JPS61209599A - Production of polysaccharide by tissue culture - Google Patents

Abstract

PURPOSE:To produce the titled polysaccharide having hypoglycemic activity, in a short time, in large guantities, independent of the weather, by carrying out the tissue culture of Panax ginseng belonging to Aradiaceae Family and extracting a specific component from the cultured product. CONSTITUTION:A part of the tissue of Panax ginseng is cut off, sterilized, cut in round slices of 5-10mm thick and cultured stationary on Murashige- Skoog medium added with 0.1-10ppm vegetable hormone (e.g. indoleacetic acid) at 20-25 deg.C to effect the induction of a callus. The obtained white callus is cultured in a medium obtained by adding 0.1-5wt% glucose and <=5ppm auxins to the above Skoog medium. The obtained callus is extracted with an aqueous organic solvent to obtain a mixture of panaxanes A-E, which is fractionated by the combination of anion exchange resin treatment and molecular weight fractionation process. The objective panaxane A having the characteristics of the Table can be separated from the mixture.

Description

[Detailed description of the invention] (Industrial application field) This invention has many advantages! ! In particular, peptidoglycans having hypoglycemic effects named panaxane A, B, C, D and E which can be isolated from medicinal ginseng.

This invention relates to a method for producing medicinal ginseng by tissue culture.

(Prior art) Medicinal ginseng belonging to the Araliaceae family, especially Panax ginseng (
Panax Ginseng, C.N. Meyer; Pa
Nax ginseng (C, A, Mayer) is used as a tonic, diuretic, anti-inflammatory, and antidiabetic drug.

Regarding the components contained in this medicinal ginseng, various saponins are known as components soluble in lower aliphatic alcohols. In addition to these saponins, the inventors have discovered a new group of peptidoglycans, which are completely different from saponins, as medicinal ingredients that are contained in the water-soluble fraction and have hypoglycemic effects.

These were named panaxan (patent application 1986-290).
83) Among the ingredients contained in medicinal carrots, it is already known that saponins are produced by tissue culture, and some knowledge has been reported regarding the culture conditions. Panaxan is not produced from plant callus obtained by conventional tissue culture, which is completely unknown.

(Problems to be Solved by the Invention) The present invention solves the above conventional problems, and its purpose is to provide a method for effectively producing vanaxanes by tissue culture of medicinal ginseng. There is a particular thing.

(Means for solving the problem) The inventor conducted repeated research on a method for producing vanaxanes through tissue culture of medicinal carrots.

A strain with panaxan productivity was successfully isolated.

We established the culture method. Vanaxanes are poly-Ii compounds contained in the water-soluble fraction of medicinal ginseng.

The method for producing polysaccharides by tissue culture of the present invention is based on Panax ginseng C, which belongs to the Araliaceae family.
.

A, Mayer) and extracting from the tissue culture at least one selected from the group consisting of panaxan A, panaxan B, panaxan C, panaxan D, panaxan, and xan E having the following characteristics. However, the above objective is thereby achieved.

■Panaxan A Molecular weight (gel filtration method): t4. oo.

Specific optical rotation: [α]. +187° (c 0223. water) Infrared absorption spectrum (KBr) νwax (cm-'
): 3370.1014IH nuclear magnetic resonance δ: 4
．． 89 (d, J3Hz) ;5.20 (d, J
3Hz) Elemental analysis value (%): C, 40, 38.

