BG66125B1 - Method for regulating the yield and the quality of seeds about the content of silymarin and unsaturated fatty acids at cultivation of the medical plant blessed milk thistle (silybum marianum l.) - Google Patents

Abstract

The invention relates to means and method for regulating the extraction and content of silymarin in seeds of the medical plant blessed milk thistle. Blessed milk thistle (Silybum Marianum L., Gaerth)is a plant used in the pharmaceutical industry. The active substance in its seeds is silymarin, which is a mixture of various flavonolignans, such as silybin, isosilybin, silydianin and silycrystin. The economic and biological properties in the cultivation of blessed milk thistle for the production of silymarin are improved by regulating its growth through the introduction of mineral fertilizers (through the roots or leaves) in combination with growth regulators. The invention aims at improving the combined effect of leaf fertilization and the application of thidiazuron, a growth regulator with cytokinin action (TDZ, Dropp(r), N-phenyl-N'-1,2,3-thiadiazol-5-yl-urea, Schering Co Germany, now produced by Aventis Crop Sci USA) on the growth and development, seed yield and their quality of blessed milk thistle (Silybum Marianum L.) plants cultivated under open-field conditions. The change is studied in some physiological parameters, such as accumulated biomass, flowering dynamics, ripening of calyces, seeds yield structure and silymarin content in the seeds of milk thistle cultivated under field microtest conditions. The foliar fertilizer Agroleaf(r) is applied as a working solution in a 0.5 percent concentration. Three different formulations of the fertilizer are applied three times during the vegetation period, such formulations differing in the NPK ratio therein. The growth regulator thidiazuron (Dropp(r)) is introduced during the rosette phase as a 0.002 percent solution. The combined TDZ treatment with foliar fertilization enhances the growth, adjusting the vegetative / reproductive mass ratio. The plants have more flower-bearing stems, form larger calyces with heavier seeds in them with higher silymarin content.

Description

Technical field

The invention relates to a method for regulating the growth, development and yield of seeds and the content of silymarin and unsaturated fatty acids in them when grown in field conditions of the medicinal thistle Silybum marianum L.

BACKGROUND OF THE INVENTION

The white thorn is a representative of the Asteraceae family and has been known since ancient times as a medicinal plant. White thorn seeds contain known amounts of biologically active compounds in the flavanolignans group, known as silymarin. Silymarin is a group of compounds of similar chemical structure silibin, isosilibin, silidianine and silicristin and their isomers. Extracted from seeds, silymarin is widely used in the pharmaceutical industry for the preparation of drugs with potent antioxidant properties, mainly used to treat liver damage. The content of silymarin active ingredients in dried seeds of cultivated plants of white thorns averages between 1 and 3%. White thorn seeds also contain some 20% reserve lipid. The triglyceride fraction mainly is used in cosmetic and medicinal chemistry as a source of unsaturated fatty acids. Usually, in cultivated cultivation, yields of white thorn seed are low, with the reasons for the high variability of yield and quality being the influence of adverse environmental conditions on its development.

One of the ways of regulating the yield and quality of plants is the conditions of mineral nutrition. So far, the most widely used in this aspect are root and leaf nutrition of plants. Leaf feeding of the mineral elements has advantages over the root, due to the faster absorption of plant elements, especially in plants with low nutrition efficiency. This approach is also associated with a more rapid response to plant nitrogen, carbohydrate and amino acid metabolism. Enhanced amino acid and carbohydrate metabolism of plant overgrowth is particularly important for the regulation of the reproductive period and the flowering phase of plants. The change in the flowering dynamics of the white thorn affects the formation of flower baskets and the seed placement in them. It is known that individual mineral elements such as nitrogen, phosphorus and potassium are absorbed at the same rate by plants during different periods of vegetation. Therefore, fine-tuning the elements or applying foliar feeds is significantly more efficient.

One of the important aspects in the regulation of white thorn flowering is related to the role of endogenous hormones in plants. Among them, gibberellins and cytokinins are most important for flowering. Often, the abortion of flowers or the development of immature embryos is associated with changes in ethylene and auxin content.

Another major problem with cultivated cultivation of white thorns is the irregularity of flower maturation during the flowering phase, the low seed yield and the low content of silymarin in them.

Regarding the regulation of seed quality and the content of silymarin in them, it must be said that any changes that lead to an improvement in phenolic metabolism in the seeds can assist these processes.

SUMMARY OF THE INVENTION

Based on these assumptions, the problem is solved by applying a suitable combination of fertilization and treatment with a growth regulator in cultured cultivation of white thorns. Fertilization was accomplished through a combination of soil mineral fertilization and leaf feeding with liquid mineral fertilizer. Mineral fertilization is carried out by a single sowing application of mineral fertilizer containing optimal amounts for the development of the white thorn.

66125 B1 nitrogen, phosphorus and potassium, and the foliar nourishment is carried out during the growing season by 5 times leaf spray with liquid mineral fertilizer Agroleaf (Scotts ltd, USA). The individual foliar feeds are carried out with different fertilizer formulations, differing in the ratio of nitrogen, phosphorus and potassium elements in the composition of the liquid fertilizer.

