CN86105166A - Produce a kind of method of ginsenoside-rd - Google Patents
Produce a kind of method of ginsenoside-rd Download PDFInfo
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- CN86105166A CN86105166A CN198686105166A CN86105166A CN86105166A CN 86105166 A CN86105166 A CN 86105166A CN 198686105166 A CN198686105166 A CN 198686105166A CN 86105166 A CN86105166 A CN 86105166A CN 86105166 A CN86105166 A CN 86105166A
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Abstract
The present invention relates to a kind of method that helps industrial preparation ginsenoside-rd (ginsenoside a kind of).According to method of the present invention, can and produce ginsenoside-rd at an easy rate with high yield, high purity.Prepared ginsenoside-rd can be used as pain killer, tranquilizer, improve metabolism or improve the medicament of cardiovascular systems.
Description
The present invention relates to a kind of method of being convenient to industrial preparation ginsenoside-rd (in the ginsenoside a kind of).
Composition to genseng (for example root of genseng Panax ginseng C.A.Meyer) has carried out extensive studies in recent years, and has isolated the dissimilar ginsenoside as the ginseng effective component.In these ginsenosides, ginsenoside-rd (following claim that it is compound (I)) and ginsenoside-Rb
1(but is compound (II) to call it in the following text) belongs to the effective constituent that mainly contains of genseng.Pharmacological action to them is illustrated, known now these two kinds of ginsenoside-rds and-Rb
1, among many kind activity that they had, at the pharmacologically active of practicality, for example analgesic activity, sedative activity, the metabolic activity of improvement or improve the activity of cardiovascular systems.The pharmacologically active that has shown genseng soap-Rd is than ginsenoside-Rb
1Pharmacologically active much better than.But, in genseng (for example root of genseng Panax ginseng C.A.Meyer) content of ginsenoside-rd seldom so that from plant extracting it far be not industrial favourable method for making.In addition, genseng is very expensive.Therefore need a kind of productive rate height of development, purity is good, and the simple method of producing ginsenoside-rd.
Report is arranged recently from strand bone indigo plant (Gynostemma pentaphyllum Makino), with ginsenoside-Rb
1Extract ginsenoside-rd together, strand bone indigo plant is a kind of perennial vine, and spontaneous growth is in the various places, Asia, (Japanese pharmaceutical journal (Yakugaku zasshi), 103, the 173 pages (1983)); The not unexamined patent application number No.56-12739(12739/1981 of Japanese publication).But main saponin is not a ginsenoside-rd in the strand bone basket, but gypenoside V.
Ginsenoside-rd, ginsenoside-Rb
1With can claim below the gypenoside V(that it is compound (III)) structural formula below all available represents that their chemical name is as follows.
From the said structure formula, can find out, at ginsenoside-rd, ginsenoside-Rb
1, and gypenoside V in chemical structural formula on have only substituent R to have any different, so present inventors think that ginsenoside-rd can be from ginsenoside-Rb
1Or gype-noside V derives with chemical process.More particularly, think that the selectivity of R-O key ruptures, promptly at ginsenoside-Rb
1The alpha-L-rhamnoside bond rupture of the rutinose base section on the terminal β-D-glucoside bond of the gentiobiose base section on the 20-position or the gypenoside V 20-position will produce ginsenoside-rd.But, at ginsenoside-Rb
1Or among the gypenoside V, except that the R-O part, also have three glycosidic links to the hydrolysis sensitivity, only optionally this R-O of hydrolysis partly is difficult.For example, when under different acidic conditionss with ginsenoside-Rb
1Or gypenoside V is when being hydrolyzed,
Can not obtain ginsenoside-rd.
The hydrolysis reaction of highly selective has carried out deep research to present inventors for have more than acid hydrolysis, with a kind of enzyme reaction of specific enzymes from ginsenoside-Rb
1And/or gype-noside V produces ginsenoside-rd effectively and succeeds, so finished the present invention.
In other words, the present invention relates to the technological process of producing ginsenoside-rd, it comprises that rhamnosidase and/or Polyglucosidase are to gypenoside V and/or ginsenoside-Rb
1Effect.More particularly, according to technology of the present invention, ginsenoside-rd be with the rhamnoside enzyme to gypenoside V effect, or glucosidase is to ginsenoside-Rb
1Effect is produced.
Gypenoside V and ginsenoside-Rb as the starting raw material (substrate) of method of the present invention
1, can respectively it be separated, perhaps separate with its form of mixtures.From industrial point, preferably use and contain gypenoside V, ginsenoside-Rb
1With the blue extract of the strand bone of ginsenoside-rd, or contain ginsenoside-Rb
1With the extract of the ginseng of ginsenoside-rd as raw material (substrate).According to method of the present invention with gypenoside V or/and ginsenoside-Rb
1Be transformed into ginsenoside-Re, but the ginsenoside-rd in raw material does not change.Really, be best with the blue extract of strand bone as raw material (substrate), can buy very cheaply because twist bone indigo plant, and genseng is very expensive.The extract of blue extract of strand bone or ginseng is carried out the extract that enzymolysis can provide a kind of enrichment ginsenoside-rd as substrate according to method of the present invention.The material that extracts is like this carried out common separation and/or purification process again, chromatography for example, recrystallizations etc. obtain highly purified ginsenoside-rd soon.With extracting from strand bone indigo plant or ginseng, separate and/or the usual way of purifying ginsenoside-rd is compared, the ginsenoside-rd output that method of the present invention provides is big, purity height, and much easier.