H, 5,97iN, 0.15 Elution time for gel chromatography (TSK Gel G 4000W column): 29.3 minutes Elution time for DEAE-cellulose chromatography:
3.3 hours■Panaxane B Molecular weight (gel filtration method): 4. OOO,000 Specific optical rotation: [α]. +180° (c O, 12, water) Infrared absorption spectrum (KBr) νffiax (cm-
') :3430.1012IH nuclear magnetic resonance δ: 4
．． 88 (d, J3Hz) :5.24 (d, J
3Hz) Elemental analysis value (%”): C, 43, 41; H, 5,
99;N, 0.42 Elution time for gel chromatography (TSK Gel G 4000W column): 18.1 minutes Elution time for DEAE-cellulose chromatography:
3.3 hours ■Panaxane C Molecular weight (gel filtration method): 34.000 Specific optical rotation:
[α) e +96.1@(c O, 10, water) Infrared absorption spectrum (KBr) νWaX (cm-'
): 3390.1021'H nuclear magnetic resonance δ: 4
．． 88 (d, J3Hz) ;5.18 (d, J
3Hz) Elemental analysis value (%”): C, 35, 37: H, 5,
35:N, 1.06 Elution time of gel chromatography (TSK Gel G 4000W column): 28.9 minutes Elution time of DEAE-cellulose chromatography =
7.3 hours ■ Panaxan D Molecular weight (gel filtration method) = 350. OOO specific rotation:
[α]. +126' (c 1.01. water) Infrared absorption spectrum (KBr) νIIIax (cm-
') :3420.1035IH nuclear magnetic resonance δ: 4
．． 92 (d, J3H2): 5.23 (d, J
3Hz) Elemental analysis value (%): C, 39,20; H, 6,0
2, N, 1.23 Gel chromatography (TSK Gel G 4000W column) elution time: 28.0 minutes DEAll! -Cellulose Chromatography elution time: 3 hours ■Panaxane E Molecular weight (gel filtration method): 1,500. OOO specific optical rotation: [α]. +188@ (CO, 20, water) Infrared absorption spectrum (KBr) νa+ax (cm-'
): 3390.10171) 1 nuclear magnetic resonance δ:
4.87 (d, J3Hz) ;5.22 (d,
J3Hz) Elemental analysis value (%): C, 37, 41.

, H, 6,25:N, 0.68 Elution time of gel chromatography (TSK Gel G 4000W column): 17.7 minutes Elution time of DEAH-cellulose chromatography Near, 3 hours According to the method of the present invention, first, A part of the medicinal carrot tissue is cultured in a conventional manner to induce callus. Panax ginseng is preferably used as the medicinal ginseng. For example, a part of the tissue (root, stem, etc.) of this Panax ginseng is cut out, the cut end and surface are thoroughly sterilized using an aqueous disinfectant solution, and then washed with sterile water. Add this to a thickness of 5 to 10
Cut into slices, Murashige-3koog
using an agar medium etc.

Static culture is performed at 20-25°C. The medium used does not need to be special at all, and the above-mentioned Mu, rashige-5
% of Koog's medium commonly used for baka plant tissue culture
1hite medium, Lin5s+aier-3koo
g's medium, GHtheret's medium, Tuleck
E's medium, Morel's medium, etc. can be used. In order to promote callus induction, plant hormones (auxins and cytokinins) are added to the medium in advance by a conventional method.

Examples of auxins include 2,4-dichlorophenoxyacetic acid (2,4-D), indoleacetic acid (IAA), and naphthaleneacetic acid (NAA), and 0.1 to 10 p
It is added at a ratio of p+w. Examples of cytokinins include kinetin and benzyladenine, which are added to the medium at a rate of 5 ppm to 5 ppm of O,OS.

The callus induced in this way has a white color. This white callus is transferred to a callus culture medium and statically cultured.

The callus culture medium is Mura, similar to the callus induction medium described above.
A known medium (a solid medium such as an agar medium or a liquid medium) such as Shige-3koog's medium is used. In addition, 0.1 to 5% by weight of glucose, preferably 0.
．． It is added in a proportion of 1 to 2% by weight. The amount of shakerose contained in the medium may be reduced depending on the amount of glucose added.

The added glucose is taken up into the callus and acts as a precursor of panaxan, so that panaxan can be advantageously produced within the callus. If the amount of glucose is too low, the production of panaxan will be low. Even if it is in excess, it has no further effect. The amount of plant hormones contained in the callus culture medium is adjusted to be lower than the amount of plant hormones in the callus induction medium.Normally, the amount of auxins is 5 ppm or less,
Contains cytokinins at a rate of 2 ppm or less. It may not contain any plant hormones at all. When the proliferated white callus is subcultured, calli with different colors and shapes appear. From white callus to brown callus (type B strain callus) and differentiated callus with a shape similar to a leaf (L type strain callus) are produced. From the L-type callus, a callus (R-type callus) having a shape closer to a root is generated. Four types of callus are obtained, including a white callus (W-type strain callus) obtained by subculture in a callus culture medium.

Type W strain callus has almost the same properties as white callus derived from Panax ginseng tissue, but has a higher vanaxane production ability than white callus. No ability to produce alkaloids. B type strain callus, L type strain callus and R type strain callus
Type stock callus.

