GB2179042A - Ginsenoside-Rd - Google Patents

Abstract

Ginsenoside-Rd (I, R=H) is selectively produced from ginsenoside-Rb, (I, R= beta -D-glucopyranosyl) and/or gypenoside V (I, R=???-L-rhamnopyranosyl) by enzymolysis with glucosidase and/or rhamnnosidase. The starting saponins may be obtained by extraction of Gynostemma pentaphyllum Makino or Panax ginseng C.A. Meyer. The obtained ginsenoside-Rd may be used as an analgesic, sedative, metabolism-improving or cardiovascular system-improving agent. <IMAGE>

Description

SPECIFICATION A process of producing Ginsenoside-Rd This invention relates to an industrially advantageous process of preparing ginsenoside-Rd, one of the ginseng saponins.

Studies on the components of ginseng, such as the root of Panaxginseng C.A. Meyer, have recently been conducted extensively, and various types of ginseng saponins as effective components of ginseng have been isolated. Among those ginseng saponins ginsenoside-Rd (hereinafter it may be referred to as the compound [I]) and ginsenoside-Rb (hereinafter it may be referred to as the compound [11]) belong to principal effective components of ginseng. Pharmocological actions of them have been placed under elucidation, and it has now come to be known that these ginsenosides-Rd and -Rb,, have, among many others, useful pharmacological activities, such as analgesic activity, sedative activity, metabolism improving activity, or cardiovascular system improving activity.It has been shown that pharmacological activities of ginsenoside-Rd are much stronger than that of ginsenoside-Rb,. However, the content of ginsenoside-Rd in ginseng such as the root of Panax ginseng C.A. Meyer is so little that the extraction thereof from the plant is far from an industrially advantageous process.

Besides, ginseng is remarkably expensive. Circumstances being such, development of a process of obtaining ginsenoside-Rd in high yield, in high purity and with ease has been desired.

It was recently reported that the ginsenoside-Rd was isolated together with ginsenoside-Rb, from Gynostemma pentaphyllum Makino which is a perennial liane growing spontaneously all over Asia, [Yakugaku Zasshi, 103, P. 173 (1983)]; Japanese unexamined published patent application No. 56-12739 (12739/1981).

However, the principal saponin of Gynostemma pentaphyllum Makino is not ginsenoside-Rd, but gypenoside V.

Ginsenoside-Rd, ginsenoside-Rb and gypenoside V (hereinafter it may be referred to as the compound [III] ) are shown by the following structural formula and their chemical names are as follows.

Comp.

No. R Chemical Name [I] Ginsenoside-Rd H 20 (S) -protopanaxadiol-3-O- ['3- D-glucopyranosyl (1 -t2)-p- D-glucopyranoside] -20-O-P-D-glucopyranoside [I I] Ginsenoside- Rba ss- D-gluco- 20 (S) -protopanaxadiol-3-O- [ss- D-glucopyranosyl pyranosyl (1-t2) -P-D-glucopyranoside] -20-0-[P- D-glucopyranosyl (1 ~6)-ss-D-glucopyranoside] [III] Gypenoside V a- L-rhamno- 20 (S) -protopanaxadiol-3-O- [ss- D-glucopyranosyl pyranosyl (1 -t2) -P-D-glucopyranoside] -20-0- [a- L-rhamnopyranosyl (1 6)-'3-D-glucopyrnnoside] As is clear from the above structural formulae, the substituent R only is different in the chemical structure among ginsenoside-Rd, ginsenoside-Rb, and gypenoside V, and it was considered by the present inventors that gynsenoside-Rd would be derived chemically from ginsenoside-Rb, or gypenoside V. More specifically, it was considered that selective fission of R-O bond, i.e. the terminal ss-D-glucosidic bond of gentiobiosyl moiety at the 20-position of ginsenoside-Rb, or a-L-rhamnosidic bond of rutinosyl moiety at the 20-position of gypenoside V would give ginsenoside-Rd. However, in ginsenoside-Rb, and gypenoside V, there are three glycosidic bonds susceptible to hydrolysis other than R-O moiety, and it is difficult to hydrolyze selectively only this R-O moiety.

When, for instance, ginsenoside-Rb, or gypenoside V was subjected to hydrolyses under various acid conditions, no ginsenoside-Rd was obtained.

The present inventors conducted intensive research for hydrolysis reaction having higher selectivity than acid hydrolysis, and succeeded in efficiently producing ginsenoside-Rd from ginsenoside-Rb and/or gypenoside V by an enzymatic reaction using a specific enzyme, thus completing the present invention.

