JPH01285116A - Method for producing root of 'saiko' - Google Patents

Abstract

PURPOSE:To increase the content of saikosaponins in SAIKO (root of Bupleurum falcatum) obtained by the tissue-culture of the root by restricting the concentration of nitrogen and the ratio of nitrate nitrogen to ammoniacal nitrogen in the medium. CONSTITUTION:A root tissue of a plant belonging to genus Bupleurum, e.g., Bupleurum falcatum is cultured in a nutrient medium having total nitrogen content of 100-500ppm and a ratio of nitric nitrogen to ammoniacal nitrogen of 95/5-70/30. The SAIKO produced in the above nutrient medium has high sailosaponin content and high reproductive rate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to tissue culture of roots of plants belonging to the genus Bupleurum. The present invention relates to a method for producing saiko root by propagating taproots with high saikosaponin content.

The present invention can be used to produce saiko root, which is used in Chinese medicine formulations such as Daisaikoto and Shosaikoto, and dried saikoroot can be used as an antipyretic agent in Chinese medicine formulations, or as an effective treatment for arteriosclerosis and liver diseases. Used as a herbal medicine.

[Technical background and explanation of conventional technology]

Synthetic drugs are widely used in the treatment of various diseases due to their rapid and accurate therapeutic effects, but there are side effects such as the side effects of many cancer treatment drugs, hearing loss caused by large doses of streptomycin, etc. Due to side effects such as penicillin shock caused by administration of penicillin or drug damage caused by administration of chinoform, traditional Chinese herbal medicines that have been used for a long time are being reconsidered in terms of clinical trials to ensure safety for the human body. Saiko is a commonly used medicine used in many Chinese herbal medicines such as Daisaikoto and Shosaikoto, and is widely used for anti-inflammatory, antipyretic, analgesic, and detoxifying purposes.

Mishima Saiko (Bupleurum falcatum)
L, ) is known as high-quality saiko from Japan,
Its production is not enough to meet demand, and large quantities are imported from China and South Korea. It is already known that the active ingredients of Saiko are saikosaponins (saikosaponins a, c, and d) [Japanese Patent Publication No. 10723/1983, Tet
rahedron Letters 303 (+96
8), Planta medica 40+ 366
(1980), J. Pharm.

Dyn, 3.269 (+980) ), a plant of the genus Bupurulum is extracted with an alkaline solution, and the alkaline solution extract is extracted with water and an organic solvent that is hardly miscible with water, or a lead salt is added to the alkaline solution extract. In addition, a method for separating the saiko component is known, in which it is precipitated and the precipitate is extracted with a water-based organic solvent (Japanese Patent Application Laid-open No. 76213/1983).
In addition, a method for obtaining regenerated plants from callus by propagating callus derived from plants of the genus Bupurulum by suspension culture and then lowering the auxin concentration of the culture solution in the dark (Japanese Patent Application Laid-Open No. 1983-1999) 12988) has been proposed.

In addition, in conventional plant tissue culture, a method of culturing specific organs of seedlings obtained by germinating plant seeds without inducing callus and obtaining a large amount of the specific organs (Japanese Patent Laid-Open No. 59-95816) is known.

The present inventors have been conducting research on tissue culture of plants for many years, and in research on tissue culture of plants of Mishimasaiko, it was found that the tap root of Mishimasaiko contains a significant amount of saikosaponins, but the content of saikosaponins in the lateral roots is Furthermore, in tissue culture of the roots of Cycosaponins, we found that replacing ammonia nitrogen in the medium with nitrate nitrogen increased the proliferation of taproots with a high sacosaponin content.Based on these findings, we developed this book. invention has been achieved.

[Object of the invention and summary of the invention]

An object of the present invention is to provide a method for producing saiko root, and more specifically, to provide a method for producing saiko root by tissue culture. An object of the present invention is to provide a method for producing peppermint by tissue culture.

The present invention provides a method for producing sapiens roots which are grown by tissue culturing the root tissue of a plant belonging to the genus Bupululum in a nutrient medium, in which the nutrient medium has a ratio of nitrate nitrogen as a nitrogen source greater than that of ammonia nitrogen. This is a method for producing saiko root characterized by its composition.

In the tissue culture of sapiens root in the nitrate-containing nutrient medium of the present invention, a nutrient medium containing a total nitrogen content of at least 100 to 500 ppm can be used, and 951
Using a nutrient medium containing nitrate nitrogen and ammonia nitrogen in a ratio of 5 to 70/30 nitrate nitrogen/ammonium nitrogen, thereby improving the multiplication factor of root tissue, and also plants belonging to the genus Bupululum. can use the root tissue of a plant belonging to the genus Bupululum, and use the root tissue of a young plant obtained by germinating the seeds of a plant belonging to the genus Bupululum, thereby reducing the saikosaponin content. It is possible to produce high quality saiko root. Furthermore, the nitrate nitrogen in the nutrient medium is added to the nutrient medium by potassium nitrate, thereby making it possible to obtain chai root with a high saikosaponin content and improved commercial value.

