KR20040027818A - Mass Production Method of the Ginseng Saponin by the Treatment with Mevalonic Acid - Google Patents

Abstract

PURPOSE: A method for increasing the content of ginseng saponin by treatment of ginseng with mevalonic acid is provided, thereby increasing panaxadiol saponin content as well as ginseng saponin, and regulating the quality of saponin. CONSTITUTION: A method for increasing the content of ginseng saponin comprises the steps of: inducing a hairy root of ginseng and cutting it; culturing the cut pieces of the hairy root in an MS medium for 2 to 3 weeks; adding 1 to 200 micromole of mevalonic acid(mevalolactone, (±)-3-hydroxy-3-methyl-5-pentanolide) into the medium and culturing them for 2 to 3 weeks; and washing the cultured hairy roots of ginseng with water and drying them.

Description

Mass Production Method of the Ginseng Saponin by the Treatment with Mevalonic Acid}

Ginseng is a perennial herb belonging to the Panax Genus of the Araliaceae, and has been used for a long time. The study of ginseng's ingredients began with the first scientific study by Garriques (1854) of the United States, which separated an amorphous glycoside mixture from American ginseng and named it Panaquilon. Ginseng has been scientifically proven to exist in a variety of pharmacological effects, such action is known to be mainly due to ginseng glycosides (saponin) components.

Saponins exist as glycosides in plants, and when hydrolyzed, sugars separate and produce genins. The saponins of Korean ginseng are glycosides in which panaxadiol (PD) and panaxatriol (PT) of the genin are combined with sugars and are called ginsenosides. It is expected to be similar to the sterol synthesis process in. In the PD system, ginsenoside-Ra, -Rb ₁ , -Rb ₂ , -Rc, and -Rd are present, and in the PT system, ginsenoside-Re, -Rf, -Rg ₁ , and -Rg ₂ are present. The proportions vary depending on the area, region and species of ginseng.

Ginsenoside-specific pharmacological effects of ginseng have been identified by many scientists, but the results of systematic clinical studies using 10 or more ginsenosides of human ginsenosides that have been identified through physiological activities have shown that G-Rb ₁ is a central inhibitor, behavioral inhibitor, and anticonvulsant. Action, G-Rb ₂ promotes sugar, fat metabolism and anti-diabetic action, hepatocellular proliferation and DNA synthesis, G-Rc analgesic, protein and lipid synthesis, RNA synthesis, G-Rd is adrenal cortex stimulating hormone , Corticosterone secretion promoting effect, G-Re is adrenal cortical stimulating hormone, DNA, RNA, protein, lipid synthesis promoting effect of bone marrow cells, G-Rf is pain suppression, lipid peroxidation inhibitory and alcohol-induced brain development action, G-Rg ₁ is immunity enhancing action, memory and learning function promoting action, _G-2 Rg is proven that the effects such as inhibition of platelet aggregation and improving memory loss, but still undisclosed its pharmacological action It is reported that there are lots of amount of saponins. Based on these results, it can be said that the in vivo effect of saponin is determined by the ratio of PD-based saponin and PT-based saponin.

As such, ginseng is recognized for its pharmacological efficacy, and its demand is gradually increasing not only as a medicine but also in the preparation of food, cosmetics, soap products, and the like. However, because ginseng is a perennial plant, it takes not only a long time to cultivate it, but also changes in its contents due to climate, climate, disease, cultivation conditions, irregular productivity, and increase in production cost due to labor-intensive production. As a result, mass production of plant cells using bioreactors is now being carried out by some companies, especially in wild ginseng roots (Tec. 2001-0044236, T. 2001-0070932).

Recently, a method for improving the content of ginseng saponin was proposed by treating jasmonic acid in Korea (10-0353636-0000), but a method for enhancing the biosynthesis of ginseng saponin through mevalonic acid treatment, a precursor of saponin biosynthesis, was proposed. There is no research.

In view of the above, the present invention is a precursor of saponin biosynthesis in a liquid nutrient medium when a large amount of negative roots derived from the roots of cultivated ginseng, goat, ginseng, camphor ginseng, and cheonsam ginseng are produced using a bioreactor. Based on the results of the addition of mevalonic acid by concentration and treatment time, the most efficient method of remarkably increasing saponin biosynthesis was achieved.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The method for enhancing ginseng saponin biosynthesis of the present invention comprises the steps of preparing a liquid nutrient medium for culture of intestinal root muscle, treatment of mevalolactone while inoculating atypical root muscle aseptically, incubating for 2-3 weeks and then treating mevalolactone. It consists of steps.

Hereinafter, a preferred embodiment of the present invention will be described in detail.

When ginseng roots are grown to a suitable length, the apical roots are cut off, transferred to a liquid nutrient medium containing IBA, and then cultured in a shaker incubator and passaged every 3-4 weeks. The 1/2 MS medium used in the experiment was prepared by adding the corresponding amount of stock solution to each, followed by 3% sucrose, and then adjusting the pH to 5.8 using 0.5 N NaOH solution, and adding the appropriate amount to each bioreactor (18.3 L). After dispensing, autoclaving at 121 ° C. for 20 minutes is used.

Mevalonic acid ((±) -Mevalonolactone, Sigma) is made of 1 M stock solution and stored frozen. When ginseng root muscle is incubated, incubate 1-200μM of mevalonic acid in 1/2 MS liquid medium or inoculate ginseng root muscle for 2-3 weeks, and then incubate for 2-3 weeks after adding mevalonic acid.

