RU2296154C1 - Strain of the cultured cells of plants ajuga reptans l - Google Patents

Abstract

FIELD: biotechnological methods.

SUBSTANCE: invention is intended for use in medicinal, food processing, and perfumery industries and provides strain E IVk of suspension culture of cells of plants Ajuga reptans L. N 63 is producer of ecdisteroids.

EFFECT: enabled 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 / Edited 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 No. 2155599, MKI A 61 K 35/78. Method for the isolation of 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 pp .; RF Patent No. 2119331, MKI A 61 K 9/06, 35/78. "Vitaderm" for the treatment of burn wounds / V. N. Darmograi, S. M. Potechinsky et al .; Patent EP 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. Method for the treatment of insulin-dependent diabetes mellitus. / M.I. Kos ovsky, V.N.Syrov, etc.)

Cultured 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 (Tomas J., Camps F., Claveria E., Coll J., Mele E., Messeguer J. Composition and location of phytoecdysteroids in Ajuga reptans in vivo and vitro cultures // Phytochemistry. 1992. V.31. No.5. P.1585-1591). 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 suspension culture of plant cells of a tenacious creeping survivor (Ajuga reptans L.) characterized by a high content of phytoecdysteroids.

The strain Ajuga reptans E IVk was deposited in the Russian collection of higher plant cells at the Institute of Physiology. K.A. Timiryazev RAS under collection number 63.

Description of the source material. The source of the strain of the suspension culture Ajuga reptans 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 root of a sterile creeping tenacious plant grown in the scientific collection of the said 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 is 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 weeks, the 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: 12 ± 1.5 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 - 13.02 ± 0.32, for dry biomass - 12.69 ± 0.83. The specific growth rate for crude biomass is 0.36 ± 0.03 days ^-1 , for dry biomass - 0.32 ± 0.03 days ^-1 , for the number of cells - 0.31 ± 0.03 days ^-1 . The number of living cells is 88-92%.

Cytological characteristic. In suspension creeping survivor culture, the content of large aggregates (> 50 cells in the aggregate) during the cultivation cycle varied from 14.6 to 63.5%, and the proportion of small aggregates (1-4 and 5-20 cells in the aggregate), on the contrary, decreased. Figure 1 shows the aggregation of the suspension culture of Ajuga reptans with a culture cycle of one week

Karyological characteristic.

According to literature data (Bolkhovskikh Z.V., Grif V.G., Zakharyeva O.I., Matveeva T.S. Chromosomal numbers of flowering plants. L .: Nauka, - 1969) the number of chromosomes in the cells of the root meristem of the intact plant Ajuga reptans 2n = 32. Analysis of the distribution of the number of chromosomes in the cells of the suspension culture of the root of Ajuga reptans revealed a large variability in the level of ploidy. Figure 2 presents the distribution of the number of chromosomes in cells of a suspension culture of Ajuga reptans.

In the population, cells with the number of chromosomes close to diploid (2n = 30-34) make up only 11%. During long-term cultivation, a reduction in the number of chromosomes occurs: the modal class (40% of the population) is represented by aneuploid cells with a number of chromosomes 25-29. Cells with a lower number of chromosomes of 20-24 and <20 chromosomes were also found. In the suspension culture population, there are also aneuploid cells with the number of chromosomes significantly exceeding the diploid one: from 35 to 55 and higher. The proportion of such cells is approximately 30%.

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"). Minor amounts of polypodine B, 29-norgsengosterone and ayugolactone, also characteristic of intact creeping tenacious plants, were present in the analyzed biomass samples. The dynamics of the accumulation of ecdysteroids in the growing cycle is periodic. The maximum synthesis of ecdysteroids was, as a rule, at the end of the exponential phase (on average 0.5%). In the early days of cultivation, a decrease in the level of 20E biosynthesis was observed. Bursts of biosynthetic activity were observed during the exponential growth phase.

An example of a specific implementation.

The strain of the suspension culture of plant cells of a tenacious creep 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 extraction of the biomass with aqueous ethanol or methanol is carried out. 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. The determination of the target fractions containing the sum of ecdysteroids is carried out using TLC and HPLC chromatography. The desired fractions were evaporated and recrystallized in a 9: 1 ethyl acetate-methanol mixture. Obtain 0.03 g of 20-hydroxyecdysone (product purity - 92%) containing, as concomitant ecdysteroids, polypodine B, 29-norgsengosterone and ayugolactone, in minor concentrations. 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 culture of cells as an alternative source of 20-hydroxyecdysone, a substance of actoprotective and tonic ecdysteroid-containing drugs.

Claims (1)

Strain E IVk of the suspension culture of plant cells Ajuga reptans L. N 63 - 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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