It has the ability to produce saponins and alkaloids in addition to vanaxanes.

To obtain panaxanes from these calli.

First, callus is extracted with water or an aqueous organic solvent, either as it is or after drying. Extracting after drying the callus increases the extraction rate of vanaxane. This is thought to be because panaxanes are contained in the cell walls of callus, and therefore panaxanes can be extracted advantageously by dying the callus cells. Although water is sufficient for extraction, an aqueous organic solvent may be used to prevent spoilage of the extract and to promote extraction, or extraction may be performed with both.

Examples of aqueous organic solvents include lower alcohols such as methanol and ethanol, acetone, and acetonitrile.

A mixed solution of water and a water-soluble organic solvent such as dimethylformamide or dimethylacetamide is used. Its concentration varies depending on the dry state of the callus, but it is approximately 1 to 6
0%, preferably about 1-20%. Heating is preferable because it accelerates extraction.

The above extract is subjected to dialysis or ultrafiltration to obtain a panaxane mixture. When subjecting to dialysis or ultrafiltration, the above extract may be used as it is, or may be concentrated by distilling off the solvent. In addition, a water-soluble organic solvent such as methanol or ethanol is added to the above extract to precipitate a precipitate containing the desired panaxane.

The obtained precipitate may be diluted by adding an appropriate amount of water or the above-mentioned aqueous organic solvent.

The panaxane mixture thus obtained usually contains at least 60% of the total amount of panaxanes A, B, C, D and E. Preferably, the panaxane content is 80% or more; such a vanaxane mixture can itself be used as a hypoglycemic agent. However, this mixture may be further subjected to the following fractionation treatment to isolate each panaxane component and use it as a hypoglycemic agent.

First, the above panaxane mixture is treated with an anion exchange resin 9 such as DEAE Sephadex and DEAE Toyopearl (both trade names) to separate a fraction containing panaxane A and B and a fraction containing panaxane C, D and E. Fractionate into. The fraction containing panaxane A and B thus obtained is then subjected to a molecular weight fractionation method, which uses Sepharose, Sephacryl, Sephadex, agarose beads (all trade names), etc. Gel filtration method: A method using an ultrafiltration membrane having a molecular weight cut-off of 20,000 to 100,000 is exemplified. In this manner, it is fractionated into a fraction containing panaxane A and a fraction containing vanaxane B by the molecular weight fractionation method 9, for example, column chromatography using Sepharose 6B. The fraction containing Panaxane B is further purified using Sephacryl S-500 (trade name) or the like to obtain Panaxane B.

The previously obtained fractions containing panaxane C, D and E were further purified by molecular weight fractionation method 9, for example column chromatography using Sepharose 6B, into a fraction containing vanaxane C and D and a fraction containing panaxane E. fractionated.

The fractions containing panaxane C and D are further purified by molecular weight fractionation method 2, such as column chromatography using Sephacryl S-200 (trade name).
It is separated into a fraction containing vanaxane D and a fraction containing vanaxane D.

(Effect) In this way, panaxanes are advantageously produced by tissue culture of medicinal ginseng. B type strain callus, L type strain callus, R type strain callus
Panaxanes can be advantageously extracted from any type of callus, although it differs somewhat depending on the type of callus. Separated panaxane A and B.

C, D and E all have excellent hypoglycemic effects,
And almost no side effects are observed.

(Example) The present invention will be described below with reference to Examples.

Induction and culture of callus (A) A portion of the root of Panax ginseng was cut out and immersed in a 70% ethanol solution for 3 minutes. Furthermore, it was immersed in an aqueous sodium hypochlorite solution (CI content 1.5%) for 20 minutes, and then washed with sterilized water. 5 to 10 ginseng roots sterilized in this way
Slice into m-thick rounds.

The cells were placed on a callus induction medium prepared in advance.

+ Murashige-5 as a callus induction medium
Koog's medium contains 10 ppm of IAA as auxin.
, and 2 pp of kinetin as cytokinin.
A conventional agar medium for callus induction supplemented with a+ was used. When this was cultured in the dark at 20-25°C for 30 days, a white callus was formed.

