doi: 10.15389/agrobiology.2023.1.114eng

UDC: 633.8:58.144:58.01:57.017.6:574.45:581.192



N.P. Timofeev

KKh BIO, 47, ul, Lenina, Koryazhma, 165650 Russia, e-mail (✉ corresponding author)

Timofeev N.P.

Final revision received May 15, 2022
Accepted July 7, 2022

In 2021, the global market for ecdysterone-containing substances amounted to $100 billion and is expected to grow significantly in the next 5 years. The aboveground and underground parts of Rhaponticum carthamoides (Willd.) Iliin are primarily suitable to obtain ecdysterone-containing products of pharmaceutical quality. However, both in Russia and abroad, сommon technologies for R. carthamoides cultivation and study are not focused on the quality of the medicinal raw materials. In this paper, we have implemented for the first time an alternative technology for the production of ecdysterone-containing substance from the leaf parts of R. carthamoides. The technology is simple and can be scaled up in agropopulations of the Northern European Russia and meets the key requirements for industrial raw materials and end ecdysterone products set by international experts. Our goal was to summarize 32 years of experience in growing R. carthamoides on a plantation located in the European Northeast of Russia (Arkhangelsk Province). We assessed the potential for longevity and productivity of the agricultural population by life cycle stages and age, the regularities of ecdysterone accumulation in annually harvested aboveground plant parts, and the quality of obtained plant raw material for the content of standardized substances. The study was performed in the southeast of the Arkhangelsk Province (the Middle Taiga subzone, Kotlass District; 61°20'' N, 47°E) in an agropopulation of R. carthamoides (the field area of 1 ha) in 1989-2022. The seeds obtained from the Botanical Garden of the Komi Scientific Center, UB RAS (Syktyvkar) were initially originated from the Altai natural population (first collected in 1956). Autumn sowing was performed in mid-October after the beginning of autumn frosts (row spacing of 70 cm, seed embedding depth of 2-3 cm, seed rate of 2.7 kg/ha; 58 % field germination of seeds). Mineral fertilizers (NPK60-90) were applied during the first three years after sowing followed by organic farming without use of mineral and organic fertilizers, chemical pesticides, and plant growth regulators. The aboveground parts were annually harvested during budding. A set of population, agrochemical, morphoanatomical, biochemical and statistical methods were used to asses parameters of the plant population (age states in ontogenesis, population density, gross production of above-ground and under-ground organs, seed yield) and individual plants (growth, development, morphological structure, productivity of roots, leaves, and seeds). Samples were collected at optimal phases of plant development, the aboveground phytomass during budding (I-II decade of June), rhizomes in autumn, after the end of vegetation (October), or in early spring, before the beginning of vegetation (April). Shortened vegetative (rosette) and stem generative (reproductive) shoots with inflorescences were morphologically heterogeneous organs separated in the aboveground part. Industrial harvesting of plant raw materials was carried out in late May-early June, during budding. Plant material (organs, elements and fractions) was dried at 23-25 to 35-40 °С and relative humidity of 25-40 % in accordance with the procedure for harvesting and drying medicinal raw materials. The samples of air-dry raw materials for further determination of the content of primary and secondary metabolites were formed by quartering method. The amount of ecdysterone in dry samples was determined by reverse phase high performance liquid chromatography with the internal standards (a liquid microcolumn chromatograph Milichrom-5, column 80½2 mm, Nucleosil C18 sorbent with a particle size of 5 μm; LLC «Medicant», Russia). Potentially dangerous substances were also assessed. Duration of agropopulation ontogenesis was close to parameters of natural populations in subalpine meadows and is more than 30 years without transition to senile age on the 33rd year of life, plant density reached optimum values of 28-23 thousand pcs/ha, starting from the 3-4 years of life. Diseases and pests did not affect vegetative aboveground mass and roots with rhizomes. The average root mass with rhizomes during the period of relatively stable mass production (from the 5th to the 32nd year of life) was 246.3 g/plant, aboveground mass was 223.4 g/plant. Calculated annual productivity of the agropopulation during the same period averaged approximately 5300 kg/ha for aboveground parts, and approximately 6100 kg/ha for underground parts. Vegetative type of reproduction was most pronounced for subsenile age state (from the 13th to the 32nd year and onwards), when seed production was extremely low, 1.3 kg/ha. The seed type of reproduction was characteristic of the generative period from years 4-5 (8-30 kg/ha) with a peak during years 6-7 (108 and 78 kg/ha). In general, the percent of generative shoots in total plant biomass was insignificant throughout the life cycle. Ecdysterone biosynthesis and accumulation in the leaves of rosellate shoots were directly related to vegetative reproduction, namely to the aboveground mass value (R2 = 0,768, or close to 80 %). More than 90 % of annually synthesized ecdysterone (22 kg/ha) was concentrated in the aboveground part of plants at optimal harvesting age (from year 5 to year 32), or about 600 kg of ecdysterone for 27 years of operation. Qualitative indicators of medicinal raw materials from leaves of R. carthamoides were high and met the requirements for the manufacture of pharmaceuticals with a relative purity of ecdysterone 97 %. The plant material met all the regulatory requirements of the supervisory authorities. The levels of heavy metals (Hg, Cd, As, Zn; Ni, Cu, Cr) did no exceeded the permissible level, there were no prohibited organochlorine and phosphorus compounds, the content of radionuclides 90Sr and 137Cs and nitrites were below the permissible limit. Ecdysterone from the dried flour of the R. carthamoides leaves was well extracted into aqueous and alcohol solutions and was well preserved (up to 93-98 % within 24 hours) without preservatives. Its use significantly improved animal health (a 1.6-2.5-fold decrease in mortality), had an anabolic and economic effects (i.e., a 24-33 % higher daily weight gain and a decrease in feed consumption by 11-17 %).

Keywords: phytoecdysteroids, 20-hydroxyecdysone, Rhaponticum carthamoides, Leuzea safflower, maral root, feed additives, anabolic substances.



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