Employed in the present invention enzyme is rhamnosidase and/or Polyglucosidase.Rhamnosidase and/or Polyglucosidase are to produce with the method for microorganisms, and this microorganism can produce rhamnosidase and/or Polyglucosidase in the liquid medium within, and accumulation rhamnosidase and/or Polyglucosidase.Comprise fungi and yeast in the example of these microorganisms.Come hydrolysis ginsenoside-Rb with these enzymes
1Glucoside bond and/or the rhamnosyl glycosidic bond of gypenoside V.Specifically, be the alpha-L-rhamnoside key on the rhamnoside enzymic hydrolysis gypenoside V, glucoside ginsenoside enzymatically hydrolyzing-Rb
1Terminal β-D-the glucoside bond of the gentiobiose base section of 20-position.Therefore, employed in the present invention rhamnosidase can be to the activated any enzyme of gypenoside V, and employed in the present invention Polyglucosidase can be can hydrolysis ginsenoside-Rb
1Any enzyme of terminal β-D-glucoside bond.These two kinds of enzymes use behind the purifying separately, also can be with their mixture.When doing substrate, can use the mixture of rhamnosidase or use rhamnosidase and Polyglucosidase, when using ginsenoside-Rb with gypenoside V or the blue extract of strand bone
1Or the extract of ginseng is when doing substrate, can use Polyglucosidase or use the mixture of Polyglucosidase and rhamnosidase.But will obtain pure enzyme generally needs a large amount of work and cost.In the present invention, this two kinds of enzymes even also can use at the raw product state are as long as rhamnosidase and/or Polyglucosidase that they contain have the activity that enough satisfies needs of the present invention.In addition, also can use to contain and have the liquid nutrient medium that is suitable for active rhamnosidase required for the present invention and/or Polyglucosidase.From industrial point, use the blue extract of strand bone as substrate and with thick enzyme or liquid nutrient medium as enzyme, be production ginsenoside-rd advantageous method the most.Enzymolysis of the present invention generally the pH value from about 4 to 7 and temperature carry out under about 40 to 65 ℃ condition.
As the thick enzyme that is applicable to effectiveness of the present invention, commercial commercially available crude zyme preparation all can use, for example " solubility hesperidinase " (being produced by Japanese Tanabe Seiyaku company).This " solubility hesperidinase " is a kind of thick enzyme, except that containing the activated rhamnosidase of gypenoside V, also contains Polyglucosidase.For example, when being added to " solubility hesperidinase " on the gypenoside V or add in the aqueous extract that twists bone indigo plant, mixture is remained on the pH value to be preferably in the buffered soln from about 6.8 to 7(), temperature is under about 50 to 60 ℃ condition, and productive rate that then can be very high produces ginsenoside-rd.When buffered soln uses common buffered soln commonly used, during as phthalate buffer, phosphate buffered saline buffer, acetate buffer etc., " solubility hesperidinase " is a kind of enzyme that is specially adapted to gypenoside V is changed into ginsenoside-rd.
The liquid nutrient medium that is suitable for effectiveness of the present invention can prepare like this, (for example: fungi, yeast) some bacterial strain is placed in the substratum that contains L-rhamnosyl or a kind of material (as carbon source) that contains the alpha-L-rhamnoside key and cultivates to be about to belong to Mycophyta, according to the method for following introduction, selection can be with gypenoside V and/or ginsenoside-Rb then
1Convert the bacterial strain of ginsenoside-rd to high yield.
In this liquid nutrient medium, except that Polyglucosidase, also accumulated activated rhamnosidase to gypenosi-de V, liquid nutrient medium itself also can be used as gypeno-side V and/or ginsenoside-Rb
1Be transformed into the enzyme source of ginsenoside-rd.For example, gypenoside V or the blue extracting solution of strand bone are added in this liquid nutrient medium, make mixture pH value be maintained at about 4 to 7, temperature is from about 40 to 65 ℃, be preferably in the pH value from about 5 to 7 and in temperature under about 40 to 55 ℃ condition, then can obtain ginsenoside-rd with high yield.Also have, with ginsenoside-Rb
1Or the extract of ginseng is added in this liquid nutrient medium, makes mixture remain on the pH value from about 4 to 7, and temperature is from about 40 to 65 ℃, and preferably the pH value is from about 5 to 7, and temperature then obtains ginsenoside-rd with high yield under about 40 to 55 ℃ condition.
