RU2281779C2 - Method for preparing natural melanoid antioxidant - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to a method for preparing the natural melanoid antioxidant. Method for preparing the natural melanoid antioxidant involves washing out with water sunflower non-milled husk, its drying at the definite temperature, extraction of sunflower non-milled husk with water at boiling taken in the definite ratio, filtration, treatment with food adsorbent KCM № 6С and evaporation up to gel-like state. The natural melanoid antioxidant prepared by abovementioned method elicits the enhanced antioxidant properties and absence of negative hemolytic properties.

EFFECT: improved preparing method of antioxidant.

3 tbl, 1 ex

Description

The invention relates primarily to medicine and can also be used to inhibit oxidative processes in the food industry (including oil and fat), in veterinary medicine to enhance the immunity of animals and birds.

In connection with the development of a number of industries (chemical, nuclear, etc.), accompanied by environmental pollution, it has become increasingly difficult to maintain the health of biological objects living on the earth. The number of exogenous diseases is growing rapidly against the background of a deteriorating immune state of people, animals, birds.

To correct the situation in recent years, it has been used as immunomodulators of synthetic and natural antioxidants (AO), including melanoid AO [7]. The literature describes the positive use of AO in various (over 200) pathologies.

Given the high cost of the starting material for the production of melanoid AO by chemical means, the production of a cheap natural antioxidant will expand the range of its application in connection with the cheapening of AO.

Exotic sources of obtaining similar drugs are known - from the sclera of the bull’s eye, from squid ink, from the tails of horses [1]. The source of information does not disclose the production technology, however, the drawback is obvious: it is unlikely that the method is feasible on an industrial scale.

It was proposed to organize microbiological synthesis. The method intended to grow melaninogenic microorganisms and then extract the necessary substance with 0.1 N NaOH (followed by neutralization of the alkali with hydrochloric acid).

The stepwise path of the first synthesis of microorganisms, and then the isolation of substances from them, is considered very difficult.

It is more advisable to obtain a water-soluble antioxidant using cheap natural raw materials - husk (waste from the established production of sunflower oil).

For the prototype adopted "Method for the production of natural antioxidant" [3].

The method is as follows. Unfinished sunflower husk obtained after the seeds were crushed in the technological cycle for the production of sunflower oil is mixed with water in a mass ratio of water: husk = 10: 1, brought to a boil and boiled for 30-55 min, and then filtered. The solvent (water) is evaporated by boiling, bringing the extract to a jelly-like state.

It has been established that from 30 to 100% of the trace elements found in the husk go into the antioxidant, the main of which is potassium. In addition, AO is rich in phosphorus, magnesium, calcium, etc. According to the degree of mineralization, unpaired AO is similar to Essentuki 4 mineral water.

The melanoid nature of the indicated water-soluble AO was revealed, since a water-insoluble black powder with a metallic luster obtained from sunflower husks according to the known method [4], with respect to organic solvents, acids, alkalis, interaction with oxidizing agents, spectra in the visible and IR regions The EPR signal and elemental composition corresponded to the concept of “melanin” [4].

By the method of selecting radioprotective properties adopted in radiobiology [5], the radioprotective properties of the obtained AO were revealed (a blood sample irradiated in UV in its presence turned out to be 6 times more stable compared to the control where the AO was not added) [6].

AO from husks has a positive effect on the bactericidal and energy systems of neutrophilic white blood cells (in white rats with paratyphoid patients), which are responsible for maintaining the body's immune-structural homeostasis [7].

The animals taken in the experiment had a reduced content of glycogen, a bactericidal lysosomal cationic protein in neutrophils. The activity of myeloperoxidase, another component of the bactericidal system, was sharply reduced. Cells were unable to fully realize their main function - phagocytosis. After 10 days of treatment, these indicators were significantly (P = 0.01) different in the control and treated animals, and after 20 days of treatment, the latter recovered (indicators corresponded to the norm). In the control group, the content of glycogen and cationic protein remained low, the activity of myeloperoxidoses continued to decrease, the clinical manifestations of the disease increased, the percentage of dead animals increased, and as a result reached 20%.