Collect ginseng roots incubated for 4-6 weeks, wash them three times with tap water, and store them in hot air. Ginsenosides content is extracted by saturated 1-butanol extraction. The hot-dried or lyophilized wild ginseng roots were extracted three times with 30 ml of 80% methanol in hot water at 80 ° C, dried, and then extracted with ether, degreased with ether, and extracted three times with saturated 1-butanol. The mixture was washed once with distilled water, discarded the aqueous layer, dried only 1-butanol layer, dissolved in 500 µl of methanol for HPLC, filtered through a 0.45 µm millipore syringe filter, and injected into 10 µl of High Performance Liquid Chromatograph (HPLC) to ginsenosides. Separate and quantify

Hereinafter, the present invention will be described in detail with reference to the following examples.

Example 1 Effect of Mevalonic Acid Treatment on Growth and Saponin Biosynthesis of Ginseng Nerve Muscle

1/2 MS medium used for the experiment was prepared using a commercial product, 3% sucrose, and after adjusting the pH to 5.8 using 0.5N NaOH solution and 40 ml each of 100 ml Erlenmeyer flask, Autoclave 20 minutes at 121 ℃ was used. Aseptically cut ginseng root roots into 1-2cm lengths and transfer them to 1/2 MS medium containing IBA, and incubate for 4 weeks with mevalonic acid to examine growth and saponin content of root muscles. 1 is shown.

[Table 1] Effect of mevalonic acid treatment on saponin biosynthesis of ginseng root muscle

Mevalonic acid (uM) Live weight (g / flask) Under building (g / flask) Panaxa Triol (PT) Panaxa diol (PD) TOTAL Saponin (in mg / g buildings) productivity 0 10.84 0.86 5.70 6.32 12.02 10.29 0.5 10.75 0.85 6.24 7.17 13.41 11.38 One 10.89 0.86 6.63 7.81 14.44 12.45 5 10.43 0.83 6.06 9.88 15.96 13.21 10 10.77 0.85 7.57 12.97 20.54 17.46 30 9.00 0.72 7.69 14.97 22.66 16.32 50 8.31 0.70 7.22 14.92 22.14 15.55 100 8.04 0.68 6.92 13.75 20.68 14.14

At the initial stage of incubation, the treatment of mevalonic acid, a precursor of saponin biosynthesis at various concentrations, showed a tendency of inhibiting the growth of the myosoid muscle, but the saponin content tended to increase. Characteristically, the concentration of Panaxadiol-based saponins was significantly increased at concentrations above 10 uM. In terms of productivity, 10 uM treatment was the highest, and it was suggested that 10 uM of mevalonic acid should be used for the mass cultivation of the root muscle using bioreactor.

Example 2 Effect of Mevalonic Acid Treatment on Growth and Saponin Biosynthesis of Ginseng Nerves

When treated with a high concentration of mevalonic acid, a precursor of saponin biosynthesis, negative myocyte growth was inhibited as shown in Table 1. In order to solve this problem, the results of incubation with 30 uM mevalonic acid were added to the incubated roots of 2-3 weeks, which were actively inoculated by involuntary roots.

[Table 2] Effect on Saponin Biosynthesis by Mevalonic Acid Treatment

Processing time (days) Live weight (g / flask) Under building (g / flask) Panaxa Triol (PT) Panaxa diol (PD) TOTAL Saponin (in mg / g buildings) productivity control 10.84 5.70 6.32 1.11 12.02 10.29 0 10.19 6.36 7.15 1.12 13.52 10.98 7 10.76 6.74 9.26 1.37 16.00 13.53 14 10.51 7.59 12.97 1.71 20.56 17.11 21 10.32 7.33 12.01 1.64 19.34 15.90

When ginseng roots are inoculated into a new medium, the growth of the roots of the roots of the roots of the root of the ginseng begins to grow in earnest after 7 days of culture. It starts to occur. Therefore, by properly adjusting the timing of mevalonic acid treatment, saponin biosynthesis can be effectively promoted, and the highest concentration of saponin was found on the 14th day after 30 μM concentration was selected. It appeared best. However, after 21 days of treatment, the productivity was low, and after 14 days of incubation, mevalonic acid was effectively treated with saponin biosynthesis.

As described above, the method of the present invention by treating mevalonic acid, a precursor of saponin biosynthesis, in mass production using bioreactors of involuntary roots derived from the roots of cultivated ginseng, camphor ginseng, goat ginseng, cheonjong ginseng, etc. In addition to increasing saponin productivity, the qualitative changes in saponin can be controlled to some extent, and thus, new drugs can be developed based on hidden pharmacology and efficacy by supplying a large amount of research samples to the pharmacology researching pharmacological effects of ginseng and wild ginseng.

Claims (2)

Mevalonic acid (mevalolactone, (±) -3-hydroxy-), a precursor of saponin biosynthesis, to enhance saponin biosynthesis when growing and producing ginseng roots, cultivated ginseng, camphor ginseng, goat ginseng, and wild ginseng in liquid culture using a bioreactor How to add 1-200 uM of 3-methyl-5-pentanolide) Mevalonic, a precursor of saponin biosynthesis, to enhance the saponin biosynthesis when growing and producing hairy roots from cultivated ginseng, camphor ginseng, goat ginseng, and wild ginseng in liquid culture using bioreactors. Add 1-200 uM of acid (mevalolactone, (±) -3-hydroxy-3-methyl-5-pentanolide)