That is to say, the present invention relates to a process of producing ginsenoside-Rd, which comprises allowing rhamnosidase and/or glucosidase to act on gypenoside V and/or ginsenoside-Rb. More concretely, according to the process of this invention ginsenoside-Rd is produced by allowing rhamnosidase to act on gypenoside V, or allowing glucosidase to act on ginsenoside-Rb1.

Gypenoside V and ginsenoside-Rb1 as the starting materials (substrates) of the process of this invention may respectively be isolated ones or a mixture thereof. From the industrial viewpoint, it is advantageous to use the extract of Gynostemma pentaphyllum Makino containing gypenoside V, ginsenoside-Rb1 and ginsenoside-Rd or the extract of the root of Panax ginseng C.A. Meyer containing ginsenoside-Rb, and ginsenoside-Rd as it is, for the starting material (substrate). Gypenoside V or/and ginsenoside-Rb1 are changed into ginsenoside-Rd by the method of the present invention, but the ginsenoside-Rd in the starting material is not changed.However, use of the Gynostemma pentaphyllum Makino extract as the starting material (substrate) is most advantageous, because Gynostemma pentaphyllum Makino is available at low cost while Panax ginseng C.A. Meyer is expensive. The extract of Gynostemma pentaphyllum Makino or the extract of the root of Panax ginseng C.A.

Meyer as the substrate is subjected to enzymatic hydrolysis according to the process of this invention to afford an extract rich in ginsenoside-Rd.

Thus extracted material is subjected to conventional separation and/or purification procedures, such as chromatography, recrystallization, etc. to readily give ginsenoside-Rd in high purity. As compared with conventional processes of extracting, separating and/or purifying ginsenoside-Rd from Gynostemma pentaphyllum Makino or the root of Panax ginseng C.A. Meyer, the process of this invention provides ginsenoside-Rd in a larger yield, in higher purity and far more readily.

The enzymes employed in this invention are rhamnosidase and/or glucosidase. The rhamnosidase and/or glucosidase are produced by cultivating a microorganism which is capable of producing rhamnosidase and/or glucosidase to accumulate rhamnosidase and/or glucosidase in the culture broth. The examples of the microorganism include among others a fungus and a yeast These enzymes are used for hydrolyzing the glucosidic bonds of ginsenoside-Rb, and/or the rhamnosidic bond of gypenoside V. Concretely rhamnosidase hydrolyzes a-L-rhamnosidic linkage of gypenoside V, and glucosidase hydrolyzes the terminal ss-D-glucosidic linkage of gentiobiosyl moiety at the 20-position of ginsenoside-Rb1.Therefore, rhamnosidase employed in the present invention can be any one which is active on gypenoside V, and glucosidase employed in the present invention can be any one which can hydrolyze the terminal ss-D-glucosidic linkage of ginsenoside-Rb1. These two enzymes may be used as each purified single one or in the mixture of them. When gypenoside V or an extract of Gynostemma pentaphyllum Makino is employed as the substrate, rhamnosidase or a mixture of rhamnosidase and glucosidase is used, and, when ginsenoside-Rb1 or an extract of the root of Panax ginseng C.A. Meyer is employed as the substrate, glucosidase or a mixture of glucosidase and rhamnosidase is used.

However, to obtain a purified enzyme requires in general a great deal of labour and expense. In the present invention, these enzymes can be employed even in a crude state, so long as they contain rhamnosidase and/or glucosidase having activities enough for satisfying the purpose of this invention. Furthermore, a culture broth containing rhamnosidase and/or glucosidase having activities suitable for the purpose of this invention may be employed. To use an extract of Gynostemma pentaphyllum Makino as the substrate and a crude enzyme or a culture broth as the enzyme is, from the industrial viewpoint, the most advantageous method for producing ginsenoside-Rd. The enzymatic hydrolysis of this invention is usually conducted at a pH value of from about 4 to about 7 and at a temperature of from about 40 to about 65"C.

As the crude enzyme suitable for the purpose of this invention, commercially available crude enzyme preparations may be conveniently employed, for example "Soluble Hesperidinase" (manufactured by Tanabe Seiyaku Co., Ltd., Japan). This "Soluble Hesperidinase" is a crude enzyme containing glucosidase besides rhamnosidase active on gypenoside V. When, for example, the "Soluble Hesperidinase" is added to gypenoside V or an aqueous extract of Gynostemma pentaphyllum Makino and the mixture is maintained, preferably in a buffer solution, at a pH value of from abount 6.8 to about 7 at a temperature of from about 50 to about 60"C, ginsenoside-Rd is produced in high yield. As the buffer solution are employed conventional buffer solution e.g.

phthalate buffer solution, phosphate buffer solution, acetate buffer solution, etc. "Soluble Hesperidinase" is an enzyme especially suitable for converting gypenoside V into ginsenoside-Rd.