In agriculture, the immunomodulatory role of AO was tested in the conditions of the Krasnodar poultry farm.

Birds were observed from 5 to 52 days old and found that in the experimental group broilers that consumed an antioxidant had a gain of 9% higher, in this group there were no weak birds (in the control group there were 35%), broilers with reduced immunity in the experimental group was 1/3 less than in the control.

A study of the activity of enzymes and melanoid AO in the husk by the phases of sunflower maturation [8] showed that of the studied enzymes (tyrosinase, catalase, laccase, peroxidase), tyrosinase has a noticeable effect on the accumulation of AO, i.e. AO owes its synthesis in husks to tyrosine, an expensive raw material in the chemical process for producing melanin.

The specified method has the disadvantage that the obtained AO simultaneously has the ability to destroy blood, is a hemolytic [3]. The presence of hemolytic activity not only reduces the beneficial properties of AO, but can lead to damage to health.

The task of the invention is to increase beneficial antioxidant properties and eliminate negative - hemolytic.

The technical result of the proposal is to remove from the husk and liquid AO the ingredients responsible for hemolysis.

A significant novelty of the solution is the preliminary (before AO extraction) washing and drying of husk at a temperature of 90-110 ° С and before evaporation to a jelly-like state - processing the obtained liquid antioxidant with a food adsorbent (silica gels, zeolites, etc.).

The proposed method is as follows. Unmilled sunflower husk is washed with water, the washed husk is dried at a temperature of 90-110 ° C to a friable state; mix water with husk in a mass ratio of water: husk = 10: 1, bring to a boil and boil for 30-55 minutes, then filter. The filtrate is a liquid antioxidant. An adsorbent is added to the obtained AO, which, after interaction with the substance, is separated; the solvent (water) from AO is evaporated by boiling, bringing the extract to a jelly-like state.

The reliability of the stated material is confirmed by Tables 1, 2 and 3.

Table 1
The influence of the method of preparation of AO on its antioxidant activity * Oil Experience No. not protected by JSC protected by AO prepared by the method prototype proposed one 90 min 150 min 225 min 2 125 min 300 min 420 min 3 105 min 270 min 410 min four 140 min 370 min 480 min 5 150 min 410 min 525 min Note: in all variants of the experiments, the advantage of the proposed method is shown.
* Antioxidant activity was evaluated by the duration of the induction period of oxidation of sunflower oil [3]. table 2
Effect of washing and temperature conditions of husk drying on radioprotective ** and hemolytic properties of melanoid AO Experience No. Object of study AO from husks of sunflower seeds AO Properties radiation protective hemolytic "0" hemolysis time, min one unwashed (prepared by the prototype method) 1.9 40 2 washed, dried 0.3 8 3 washed, dried with 30-50 ° C 0.8 twenty four 50-70 ° C 0.9 40 5 70-90 ° C 1,5 60 6 90-110 ° C 3.6 not hemolytic 7 110-130 ° C 3.6 not hemolytic Note: the drying result at 90-110 ° С showed the best result; higher temperature is not required.
** Radioprotective properties were studied using erythrograms. If "0" is the ratio of the time of half hemolysis of erythrocytes with the addition of the studied substance to the time of half hemolysis of erythrocytes of the control is more than 1 - the substance is a radioprotector, if less - hemolytic. To characterize hemolytic activity proper, hemolysis time was taken into account. A photocolorimetric method was used, which makes it possible to evaluate changes in optical density due to hemolysis over time.
Table 3
The effect of adsorption treatment *** on the radioprotective and hemolytic properties of AO. Experience No. Brand of adsorbent; AO concentration,% "0" AO properties hemolytic, optical density of a suspension of red blood cells in 1 hour of experience one KSS No. 3; 0.1 2.9 0.690 2 KSS No. 4; 0.2 2,3 0.700 3 KSM No. 5; 0.2-0.4 3.3 0.640 four KSM No. 6P; 0.2-0.4 4.8 0.590 5 KSM No. 6C; 0.2-0.4 4,5 0.700 is not a hemolytic Note: the best result for AO activity and a decrease in hemolytic properties was shown by option "5".
*** A prerequisite for the choice of adsorption purification is the preliminary experience of double thin layer chromatography, first in the chloroform – methanol system, then in the n. butanol - acetic acid - water in a ratio of 40:10:80. He allowed to divide AO into three components with an activity of 1.3; 15.3 and 6.3. Those. The fundamental possibility of rejecting unwanted ingredients by adsorption purification with a simultaneous sharp increase in useful properties was revealed.