Mura with a 5ucrose content of 2% by weight
A callus culture medium (agar medium) was prepared by adding 0.5% by weight of glucose, 5 pp+s of IAA as auxin, and 1 pp-s of kinetin as cytokinin to a Shige-Skoog medium. The above white callus was transplanted onto this callus culture agar medium and cultured under the same conditions. When the proliferated callus was subcultured, a brown callus (type B strain callus) and a differentiated callus with a leaf shape (L type strain callus) appeared. White callus (W-type callus), B-type callus, and L-type callus were separated and subsequently cultured. The L-type strain was further differentiated into a callus with root morphology (R-type strain).

R-type strains were also isolated, and the respective calli were subsequently cultured.

Next, prepare a liquid medium with the same components as the above callus culture medium, and add 500 ml to each of four 11 Erlenmeyer flasks.
The solution was divided into portions and sterilized using a conventional method.

Approximately 20 calli of the above four types were added to this callus culture liquid medium.
The cells were transplanted and cultured with shaking at 25° C. for 4 weeks. The medium was filtered, and 120 g of W-type callus and 105 g of B-type callus were collected.
Type strain callus, 110g L type strain callus, and 93
R-type strain callus of g was obtained.

(B) Extraction of panaxanes from W type strain callus: (A)
After drying the W-type strain callus obtained in Section.

This was extracted once with 50% ethanol 50II11 and then twice with water. After the extracts were combined and concentrated, dialysis was performed for 2 days using a cellulose tube. When the obtained dialyzed fluid was subjected to ethanol precipitation and its weight was measured, it was found that 592 μ of crude panaxane was contained per 100 g (in terms of dry matter) of callus. This dialysis solution was applied to a DEAE cellulose column (2aaφX 100cm).
), then 1 and then 0.
Fraction P eluted with 1-0.5M NaCl aqueous solution
-1 and fraction P-2 were obtained. Fraction P-
The vanaxanes contained in Fraction 1 and Fraction P-2 are each 99 μg per 100 g of callus (in terms of dry matter).
and 234 ■.

This fraction P-1 was transferred to a Sepharose 6B column (2
CIlφX95C1l; 0. Fractionation was performed using IM NaC1) to obtain fraction P-3 and fraction P-4. Fraction P-3 was concentrated to obtain 63 ml of white powder. When the physicochemical properties of this white powder were investigated, the following measured values were obtained.

Molecular weight (gel filtration method): 14.000 Specific optical rotation:
[α]. +187° (c O, 23, water) Infrared absorption spectrum (KBr) νIIIax (cm-
') :3370. 10141H nuclear magnetic resonance δ:
4.89 (d, J3Hz); 5.20 (d,
J3Hz) Elemental analysis value (%): C, 40,38; H, 5,9
7SN, 0.15 Glass fiber filter paper electrophoretic mobility: 19.7 cm (glucose 13.0m) Polyacrylamide electrophoretic mobility: Qai gel chromatography (T
Elution time of SK gel G 4000W column: 29.
3 minutes DEAR-cellulose chromatography elution time:
3.3 hours The white powder from the above data is a single substance.

It turned out to be vanaxane A. Fraction P-
4 was applied to a Sephacryl S-500 column (2 aaφ x 95 pieces, 0.1 M Tris-HCl buffer pH 7.0) to obtain 34 μm of white powder. When the physicochemical properties of this white powder were investigated, the following measured values were obtained.

Molecular weight (gel filtration method): 4,000.000 Specific optical rotation: [α] o + 180° (c O, 12,
Water) Infrared absorption spectrum (KBr) νwax (cm-'
): 3430.1012IH nuclear magnetic resonance δ: 4
．． 8B (d, J3Hz) ;5.24 (d, J
3H2) Elemental analysis value (%): C, 43, 41: H, 5, 9
9, N, 0.42 Glass fiber filter paper electrophoretic mobility: 19.0 C1l (glucose 13.0 cm) polyacrylamide electrophoretic mobility: 03 Gel chromatography (TSK Gel G 4000W column) elution time: 1B, 1 minute DEAE - Cellulose chromatography elution time:
This white powder was found to be Panaxane B based on the above data for 3.3 hours.The next fraction P-2 was transferred to a Sepharose 6B column (21φ
) to obtain fraction P-5 and fraction P-6. Fraction P-5 was prepared using a Sephacryl S-200 column (2CI1φX95cm, 0.1M).
Hydrochloric acid buffer pH 7, 0, 0, 5M NaCl),
86■ white powder (I) and 93■white powder (II
) was obtained. When the physicochemical properties of each were investigated, the following measured values were obtained.