[Detailed description of the invention]

The plants belonging to the genus Bupleurum used in the production of the saiko root of the present invention include Bupleurumfalc.
atu+s) + Hotaru Psycho (B, 5acha
linense) + Manshuumishima Saiko (B,
chinense) + Dafurya Psycho (B, dah
uricum) + hosoba mishima wallet (B, sco
rzoneraefolium) + towpsycho (
B, jeholense) + Kohoku Saiko (B,
Microcepha-ium ) + B. euphorbioides are known (Encyclopedia of Medicinal Plants, Koyo Shoten) 9 In the present invention, root tissue pieces of these plants belonging to the genus Bupululum can be used. , it is preferable to use root tissue pieces of Mishimasaiko.

The root tissue piece can be used by cutting the root part of an existing plant body, or by cutting the root part of a seedling obtained by germinating a plant seed. A piece of root tissue obtained by culturing callus derived from the body and redifferentiating into root tissue can be used.

The nutrient medium used for tissue culture of root tissue pieces in the present invention must contain nitrate nitrogen (NO-N) as a nitrogen source.

If the nutrient medium is one that does not contain nitrate nitrogen and only contains ammonia nitrogen (NH-N) as a nutrient source, the root tissue will not only not grow normally, but will also be in poor condition during life, and the content of psychosaponins will decrease. This results in a decrease in the production of the active ingredient psychosaponins.

When ammonia nitrogen in the nutrient medium was replaced with nitrate nitrogen, normal growth was observed, and the shape of the cultured roots changed from having many lateral roots (bearded roots) to having fewer lateral roots and an enlarged main root. As a result, the content of saikosaponins also increases. For cultured root production with a balance of root proliferation, root morphology, and saikosaponin content, a total nitrogen content of 100 to 500 is required.
ppm, nitrate nitrogen/ammonia nitrogen ratio is 9
A range of 515 to 70/30 is preferred, thereby increasing the yield of root tissue of plants belonging to the genus Bupululum. Tissue culture of root tissue pieces in the present invention is performed at 18°C to 3°C.
It is preferable to carry out the cultivation under aerobic conditions in the dark at a temperature of 0° C., and it is more preferable to use aeration stirring or shaking culture.

The root tissues of plants belonging to the Bupurulum genus grown by the tissue culture of the present invention can be washed with water and dried to be used as a material for Chinese herbal medicine formulations, and psychosaponins can also be extracted by known methods. It can also be used as a raw material for separation.

The present invention will be explained in more detail below using test examples and examples.

Test Example 1 In tissue culture of root tissue pieces of P. japonica, a test was conducted to examine the influence of the form of the nitrogen source in the medium (nitrate form and ammonia form) on its growth.

H) Preparation of root tissue pieces of Mishimasaiko It was carried out in the same manner as in the example.

(2) NH in the MS (Murashige-Skoog) basic medium used in the test
Instead of NO, an amount of NHC1 corresponding to its nitrogen content
and KNO to constitute a modified medium with the composition shown in Table 1, and the amounts of NHC1 and KNO in the modified medium were changed as shown in Table 2 according to their respective nitrogen contents, respectively. A medium was prepared.

(Leaving space below) Table 1 Composition of modified culture medium in Test Example 1 Table 2 Amount of nitrogen source in culture medium in Test Example 1 (ppm) (3)
Test method In a modified medium with the composition shown in Table 1, the nitrogen source KNO
A piece of root tissue of P. aeruginosa was cultured in the same manner as in Example, except that a medium containing 3 and NH4Cl as nitrogen sources as shown in Table 2 was used, and the culture period was 50 days.

In addition, culture of the root tissue pieces of P. aeruginosa was carried out in the same manner as above, using media containing i ppm and 2 ppm of indolebutyric acid as the plant hormone in each medium.

(4) Results The multiplication factor (dry type) of the root tissue fragments of Mishimasaiko was as shown in Figure 1. As the ratio of nitrate nitrogen/ammonium nitrogen increases, the main root of the multiplied root tissue fibers increases. It became enlarged and had fewer lateral roots (beard roots).