The examples of substances that contains the alpha-L-rhamnoside key comprises the oligosaccharides (disaccharide is to tetrose) with rhamanopyranosyl, and the glucosides with rhamanopyranosyl, and the former can rutinose, neohesperidose etc. is an example, and the latter can Hesperidin, rutin, and citrus glycosides etc. is an example.
In order to obtain being suitable for the liquid nutrient medium of effectiveness of the present invention, when some belongs to the bacterial strain of Mycophyta in cultivation, can use a kind of aqueous solution substratum, the concentration of interior carbonaceous sources is from about 0.1 to about 10 mg/ml, preferably uses from about 0.5 to the L-rhamnosyl of about 3 mg/ml or have a kind of material of alpha-L-rhamnoside key.In addition, liquid nutrient medium can contain nitrogenous source and can be used for the inorganic salt of general microorganism culturing, also can make the liquid nutrient medium that is suitable for effectiveness of the present invention.
Available organic compound or mineral compound be as nitrogenous source, and for example peptone, urea, ammonium sulfate, ammonium chloride or ammonium nitrate use separately or unites use.
The salt of available potassium, sodium, magnesium, calcium, zinc, iron, manganese, cobalt, copper and phosphoric acid etc. is as inorganic salt.
The concentration of nitrogenous source and inorganic salt is undemanding in substratum, as long as can obtain being suitable for the liquid nutrient medium of effectiveness of the present invention.Usually used concentration range also can be used for the present invention in the fermentation process.
Can under aerobic conditions, cultivate for example jolting or stirring under the aerobic situation.
The temperature of cultivating is preferably from about 20 ° to 40 ℃.The pH value of cultivating is normally from about 5.0 to 7.5, preferably from about 5.5 to 6.0.
PH regulator is by adding the aqueous solution (from about 5 to 40%(weight ratios) such as damping fluid, sodium hydroxide, potassium hydroxide or sodium hydroxide or potassium hydroxide, preferably from about 30 to 40%(weight ratios) result that can both obtain.Under these conditions, cultivate and to carry out usually about 2 days to 10 days, preferably from about 4 days by about 7 days, and from the liquid nutrient medium of making, select to be suitable for the liquid nutrient medium of effectiveness of the present invention.
The microorganism strains of selecting to be fit to can carry out like this, is about to a kind of to gypeno-side V and/or ginsenoside-Rb
1Be transformed into panaquilon-Rd and have the liquid nutrient medium of high conversion as standard substance.For example, in resulting about 2 milliliters of liquid substratum, add about 1 milliliter to about 10 milliliters (preferably about 3 milliliters to about 5 milliliters) phosphate buffered saline buffer with aforesaid method, its pH value is from about 4.0 to about 8.0, adds from about 0.05 to 0.2 milliliter (preferably about 0.1 milliliter) every milliliter to include about 10 to 30 milligrams (preferably about 20 milligrams) gypenoside V or ginsenoside-Rb in damping fluid
1Alcoholic solution (for example a kind of C
1-4Alcohol is as methyl alcohol, ethanol, propyl alcohol, Virahol or butanols).
Mixture is incubated about 10 to 30 hours, preferably about 15 to 20 hours down in about 30 ° to 80 ℃.Be chosen in wherein gypenoside V or ginsenoside-Rb
1The less and ginsenoside-rd of residual content by the liquid nutrient medium of enrichment, because it can satisfy effectiveness of the present invention.Compound in the liquid medium within (I), the available for example high-efficient liquid spectrum of (II) and (III) amount separately method are measured.
Employed bacterial strain can repeat to do above-mentioned cultivation in those not selected liquid nutrient mediums, and through select once more or repeatedly with obtain a kind of can be with high yield with gypenoside V and/or ginsenoside-Rb
1Convert the liquid nutrient medium of ginsenoside-rd to.More particularly, can be with gypenoside V and/or ginsenoside-Rb
1The liquid nutrient medium that changes into ginsenoside-rd can prepare with following method.
(1) 6.0 gram ammonium sulfate, 1.0 being restrained the sal epsom, 2.0 gram potassium primary phosphates and the 1.0 gram yeast extracts that contain 7 molecular crystal water is dissolved in 2.0 premium on currency, (this liquid nutrient medium is hereinafter referred to as " basic medium "), as mentioned above to the material that wherein adds L-rhamnosyl or a kind of α of containing-L-rhamnosyl key, make its concentration become 1 mg/ml, then the pH value is adjusted to about 5.7 and does the liquid nutrient medium of cultivating usefulness with preparation.
(2) with the inoculation that will do experiment of liquid nutrient medium, place 28 ℃ of following joltings to cultivate a week then.