Physicochemical characteristics of H) Molecular weight (gel filtration method): 34,000 Specific optical rotation:
[α) o +96.1' (c O, 10, water) Infrared absorption spectrum (KBr) νmax (cm-'
): 3390.1021tH nuclear magnetic resonance δ: 4
．． 8B (d, J3Hz) ;5.18 (d, J
3Hz) Elemental analysis value (%): C, 35, 37: H, 5, 3
5; N+ 1.06 Glass fiber-filter paper electrophoretic mobility: 19.0 Bm (glucose 13.0 Bm) Polyacrylamide electrophoretic mobility: Qaa gel chromatography (
Elution time of TSK gel G 4000W column: 28
．． 9 minutes DEAE-Cellulose Chromatography elution time Near, 3 hours (I[) Physicochemical properties Molecular weight (gel filtration method): 350,000 Specific optical rotation:
[α) D+126” (c 1.01. Water) Infrared absorption spectrum (KBr) νmax (cm-'
): 3420.1035IH nuclear magnetic resonance δ: 4
．． 92 (d, J3Hz) ;5.23 (d, J
3Hz) Elemental analysis value (%): C, 39,20; H, 6,0
2:N, 1.23 Glass fiber filter paper electrophoretic mobility: 19.0Bm (glucose 13.OClm) Polyacrylamide electrophoretic mobility: 0cIIl gel chromatography (TSK gel G 4000W column) elution time:
28.0 min DEAE-cellulose chromatography elution time near, 3 hours From the above data these white powders (I) and (n)
were found to be vanaxane C and vanaxane D, respectively. When fraction P-6 was concentrated, 23
A white powder (2) was obtained. When the physicochemical properties of this white powder were investigated, the following measured values were obtained.

Molecular weight (gel filtration method): 1.500,000 Specific optical rotation: [α]・+188° (cO, 20, water) Infrared absorption spectrum (KBr) νwax (C′)
:3390. 10171H nuclear magnetic resonance δ: 4
．． 87 (d, J3H2) ;5.22 (d, J
3Hz) Elemental analysis value (%): C, 37, 41.

H, 6,25, N, 0.68 Glass fiber filter paper electrophoretic mobility: 19.3C11 (glucose 13.0Bm) polyacrylamide electrophoretic mobility: 0C11 gel chromatography
(TS) (Gel G 4000W column) elution time:
17.7 minutes Dll! AI! - Cellulose Chromatography elution time: Near, 3 hours From the above data, this white powder was found to be Panaxane E.

(The following is a blank space) When the decomposition properties of Panaxane A, B, C, D, and E to acids were investigated, the following results were obtained.

Each of acid-hydrolysable panaxanes A, B, C, D and E had 2N
Sulfuric acid was added and the mixture was heated at 100° C. for 6 hours to perform hydrolysis. Thin layer chromatography (developing solvent: n-butanol)
Pyridine-water (6:4:3) mixture) and p
- When the color was developed with anisidine hydrochloric acid solution, Rf O,
A single spot was observed in 29. This spot matched the control glucose spot. In addition, 6N hydrochloric acid was added to each of Panaxane A-E, and the mixture was heated at 100°C for 24 hours.
Hydrolysis was carried out by heating for hours. Thin layer chromatography (
Developing solvent: n-butanol-acetic acid-water (12:3:
5) Mixture) was carried out, and several spots were observed when color was developed with ninhydrin.

Among the above data, the molecular weight, glass fiber filter paper electrophoretic mobility, polyacrylamide electrophoretic mobility, gel chromatography elution time, and DEAE-cellulose chromatography elution time were measured as follows. The component other than C9H and N in the elemental analysis value is O.

1) Molecular weight Each vanaxane was subjected to gel filtration using Sephacryl S-200, 300, or 500 to determine its retention capacity, and the molecular weight was calculated from a standard curve prepared using Dextran T series.