Saikosaponin (a, c), the active ingredient of Mishima Saiko
, d, ttffi) (with respect to the dry type) was analyzed separately in the main root and lateral roots, and it was found that (psychosaponin content in the main root) / (psychosaponin content in the lateral root) It was 8 to 14 times. In FIG. 1, the vertical axis is the multiplication factor of the dry form of the root tissue of P. aeruginosa, and the horizontal axis is the nitrate nitrogen/ammonia nitrogen (weight ratio) in the nitrogen source of the culture medium.

A C solid line (○)] indicates that the indole butyric acid concentration is ippm.
B [dotted line (x)] is the result in a medium with an indolebutyric acid concentration of 2 ppm.

(5) Discussion According to Figure 1, when using a medium in which the ratio of nitrate nitrogen/ammonium nitrogen in the nitrogen source of the medium was adjusted to 7V30 or higher, the multiplication factor of the root tissue pieces of Mishima rhinoceros was improved, and It can be seen that the proportion of

Test Example 2 In tissue culture of root tissue pieces of P. japonica, a test was conducted to examine the influence of the form of the nitrogen source (ammonium nitrogen and nitrate nitrogen) in the medium on saikosaponin production.

(1) Preparation of root tissue piece of Mishima Saiko The same procedure as in Example was carried out.

(2) Medium LS (Linsmeyer-Skoog) used in the test Instead of NHNo in the basic medium, NHC1 and KNO were used in amounts corresponding to the nitrogen content to prepare a modified LS medium with the composition shown in Table 3. Construct and NHC in modified LS medium
1 and KNO according to their respective nitrogen contents,
Each medium was prepared with the changes shown in Table 4.

Table 3 Composition of the modified LS medium of Test Example 3 Table 4 Amount of nitrogen source in the medium of Test Example 2 (ppm) (3) Test method In the modified LS medium with the composition shown in Table 3, the nitrogen source K
Using a medium containing NO and NHC1 as nitrogen sources as shown in Table 4, the root tissue pieces of P. japonica were cultured in the same manner as in the examples.

(4) Results The content of saikosaponin in the root tissue of Mishima saiko (
(based on dry weight) was higher when an appropriate amount of ammonia nitrogen was added than when nitrate nitrogen was the only nitrogen source in the medium. Although ammonia nitrogen has the effect of suppressing proliferation, it was recognized that it is a necessary component for producing saikosaponin. Table 5 shows the relationship between the ratio of nitrate nitrogen and ammonia nitrogen as nitrogen groups and the production amount of saikosaponin by the root tissue per liter of medium volume.

Table 5 Results of Test Example 2 (5) Discussion According to Table 5, culturing the root tissue pieces of P. aeruginosa in a medium in which the ratio of nitrate nitrogen/ammonium nitrogen in the nitrogen source of the medium was adjusted to 7V30 to 9515 resulted in It can be seen that the production amount of saponin is improved.

Test Example 3 Using an LS basic medium, a test was conducted on the influence of the total nitrogen content of the medium on the growth and production of saikosaponin in tissue culture of root tissue pieces of P. aeruginosa.

(+) Preparation of root tissue piece of Mishima Saiko The same procedure as in Example was carried out.

(2) Instead of NHNo in the medium LS basic medium used in the test, a modified LS medium with the composition shown in Table 3 was constructed using NHC1 and KNO in an amount equivalent to the nitrogen content, and The amounts of NHC1 and KNO were changed so that the total amount of nitrate nitrogen and ammonia nitrogen was the amount shown in Table 6 at a nitrate nitrogen/ammonia nitrogen ratio of 85/15 and 10010, and each culture medium was Prepared.

Table 6 Nitrogen (3) test method in the culture medium of Test Example 3 A culture medium with KNO and NHC1 in the modified LS medium of Table 3 as shown in Table 6 was used, and the culture was carried out for 5 days.
The root tissue pieces of P. japonica were cultured in the same manner as in the example except that the culture was carried out for 1 day.

(4) Results The multiplication factor (dry weight) of the root tissue fragments of Mishimasaiko was 2nd.
As shown in the figure, all of the proliferated root tissues had enlarged taproots and few lateral roots (bearded roots). The growth of root tissue fragments when the nitrate nitrogen/ammonia nitrogen ratio was 85/15 was slightly lower than that with nitrate nitrogen alone due to the growth-inhibiting effect of ammonia nitrogen, but as shown in Figure 3, A significant effect on saponin production was observed. In Fig. 2, the vertical axis is the multiplication factor of the root tissue piece of P. aeruginosa, and the horizontal axis is the total nitrogen concentration in the medium. In FIG. 3, the vertical axis is the production amount (y) of saikosaponin in the root tissue piece, and the horizontal axis is the total nitrogen concentration in the medium. In Figures 2 and 3, C [solid line (○)] indicates that nitrate nitrogen/ammonia nitrogen is 85/1.
5, and D [dotted line (x)] is 10
010 medium.