The culture broth suitable for the purpose of this invention can be prepared by cultivating certain strains belonging to Eumycetes (e.g. a fungus, a yeast) in a medium containing, as the carbon source, L-rhamnose or a substance containing a-L-rhamnosidic linkage, followed by selecting the strains capable of converting gypenoside V and/or ginsenoside-Rb1 into ginsenoside-Rd in high yield according to the manner as described below.

In this culture broth, are accumulated, besides glucosidase, rhamnosidase which is active on gypenoside V, and the culture broth itself can be utilized as the enzymic source to convert gypenoside V and/or ginsenoside-Rb1 into ginsenoside-Rd. For example, to this culture broth is added gypenoside V or an extract of Gynostemma pentaphyllum Makino, and the mixture is maintained at a pH value from about 4 to about 7 and at a temperature of from about 40 to about 65"C, preferably at a pH value of from about 5 to about 7 and at a temperature of from about 40 to about 55"C to give ginsenoside-Rd in high yield. And, to this culture broth is added ginsenoside Rb1 or an extract of the root of Panax ginseng C.A.Meyer, and the mixture is maintained at a pH value of from about 4 to about 7 and at a temperature of from about 40 to about 65"C, preferably at a pH value of from about 5 to about 7 and at a temperature of about 40 to about 55"C to give ginsenoside-Rd in a high yield.

Examples of the substance containing a-L-rhamnosidic bond include oligosaccharides (di- to tetra-saccharides) having rhamnosyl group, and glycosides having rhamnosyl group, the former being exemplified by rutinose, neohesperidose, etc., and the latter being exemplified by hesperidin, rutin, naringin, etc.

In cultivating certain strains belonging to Eumycetes for obtaining the culture broth suitable for purpose of this invention, an aqueous medium containing as the carbon source in the concentration from about of 0.1 to about 10 mg/ml, preferably from about 0.5 to about 3 mg/ml, L-rhamnose or a substance having x-L-rhamnosidic linkage may be used. In addition a liquid medium may contain nitrogen source and inorganic salts which can be employed for the conventional cultivation of microorganism, as far as the culture broth suitable for the purpose of this invention is obtained.

As the nitrogen source, use may be made of organic or inorganic compounds, such as peptone, urea, ammonium sulfate, ammonium chloride or ammonium nitrate, alone or in the combination.

As the inorganic salts, use may be made of salts with potassium, sodium, magnesium, calcium, zinc, iron, manganese, cobalt, copper, phosphoric acid and so on.

The concentration of nitrogen source and inorganic salts in the medium is optional, so long as the culture broth suitable for the purpose of this invention can be obtained. And the concentration scope used in the usual fermentation method may be employed for this invention.

Cultivation may be conducted in an aerobic condition, such as shaking or stirring under aeration.

The temperature of cultivation is preferably about 20 to about 40"C. The pH value in the cultivation is usually from about 5.0 to about 7.5, preferably from about 5.5 to about 6.0.

The pH adjustment by adding, for example a buffer solution, sodium hydroxide, potassium hydroxide, or an aqueous solution (about 5 to about 40% w/w, preferably about 30 to about 40% w/w) of sodium hydroxide or potassium hydroxide can provide good result. Under the conditions as above, cultivation is conducted for usually about 2 to about 10 days, preferably about 4 to about 7 days, and from the resultant culture broth is selected a culture broth suitable for the purpose of this invention. The selection of the suitable strain of the microorganism can be conducted by taking as the standard a culture broth in which a high conversion rate of gypenoside V and/or ginsenoside-Rb1 to ginsenoside-Rd is performed.For example, to about 2 ml of a culture broth obtained by the above-mentioned procedure is added about 1 to about 10 ml, preferably about 3 to about 5 ml of a phosphate buffer whose pH value is from about 4.0 to about 8.0, to which is added from about 0.05 to about 0.2 ml, preferably about 0.1 ml of a solution of from about 10 to about 30 mg, preferably about 20 mg of gypenoside V or ginsenoside-Rb1 in 1 ml of an alcohol (e.g. a C14 alcohol, such as methanol, ethanol, propanol, isopropanol or butanol).

The mixture is maintained at from about 30 to about 80"C for about 10 to about 30 hours, preferably about 15 to about 20 hours. A culture broth in which gypenoside V or ginsenoside-Rb1 remains less in amount and ginsenoside-Rd is accumulated abundantly is selected as that meeting the purpose of this invention. The respective amounts of the compounds [I], [II] and [III] in the culture broth can be determined by, for example a high performance liquid chromatography.

Strains employed in the culture broth which was not selected may be subjected to repeating the above-mentioned cultivation and selection once or more to obtain a culture broth which is capable of converting gypenoside V and/or ginsenoside-Rb1 into ginsenoside-Rd in high yield. More concretely, the culture broth which can convert gypenoside V and/or ginsenoside-Rb1 into ginsenoside-Rd may be prepared by the following method.