Molecular weight used for measurement Cephacrylpanaxa 7 A 14.000 S-20
0 Panaxan B 4,000.00OS-500 Panaxa7 C34,00OS-200 Banaxa7 D
350.00OS-300 Panaxa 7E 1
．． 500.00OS-5002) Glass fiber filter paper Electrophoretic mobility Glass fiber filter paper (Whatman GF/C, 15X4
The distance traveled was measured using 0aa). (Conditions: alkaline borate buffer pH 9, 2,450V, 2 hours)
3) Polyacrylamide electrophoretic mobility 30% polyacrylamide gel column (inner diameter 10 cm)
) was used to measure the distance traveled. (Conditions: diborate buffer pH 9, 3, 2 mA, 2 hours, color development using thymol sulfuric acid method) 4) Gel chromatography elution time TSK Gel G 4000W column (inner diameter 0.75C1)
1, length 600 m), and elution (0.5 tail/a+in) was performed with a 0.1 M aqueous sodium chloride solution.

5) Elution time of DEAE-cellulose chromatography Using a DEAE-cellulose column (inner diameter 2cIII, length 50al), elution was performed with 0.05M Tris-HCl buffer (pi 8.0) for 5 hours, and then the above buffer Elution was performed using a buffer solution containing sodium chloride. (0-0.5M NaCl 1,20 hours,
Elution rate 1 @j! /5in-) * (C) Extraction of panaxanes from type B strain callus: (A)
After drying the type B strain callus obtained in Section 1.

Using this, extraction was performed in the same manner as in section (B). The panaxanes contained in the dialysate fluid after dialysis treatment, fraction P-1 and fraction P-2, are called callus 100.
They were 729 ■, 68 ■ and 370 ■ per g (dry matter equivalent), respectively. The dialysis fluid was treated in the same manner as in section (B), and the white powders obtained were panaxane A and panaxane A, respectively.
B, C, D and E were confirmed. The yield was 28cm of vanaxane A.

The weight of vanaxane B was 22cm, the weight of vanaxane C was 86cm, the weight of panaxane D was 93cm, and the weight of panaxane E was 45cm.

CD) Extraction of panaxanes from type L strain callus = (A)
After drying the L-type strain callus obtained in Section.

Using this, extraction was performed in the same manner as in section CB). The panaxanes contained in the dialysate fluid after dialysis treatment, fraction P-1 and fraction P-2, are called callus 100.
They were 524 ■, 67 ■ and 482 ■ per g (dry matter equivalent), respectively. The dialysis fluid was treated in the same manner as in section (B), and the white powders obtained were panaxane A and panaxane A, respectively.
B, C, D and E were confirmed. The yield was 22cm of Panaxane A.

Panaxane B was 18 ■, Panaxane C was 128 ■, Vanaxane D was 86 ■, and Panaxane E was 55 ■.

(E) Extraction of panaxanes from R type strain callus = (A)
After drying the R-type strain callus obtained in Section.

Using this, extraction was performed in the same manner as in section (B). The panaxanes contained in the dialysate fluid after dialysis treatment, fraction P-1 and fraction P-2, are called callus 100.
They were 268 ■, 105 ■ and 133 ■ per g (dry matter equivalent), respectively. The dialysis fluid was treated in the same manner as in section (B), and the resulting white powder was treated with panaxan A.
, B, C, D and E. The yield was 36 cm for panaxane A, 21 cm for panaxane B, and 130 cm for vanaxane C.

Panaxane D was 172 Illr and Panaxane E was 46 ■.

Grind 100g of the dried root of Kitagogo Panax ginseng, mix once with 200wl of 50% methanol aqueous solution, and then add 300v of water.
Extraction was performed twice with all. The extracts were combined and concentrated under reduced pressure to obtain 38 g of extract. Add this extract to 5゜g of cellulose powder (Whatman CFII; trade name)
mixed with. The above mixture was layered on the cellulose layer of a column previously filled with cellulose powder of the same quality, and washed by injecting methanol. After further washing with a 50% methanol aqueous solution, elution was performed with water.

The obtained fraction was concentrated to obtain 3.8 g of a water-soluble fraction. This water-soluble fraction was collected in a cellulose tube (Vi
skin Co.

Dialysis was performed for 4 days using The resulting dialysis fluid contained 1082 ml of vanaxanes per 100 g of dried Panax ginseng root. The dialyzed fluid was subjected to molecular weight fractionation using a DEAE cellulose column (2 units φX 100a1) (pH 8.0 Tris-HCl buffer, 0.0.
1-0.5M NaCl), fraction P-1 and fraction P-2 were obtained. Fraction P-1 contains a total of 619 μ of panaxane per 100 g of dried Panax ginseng root, and Fraction P-2 contains:
A total of 412 parts of panaxane was contained per 100 g of dried Panax ginseng root.