(3) liquid nutrient medium is filtered through tampon.Filtrate is poured into 4 in vitro by 2 milliliters every part.These test tubes are further divided into two groups, and every group has two test tubes.In every group test tube, add 3 milliliters every part pH7.0 or the phosphate buffered saline buffer of pH5.0.The methanol solution (10 mg/ml) that in each test tube, adds 0.1 milliliter gypenoside V again.Get a test tube that has added the buffered soln of pH7.0 or 5.0 in every group, place 60 ℃ to be incubated 18 hours down.Two remaining test tubes place under 35 ℃ and react.
(4) with ginsenoside-Rb
1Replace gypenoside V to carry out (3) operation step by step.
(5) reaction mixture that obtains from (3) and (4) is carried out the high-efficient liquid spectrum analysis respectively, to select to contain less gypenoside V or ginsenoside-Rb
1, and expect it is the liquid nutrient medium of a large amount of enrichment ginsenoside-rds, the liquid nutrient medium of this class bacterial strain just is applicable to method of the present invention.
(6) undesirable those bacterial strains in (5) are inoculated on the fresh substratum, this substratum is cultivated a week through jolting again, and then repeat the operation of (3)-(5).
(7) through after step (6), still not presenting those bacterial strains of satisfactory result, again it is cultivated three times each week.All carry out the selection of strain cultures applicatory each time.
The bacterial strain in suitable liquid nutrient medium that (8) will choose like this inoculates on the solid medium respectively, this solid medium is the liquid nutrient medium of above-mentioned steps (1) usefulness, the agar of adding 1% in 7 milliliters every part, place again under 20 to 35 ℃, preferably about 30 ℃, cultivated seven days.Then substratum being placed on about 5 ℃ cold place stores.
The bacterial strain of storing like this use the used liquid nutrient medium of above-mentioned steps (1) respectively again and under above-mentioned steps (2) condition through the cultivation in a week.The liquid nutrient medium that is suitable for effectiveness of the present invention needs to filter through tampon.Filtrate is divided in the test tube to measure optimum reaction condition.More particularly, adding concentration in 2 milliliters every part filtrate is the gypenoside V or the ginsenoside-Rb of 10 mg/ml
1Methanol solution, every part adds 3 milliliters of pH4.0,5.0,6.0 or 7.0 phosphate buffered saline buffer respectively subsequently.These test tubes are placed on 35 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃ are incubated 18 hours down.Every kind of reaction mixture is all carried out the high-efficient liquid spectrum analysis respectively, to be determined at gypenoside V or ginsenoside-Rb under any condition
1By hydrolysis most effectively, and a large amount of enrichments of ginsenoside-rd quilt.
Use and cultivate the liquid nutrient medium that selected bacterial strain makes according to the method described above, regulating suitable pH and temperature can be from gypenoside V or ginsenoside-Rb
1Produce ginsenoside-rd at an easy rate, can from the extract of blue extract of strand bone or ginseng, make the abundant mixture of ginsenoside-rd.
Be applicable to that the Mycophyta bacterial strain that the present invention does liquid nutrient medium can be exemplified below: light white latent ball yeast (IFO 0378), Cryptococcus laurentii (IFO 0609), the debaryomyces hansenii (IFO 1383) of giving birth on the tongue, Aspergillus awamori (IFO 4033), carbon black aspergillus (IFO 4038), aspergillus niger (Aspergillus niger var.fermentarius) (IFO 4068) and golden yellow aspergillus (Aspergillus aureus var.minor) (IFO 4118).Bacterial strain is light white latent ball yeast (IFO 0378) preferably, Cryptococcus laurentii (IFO 0609), carbon black aspergillus (IFO 4038) and aspergillus niger (IFO 4068).But as indicated above, can use any bacterial strain, as long as can providing, they contain the active rhamnosidase that can satisfy effectiveness of the present invention and/or the liquid nutrient medium of Polyglucosidase.
In substratum, cultivate the microorganism that belongs to genera cryptococcus, Hansenula or Aspergillus,, also can be used for the present invention to produce the liquid nutrient medium of rhamnosidase and/or Polyglucosidase in the liquid medium within.
Some bacterial strains of mentioning above, IFO 0378, IFO 0609, IFO 1383, IFO 4033, IFO 4038, and IFO 4068 and IFO 4118 have left Osaka fermentation research institute in, and are put in " catalogue is grown in the 7th edition training in 1984 " of being published by above-mentioned institute.
According to the present invention, have such as pain relieving, calmness, improve metabolism or improve the ginsenoside-rd of many useful pharmacologically actives such as cardiovascular systems, can be with high yield, high purity, more easily produce and the mode of low expense provides.
Below all examples will more specifically explain the present invention, but be not that scope of the present invention is imposed any restrictions.
Example 1:
2 milligrams of gypenoside V and 50 milligrams " solubility hesperidinase " (Japanese Tanabe Seiyaku company produce) are joined in 5 milliliters of 0.025M phosphate buffer solns.With the sample of four parts of this solution, under specified pH of table 1 and temperature, kept 18 hours respectively.Every kind of resultant got 5 microlitres and done efficient liquid phase chromatographic analysis
*, to measure the content of raw material and ginsenoside-rd.The results are shown in table I (" % " expression in the table I and the ratio of theoretical value are represented identical meanings at table 2 in table 9).
