RU2147608C1 - Strain of cultured plant cell panax ginseng c - Google Patents

Abstract

A. MEY - PRODUCER OF BIOLOGICALLY ACTIVE SUBSTANCES. FIELD: biotechnology. SUBSTANCE: strain is obtained from leaves of plant Panax ginseng C. A. Mey of seaside population and stored in collection of vegetable cultures "ZAO 2 tsekch" at code N L-66. Isolated strain is obtained after 48 passages, it grows in concentrated nutrient medium containing sucrose at concentration 50000.0-60000.0 mg/l and has bitter taste. Strain produces biomass containing biologically active substances: triterpene saponins (panaxosides), trace elements, polyacetylenes, amino acids, sterines, flavonoids and others. Extract obtained from the strain biomass shows biological activity in hypoxic and ischemic disorders of cerebral circulation. Invention can be used in pharmaceutical, cosmetic and food industries. EFFECT: ginseng strain indicated above, increased yield by biomass, increased yield of total glycosides as compared with strains obtained from root. 1 ex

Description

 The invention relates to biotechnology, in particular to the production of biologically active substances from cultivated plant cells, and can be used in the pharmaceutical, cosmetic and food industries.

Various ginseng strains obtained from the root are known: IFRG-2 ¹ , IFRG-3 ² , or suspension strains, for example, the ginseng cell strain DAN-25 ³ . These strains have insufficient biomass productivity.

Closest to the proposed is a ginseng strain obtained from a 6-year-old ginseng root ⁴ . It is a whitish or creamy biomass, fine-grained, easily crumbling into small pieces. The bulk of the tissue is 90% composed of parenchymal cells with a size of 74.7-122.5 microns and small meristematic cells with a size of 34.9-72.9 microns. The strain is cultivated in a dense nutrient medium with transplants after 40 days. The growth index is 5, the content of the sum of glycosides is 3.0-3.5%. The disadvantage is not a very high yield of biomass and biologically active substances. In addition, all these strains produce biologically active substances (BAS), characteristic of ginseng root.

It is known that ginseng leaves also contain a lot of biologically active substances ^5.6 . They contain, in addition to triterpene saponins (panaxosides), flavonoids (kempferol, trifolin, panasenoside), carotenoids, aliphatic, aromatic, heterocyclic amino acids, etc. However, the use of ginseng leaves in industry is complicated by the seasonality of their harvest, the non-standard content of biologically active substances and, moreover, , plantation cultivation of ginseng provides for the repeated processing of leaves with pesticides, which makes it impossible to use them as raw materials.

 The objective of the invention is to obtain a strain of ginseng, producing a complex of biologically active substances characteristic of ginseng leaves, having high productivity, to expand the range of biologically active substances of plant origin.

 The task is realized by the proposed strain of cultured plant cells Panax ginseng C.A.Mey - a producer of biologically active substances.

The strain is obtained as follows:
The seeds of the Panax ginseng CAMey of the Primorsky population are thoroughly washed with soapy water, rinsed with water and sterilized in concentrated sulfuric acid for one minute. Then it is washed with water and sterilized with active chlorine (0.1%) for 30 minutes. Thus sterilized seeds are placed on a nutrient medium containing half the dose of mineral salts and trace elements according to Murasige-Skoog and supplemented with the following components (mg / l): agar - 6000.0; sucrose - 20,000.0; inositol - 100.0; thiamine bromide - 1.0; benzylaminopurin - 0.5, and incubated in a thermostat (24 ^o C) with 16 hour photoperiods. Within 30 to 40 days, the seeds germinate. The seedlings are freed from the pericarp and transferred to a shoot formation medium containing (mg / l): macro- and microelements according to Murashige-Skoog, agar - 6000.0; sucrose - 25,000; inositol - 100.0; thiamine bromide - 1.0; benzylaminopurine - 0.5. On this nutrient medium for 15 - 25 days, the formation of numerous shoots with developed leaves. The leaves of shoots propagated in vitro serve as a sterile material for receiving callus. Casting under aseptic conditions is cut into segments and placed on a nutrient medium for callus formation (mg / l): KNO ₃ -2000.0; NH ₄ NO ₃ -1000.0; MgSO ₄ • 7H ₂ O; CaCl ₂ -300.0; KH ₂ PO ₄ -100.0; agar-6000.0; sucrose-25000.0; inositol-100.0; pyridoxine-1.0; thiamine bromide-1.0; 2,4-dichlorophenoxyacetic acid-2.0; kinetin-1.0. Callus is formed within 3 days, after which it is isolated for 30 days, after which it is isolated from the leaves and subculturing is continued, regularly replacing the tissue on a fresh nutrient medium containing an increased amount of sucrose-50,000.0 - 60,000.0 mg / l.