The use of plant growth regulators in plants is intended to influence both the overall growth dynamics and specific units of plant metabolism.

In order to study the effect of combination feeding and the application of a growth regulator in cultivated cultivation of white thorns, field trials were conducted with a test plot size of 9 m ² and a plant density of 5 m 1 m ² . The soil for the experiments was treated by standard procedure. The soil was fertilized before sowing. The leaf feeding was done by spreading a solution of Agroleaf leaf mineral fertilizer (Scotts, USA) with trace elements added. Its concentration was 0.5% and the dose per decare 1001. Treatment with the growth regulator was carried out once by spraying the plant mass over the rosette phase (30 cm diameter) with a cytokinin cytokine action solution tidiazuron Dropp ^R (N -phenyl-N'-l, 2,3-thiadiazol-5yl-urea, developed by Schering Co (Germany) and now manufactured by Aventis Crop Sci (USA) .The regulator has the chemical name by SCR - (1phenyl-3- (l , 2,3-thidiazol-5-yl) urea] and, according to the Chemical Abstract, [N-phenyl-N'-1,2,3-thidiazol-5-ylurea], which was used at a concentration of 20 mg / ί (active substance per hectare is 0.85 g and the working solution consumption is 25 l Thiadiazuron (abbreviated as TD3 or Dropp) was used as a defoliant in cotton at a dose of 300 g / dka for pre-wart treatment. a phenylcarbamide type cytokinin growth regulator has been described by Mok et al., (1982). As a cytokinin, TDZ has been successfully used to regulate flowering, micro-multiplication of calluses and organogenesis, increasing resistance to salinization of wheat plants, etc. Its effective concentrations as a regulator of callus growth and microcloning are between 0.5 and 1 mg / ί in the medium in combination with indolyl acetic acid (IOC). As a commercial preparation, it produces a powdery yellow substance, slightly soluble in water with 50% active substance and applied as a cytokinin-acting PP as a working solution with concentrations in the range 0.002% to 0.0002%.

The following growth parameters of the plants during the growing season were monitored during the field and vegetation experiments with white thorns:

1. change in the vegetative and reproductive phase of growth

2. flowering dynamics

3. structure of seed production

4. determination of silymarin and fatty acids content in seeds.

The soil fertilizers used are NH ₄ NO ₃ ammonium nitrate, triple superphosphate, and potassium sulfate K ₂ SO ₄ at the following basic element concentrations (5 kg I (nitrogen) / dka, 14 kg P ₂ O ₅ (phosphorus) and 15 kg K ₂ O (potassium) / dka) are introduced pre-sown. The leafy liquid fertilizer was applied by spreading on the vegetative mass of the plants 5 times during the growing season at intervals of 15 days at a working concentration of 0.5%. During the period of vegetative growth after germination, the leaf manure was introduced twice by spreading at a basic macronutrient N: P: K ratio of 20:20:20 in complex with the necessary trace elements added to provide optimal nutrition and to accelerate growth. In the pre-flowering phase to induce flowering, the fertilizer was introduced at a ratio of N: P: K = 12: 52: 5 and a complex of trace elements. Higher phosphorus content accelerates flowering of plants. For more uniform ripening and healthy, high quality seed, a double fertilizer with a ratio of N: P: K = 15:10:31 and a complex of trace elements were introduced. Increased potassium content improves ripening. The trace elements used are in chelate form and percentage in the stock solution: 0.1% Fe, 0.06% Μη, 0.06% Cu, 0.06% Zn, 0.02% B.

The method offers spraying with the growth regulator thidiazuron through the phase of rosette formation of the plants white thorn at a dose of 0.8 g / dka active substance (working solution of de

66125 Blcar 25 1) and 5-fold feeding of the aboveground part of the plants during the growing season with liquid mineral fertilizer containing the elements nitrogen, phosphorus and potassium in different proportions according to the above combinations, all done against the background of essential mineral fertilizers introduced into the soil in the amount described above. This method is proposed in order to achieve accelerated flowering, which provides more even ripening and increased seed yield when harvesting the experiment. This technology leads to prolongation of vegetation at the expense of the reproductive phase (between 5-14 days depending on soil and climatic conditions), changes the dynamics of flowering, laying more flowering stems per plant (125-140% of control), accelerates ripening of seeds in a flower basket and increases the weight of seeds in the basket. The increased yield of white thorn seeds is achieved at the expense of the formation of heavy seeds in a basket (120-160% of the control). At the same time, chemical analysis of the seeds showed that the application of the method also led to the accumulation of more silymarin and unsaturated fatty acids in them.

Explanation of the annexed figures

Figure 1 depicts a vegetative vessel experience providing (under partially controlled environmental conditions such as humidity and nutrient composition) the choice of the optimum concentration of mineral elements for the accumulation of maximum biomass of white thorn plants under hydroponic crop conditions.