(1) In 2.0 f of water are dissolved 6.0 g of ammonium sulfate, 1.0 g of magnesium sulfate7-hydrate, 2.0 g of potassium dihydrogen phosphate and 1.0 g of yeast extract, (this liquid medium is hereinafter referred to as "basic medium"), to which is added L-rhamnose or a substance containing a-L-rhamnosidic linkage as mentioned above so that its concentration becomes 1 mg/ml, followed by adjusting the pH value to be about 5.7 to prepare a liquid medium for cultivation.

(2) The liquid medium is inoculated with a strain to be tested, which is subjected to shaking culture at 28"C for one week.

(3) The culture broth is subjected to cotton plug filtration. The filtrate is distributed to four test tubes by 2 ml each portion. These test tubes are divided into two groups, each group consisting of two test tubes. To the test tubes of each group is added 3 ml each portion of a phosphate buffer of pH 7.0 or pH 5.0. To each of the test tubes is added 0.1 ml of methanol solution of gypenoside V (10 mg/ml). One each of the test tube, to which is added a buffer solution of pH 7.0 or 5.0, is maintained at 60"C for 1 8 hours. The remaining two tubes are subjected to the reaction at 35"C.

(4) Using ginsenoside-Rb1 in place of gypenoside V, the procedure (3) is conducted.

(5) The reaction mixtures resulting from (3) and (4) are respectively subjected to high performance liquid chromatography to select a culture broth containing less gypenoside V or ginsenoside-Rb1 and, desirably, having ginsenoside-Rd accumulated in much amount, and such a culture broth of the strains is suitable for the process of this invention.

(6) Strains which showed undesirable result in (5) are inoculated on fresh media, which are subjected to shaking culture for further one week, followed by repeating procedures of (3) - (5).

(7) Strains which showed still unsatisfactory result after being subjected to the procedures up to (6) are sequently cultivated three times for one each week. Selection of the suitable culture broth of the strains is conducted each time.

(8) Thus selected strains in the suitable culture broth are respectively inoculated on the solid media prepared by adding about 1% each of agar to 7 ml of the liquid media employed in the above procedure (1), followed by cultivation at 20 to 35"C, preferably about 30"C, for 7 days. Then the media are stored at a cool place of about 5"C.

Then, thus stored strains are respectively subjected to cultivation for one week employing again the liquid media used in the above procedure (1) and under condition in the above procedure (2). The culture broth suitable for the purpose of this invention is subjected to cotton plug filtration. The filtrate is distributed into test tubes to determine the optimal reaction conditions. More concretely stating, to 2 ml each portion of the filtrate is added 0.1 ml of methanol solution of gypenoside V or ginsenoside-Rb1 in the concentration of 10 mg/ml, followed by adding 3 ml each portion of phosphate buffer of pH 4.0, 5.0, 6.0 or 7.0.These test tubes are maintained for 18 hours at 35"C, 45"C, 50"C, 55"C or 60"C. The respective reaction mixtures are subjected to high performance liquid chromatography to determine conditions under which gypenoside V or ginsenoside-Rbl is most efficiently hydrolyzed and ginsenoside-Rd is accumulated in a large amount.

Employing a culture broth obtained by cultivating strains selected by the above-mentioned procedures, ginsenoside-Rd can be easily produced from gypenoside V or ginsenoside-Rb1 by suitably adjusting pHs and temperatures, and a mixture rich in ginsenoside-Rd can be obtained from an extract of Gynostemma pentaphyllum Makino or an extract of the root of Panax ginseng C.A. Meyer.

The strains belonging to the Eumycetes suitable for providing the culture broth employed for the present invention are exemplified by Cryptococcus albidus (IFO 0378), Cryptococcus laurentii (IFO 0609), Hansenula musicola (IFO 1383), Aspergllius awamori (IFO 4033), Aspergillus carbonarius (IFO 4038), Aspergillus niger var. fermentarius (IFO 4068) and Aspergillus aureus var. minor (I FO 4118). More preferable strains are Cryptococcus albidus (IFO 0378), Cryptococcus laurentii (IFO 0609), Aspergillus carbonarius (IFO 4038) and Asperglllus niger var. fermentarius (IFO 4068).But as stated in the foregoing, any strains can be employed so long as they can provide the culture broth containing rhamnosidase and/or glucosidase having activities meeting the purpose of this invention.

The culture broth obtained by culturing in a culture medium a microorganism which belongs to the genius Cryptococcus, Hansenula or Aspergillus to produce rhamnosidase and/or glucosidase in the culture broth may be used in this invention.

