The method of obtaining compounds having antioxidant effect

 

The invention relates to pharmaceutical industry. For extraction, milk Thistle fruit using 80% ethyl alcohol in the ratio of raw material:solvent 1:9-1:10. Sequential chromatographic separation of extractives on the polyamide and silica gel using graduates suantai a mixture of water - ethanol and chloroform - ethyl alcohol respectively in relationships 100:0; 98:2; 97:3; 95:5; 93:7 get the target product after crystallization. The invention allows to increase the yield of the target product. table 1.

The invention relates to chemical-pharmaceutical industry, in particular the production of medicines in the form of substances and individual biologically active compounds (BASS), and relates to a method of producing 2,3-dehydrosilybin from fruits, milk Thistle [Silybum marianum (L.) Gaertn.].

In pharmaceutical technology known methods for producing medicines and substances based on the amount of BASS (Kars, zelibor, silimar, silymarin and other ), as well as individual substances from fruits, milk Thistle[7, 16-21, 24, 27, 34, 47, 49], however, a disadvantage of known methods is the complexity of the technological process and leg opening [5-7, 20, 26, 42, 46, 50] it was emphasized above all the importance of three main components, milk Thistle fruit - silybin, silydianin and silychristin that define gepatoprotektornaya, antioxidant, membrane-stabilizing properties of products based on raw materials of this plant to date in both domestic and foreign literature[5-7, 22-24, 26, 29-33, 44-50]. This was the basis for the development of a series of refined products on the basis of the sum of the above flavolignans called "Silymarin". For the purposes of standardization, milk Thistle fruit and domestic products on the basis of this raw material, we propose to use silybin as the state standard sample [8-11, 14, 15].

A prototype of the invention taken us way to obtain 2,3-dehydrosilybin from fruits, milk Thistle [28], however, the allocation of this substance is carried out with the use of cumbersome technology, consisting in the sequential extraction of raw materials (2.0 kg) petroleum ether, benzene, ethyl alcohol, the process of evaporation of the alcoholic extract, chromatographicaliy the obtained residue on silica gel (eluent benzene-ethyl acetate, 1:1), subsequent rechromatography on what Ecodom crystallization from methanol with a yield of the target substance is 0.2 g or 0.01% of air-dried material. This method is not only cumbersome, but also gives a low yield of the target substance 2,3-dehydrosilybin. In addition, at the stage of extraction of the raw materials used such hazardous solvents, such as petroleum ether and benzene.

The aim of the invention is to increase the yield of the target product with antioxidant effect.

This goal is achieved by the fact that as the extractant using 80% ethyl alcohol, allowing exhaustively extract the target product 2,3-dehydrosilybin (1), milk Thistle fruit, followed by chromatographic separation of extractives on the polyamide and silica gelConducted by the applicant search on scientific, technical and patent information sources and selected from the list of analogues prototype [28] allowed us to identify distinctive features in the claimed technical solution. Therefore, the inventive method of obtaining satisfies the criteria of the invention of "novelty."

Conducted by the applicant additional search of the known technical solutions[1-4, 7, 13-25, 27, 35-41, 43, 47, 49] to detect in them the characteristics similar to the characteristics of the proposed method of obtaining, showed that these signs are absent, sledovatelxno actions flavolignans, milk Thistle fruit (2,3-dehydrosilybin, silybin) and extract milk Thistle liquid conducted on rats, which under anesthesia liver was removed and it was the homogenate prepared in a glass homogenizer at a temperature of +1oWith 0.05 M phosphate buffer. In all series of experiments the homogenate was taken from one rat, diluted to five portions, which were used in five series of experiments on nine samples each, which allowed to compare the results of all series of experiments with a fairly high degree of reliability. In all samples was determined by the concentration of malondialdehyde (MDA) by the reaction with thiobarbituric acid (TBA) according to the method of O. N. Korobeinikova [12]. To 0.5 ml of liver homogenate was added 5 ml of 20% solution phosphomevalonate acid. The mixture was left out in the cold to the formation of large flakes, and then centrifuged. The supernatant liquid was decanted, the precipitate was added 2 ml of distilled water and 1 ml of TBA, which was prepared by dissolving 80 mg of TBA in 5 ml of glacial acetic acid. Samples were incubated in a boiling water bath in test tubes at a temperature of 99-100oC. the Tubes were cooled, centrifuged 10 min at 8000 rpm, then photometrically on the spectrophotometer at a wavelength of 535 nm and with a length of 580 nm.

Calculation proizvol/ml of liver homogenate;D - optical density at a wavelength of 535 nm minus the absorbance at a wavelength of 580 nm.