With ginsenoside-Rb
1Replace gypenoside V as substrate, handle by above-mentioned steps then.The results are shown in table 2.
*The condition of efficient liquid phase chromatographic analysis: (all using these conditions in each example of the following stated)
Stationary phase: tsk gel ODS-120T(by Japanese Toyo Soda manufacturing company produce, φ 4.6 * 250mm).
Mobile phase: acetonitrile-water (36: 64, (volume ratio), flow velocity-1.2 ml/min.Detect wavelength-203 millimicron.
Retention time-ginsenoside-Rb
1About 8.5 minutes,
About 15.5 minutes of gypenoside V
About 21.3 minutes of ginsenoside-rd
Example 2:
In 1 premium on currency, dissolve in the extract of the blue over-ground part of 123 gram strand bones, include 7.12 gram gypenoside V, 1.23 gram ginsenoside-Rb
1Described in 0.61 gram ginsenoside-rd such as example 1, in this solution, add 500 grams " solubility hesperidinase ", the aqueous solution with the 0.2M Sodium phosphate dibasic transfers to 7.0 with pH subsequently.Then mixture is placed on 60 ℃ and is incubated 24 hours down.
Left standstill seven days at the cold place that reaction mixture is placed on 2 ℃, can obtain 13.4 gram pale asphyxia precipitations, this precipitation is produced through Li Chroprep RP-8(U.S. Merck company) column chromatography, use the 65%(volume ratio then) the aqueous methanol wash-out, can obtain 7.01 gram ginsenoside-rds and 1.03 gram ginsenoside-Rb
1
The said extracted thing of the blue over-ground part of strand bone can prepare in the following method.In 2 kilograms of blue over-ground parts of exsiccant strand bone, add 10 liters of methyl alcohol.With mixture reflux 2 hours.Methanol extract liquid under reduced pressure is concentrated into 1 liter.
In concentrated solution, add 2 premium on currency and 2 liters of vinyl acetic monomers.The jolting mixture.Branch vibration layer also under reduced pressure is concentrated into dried.
Example 3:
The pH value that will wherein contain 1200 milliliters of basic medium solution of 1200 milligrams of L-rhamnosyls with the 0.2M phosphate buffered saline buffer transfers to 5.7, it is inoculated with aspergillus niger (Aspergillus niger var.fermentarius) (IFO 4068), place 28 ℃ then, under jolting, cultivated 7 days.Liquid nutrient medium filters through tampon, and filtrate is divided into 4 milliliters every part.Every part pH value is transferred to as specified a kind of of table 3, respectively add 4 milligrams gypenoside V or ginsenoside-Rb then
1With the insulation 18 hours under the specified temperature of table 3 of every kind of mixture.Every kind of reaction mixture is through efficient liquid phase chromatographic analysis.The results are shown in table 3.
When stating IFO 4068 liquid nutrient mediums prepared under example 3 conditions in the use, can sum up from the result of table 3, be converted into ginsenoside-rd for gypenoside V, preferably allow and be reflected at the pH value be about 7 and carry out in about 45 ℃ scope from about 40 in temperature.Also have, for ginsenoside-Rb
1Change into ginsenoside-rd, allow and be reflected at the pH value be about 5 and carry out in about 55 ℃ scope best from about 40 in temperature.
Example 4
In containing 600 milliliters of basic mediums of hesperidin of 600 milligrams, add the 0.2M phosphate buffered saline buffer to regulate pH value to 5.7, with this substratum with carbon black aspergillus (IFO 4038) inoculation, then 28 ℃ of following joltings cultivations 7 days.This liquid nutrient medium is used with the same method of example 3 again and is handled.
The results are shown in table 4.
Under making use-case 4 conditions during prepared above-mentioned IFO 4038 liquid nutrient mediums, can sum up from the result of table 4, for gypenoside V is converted into ginsenoside-rd, preferably allow be reflected at the pH value be about 7 and temperature carrying out in about 55 ℃ scope from about 45.Also have, for ginsenoside-Rb
1Change into ginsenoside-rd, obviously preferably allow be reflected at pH from about 5 to 6 and temperature under about 55 to 60 ℃ condition, carry out.
Example 5:
The extract of the blue over-ground part of dissolving 5.0 gram strand bones includes 167.7 milligrams of gypenoside V, 80.9 milligrams of ginsenoside-Rb in 15 ml waters
1With 67.1 milligrams of ginsenoside-rds.In this solution, add 350 milliliters of prepared liquid nutrient mediums from example 3, with the 0.2M phosphoric acid buffer pH value is transferred to 5.0 subsequently.This mixture is placed on 45 ℃ again and is incubated 13 hours down, mixture is measured the content of first kind of wherein contained saponin again through efficient liquid phase chromatographic analysis.The content of ginsenoside-rd is 220.3 milligrams in reaction mixture, ginsenoside-Rb
1Content be 15.8 milligrams, find no gypenoside V.
