Method for detection and quantification of ecdysteroids in plants

 

(57) Abstract:

Usage: the invention relates to Bioorganic chemistry and chemistry of natural and physiologically active substances and can be used in the search for new promising sources of ecdysteroids among the objects of the vegetable world to obtain the necessary quantities of pure hormones in the research in various fields of medicine. The invention: the proposed method allows to make a conclusion about the presence of ecdysteroids in whole plants on the basis of preliminary detection in seeds by thin-layer, high-performance liquid or gas chromatography. table 2.

The invention relates to Bioorganic chemistry and chemistry of natural and physiologically active substances and can be used in the search for new promising sources of ecdysteroids among the objects of the vegetable world to obtain the necessary quantities of pure hormones in the study in various fields of medicine.

There is a method of quantitative determination of ecdysteroids using reaction tchugaev [1] the Disadvantage of this method is that after chromatographic separation on glass layer is ncom in ice acetic acid with the addition of acetylchloride, followed by heating and spyektrofotomyetrirovaniya.

The closest to the proposed invention to the technical essence and the achieved result is chromatospectrophotometrical method for determination of ecdysone in the dry plant material (leaves, stems, roots) [2] in which the division of alcoholic extracts is carried out on glass plates with fixed layer Alusil in the solvent system chloroform-methanol-acetone in the ratio 6:2:1 with subsequent spyektrofotomyetrirovaniya eluates containing ecdysteroids.

The disadvantages of this method are: the complexity of harvesting, drying, preparation of raw material for analysis; the need for preparation of glass plates and difficulties playing the homogeneity of the layer, which affects the results of the analysis; identification of ethanol with a solution of vanillin in sulfuric acid; high consumption of solvents when chromatographicaliy and elution.

The present invention is the task of reducing the time the qualitative detection and quantitative determination of ecdysteroids.

The problem is solved in that in the proposed method of detection and quantitative determination of ecdisten flowering plants, and chromatographic analysis carried out one of the methods: thin layer chromatography (TLC), gas chromatography (GC), high performance chromatography (HPLC).

Moreover, using the TLC method, the extract analyzed by standard plates Silufol UV-254 in the solvent system chloroformmethanol (3:1) and detects ecdysteroids in UV light; using the HPLC method, the extract is analyzed using octadecylsilyl silica (C-18) as nozzles separation columns in water-ethanol eluting systems, and detection carried out by UV detector at 254 nm; GC involves the analysis of the extract on silica capillary columns with silicone phase type OV-101 and the detection carried out electron-capture detector.

The introduction of the above distinguishing features allows to obtain a technical result, which cannot be achieved in the prototype.

It is known that perennial plants in the first year of life synthesize small amounts of ecdysteroids, sometimes less than the detection limit of instrumental methods, and the maximum accumulation is observed at 2-3 year [3] in Addition, a greater proportion in the sample of above-ground parts make the stems and leaves, otlichitelnym organs (buds, flowers and seeds) [3, 4] but their contribution to the total mass of plants is very small. So in the first year of life the content of ecdysone in the aerial parts of lychnis chalcedonica is 0.15, the second and third of 0.41 and 0.36%, respectively, whereas in seeds the second year 0,76% Qualitative detection and quantitative determination of ecdysteroids in the proposed method is carried out by any chromatographic method: TLC, HPLC or GC. In the proposed method, the separation of the extracts at TLC determination is carried out on standard plates Silufol UV-254 with a thin layer of sorbent, which greatly improves the reproducibility of the results and reduces the time of analysis. The presence of fluorescent light on these plates simplifies the detection of ecdysteroids, excluding the application of the detection reagent. And because the layer of sorbent on standard plates are much thinner than on the formulation, the amount of solvent is less than, additionally, alcoholic extracts of the seeds do not require additional cleaning with organic solvents and additives to silica gel aluminum oxide, as in the prototype, due to the low content of pigments and other related substances.

The technical result of pringlesforaidan in plants, while improving the accuracy and reproducibility of the analysis. This ultimately will bring about progress in the search for promising sources of ecdysteroids among the objects of the vegetable world, which is especially important, given the wide range of pharmacological actions of these steroids.

Example 1. 0.2-0.1 grams of seeds of the genus Silene (Silene) extracted with 70% ethanol twice, concentrate. The extract is applied on the activated plate Silufol and chromatographic in the solvent system chloroform-ethanol (3:1). For the qualitative detection of ecdysteroids detected in UV-light. For the quantitative determination indicated in UV light spots on the level of standards of ecdysteroids removed and elute 96% ethanol for 4 hours. Then the eluate is filtered and spectrophotometery at 242 nm. Preparation of raw materials, preparation for analysis, analysis and the consumption of solvents are given in table 1. The results of determination of ecdysteroids are summarized in table 2.

Example 2. Extracts of seeds of the genus Silene is prepared as described in example 1, and the definition of ecdysteroids are using high-performance liquid chromatography on device "milikhrom using columns with octadecylsilyl silica gel (C-18) Lichrosorb RP-18 in the system methanol-water (8: 2). Detection osushestvleniya of atenolol in identical conditions, and quantitative prior to the calibration standards dependencies. The results are shown in tables 1, 2.