(5) Discussion According to Figure 2, the total amount of nitrogen in the medium is 100 to 500.
It can be seen that when using a medium with a total nitrogen content of 841 ppm, which corresponds to a basic medium, the proliferation of root tissue fragments of Mishimasaiko is higher. According to Figure 3, if the total nitrogen content in the medium is at a concentration of 100 to 500 pplll, the production amount of saikosaponin (bright) will also increase as the ratio of nitrate nitrogen/ammonium nitrogen is 85/15, which means ammonia nitrogen. It can be seen that when an appropriate amount of is added, the improvement is better than when using 10010.

Example (Preparation of root tissue piece) Bupleurum falcatum
A gutter in which seeds of L, ) were washed with running water for a day and night, 70
% ethanol solution for 60 seconds. Soil 509, which had been previously steam sterilized at 130°C for 30 minutes, was placed in a petri dish [1
50 mm (diameter) x 35 mm (depth)], and place 50 of the sterilized seeds of Mishimasaiko in a germination bed moistened with sterilized water.
After sowing the grains and covering them with soil, they were left standing at room temperature for one week while being sprinkled with sterile water once a day to germinate.
dling) 38 pieces were obtained.

The root part of this young plant was cut off from the base to obtain 38 pieces of root tissue from the plant.

(Culture of root tissue fragments) A 100-mL modified MS (Murashige-Skoog) medium having the composition shown in Table 7 was placed in a 300 d Meyer flask.
Add to this 1 g of the Mishima Sai root tissue piece (fresh weight)
was added, and culture with rotational shaking at 100 rpm was continued for 8 weeks. In this culture, 8.5 g (fresh weight) of root tissue of P. aeruginosa was obtained, which was branched and had lateral roots growing from an enlarged main root.

(Left below) m7 table correct Ms! 15M (Quantification of Saikosaponin) 2 g of root tissue (dry type) of Saiko saponin obtained under the above culture conditions was immersed in a 2% potassium hydroxide aqueous solution 100-
Heated on a water bath for 2 hours. 5 g of Hyfloth-Parcel was added to this extract and filtered, and the remaining solids were again extracted with 100 ml of a 2% aqueous potassium hydroxide solution while hot. The extracts were combined and extracted three times with 50° each of n-butanol, and the n-butanol layer was washed with water and then concentrated under reduced pressure of 3#LllIHg. The concentrated residue was mixed with 99% ethanol 1
After dissolving in 0- and heating under reflux for 30 minutes, insoluble matter was filtered, and ethanol was further distilled off from the ethanol extract. This extraction with ethanol was repeated twice to obtain 80 ml of a crude saikosaponin mixture. Add 5 ml of 296 hydrochloric acid/methanol to this crude saikosaponin mixture and heat at 25°C.
After leaving it in a constant temperature bath for 8 hours, 2% sodium hydroxide/methanol was added to stop the reaction, and the reaction solution was analyzed by high performance liquid chromatography to determine saikosaponin a.
+cwd was quantitatively analyzed.

(Effects of the Invention) It is possible to produce by tissue culture of the taproot with a high content of saikosaponins, and the multiplication rate is also high.

[Brief explanation of the drawing]

Figure 1 shows the results of Test Example 1, and Figures 2 and 3 show the results of Test Example 1.
The figure shows the results of Test Example 3. Applicant Sekisui Plastics Co., Ltd.

Claims (1)

[Scope of Claims] (1) A method for producing saiko root containing saikosaponin by tissue culturing the root tissue of a plant belonging to the genus Bupleurum in a nutrient medium,
A nutrient medium containing nitrate nitrogen and ammonia nitrogen as a nitrogen source, with a total nitrogen content of 100 to 500 ppm and a nitrate nitrogen to ammonia nitrogen ratio of 95/5 to 70/3.
1. A method for producing saiko root, characterized in that the medium component consists of 0. (2) A plant belonging to the Bupurulum genus is Mishimasaiko (
2. The method for producing chai root according to claim 1, characterized in that the plant is Bupleurum falcatum. (3) Claims 1 or 2, characterized in that the root tissue of a plant belonging to the genus Bupululum is the root tissue of a young plant obtained by germinating the seeds of a plant belonging to the genus Bupurulum. The method for producing saiko root described in . (4) Producing the saiko root according to any one of claims 1 to 3, wherein nitrate nitrogen is supplied by adding potassium nitrate (KNO_3) to the nutrient medium. Method.