The above-mentioned strains, IFO 0378, IFO 0609, IFO 1383, IFO 4033, IFO 4038, IFO 4068 and IFO 4118 have been deposited at Institute of Fermentation, Osaka and are listed on "List of Cultures, 1984, Seventh Edition" published by the said Institute.

By the present invention, ginsenoside-Rd having, among many other useful pharmacological activities, such as analgesic, sedative,- metabolism improving or cardio-vascular system improving activity can be provided in high yield, in high purity, more easily and with low cost.

The following examples will explain the present invention more concretely, but they are not intended to give any restriction to the scope of this invention.

Example I To 5 ml of a 0.025 M phosphate buffer solution were added 2 mg of gypenoside V and 50 mg of "Soluble Hesperidinase" (manufactured by Tanabe Seiyaku Co., Ltd., Japan). Four specimens of this solution were maintained for 18 hours, respectively at the pHs and temperatures as specified in Table 1. 5 t Each of the resultants was subjected to high performance liquid chromatography to determine the content of the starting material and ginsenoside-Rd. The results were shown in Table 1 ("%" in Table 1 shows a ratio to the theoretical value, as in Tables 2 to 9).

As substrate, ginseonside-Rb1 was employed instead of gypenoside V, followed by a procedure similar to the above. The results were shown in Table 2.

TABLE 1 pH value of Temperature of Residual rate of Conversion rate to reaction mixture reaction mixture gypenoside V (%) ginsenoside-Rd ( /OJ 6.8 60"C 0 75.6 7.0 50"C 0 70.0 7.0 55"C 0 79.9 7.0 60"C 0 100.0 TABLE 2 pH value of Temperature of Residual rate of Conversion rate to reaction mixture reaction mixture ginsenoside-Rb, ( /0) ginsenoside-Rd ( /0) 6.8 50"C 32.6 17.5 7.0 50"C 50.2 17.1 Conditions of high performance liquid chromatography: (these conditions were emplyed in the examples described hereinafter) Stationary phase: TSK Gel ODS-1 20T (manufactured by Toyo Soda Manufacturing Co., Ltd. Japan.

0 4.6 x 250 mm) Moving phase: Acetonitrile-water (36:64, v/v), flow rate - 1.2 ml/min. detected wavelength - 203 nm, retention time - ginsenoside-Rb1 ca. 8.5 min., gypenoside V ca. 15.5 min., ginsenoside-Rd ca. 21.3 min.

Example 2 In one litre of water was dissolved 123 g of the extract of the aerial parts of Gynostemma pentaphyllum Makino containing 7.12 g of gypenoside V, 1.23 g of ginsenoside-Rb1 and 0.61 g of ginsenoside-Rd. To the solution was added 500 g of "Soluble Hesperidinase" as described in Example 1, followed by adjusting to pH 7.0 with a 0.2 M aqueous solution of sodium hydrogen phosphate. The mixture was then maintained at 60"C for 24 hours.

The reaction mixture was left standing at a cool place of 2"C for 7 days to obtain 13.4 g of a whitish precipitate, which was subjected to column chromatography of LiChroprep RP-8 (manufactured by Merck & BR< Co., Ltd. U.S.A.), followed by elution with a 65% v/v aqueous methanol to obtain 7.01 g of ginsenoside-Rd and 1.03 g of ginsenoside-Rb1.

The above-mentioned extract of the aerial parts of Gynostemma pentaphyllum Makino was prepared by the following process. To 2 kg of dried aerial parts of Gynostemma pentaphyllum Makino was added 10 e of methanol. The mixture was heated under reflux for 2 hours. The methanol extract solution was concentrated to 1 e under reduced pressure.

To the concentrate were added 2 e of water and 2 e of ethyl acetate. The mixture was shaken. The aqueous layer was separated and concentrated to dryness under reduced pressure.

Example 3 With a 0.2 M phosphate buffer solution, a pH value of 1200 ml of the basic culture medium containing 1200 mg of L-rhamnose was adjusted to 5.7, which was inoculated with Asperglllus niger var. fermentarius (IFO 4068), followed by cultivation under shaking at 28"C for 7 days. The culture broth was subjected to cotton plug filtration, and the filtrate was divided into 4 ml each portions. The pH value of the respective portions was adjusted to one as specified in Table 3, followed by addition of 4 mg each of gypenoside V or ginsenoside-Rb1.

The respective mixtures were maintained for 18 hours at temperatures as specified in Table 3. The respective reaction mixtures were subjected to high performance liquid chromatography. The results were shown in Table 3.