The extract of the blue over-ground part of above-mentioned strand bone prepares with following method.In the blue over-ground part of 100 gram exsiccant strand bones, add 500 ml methanol.With mixture reflux 2 hours.Under reduced pressure methanol extract liquid is concentrated into 50 milliliters.
In concentrated solution, add 100 ml waters and 100 milliliters of vinyl acetic monomers.This mixture of jolting.Branch vibration layer also under reduced pressure is concentrated into dried.
Example 6:
In 15 ml waters, dissolve in extract substrate with the blue over-ground part of the strand bone of example 5 as much.Using 350 milliliters of prepared liquid nutrient mediums in example 4, is 7.0 and under 55 ℃ of conditions in the pH value, allows reaction carry out in the mode that is similar to example 4.Reaction mixture contains 200.5 milligrams of ginsenoside-rds, 64.8 milligrams of ginsenoside-Rb
1, no gypenoside V.
Example 7
Dissolving contains 305 milligrams of ginsenoside-Rb in 50 ml waters
1Thick saponin 2 grams of Panax genseng with 25 milligrams of ginsenoside-rds.In this solution, add the liquid nutrient medium that makes in 350 milliliters of examples 3, make mixture be 5 and 50 ℃ and be incubated 15 hours down in the pH value.The content of ginsenoside-rd is 290.6 milligrams in reaction mixture, ginsenoside-Rb
1It is 6.3 milligrams.
Above-mentioned thick saponin is to prepare with following method.Add 10 liters of methyl alcohol in the dried root of 2 kilograms of Panax gensengs, heating is 2 hours under refluxing.The extracting solution of methyl alcohol under reduced pressure is concentrated into 1 liter.In concentrated solution, add 2 premium on currency and 3 liters of butanols.Take out the butanols soluble part, it is under reduced pressure concentrated also obtain the 100 thick saponins that restrain after the drying.
Example 8
In 600 milliliters of basic mediums that include 600 milligrams of hesperidins, adding 1N sodium hydroxide, to make its pH value be 5.7.Substratum with light white latent ball yeast (IFO 0378) inoculation, is placed on 28 ℃ of following joltings 7 days again.Liquid nutrient medium is handled with the mode that is similar to example 3, be the results are shown in table 5.
When stating IFO 0378 liquid nutrient medium that example 8 conditions prepare in the use, result by table 5 can draw such conclusion, promptly for gypenoside V is changed into ginsenoside-rd, preferably allow be reflected at the pH value be about 7 and temperature be about under 40 ℃ the condition and carry out.
Example 9
In 600 milliliters of basic mediums that includes 600 milligrams of hesperidins, add 1N sodium hydroxide and make its pH value to 5.7.Substratum with Cryptococcus laurentii (IFO 0609) inoculation, is placed on 28 ℃ of following joltings 7 days again.Then this liquid nutrient medium is handled with the mode that is similar to example 3.The results are shown in table 6.
When stating IFO 0609 liquid nutrient medium that use-case 9 conditions prepare in the use, can draw such conclusion, promptly for gypenoside V is changed into ginsenoside-rd, preferably allow be reflected at the pH value be about 7 and about 40 ℃ condition under carry out.
Example 10
Adding 1N sodium hydroxide in 600 milliliters of basic mediums that include 600 milligrams of hesperidins makes its pH value reach 5.7.Substratum with Cryptococcus laurentii (IFO 0609) inoculation, is placed on 28 ℃ again and cultivated 7 days down.This liquid nutrient medium filters through tampon.The extracting solution that adds 120 milliliters of blue over-ground parts of strand bone in filtrate includes 4.45 gram gypenoside V, 2.05 gram ginsenoside-Rb
1With 1.32 gram ginsenoside-rds.In mixture, add 1N sodium hydroxide and make its pH value reach 7.0, place 40 ℃ to stir 75 hours down then.Reaction mixture is poured into 0.5 liter of Amberlite XAD-2(U.S. Rohm ﹠amp; The manufacturing of Haas company) in the post of loading.Saponin is adsorbed on the carrier, and carrier is washed with 2 premium on currency earlier, uses 2 liters of 40%(volume ratios again) methyl alcohol washes, and uses 2 liters of 60%(volume ratios subsequently) methanol-eluted fractions.Elutriant under reduced pressure is concentrated into dried, and the fallow powder of residual 10 grams is dissolved in 50 milliliters of 45%(volume ratios with it) in the methyl alcohol.This solution is added to 500 gram ODS-Q
3On the post that (Japanese Wako pure chemistry Industrial Co., Ltd produce) loaded.Pillar is with 1 liter of 55%(volume ratio) washed with methanol, use the 62.5%(volume ratio again) methanol-eluted fractions, it is (pure to make 3.12 grams
Degree: ginsenoside-rd 94%), 1.85 gram (purity: ginsenoside-Rb 91%)
1And 2.41 the gram (purity: gypenoside V 90%).