Tables 1 and 2 and Figures 2a, b, c and 3a, b, c describe the results for the productivity of the white thorn plant from three-year field trials carried out on the experimental field G. Lozen of the FMI BAS.

Examples of carrying out the invention

The invention is illustrated by the following examples.

EXAMPLE 1 Fertilizer concentration (0.5%) used in field conditions was selected by conducting a series of vessel trials with hydroponic white thorn culture under growing conditions in a growing house. Figure 1 shows the rosette diameter, leaf count and dry biomass of the plants. It has been found that when applying leaf fertilizer at a concentration of 0.5%, an optimum increase in the number of leaves, plant dry biomass and rosette diameter was observed.

EXAMPLE 2 The white thorn was grown in a field experiment on the experimental field of G. Lozen at the IGF BAS (Table 1). The fertilized soil with fertilizer and spray fertilizer was treated with the TDZ growth regulator as described above. Table 1 presents data on the number of buds, blossoms and flower buds relative to the total number of flower baskets formed by plants per unit area (1 x ² ) depending on different treatments. Our results show that foliar and soil nutrition of white thorns in combination with TDD spraying leads to an increase in the proportion of blooming flowers in the total number of flower baskets and a decrease in the number of open buds compared to soil mineral fertilization options and control untreated plants.

EXAMPLE 3 Figure 2 (a, b, c) shows the results obtained for seed production from 3 years of field experience. Treatment with the thidiazuron growth regulator in 2004 resulted in the highest seed yields for soil mineral fertilizers (root feeding). The increase in seed yield compared to control was threefold and 45% compared with untreated plants (without TDD). In TDD-treated plants against a background of leaf mineral nutrition, the yield increase was 60% relative to control and 100% to non-growth-treated plants. In the years 2005 and 2006, the highest seed yields were obtained by combining the TDZ growth regulator with leaf mineral nutrition. In the 2005 and 2006 experimental years, the increase in seed yield was 43% and 110% relative to the control and 38% and 20% respectively to the untreated TD plants. The increase in yield in these variants is mainly due to the increased number of flower baskets per unit area rather than the number and mass of seeds in a flower basket.

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Example 4. The content of silymarin components in mature seeds from 2004-2005 and 2006 (Figure 3a, b, c). These were determined by HPLC according to the conventional procedure and are presented in Figures 3, 4 and 5. Higher values were observed with respect to the silybin and isosilibin components in all variants tested. The content of silybin and isosilibin in 2004 was highest in the case of TDD and leaf mineral fertilizers. In experimental 2005, there was no significant difference in silybin and isosilibin content between leaf-fed variants with and without growth regulator. For TDD variants and foliar fertilizers, the content of the other components of silymarin - silidianin and silicristin - is much higher. The higher content of silymarin in plant seeds treated with growth regulator in combination with foliar fertilization is mainly due to the higher seed yields in these variants. In 2006, a significantly higher content of silymarin components (silidianin / silicristin and silibin / isosilibin) was reported in plant seeds treated with TDZ and combined soil and leaf mineral fertilization.

Example 5. Relative proportion of higher fatty acids in the fraction of lipophilic substances extracted from white thorn seeds grown under leaf fertilization with liquid fertilizer and treated with the growth regulator cytokinin activity tidiazuron (TD3) (Table 2). Data are average values of oils extracted from 50 g of dry seeds. The results showed that leaf fertilization alone significantly increased the amounts of saturated fatty acids in the lipophilic fraction of C16-C18 row seeds. At the same time, the percentage of unsaturated acids C18: 1, C18: 2 and C18: 3 decreased in the lipophilic extract. However, the combination of foliar fertilization and TDD leads to an increase in the unsaturated fatty acid content of the C18 and C20 rows.

Claims (2)

1. A method for regulating plant growth, seed production and quality with regard to silymarin and unsaturated fatty acid content, carried out under cultivated cultivation of white thorn in field conditions, characterized by the combined application of soil mineral fertilization and leaf mineral spraying fertilizer and treatment with growth regulator during the growing season. A method according to claim 1, characterized in that, during the rosette formation phase, the plants are sprayed with growth regulator thiadiazuron (TD3) (N-phenyl-N'-1,2,3thiadiazol-5-yl-urea) at a dose 0.85 g / dka and solution consumption 25 1 / dka), and the combined fertilization is carried out as follows - the soil is a single pre-sowing mineral fertilizer of 12 kg N (a3OT) / dka, 6 kg Р ₂ О ₅ (phosphorus) / bca and 5 kg K ₂ O (potassium) bca, and the leaf fertilization is carried out by spreading liquid mineral fertilizer at a concentration of 0.5% and consumption of solution 100 1 per dka, as follows through phase twice in 15 days with N: P: K in ratio 20:20:20 and trace elements, once in pre-flowering phase with N: P: K in ratio 12: 52: 5 and trace elements and once through budding-flowering with N: P: K at a ratio of 15:10:31 and trace elements.