TABLE 3 pH value of Temperature of Residual rate of Conversion rate to Residual rate of Conversion rate to reaction mixture reaction mixture gypenoside V ( /0) ginsenoside-Rd ginsenoside-Rb1 ginsenoside-R, (%) (o/o) (o/o) 4 40"C 0 100.0 34.3 63.0 45"C 0 97.5 11.1 84.4 50"C 0 82.4 5.6 62.5 55"C 0 71.9 3.6 67.5 5 40"C 0 100.0 38.1 60.7 45"C 0 83.2 14.8 84.5 50"C 0 75.7 7.1 91.3 55"C 0 70.6 6.0 85.8 60"C 0 53.6 25.6 66.3 6 45"C 0 84.4 30.0 64.9 50"C 0 73.3 22.2 75.8 7 40"C 0 100.0 45"C 0 100.0 50"C 0 96.6 55"C 0 100.0 60"C 12.6 87.4 In the case of employing the above-mentioned culture broth of IFO 4068 prepared under the conditions of Example 3, it is concluded from the results of Table 3 that, for converting gypenoside V into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of about 7 or at a temperature ranging from about 40 to about 45"C. And, for converting ginsenoside-Rb1 into ginsenoside-Rd, it is considered most preferable to allow the reaction to proceed at a pH value of about 5 and at a temperature ranging from about 40 to about 55"C.

Example 4 To 600 ml of a basic culture medium containing 600 mg of hesperidin was added a 0.2 M phosphate buffer solution to adjust the pH value to be 5.7, which was inoculated with Aspergillus carbonarius (IFO 4038), followed by cultivation under shaking at 28"C for 7 days. The culture broth was then processed in a manner similar to Example 3.

The results were shown in Table 4.

TABLE 4 pH value of Temperature of Residual rate of Conversion rate to Residual rate of Conversion rate to reaction mixture reaction mixture gypenoside V( /0) ginsenoside-Rd ginsenoside-Rb, ginsenoside-Rd (%) (/o) (%) 4 55"C 0 61.2 0 46.7 60"C 0 62.6 26.0 43.7 5 55"C 0 67.5 0 68.9 60"C 0 81.4 30.8 69.2 6 50"C 0 55.4 9.3 52.7 55"C 0 79.8 22.8 75.5 7 40"C 3.5 78.2 45"C 0 100.0 50'C 0 100.0 55'C 0 100.0 60"C 14.6 85.4 In the case of employing the above-mentioned culture broth of IFO 4038 prepared under the conditions of Example 4, it is concluded from the results of Table 4 that, for converting gypenoside V into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of about 7 and at a temperature ranging from about 45 to about 55"C. And, for converting ginsenoside-Rb1 into ginsenoside-Rd, it is considered most preferable to allow the reaction to proceed at a pH value of from about 5 to about 6 and at a temperature of about 55 to about 60"C.

Example 5 In 15 ml of water was dissolved 5.0 g of the extract of the aerial parts of Gynostemma pentaphyllum Makino containing 167.7 mg of gypenoside V, 80.9 mg of ginsenoside-Rb1 and 67.1 mg of ginsenoside-Rd. To the solution was added 350 ml of the culture broth obtained in Example 3, followed by adjusting to a pH value to 5.0 with a 0.2 M phosphate buffer solution. The mixture was then maintained at 45"C for 13 hours, which was then subjected to high performance liquid chromatography to determine the content of each of the saponins contained therein. The content of ginsenoside-Rd in the reaction mixture was 220.3 mg, that of ginsenoside-Rb was 1 5.8 mg, and no gypenoside V was detected.

The above-mentioned extract of the aerial parts of Gynostemma pentaphyllum Makino was prepared by the following process. To 100 g of dried aerial parts of Gynostemma pentaphyllum Makino was added 500 ml of methanol. The mixture was heated under reflux for 2 hours. The methanol extract solution was concentrated to 50 ml under reduced pressure, To the concentrate were added 100 ml of water and 100 ml of ethyl acetate. The mixture was shaken. The aqueous layer was separated and concentrated to dryness under reduced pressure.

Example 6 In 15 ml of water was dissolved the same extract of the aerial parts of Gynostemma pentaphyllum Makino employed as the substrate in Example 5. The reaction was allowed to proceed in a manner similar to Example 4, employing 350 ml of the culture broth obtained in Example 4, at a pH value of 7.0 and at 55"C. The reaction mixture contained 200.5 mg of ginsenoside-Rd, 64.8 mg of ginsenoside-Rb1 and no gypenoside V.

Example 7 In 50 ml of water were dissolved 2 g of crude saponin fraction of Panax ginseng C.A. Meyer containing 305 mg of ginsenoside-Rb1 and 25 mg of ginsenoside-Rd. To the solution was added 350 ml of the culture broth obtained in Example 3. The mixture was maintained at a pH value of 5.0 for 15 hours at 50"C. The content of ginsenoside-Rd in the reaction mixture was 290.6 mg and that of ginsenoside-Rb1 was 6.3 mg.