The extracting solution of the blue over-ground part of above-mentioned strand bone prepares with the following method.In the siccative of the blue over-ground part of 250 gram strand bones, add 2 liters of methyl alcohol.With mixture reflux 1 hour.Under reduced pressure concentrate methanol solution to 50 milliliter.In concentrated solution, add 70 ml waters and 100 milliliters of ethyl acetate, and the jolting mixture.Isolate water layer.
Example 11
In 600 milliliters of basic mediums that include 600 milligrams of rutins, add the 0.2M phosphoric acid buffer with adjust pH to 5.7, it with carbon black aspergillus (IFO 4038) inoculation, is placed on 28 ℃ and jolting again and cultivated 7 days down.Then liquid nutrient medium is handled with the mode that is similar to example 3.The results are shown in table 7 and table 8.
When stating prepared IFO 4038 liquid nutrient mediums of the condition of example 11 in the use, result by table 7 can draw such conclusion, promptly be converted into ginsenoside-rd, preferably allow be reflected at pH value from about 6 to about 7 and temperature range is carried out under about 60 ℃ condition from about 40 for gypenoside V.As can be seen from Table 8, for ginsenoside-Rb
1Change into ginsenoside-rd, preferably allow be reflected at the pH value from about 4 to 6 with under temperature range is condition about 40 ℃, carry out.
Example 12
Use the 0.2M phosphate buffered saline buffer, 600 milliliters of pH values that include the basic medium of 600 milligrams of L-rhamnosyls are transferred to 5.7, this substratum is with debaryomyces hansenii (IFO 1383) inoculation, places 28 ℃ and jolting to cultivate 7 days down then.Handle this liquid nutrient medium with the mode that is similar to example 3 again.
When stating IFO 1383 liquid nutrient mediums that example 12 conditions prepare in the use, can draw such conclusion, promptly be converted into ginsenoside-rd, preferably allow be reflected under the about 40 ℃ condition of pH value about 7 and temperature and carry out for gypenoside V from table 9.For ginsenoside-Rb
1Change into ginsenoside-rd, allow be reflected at carry out under the about 40 ℃ condition of pH value about 4 and temperature best.
Errata
Claims (12)
1, produces a kind of method of ginsenoside-rd, comprise that rhamnosidase and/or Polyglucosidase are to gypenoside V and/or ginsenoside-Rb
1Effect.
2, by the process of claim 1 wherein that rhamnosidase and/or Polyglucosidase are the method generations with culturing micro-organisms, this microorganism can produce rhamnosidase and/or Polyglucosidase in the liquid medium within.
3, by the method for claim 2, wherein microorganism is a Mycophyta.
4, by the method for claim 2, wherein microorganism is a yeast.
5, by the method for claim 3, wherein fungi belongs to fungi.
6, by the method for claim 4, wherein yeast belongs to Hansenula.
7, by the method for claim 4, wherein yeast belongs to Cryptococcus.
8, by the process of claim 1 wherein that reaction is to carry out about 40 in pH value from about 4 to about 7 and temperature under about 65 ℃ condition.
9, by the method for claim 2, wherein microorganism is aspergillus niger (Aspergillusniger var.fermentarius).
10, by the method for claim 2, wherein microorganism is a carbon black aspergillus.
11, by the method for claim 2, wherein microorganism is a light white latent ball yeast.