Conducted five series of experiments. In the first series of experiments was determined lipid peroxidation in liver homogenate without adding any chemicals. It was 2,800,064 nmol/ml of homogenate. Peroxidation in liver homogenate in the second series of experiments was increased by the addition of FeSO4at a final concentration of 0,076 mg/ml (0.5 mm). Thus we created the original background of increased lipid peroxidation with the addition of a recognized stimulant of this process is iron sulfate (II). This background served as a control for subsequent experiments. In the following the third series of experiments in test tubes was added silybin at a final concentration of 2 mm/l, in the fourth series of experiments to the liver homogenate was added dehydrosilybin also at a final concentration of 2 mm, in the fifth series of experiments to liver homogenate as well as in the previous series of experiments was added ferric sulfate, and moreover, the milk Thistle extract in an amount such that the total content in the environment silybin and dehydrosilybin contained in the extract, was 2 mm/L. the results of the investigations are given in the table.

Thus, silviosantos steps substances and drug attached) possess a strong antioxidant effect. Antioxidant activity dehydrosilybin detected for the first time and 14.0% higher than that silybin related to the main biologically active compounds of silymarin and other preparations, milk Thistle fruit.

The inventive method is illustrated by the following example.

Example 1. to 200.0 g of crushed fruits, milk Thistle [Silybum marianum (L. ) Gaertn.] fill in 80% ethyl alcohol in a ratio of 1:10 and infused for 24 h, and then the extraction of raw materials carry out 80% ethanol by heating for 30 minutes 2 times. United water-alcohol extract evaporated under vacuum to an aqueous residue, which is then put on a layer of polyamide and elute with water and then with a mixture of solvents water - ethanol with increasing concentration of the latter. Target product elute 40% ethyl alcohol. Subsequent chromatographic separation of extractives carried out on silica gel L 40/100 using gradient suantai mixture of chloroform - ethanol in different ratios(100:0,98:2,97:3,95:5,93:7), resulting crude target product (0.5 g). Final purification of the target product are carried out by crystallization from water-dry raw material.

Spectral and physico-chemical properties of 2,3-dehydrosilybin (1): yellow Crystals With25H20About10(M+480) so pl. 253-255oC. UV spectrum:max267,365 nm.1H-NMR-spectrum in deuteroacetone (200 MHz): 12,15 (s, 5-Oh), 9,74 (ush. s, 7-OH), 7,87 (D. 2,1 Hz, H-21), 7,17 (DD, of 8.3 and 2.1 Hz, H-61), 7,8-6,9 (m, 4H-AG), 6,60 (d, 2.1 Hz, H-8), 6,28 (d, 2.1 Hz, H-6), of 5.06 (d, of 8.06 Hz, HA), 4,24 (m, HIn), 3,88 (s, 3H, CH3O), 3,80 (DD, 12.5 and 3.5 Hz, HC), 3,55 (DD, 12.5, 4.3 and Hz, HD).

Example 2. to 200.0 g of crushed fruits, milk Thistle fill in 80% ethyl alcohol in a ratio of 1:9 and infused for 24 h, and then the extraction of raw materials carry out 80% ethanol by heating for 30 min 2 times, the Next process is carried out in accordance with example 1.

The yield of the desired product as individual 2,3-dehydrosilybin is 0.2 g or 0.09% of the mass of air-dried raw materials.

Example 3. to 200.0 g of crushed fruits, milk Thistle fill in 80% ethyl alcohol in a ratio of 1:11 and infused for 24 h, and then the extraction of raw materials carry out 80% ethanol by heating for 30 minutes 2 times. Next process is carried out in accordance with prisno-dry raw material.

Learning beyond parameters, namely the use of chromatography and rechromatography on the same sorbent - polyamide or silica gel - does not lead to obtaining the target product, and the implementation of the extraction 75 and 85% ethanol, and the ratio of raw materials-extractant (1:8) leads to the reduction of the yield of 2,3-dehydrosilybin to 0.6, 0.7 and 0.75%, respectively.

Therefore, the optimal parameters of the method are receiving the extraction of 80% ethyl alcohol in the ratio of raw material to the extractant in the range of 1: 8-1:10, followed by chromatographic separation of extractives on the polyamide and silica gel and crystallization of the desired product from aqueous alcohol.

Thus, the use of the claimed method of producing 2,3-dehydrosilybin provides the following benefits: 1. Use in the claimed method 80% ethanol allows exhaustively extract the target product (2,3-dehydrosilybin), milk Thistle fruit.

2. The inventive method allows 9-10 times to increase the yield of the target product from fruits, milk Thistle compared with the prototype.

3. The inventive method provides a new interpretation of the contribution BASS-based preparations, milk Thistle fruit, stove raw materials of plants, which consists in taking into account the antioxidant properties of 2,3-dehydrosilybin and therefore in need of correction traditional technologies involving obtaining the purified silimarina amount.

Claims

The way to obtain 2,3-dehydrosilybin of the fruits of milk Thistle (Silybum marianum (L.) Gaertn.), including extraction of organic solvents, including ethyl alcohol, chromatographic separation and crystallization, wherein the method is carried out using 80% ethyl alcohol in the ratio of raw materials - extractant 1:9-1:10 and sequential chromatographic separation on the first polyamide using suantai a mixture of water, ethyl alcohol and then on silica gel using a mixture of chloroform - ethyl alcohol, respectively, in the relations 100:0,98,:2,97: 3,95:5,93:7.

 

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