RU2296155C1 - Strain of cultured cells of plants serratula coronata l - Google Patents

Abstract

FIELD: biotechnological methods.

SUBSTANCE: invention is intended for use in medicinal, food processing, and perfumery industries and provides strain GI 1.1 of suspension culture of cells of plants Serratula coronata L. N 64 is producer of ecdisteroids.

EFFECT: enabled effective preparation of ecdisteroids.

2 dwg

Description

The invention relates to biotechnology and can be used in the medical, food and perfume industries.

Currently, intensive studies are being conducted to obtain phytoecdysteroids from medicinal plant materials (Phytoecdysteroids / Ed. By V.V. Volodin. St. Petersburg: Nauka, 2003. 293 pp .; RF Patent No. 2153346, MKI A 61 K 35/78. A method of producing ecdysteroids / VVVolodin, S.O. Volodin; RF Patent N21555 99, MKI A 61 K 35/78. A method of isolating individual compounds from a mixture of ecdysteroids from the aerial parts of plants Serratula coronata / VVVolodin, C .O. Volodina).

For these compounds, the perspectives of the use of actoprotective, sugar-lowering and wound healing preparations, tonic food additives and cosmetic compositions (Phytoecdysteroids / Edited by V.V. Volodin. St. Petersburg: Nauka, 2003.293 s., Russian Federation Patent No. 2119331, MKI A 61 K 9/06, 35/78. Means for the treatment of burn wounds "Vitaderm" / V. N. Darmograi, S. M. Potechinsky et al .; Patent EP No. 0436650, MKI A 61 K N 7/00. Meybeck A., Bonte F. Phases lamellaires lipidiques hydratees on liposomes a base d'ecdysteroides; RF patent No. 1561263, MKI A 61 K 35/78. A method for the treatment of insulin-dependent diabetes mellitus. / M.I. Kos ovsky, V.N. Syrov, M.M. Mirakhmedov, S.P. Katkova and Z.A. Khushbaktova.Cultivated plant cells can serve as an alternative to plant materials.

Known strains of cultured cells of ecdysteroid-containing plants described in the literature do not produce these compounds in vitro or produce at concentrations lower than wild or introduced plant species (Saad M.L., Kovalenko P.G., Medvedeva T.V. ., Kornets G.V., Shuman N.V., Kholodova Yu.D., Galkin A.P. Culture of isolated cells and serpuvencenous tissues as a source of biologically active phytoecdysteroids // Physiology and Biochemistry of Cultivated Plants. 1992. V.24 No 6. S.611-615). Links to the deposit of strains of ecdysteroid-containing plants in the available collections are missing.

An object of the present invention is to provide an actively growing strain of a suspension culture of plant cells of Crowned Serpaula (Serratula coronata L.), characterized by a high content of phytoecdysteroids.

The strain Serratula coronata GI 1.1 is deposited in the Russian collection of higher plant cells at the Institute of Physiology. K.A. Timiryazev of the Russian Academy of Sciences under collector number 64.

Description of the source material.

The source of the strain of the suspension culture Serratula coronata was a long-cultivated callus culture, which was obtained in 1993 in the laboratory of plant biochemistry and biotechnology of the Institute of Biology of the Komi Scientific Center of the Ural Branch of the Russian Academy of Sciences from the hypocotyl seedling of crown-bearing serpent plants grown in the scientific collection of this Institute. Callus culture was grown on a modified Murashige-Skoog medium with the addition of sucrose 30 g / l; 2,4-D 1 mg / ml; BAP 0.2 g / ml; meso-inositol 100 mg / l and Stab vitamins, mg / l: folic acid 0.5; riboflavin (B2) 0.5; biotin 1.0; Ca pantothenate 1.0; cobalamin (B 12) 0.0015. pH before autoclaving 5.8. To obtain suspension cultures, the callus culture was transferred to a liquid nutrient medium identical in composition to the medium on which the callus cultures were cultured, with the exception of agar. After about two weekly suspension culture was fractionated using sedimentation for 1-2 minutes (the middle fraction was selected). Suspensions were grown in 500 ml conical flasks with 60-70 ml of medium at 25-26 ° C, relative humidity 60%, in the dark, on a rocking chair, 90-100 swings per minute. The following reseeding regimen was established: 17.5 ± 1.8 ml of inoculum per 100 ml of medium. The subculture interval is 12-14 days.

The cultural properties of the strain.