In Examples and Comparative Examples, dialysis fluid, fraction P
The contents of panaxanes contained in -1 and fraction P-2 are summarized in the table below.

(■/100g) (Effects of the Invention) According to the present invention, callus having a high production ability of polyclass II and panaxane contained in medicinal ginseng can be effectively tissue cultured in a medium containing glucose. be done. Therefore, since the zero-tissue culture is used, which can produce a large amount of vanaxanes in a short period of time without requiring a long period of time to cultivate medicinal ginseng as in the past, it is not affected by natural conditions such as weather. The obtained vanaxanes have a hypoglycemic effect.

It can be used as a drug in various forms. 'that's all

Claims (1)

[Scope of Claims] 1. Panax ginseng (Panaxgi), which belongs to the Araliaceae family.
nseng C. A. Panaxan A, Panaxan B, Panaxan C, Panaxan D, and Panaxan E having the following properties were obtained from the tissue culture.
A method for producing a polysaccharide having a hypoglycemic effect, which comprises extracting at least one selected from the group consisting of: (1) Panaxane A Molecular weight (gel filtration method): 14,000 Specific rotation: [α]_D+187° (c0.23, water) Infrared absorption spectrum (KBr) νmax (cm^-^1:3370,1014^1H Nuclear magnetic resonance δ: 4.89 (d, J3Hz): 5.
20 (d, J3Hz) Elemental analysis value (%): C, 40.38: H, 5.97: N, 0.15 Elution time of gel chromatography (TSK gel G4000W column): 29.3 minutes DEAE-cellulose chromatography Elution time: 3
．． 3 hours (2) Panaxane B Molecular weight (gel filtration method): 4,000,000 Specific optical rotation:
[α]_D+180° (c0.12, water) Infrared absorption spectrum (KBr) νmax (cm^-^1): 3430, 1012^1H nuclear magnetic resonance δ: 4.88 (d, J3Hz): 5.
24 (d, J3Hz) Elemental analysis value (%): C, 43.41: H, 5.99: N, 0.42 Elution time of gel chromatography (TSK gel G4000W column): 18.1 minutes DEAE-cellulose chromatography Elution time: 3
．． 3 hours (3) Panaxane C Molecular weight (gel filtration method): 34,000 Specific optical rotation: [α]_D+96.1° (c0.10, water) Infrared absorption spectrum (KBr) νmax (cm^-^1): 3390, 1021 ^1H nuclear magnetic resonance δ: 4.88 (d, J3Hz): 5.
18 (d, J3Hz) Elemental analysis value (%): C, 35.37: H, 5.35: N, 1.06 Elution time of gel chromatography (TSK gel G4000W column): 28.9 minutes DEAE-cellulose chromatography Elution time: 7
．． 3 hours (4) Panaxane D Molecular weight (gel filtration method): 350,000 Specific optical rotation: [α]_D+126° (c1.01, water) Infrared absorption spectrum (KBr) νmax (cm^-^1): 3420, 1035 ^1H nuclear magnetic resonance δ: 4.92 (d, J3Hz): 5.
23 (d, J3Hz) Elemental analysis value (%): C, 39.20: H, 6.02: N, 1.23 Elution time of gel chromatography (TSK gel G4000W column): 28.0 minutes DEAE-cellulose chromatography Elution time: 7
．． 3 hours (5) Panaxane E Molecular weight (gel filtration method): 1,500,000 Specific optical rotation:
[α]_D+188° (c0.20, water) Infrared absorption spectrum (KBr) νmax (cm^-^1): 3390,1017^1H nuclear magnetic resonance δ: 4.87 (d, J3Hz): 5.
22 (d, J3Hz) Elemental analysis value (%): C, 37.41: H, 6.25: N, 0.68 Elution time of gel chromatography (TSK gel G4000W column): 17.7 minutes DEAE-cellulose chromatography Elution time: 7
．． 2. The manufacturing method according to claim 1, wherein the medium used for the tissue culture contains 0.1 to 5.0% by weight of glucose.