The above-mentioned crude saponin fraction was prepared by the following process. To 2 kg of dried root of Panaxginseng C.A. Meyer was added lOt of methanol, which was heated under reflux for 2 hours. The methanol extract solution was concentrated to 1 e under reduced pressure. To the concentrate were added 2 t of water and 3 t of butanol. The butanol-soluble portion was taken, which was concentrated under reduced pressure and dried up to give 100 9 of crude saponin.

Example 8 To 600 ml of basic culture medium containing 600 mg of hesperidin was added 1 Nsodium hydroxide to render its pH value to 5.7. The medium was inoculated with Cryptococcus albidus (IFO 0378), which was incubated under shaking at 28"C for 7 days. The culture broth was processed in a manner similar to Example 3.

The results were shown in Table 5.

In the case of employing the above-mentioned culture broth of IFO 0378 prepared under the conditions of Example 8, it is concluded from the results of Table 5 that for converting gypenoside V into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of about 7 or at a temperature of about 40"C.

TABLE 5 pH value of Temperature of Residual ratio of Conversion ratio to reaction mixture reaction mixture gypenoside V (%) ginsenoside-Rd (%) 6 20"C 74.7 30.4 30"C 45.4 58.4 40"C 18.6 79.6 50"C 72.7 30.1 7 20"C 71.9 33.2 30"C 39.7 69.7 40"C 5.9 98.5 50"C 13.4 90.1 8 20"C 73.5 32.0 30"C 44.7 57.9 40"C 6.0 95.3 50"C 12.1 89.2 Example 9 To 600 ml of a basic culture medium containing 600 mg of hesperidin was added 1N sodium hydroxide to render its pH value to 5.7.The medium was inoculated with Cryptococcus laurentii (IFO 0609), which was incubated under shaking at 28"C for 7 days. The culture broth was then processed in a manner similar to Example 3. The results were shown in Table 6.

In the case of employing the above-mentioned culture broth of IFO 0609 prepared under the conditions of Example 9, it is concluded that, for converting gypenoside V into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of about 7 or at about 40"C.

TABLE 6 pH value of Temperature of Residual ratio of Conversion ratio to reaction mixture reaction mixture gypenoside V ( /0) ginsenoside-Rd (%) 6 20"C 9.9 89.3 30"C 0 100 40"C 0 100 50"C 24.4 79.3 7 20"C 10.1 90.1 30"C 0 100 40"C 0 100 50"C 0 100 8 20"C 15.3 85.6 30"C 0 100 40"C 0 100 50"C 4.7 95.2 Example 10 To 600 ml of basic culture medium containing 600 mg of hesperidin was added 1N sodium hydroxide to render its pH value to 5.7. The medium was inoculated with Cryptococcus laurentii (IFO 0609), which was incubated at 28"C for 7 days. The culture broth was subjected to cotton plug filtration. To the filtrate was added 120 ml of an extract solution of aerial parts of Gynostemma pentaphyllum Makino containing 4.45 g of 3ypenoside V, 2.05 g of ginsenoside-Rb, and 1.32g of ginsenoside-Rd. To the mixtue was added 1sodium hydroxide to render its pH value to 7.0, followed by stirring at 40"C for 75 hours. The reaction mixture was soured onto a column packed with 0.5 t of Amberlite XAD-2 (manufactured by Rohm & Haas Co., U.S.A.).The farrier on which saponin was adsorbed was washed with 2 e of water and then with 2 t of 40% v/v methanol, Followed by elution with 2t of 60% v/v methanol. The eluate was concentrated to dryness under reduced pressure to leave 10 g of yellowish brown powder, which was dissolved in 50 ml of 45% v/v methanol. The solution was poured onto a column packed with 500 g of ODS - Q3 (manufactured by Wako Pure Chemical Industries Ltd. Japan). The column was washed with 1 t of 55% v/v methanol, followed by elution with 62.5% v/v methanol to obtain 3.12 g (purity: 94%) of ginsenoside-Rd, 1.85 g (purity: 91%) of ginsenoside- Rb1 and 2.41 g (purity: 90%) of gypenoside V.

The above-mentioned extract solution of the aerial parts of Gynostemma pentaphyllum Makino was prepared by the following process. To 250 g of dried aerial parts of Gynostemma pentaphyllum Makino was added 2 t of methanol. The mixture was heated under reflux for 1 hour. The methanol solution wad concentrated to 50 ml under reduced pressure. To the concentrate were added 70 ml of water and 100 ml of ethyl acetate, and the mixture was shaken. The aqueous layer was separated.