 At the time of application, an isolated strain called L-66 had passed 48 passages. It is stored in the collection of plant crops of JSC "2nd workshop".

Strain L-66 has the following characteristics:
Morphological and anatomical signs.

 The strain is a loose mass of yellowish-cream color with a specific smell and bitter taste. Culture cells, as a rule, are round, thin-walled, contain one nucleus with one, less often with several nucleoli, the size of most cells is 50 - 120 microns. Conducting tissues represented by tracheids, partially lignified, and mechanical fibers were found in biomass.

 Physiological and cytological signs.

 The growth cycle of strain L-66 is characterized by an S-shaped curve with a 3–5 day lag phase, an exponential growth phase of 5–12 days, a linear phase of 15–20 days, and a stationary phase of 25–30 days.

 The growth index at the end of cultivation is 20 in raw biomass and 10 in dry biomass. The mitotic activity curve has a three-vertex character, but dividing cells also occur at the end of the growth cycle. The strain is represented mainly by aneuploid cells, while the frequency of haploid cells is 75%, diploid - 23%, triploid - 2%. Marker chromosomes are absent, but binuclear metaphases, including those with diplochromosomes, are often found.

 Characterization of secondary metabolic products.

Strain L-66 is a producer of biomass containing biologically active substances: triterpene saponins (panaxosides), of which ginsenosides Rg ₁ , Rf, Re, Rc, microelements, polyacetylenes, amino acids, sterols, flavonoids, etc. are identified. Its distinctive feature is its bitter taste. It is able to grow in a concentrated medium containing 5 - 6% sucrose, at the end of the growing cycle gives at least 3.4 g of dry biomass per 1 bottle (100 ml) or 34 g per 1 liter of medium. The content of the sum of glycosides per 1 liter of medium is 1080 ± 52 mg, in addition, it synthesizes flavonoids. No biosynthesis products of the strain were found in the nutrient medium.

Example 1. In culture jars, 100 ml of culture medium with an increased sucrose content of up to 50,000-60000 mg / l is poured. Banks with medium are sterilized at 1 atm for 15 minutes. Landing is carried out with an inoculum weighing 3 - 5 g per 1 vial. Banks with inoculum are incubated at 24 ^o C and relative humidity of 70%. After 25 days of incubation, the biomass is separated from the agar residues, weighed and dried.

 Comparative characteristics of the biomass of strain L-66 and strain analogue are presented in the table.

The pharmacological activity of the alcohol tincture made from the biomass of strain L-66 was studied according to the traditional method of ^7.8 .

 In animal experiments, it was found that preliminary administration of 50 drops of tincture in a daily dose of 50 drops during the week increased the survival of rats by 30% after modeling circulatory hypoxia of the brain by ligation of the carotid arteries compared with the control. A 3-week course daily administration of tincture inside at the same dose prevented the development of postischemic hypoperfusion and helped to restore the autoregulatory responses of cerebral vessels impaired by ischemia. The decrease in vascular resistance of cerebral vessels after ischemia indicates a direct selective effect of biologically active substances on the vessels of the brain, which, apparently, leads to an improvement in cerebral circulation. Thus, a complex of biologically active substances from the biomass of strain L-66 can be tested as a prophylactic for hypoxic and ischemic disorders of cerebral circulation.

Thus, the inventive strain L-66, obtained from ginseng leaves, has a number of significant differences from the known and has several advantages:
- synthesizes flavonoids in contrast to strains obtained from the root;
- grows on a concentrated nutrient medium containing 50,000.0-60000.0 mg / l of sucrose;
- has a specific bitter taste, different from the taste of the biomass of analog strains obtained from the root;
- has a high productivity: biomass 3 times higher and 3 times higher in the output of the sum of glycosides.

 In addition, an extract (infusion) of biologically active substances obtained from biomass exhibits biological activity in hypoxic and ischemic disorders of cerebral circulation, not found in preparations from ginseng root and analog strains.

Literature
1. A.S. USSR N 1058281, C 12 N 5/00, 1982.

 2. A.S. USSR N 1058282, C 12 N 5/00, 1982.

 3. RF patent N 2052925, A 01 H 4/00, BI N 3 1996.

 4. Slepyan L. I. Ginseng in the early stages of ontogenesis and in the culture of isolated tissues. Abstract of Cand. for the degree of candidate of biological sciences, Leningrad, 1969, p. 19.

 5. Legosteva A.B. and other plant resources. 1986. N 4.P. 523 - 26.

 6. Brigikhach B. et al. Plant resources. 1982.Vol. 18. Issue. 3, p. 357-363.

 7. Mashkovsky M.D. Medicines M. "Medicine". 1993. Part 1. P. 162.

 8. VFS 42-1890. Infusion of ginseng.

Claims (1)

 The strain of cultured plant cells Panax ginseng C.A.Mey L-66 is a producer of biologically active substances.

Images