RU2139710C1 - Method of preparing vitamin e from hexane fraction of natural sapropel - Google Patents

Abstract

FIELD: pharmaceutical chemistry. SUBSTANCE: water- soluble substances are isolated from native sapropel by successive extraction with 96% ethanol and hexane (40-45 min) followed by centrifugation at 1600-2000 rpm for 8-10 min. According to invention, hexane extract is concentrated and successively chromatographed in hexane and hexane-ethylacetate (37:3). EFFECT: accelerated process and increased yield. 2 ex

Description

 The invention relates to balneology, and in particular to methods for producing individual components of lipid complex concentrates containing mixtures of fat-soluble vitamins, and can be used in the manufacture of individual components of an ointment-based hexane fraction in the treatment of burns and skin diseases in the practice of medical institutions.

 Vitamin E in the lipid complex of therapeutic sapropels is a fat-soluble antioxidant of biological origin or bioantioxidant, the presence of which can control the quality of natural raw materials. A large role in creating the antioxidant background of vitamin E sapropels, a phenolic antioxidant, is determined by its ability to bind active free radicals, along with ascorbic acid and rutin, it is an important component of the antioxidant system of mud substrates. The most common form of vitamin E in plants is known to be α-tocopherol. The bioproducers of vitamin E healing mud are char and algae algae, green parts of coastal macrophytes, etc.

Most often, an increased concentration of vitamin E in sapropels is combined with a high content of lipid fraction, which has antimicrobial activity. In sapropelic mud, unsaponifiable substances of the lipid complex have anti-inflammatory and antibacterial activity against certain strains of microorganisms (Staphylococcus aureus N 209, Pseudomonas aeruginosa and Escherichia coli N 675 and M ₁₇ , sarcin and mycoid). The hexane insoluble fraction (chloroform or non-polar) consists of phospholipids, alcohols, sterols, fatty acids, pigments. Experimental studies indicate a pronounced anti-inflammatory, absorbable and stimulating effect of total lipids and chloroform fraction of silt sulfide mud.

 A known method of producing vitamin E from the total lipid complex of native sapropel by 3-times extraction with ethanol-chloroform mixture and subsequent extraction of the α-form of vitamin E from it. The disadvantages of the method are multi-stage operations and high consumption of the initial volume of therapeutic mud [1].

 There is also a known method of producing a hexane fraction from silt sulfide mud selected by the author as a prototype, according to which, only 30-50% of all lipids, among which mainly hydrocarbon compounds (paraffins, squalene, carotenes) are identified in the hexano-soluble (non-polar part), are identified . {1}. However, this method has several disadvantages, namely: only half of the amount of all lipids is extracted, the balneologically valuable components of which are not separated. The method is incomplete with respect to a narrower separation of chloroform and hexane fractions and the isolation from them of certain classes that are known to cause a therapeutic effect, which involves the search for drugs with a specific effect.

 The objective of the invention is to reduce the time of learning the total lipid complex and extract from it one of the forms of vitamin E. The goal is achieved by the fact that from the native sapropel water-soluble substances are removed with the addition of 96% ethanol, incubated for 24 hours and vigorously shaken for 10-15 minutes, then get the hexane fraction by adding n-hexane and extracting the mixture for 40-45 min; the contents are transferred to centrifugation tubes and centrifuged for 8-10 minutes at 1500-2000 rpm, after separation of the mixture, the hexane extract is recovered. All operations are performed in dark glass dishes. The result of these operations is to obtain the total lipid complex, which contains the main fat-soluble LHC-chlorophylls, carotenoids, carotenoids, vitamins A and E, etc.

 An aliquot of the concentrated hexane extract is applied to a plate with a fixed layer of siloufol and placed in a chromatographic chamber with a moving phase - hexane. After the solvent front reaches the upper edge, the plate is removed, dried under natural conditions, then placed in a chamber with the hexane-ethyl acetate system (37: 3) a second time [2]. After drying for 5 minutes at room temperature, a standard witness solution (tocopherol solution in oil) is developed after staining it pink with a solution of dipyridyl and ferric chloride in absolute ethanol. The chromatogram was viewed at a wavelength of 260-290 nm under a KFK-50 mercury-quartz lamp. The spots were purple and Rf at witness level. Extraction with hexane for less than 40 min is undesirable due to incomplete extraction of the sapropel lipid complex. When using a centrifugation speed of 1000 rpm, the final result was not achieved, namely: the separation of carotenes and vitamin E during chromatography did not occur. In the case of using single chromatography in any of the above systems, vitamin E spots were not detected during identification, and drying chromatographic plates in ovens led to the loss of balneologically valuable components.

 Example 1. 100 g of therapeutic mud of medium or high-ash algal origin is poured, approximately 100 ml of 96% ethanol to cover the mud, incubated for a day, divided into several portions and continuously shaken for 15 minutes. The liquid phase is carefully drained and filtered through a pre-defatted filter with sodium sulfate until completely dehydrated, then combined with the solid phase and the combined extracts are extracted after adding 5 ml of n-hexane for 45 minutes. The contents, separated into phases, are transferred to centrifuge tubes and centrifuged for 10 minutes at 2 thousand rpm. The resulting volume of hexane extract (about 50 ml) is concentrated in a rotary evaporator to a volume of 3-5 ml and aliquots of 0.1 - 0.2 ml are prepared for chromatography.

 Example 2. To 100 g of native sapropel oily consistency with a high content of fat-like and waxy substances add about 10 ml of 96% ethanol and carry out vigorous shaking for 15 min in a test tube of dark glass. Then 5 ml of n-hexane is added in a quick motion and extraction is carried out for 45 minutes. Separation of the extract was observed, the resulting mixture was centrifuged for 8 minutes at 1500 rpm. A hexane layer was taken with a pipette, which was analyzed by thin layer chromatography.

 Result: the method significantly reduces the time of operations to isolate the total lipid complex and reduces the consumption of the initial volume of therapeutic mud in comparison with known methods.

 The advantages of the proposed method are as follows.

 The method allows several times to reduce the initial volume of therapeutic mud, reduce material costs for the purchase of reagents, reduce the time to get vitamin E from the total lipid complex to create preparations on an ointment basis for the treatment of patients with burns, skin and other diseases.

Literature
1. Matis E.Ya., Starikov NM, Kurakolova EA, Burkova VN, Titov VI, Nizkodubova CB Obtaining a lipid concentrate from peloids // Issues of balneology and physiotherapy, N 2.- 1985.- S. 63-65.

 2. E. Heftman // Chromatography, part 1, M., 1986.- p. 327.

Claims (1)

 A method of producing vitamin E from the hexane fraction of native sapropel, comprising extracting the lipid concentrate with organic solvents, characterized in that water-soluble substances are sequentially removed from sapropel with 96% ethanol, then the hexane fraction is obtained by extraction for 40-45 min at a speed of 1600-2000 rpm , which is concentrated, chromatographic to isolate vitamin E in two systems, hexane and hexane - ethyl acetate (37: 3).