Growth pattern: white suspension, growth index for raw biomass 10.54 ± 0.53, for dry biomass 10.65 ± 0.72. The specific growth rate for crude biomass is 0.35 ± 0.08 days ^-1 , for dry biomass 0.31 ± 0.05 days ^-1 , for the number of cells 0.58 ± 0.16 days ^-1 . The number of living cells is 75.44 ± 8.77%.

Cytological characteristic.

Suspension of sickle-venous cells consists of four types of cells: meristematic type (size 40-80 microns), parenchymal type (size 100-200 microns), elongated cells (transverse size varies from 4 to 80 microns, length 200-350 microns), budding cells (cells of irregular shape, having large sizes, whose diameter exceeds 200 microns). During the entire period of subculture, large aggregates consisting of 20-50 cells prevail. At the beginning of exponential growth, the number of small clusters consisting of 6-10 cells increases. Throughout the entire period of subculturing, aggregates with a cell number of 21-50 prevail. By the end of the passage, the number of single cells increases and aggregation in suspension occurs. Figure 1, shows the aggregation of a suspension culture of Serratula coronata L. with a culture cycle of two weeks.

Karyological characteristic.

The number of chromosomes in the meristem of the root of the intact plant of the serpent crown is 22, which corresponds to published data. The population of long-cultured cells is characterized by a significant variety of karyotypes. The modal class is represented by cells with the number of chromosomes 49-68 (58%). There are cells by the number of chromosomes close to the diploid level (5%). There are also cells with a chromosome number of more than 8n (10%). Figure 2 presents the distribution of the number of chromosomes in cells of a suspension culture of Serratula coronata L.

Characterization of ecdysteroid biosynthesis in cell suspension culture.

Analysis of cell biomass for the content of ecdysteroids was carried out by reverse phase HPLC on an analytical HPLC system Varian, Pro Star (USA).

The composition of the eluent: water - acetonitrile (100: 20), the elution rate of 1.5 ml / min; λ = 242 nm; Diasorb C ₁₆ / T column (150 × 4 mm; 7 μm). The main component of cellular biomass, as in intact plants, is 20-hydroxyecdysone (used as a substance of the famous tonic drug "Ecdisten"). In the analyzed biomass samples, minor amounts of inocosterone, ecdysone, and macisterone A were present, which are also characteristic of intact plants of the crowned serpent. The dynamics of the accumulation of ecdysteroids in the growing cycle is periodic. The maximum synthesis of ecdysteroids is observed at the beginning of the stationary phase and at the stage of growth retardation (0.4-0.5% by dry biomass). The results of the analysis of samples of the culture fluid showed that throughout the growing cycle, the release of ecdysteroids into the medium does not occur.

An example of a specific implementation.

The strain of the suspension culture of plant cells of the serpent crown is cultured on a medium of the above composition at 26 ° C in the dark. In the phase of growth retardation (12-14 days), the biomass of the cells is separated from the liquid medium by filtration under vacuum. 500 g of crude biomass is washed with distilled water and destroyed by a homogenizer at a speed of 15 thousand rpm for 5 minutes at room temperature. A double volume of aqueous ethanol or methanol is added to the homogenate. The extraction is carried out for 2 hours with stirring at a temperature of 20-40 ° C. After separation of the sediment, secondary biomass extraction is carried out with aqueous ethanol or methanol. The extracts are combined and evaporated in vacuo. The extract is not concentrated to dryness, but 10% of the initial amount of the liquid phase is left. The residue was extracted three times with hexane to remove lipid by-products, and then twice with ethyl acetate: methanol to recover the desired product. The organic extracts are evaporated to dryness, applied to alumina and column chromatography is carried out with a mixture of increasing polarity chloroform-methanol. Target fractions containing 20-hydroxyecdysone are determined by TLC and HPLC. The desired fractions are evaporated and recrystallized in a mixture of ethyl acetate: methanol 9: 1. 0.05 g of 20-hydroxyecdysone is obtained (product purity - 92%), which contains 25S-inocosterone and ecdysone in minor concentrations as concomitant ecdysteroids. In its composition, the ecdysteroid preparation isolated from the biomass of cultured cells is identical to the preparation obtained from native plants of crowned serpent, which indicates the possibility of using a suspension cell culture as an alternative source of 20-hydroxyecdysone, a substance of actoprotective and tonic ecdysteroid-containing drugs.

Claims (1)

Strain GI 1.1 of the suspension culture of plant cells Serratula coronata L. N 64 is a producer of ecdysteroids (Russian collection of higher plant cells at the K.A. Timiryazev Institute of Physiology, Russian Academy of Sciences).

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