Example 11 To 600 ml of a basic culture medium containing 600 mg of rutin was added a 0.2 M phosphate buffer solution to adjust the pH value at 5.7, which was inoculated with Aspergillus carbonarius (IFO 4038), followed by cultivation under shaking at 28"C for 7 days. The culture broth was then processed in a manner similar to Example 3. The results were shown in Tables 7 and 8.

In the case of employing the above-mentioned culture broth of I FO 4038 prepared under the conditions of Example 11, it is concluded from the results of Table 7 that, for converting gypenoside V into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of from about 6 to about 7 and at a temperature ranging from about 40 to about 60"C. And from Table 8 for converting ginsenoside-Rb, into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of from about 4 to about 6 and at a temperature ranging from about 40"C.

TABLE 7 pH value of Temperature of Residual rate of Conversion rate to reaction mixture reaction mixture gypenoside V ginsenoside-Rd (o/o) ( /0) 30"C 0 50.8 40"C 0 37.9 4 50"C 0 23.7 60"C 0 6.5 30"C 0 75.7 40"C 0 68.8 5 50"C 0 51.6 60"C 0 26.6 30"C 15.8 83.9 40"C 0 99.7 6 50"C 0 88.0 60"C 0 78.5 30"C 70.3 33.3 40"C 37.3 65.9 7 50 C 5.9 95.0 60"C 0 100 TABLE 8 pH value of Temperature of Residual rate of Conversion rate to reaction mixture reaction mixture ginsenoside-Rb, ginsenoside-Rd (o/o) ( /0) 30"C 16.6 73.0 40"C 0 80.7 4 SOC 1 1.1 44.4 60"C 12.2 0 30"C 15.1 71.2 40"C 0 76.3 50"C 7.1 64.2 60"C 26.3 19.1 30"C 42.3 60.1 40"C 28.6 74.1 6 50'C 59.5 39.6 60"C 72.3 5.1 30"C 89.2 12.6 40"C 93.1 8.1 50"C 100 0 60"C 97.4 0 Example 12 With a 0.2 M phosphate buffer solution, pH of 600 ml of the basic culture medium containing 600 mg of L-rhamnose was adjusted to 5.7, which was inoculated with Hansenula musicola (IFO 1383), followed by cultivation under shaking at 28"C for 7 days. The culture broth was then processed in a manner similar to Example 3.

The results were shown in Table 9.

TABLE 9 pH value of Temperature of Residual Conversion Residual Conversion reaction reaction rate of rate to rate of rate to mixture mixture gypenoside V ginsenoside-Rd ginsenoside-Rb, ginsenoside-Rd (0/) (0/o) ( /0) (/ ) 40"C 0 30.8 0 49.8 4 60"C 0 0 4.9 8.9 40"C 4.4 95.6 56.1 8.9 7 60"C 30.0 68.1 85.7 0 In the case of employing the above-mentioned culture broth of IFO 1383 prepared under the conditions of Example 12, it is concluded from the results of Table 9 that, for converting gypenoside V into ginsenoside-Rd, it is most preferable to allow the reaction to proceed at a pH value of about 7 and at a temperature of about 40"C. And, for converting ginsenoside-Rb, into ginsenoside-Rd, it is considered most preferable to allow the reaction to proceed at a pH value of about 4 and at a temperature of about 40"C.

Claims (14)

1. A process of producing ginsenoside-Rd which comprises allowing rhamnosidase and/or glucosidase to act on gypenoside V and/or ginsenoside-Rb,.

2. A process as claimed in claim 1, wherein the rhamnosidase and/or glucosidase are produced by cultivating microorganism which is capable of producing rhamnosidase and/or glucosidase to produce rhamnosidase and/or glucosidase in the culture broth.

3. A process as claimed in claim 2, wherein the microorganism is a fungus.

4. A process as claimed in claim 2, wherein the microorganism is a yeast.

5. A process as claimed in claim 3, wherein the fungus belongs to genus Aspergillus.

6. A process as claimed in claim 4, wherein the yeast belongs to genus Hansenula.

7. A process as claimed in claim 4, wherein the yeast belongs to genus Cryptococcus.

8. A process as claimed in claim 1, wherein the reaction is carried out at a pH value of from about 4 to about 7 at a temperature of from about 40 to about 65"C.

9. A process as claimed in claim 2, wherein the microorganism is Aspergillus niger var. fermentarius.

10. A process as claimed in claim 2, wherein the microorganism is Aspergillus carbonarius.

11. A process as claimed in claim 2, wherein the microorganism is Cryptococcus albidus.

12. A process as claimed in claim 2, wherein the microorganism is Cryptococcus laurentii.

13. A process of producing ginsenoside-Rd substantially as hereinbefore described with reference to any of the Examples.

14. Ginsenoside-Rd when prepared by a process as claimed in any of claims 1 to 13.