates; Two dimension hydrogen spectrum COSY, two-dimentional hydrocarbon Correlated Spectroscopy HSQC and HMBC; One-dimensional1The observing frequency of HNMR spectrum is 600MHz,13When the observing frequency of CNMR spectrum is 150MHz. That obtain, Radix Ginseng rare saponin C-K and F1, each sapogenin by measuring respectively1HNMR��13CNMR, DEPT45, DEPT90, DEPT135, COSY, HSQC, HMBC nuclear magnetic resonance map, and the comparing of list of references 1 and 2, confirm the structure of Radix Ginseng rare saponin C-K and the F1 that enzymatic conversion obtains, each sapogenin.
Claims (8)
1. a preparation method for Radix Ginseng rare saponin C-K, F1 and four kinds of isomer ginsengenins, it is special
Levy and be in that to carry out in accordance with the following steps:
A. with Radix Ginseng for raw material, separation and Extraction panoxadiol's saponins mixture, panaxatriol's saponins mixture;
B. respectively with panoxadiol's saponins mixture or panaxatriol's saponins mixture for raw material, become substrate solution with organic solvent and buffer, then the crude enzyme liquid that substrate solution and aspergillosis fermentable obtain is reacted;
C. extract saponin in reactant liquor respectively, obtain Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng.
2. the preparation method of Radix Ginseng rare saponin C-K, F1 and four kinds of isomer ginsengenins according to claim 1, it is characterized in that in described substrate solution, the pH of buffer is 4.5��6.0, concentration is 0.01��0.05M, the mass concentration of organic solvent is 5��40%, and the mass concentration of panoxadiol's saponins mixture or panaxatriol's saponins mixture is 0.5��8%; Described buffer is acetate buffer solution, citrate buffer solution or phosphate buffer, and described organic solvent is propanol, acetone, ethanol or methanol.
3. the preparation method of Radix Ginseng rare saponin C-K, F1 and four kinds of isomer ginsengenins according to claim 2, it is characterized in that described crude enzyme liquid is to adopt Aspergillus niger or aspergillus oryzae liquid fermentation or solid fermentation, the product enzyme induction thing of enzyme fermentation culture medium is Radix Ginseng powder, Radix Ginseng total saponins or Flos Sophorae powder; To the centrifugal slagging-off of fermentation enzyme liquid or solid fermentation zyme extract, collect pheron precipitation, pheron is precipitated and dissolved in buffer.
4. the preparation method of Radix Ginseng rare saponin C-K, F1 and four kinds of isomer ginsengenins according to claim 3, it is characterised in that it is mixed with crude enzyme liquid by substrate solution that described substrate solution reacts with crude enzyme liquid, reacts 16��48 hours at 35��60 DEG C; The volume ratio of described substrate solution and crude enzyme liquid is 1:0.1 ~ 1.
5. according to claim 1, 2, the rare saponin C-K of Radix Ginseng described in 3 or 4, the preparation method of F1 and four kinds of isomer ginsengenins, it is characterized in that described step c extracts saponin in reactant liquor as follows: add the organic solvent deposit pheron of 2��4 times of volumes of reactant liquor, obtain supernatant, through decolouring macroporous resin column decolouring, concentrating under reduced pressure and recovery organic solvent obtain concentrated solution, again gained concentrated solution is adsorbed saponin repeatedly through macroporous resin column, with the deionized water eluting impurity of 5��8 times of column volumes, again with the organic solvent eluting saponin that the mass concentration of 4��6 times of column volumes is 80��96%, concentration, crystallization and dry, obtain Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng.
6. the rare saponin C-K of Radix Ginseng according to claim 1, the preparation method of F1 and four kinds of isomer ginsengenins, it is characterized in that reacting obtained Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng with Radix Ginseng self the saponin multienzyme complex extracted from Radix Ginseng, react 1.5��10 hours at 60��85 DEG C, extract sapogenin in reactant liquor respectively, obtain by 20 (S)-Panaxadiol saponin units, 20 (R)-Panaxadiol saponin units, the 20 (21) of its 20-hydroxyls dehydrate, 24-diene Panaxadiol saponin unit and 20 (22), the Panaxadiol saponin unit of four kinds of isomer compositions of 24-diene Panaxadiol saponin unit, or obtain 20 (S)-protopanaxatriol ginsenoside unit, 20 (R)-protopanaxatriol ginsenoside unit, its 20-hydroxyls dehydrate 20 (21), 24-diene protopanaxatriol ginsenoside unit and 20 (22), the protopanaxatriol ginsenoside unit of four kinds of isomer compositions of 24-diene protopanaxatriol ginsenoside unit.
7. the preparation method of Radix Ginseng according to claim 6 rare saponin C-K, F1 and four kinds of isomer ginsengenins, it is characterized in that described Radix Ginseng rare saponin C-K or the rare saponin F1 of Radix Ginseng is 1:0.1��10 with the mass ratio of Radix Ginseng self the saponin multienzyme complex of extraction from Radix Ginseng, added with the formic acid of reaction volume 0.1��50%, acetic acid or citric acid in described reaction system, personal rare saponin C-K or Radix Ginseng rare saponin F1 reaction mass concentration 0.1��10%.
8. the preparation method of Radix Ginseng rare saponin C-K, F1 and four kinds of isomer ginsengenins according to claim 7, it is characterized in that described Radix Ginseng self saponin multienzyme complex is prepared as follows: extract in the ginseng residue after panoxadiol's saponins mixture and panaxatriol's saponins mixture in described a step, add 5��6 times of water of ginseng residue's dry weight, stir 2��3 hours at 55��75 DEG C, centrifugal collection supernatant, below 65 DEG C, fine vacuum is concentrated into Baume degrees 20��25, is Radix Ginseng self saponin multienzyme complex.
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CN111840121A (en) * | 2019-04-26 | 2020-10-30 | 东莞自然衡健康科技有限公司 | Cosmetic composition and method for preparing the same |
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CN113940952A (en) * | 2021-11-25 | 2022-01-18 | 瑞莱茵(北京)生物科技有限责任公司 | Ginseng fermented extract and preparation method thereof |
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