12, by the method for claim 2, wherein microorganism is a Cryptococcus laurentii.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16226885 | 1985-07-22 | ||
| JP162268/1985 | 1985-07-22 | ||
| JP5434186 | 1986-03-11 | ||
| JP54341/1986 | 1986-03-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN86105166A true CN86105166A (en) | 1987-03-04 |
Family
ID=26395096
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198686105166A Pending CN86105166A (en) | 1985-07-22 | 1986-07-22 | Produce a kind of method of ginsenoside-rd |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR870001227A (en) |
| CN (1) | CN86105166A (en) |
| GB (1) | GB2179042B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001049704A1 (en) * | 2000-01-03 | 2001-07-12 | Guangdong Taihe Biopharmaceutical Co. Ltd. | Compound (i), a method for extracting it and a pharmaceutical composition containing it |
| CN1092203C (en) * | 1998-07-22 | 2002-10-09 | 北京鑫利恒医药科技发展有限公司 | Process for extracting ginsenoside Re, and use of medicine thereof |
| CN1092204C (en) * | 1998-07-28 | 2002-10-09 | 吉林大学基础医学院科技开发中心 | Semisynthesizing method for 20(S)-ginsenoside Rg3, and use in medicine |
| CN1105781C (en) * | 1999-03-17 | 2003-04-16 | 金凤燮 | Method for preparing rare ginsengoside using enzymatic method to modify ginsenoside glycoside |
| CN101768619A (en) * | 2010-02-10 | 2010-07-07 | 华侨大学 | Method for preparing rare ginsenoside IH-901 with Rd as substrate |
| CN101333549B (en) * | 2008-03-04 | 2011-08-24 | 常景玲 | Method for directly transforming active dry yeast to be ginsenoside |
| CN101619340B (en) * | 2009-08-13 | 2012-06-13 | 安徽农业大学 | Method for preparing saponins compounds by fermenting and culturing gen-seng fruits |
| CN103037879A (en) * | 2010-05-14 | 2013-04-10 | 株式会社Gch&P | Method for preparing novel processed ginseng or an extract thereof, the usually minute ginsenoside content of which is increased |
| CN103525892A (en) * | 2013-09-20 | 2014-01-22 | 吉林大学 | Method for quickly preparing rare panaxoside Rd |
| CN105199966A (en) * | 2015-08-28 | 2015-12-30 | 天津大学 | Aspergillus for converting ginsenoside Rb1 to produce Rd and application |
| CN112646737A (en) * | 2021-01-22 | 2021-04-13 | 宁夏农产品质量标准与检测技术研究所(宁夏农产品质量监测中心) | Cryptococcus albidus strain YN14 capable of producing aroma substances at high yield and application thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4953547B2 (en) | 2000-12-29 | 2012-06-13 | ▲鳳▼燮 金 | Ginsenoside glycosidase hydrolyzing ginsenoside sugar group and use thereof |
| DE10158281A1 (en) * | 2001-11-19 | 2003-05-28 | Mediwirk Gmbh | Pharmaceutical preparation comprises ginsenoside enclosed in micro-shell, e.g. liposome, providing targeted and controlled delivery, useful e.g. in treatment of liver tumors, diabetes mellitus or hypertension |
| CN115368425B (en) * | 2021-08-06 | 2023-07-18 | 遵义医科大学 | Triterpene compound, preparation method and anti-inflammatory application thereof |
-
1986
- 1986-07-18 GB GB08617616A patent/GB2179042B/en not_active Expired
- 1986-07-22 KR KR1019860005959A patent/KR870001227A/en not_active Application Discontinuation
- 1986-07-22 CN CN198686105166A patent/CN86105166A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1092203C (en) * | 1998-07-22 | 2002-10-09 | 北京鑫利恒医药科技发展有限公司 | Process for extracting ginsenoside Re, and use of medicine thereof |
| CN1092204C (en) * | 1998-07-28 | 2002-10-09 | 吉林大学基础医学院科技开发中心 | Semisynthesizing method for 20(S)-ginsenoside Rg3, and use in medicine |
| CN1105781C (en) * | 1999-03-17 | 2003-04-16 | 金凤燮 | Method for preparing rare ginsengoside using enzymatic method to modify ginsenoside glycoside |
| WO2001049704A1 (en) * | 2000-01-03 | 2001-07-12 | Guangdong Taihe Biopharmaceutical Co. Ltd. | Compound (i), a method for extracting it and a pharmaceutical composition containing it |
| CN101333549B (en) * | 2008-03-04 | 2011-08-24 | 常景玲 | Method for directly transforming active dry yeast to be ginsenoside |
| CN101619340B (en) * | 2009-08-13 | 2012-06-13 | 安徽农业大学 | Method for preparing saponins compounds by fermenting and culturing gen-seng fruits |
| CN101768619A (en) * | 2010-02-10 | 2010-07-07 | 华侨大学 | Method for preparing rare ginsenoside IH-901 with Rd as substrate |
| CN103037879A (en) * | 2010-05-14 | 2013-04-10 | 株式会社Gch&P | Method for preparing novel processed ginseng or an extract thereof, the usually minute ginsenoside content of which is increased |
| CN103037879B (en) * | 2010-05-14 | 2016-06-01 | 绿十字生命健康有限公司 | The preparation method that Tiny Panax ginseng saponin constituent obtains novel processed ginseng or the processed ginseng extract increased |
| CN103525892A (en) * | 2013-09-20 | 2014-01-22 | 吉林大学 | Method for quickly preparing rare panaxoside Rd |
| CN105199966A (en) * | 2015-08-28 | 2015-12-30 | 天津大学 | Aspergillus for converting ginsenoside Rb1 to produce Rd and application |
| CN105199966B (en) * | 2015-08-28 | 2018-08-17 | 天津大学 | A kind of conversion ginsenoside Rb1 produces aspergillus and the application of Rd |
| CN112646737A (en) * | 2021-01-22 | 2021-04-13 | 宁夏农产品质量标准与检测技术研究所(宁夏农产品质量监测中心) | Cryptococcus albidus strain YN14 capable of producing aroma substances at high yield and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2179042A (en) | 1987-02-25 |
| KR870001227A (en) | 1987-03-12 |
| GB8617616D0 (en) | 1986-08-28 |
| GB2179042B (en) | 1988-11-23 |
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