Example 3. Seed extract is prepared as described in example 1 were then added N-(trimethylsilyl)imidazole in an amount of 1 ml per 10 ml of the extract and shake for 30-45 minutes. Analysis of ecdysteroids in the form trimethylsilyl esters carried out by means of gas-liquid chromatography on the device model "3700" quartz capillary column (25 m x 0.22 mm) with silicone immobilized phase OV-101 (thickness of 0.1 μm) and electron capture detector. The separation conditions: carrier gas, nitrogen (C. H.) 0.8-1.0 cm3/min, column temperature 270-310oC. Qualitative analysis is performed according to the retention times of TMS-ethers of ecdysteroids-standards, quantitative prior calibration dependencies in identical conditions analysis.

The results are shown in tables 1 and 2.

As can be seen from the data presented in table 1, the use of the proposed method in examples 1-3 will greatly expedite the conduct of all stages of preparation and analysis of the extracts and significantly reduce the consumption of the used solvents.

As can be seen from the data, n is to divide the ecdysteroids quantitatively with greater precision than that of the prototype.

Thus, using the proposed method allows to make a conclusion about the presence of ecdysteroids in whole plants in any phase of development on the basis of a preliminary detection and quantitative determination in seeds. The application of this method to search for ecdysteroids in plants accelerates chemical screening increases the likelihood of detection, easier way to determine improves the accuracy and reproducibility of quantitative determination. 15-20 years of active study of phytoecdysteroids researchers found 18 species of the genus Silene and the use of the proposed solution has allowed for 1 year to analyze 60 species and to identify 20 new producers of ecdysteroids from the specified type.

The technical feasibility of the invention is obvious, as is used commercially available domestic equipment, commodity-produced plates and organic solvents.

Sources of information

1. Kholodova Y. D. Application of reaction tchugaev for the quantitative determination of ecdysone //Chemistry of natural. connect. 1977. C. 227-230.

2. Chromatospectrophotometrical method definitions. 978. C. 737-740 (prototype).

3. Zibarev L. N. The phytoecdysteroids and other chemical components of plants of the genus Lychnis: author. dis. Kida. chem. Sciences. Novosibirsk, 1991. 19 C.

4. Ereskovsky centuries Chekalinsky And. And. flap, Century Dynamics of the content of ecdysone in species of the genus Rhaponticum Ludw. //Rastitel. resources. 1983. - Vol.1 C. 60-65.

Method for detection and quantification of ecdysteroids in plants by chromatographic analysis of the feedstock, wherein the feedstock used the seeds of flowering plants, and the analysis is carried out by thin-layer high-performance liquid or gas chromatography.

 

Same patents:

The invention relates to a device for performing immunochemical determination of containing at least one cartridge, the top of which has a hole, and inside the put immunochemical active substance (1AS1)

The invention relates to medicine, namely to obstetrics, and can be used to predict the development of late gestosis pregnancy

The invention relates to aquatic toxicology, testing adverse toxic effects of pharmaceutical products, food concentrates, feed additives, testing of environmental clean technologies associated with wastewater discharge, the forecast changes in populations of aquatic animals transformed by wastewater ecosystems

The invention relates to the field of analysis, and in particular to methods of enzymatic determination of phenols and can be used both in laboratory practice and research, and to identify these toxic compounds in natural and waste waters, soil, etc
The invention relates to medicine, in particular to biotechnology, and relates to methods of obtaining new drugs from the blood of humans and animals
The invention relates to medicine, namely to the study of blood and urine, and can be used in urology

The invention relates to the field of chromatography and can be used to separate mixtures of substances in a highly efficient microtunneling chromatography

The invention relates to the measurement of the sorption properties by the interaction of amino acids with optionalinfo ore
The invention relates to analytical chemistry, in particular to methods for determining benzoxazolone-2 and 6-chlorobenzoxazole-2 in waste water production of pesticide phosalone of benzoxazolone-2 (phosalone) thin-layer chromatography (TLC)

The invention relates to analytical chemistry and can be used to determine the content as I ammoxidation furadonin, furagin in pure form, in mixtures, in dosage forms, using chromatographic separation techniques, in particular thin-layer chromatography (TLC) in practice, control and analytical laboratories, factories, pharmacies

The invention relates to gas chromatography and can be used to determine the qualitative composition of multicomponent mixtures of organic compounds

The invention relates to the medical industry and can be used to obtain preparations of insulin used in the treatment of diabetes

The invention relates to a developing method of analysis of various toxic substances, and the proposed method can detect them in the trace

The invention relates to the field of physico-chemical applications gaschromatographie, in particular, to methods of determining the adsorption capacity of the solid materials used in the computation of the structural parameters of the surface of solid materials, as well as in the ceramics industry in defining the parameters of porous structure of raw materials and finished products and in the production of solid polymeric materials
Up!