The proposed method allowed to increase the radioprotective properties of AO from 1.9 (table 2) to 4.5 (table 3), i.e. 2.4 times and completely eliminate hemolytic properties. If in the sample prepared by the prototype method under the influence of AO, the complete hemolysis of erythrocytes proceeded in 40 minutes (Table 2), then in the sample prepared by the proposed method, hemolytic properties were not detected (Table 3).

Thus, having all the advantages of the antioxidant obtained in the prototype method specified in this description, the proposed method allowed them to be enhanced by eliminating hemolytic properties and increasing antioxidant properties.

Example.

10.0 kg of unrefined sunflower husk was washed with water, the water was drained; the husk was dried at a temperature of 90-110 ° C to a free-flowing state, 100 kg of water was added to the husk and boiled for 30 minutes (from the moment of boiling), the mixture was filtered and the filtrate was obtained - liquid AO; KSM silica gel adsorbent No. 6C was added to the AO solution with stirring, which was separated after interaction with the substance. Got 55 kg of liquid AO concentration of 0.67%; liquid AO was evaporated by boiling to a gel-like AO.

The output of AO was 3.7% by weight of husk.

The obtained trial batch of AO is tested on a number of patients for health reasons.

The low level of production of a similar synthetic drug in the world and its high cost [1] indicate the feasibility of organizing the production of natural AO in Russia on an industrial scale.

Due to the fact that AO has a positive effect on the bactericidal and energy systems of neutrophilic blood lymphocytes, it can be used to correct the immune status of biological objects, which is impaired in a wide range of diseases - radiation damage, AIDS, tuberculosis, cardiovascular, gastrointestinal metabolic diseases of the respiratory organs, etc.

The resulting liquid AO can be used as a remedy, because, according to the provisions of the World Health Organization, a substance obtained from the raw materials traditionally used for food production can be used as food [9].

At the same time, the issue of waste disposal of the food (oil and fat) industry is being resolved.

Literature

1. L. Cancer. “Bulletproof vest from radiation”, the newspaper Trud No. 119 for 04/30/2000

2. S.P. Lyakh, E.L. Ruban. Microbial melanins. M .: Nauka, 1972.

3. Copyright certificate No. 502012, published on 02/05/1976, BI No. 5.

4. O.V. Khakhova, R. N. Kashevatskaya, L. A. Zhorina, V. Ya. Menshinin, L. A. Kizilova. Physico-chemical and biological properties of the melanoid pigment and melanin from the pericarp of sunflower. Scientific and Technical Bulletin of the All-Union Scientific Research Institute of Oilseeds, 1987, issue II (97), Krasnodar, 1987, p.15.

5. Filenko O.F. The use of some model systems for the primary selection of radioprotective substances. Abstract. Cand. dis. M., 1969.

6. L.A. Zhorina, L.M. Matygina, G.E. Mihio, R.N.Kashevatskaya. Properties and possibilities of using water-soluble fractions of sunflower husk. Scientific and Technical Bulletin of the All-Union Scientific Research Institute of Oilseeds, 1987 issue II (97), Krasnodar, 1987, p.20.

7. E.A. Venglinskaya, R.N.Kashevatskaya, L.A. Zhorina. The effect of melanoid antioxidant on the interleukocyte bactericidal system. Scientific and Technical Bulletin of the All-Union Scientific Research Institute of Oilseeds, 1988, issue 4 (103), Krasnodar, 1988, p. 61.

8. L.A. Zhorina, L.M. Matygina, G.E. Mihio, S.S. Malkhasyan. Enzymatic activity of sunflower husks. Oil and fat industry, No. 8, 1985, p. 11.

9. Evaluation of nutritional supplements. Fifteenth report of the Joint FAS WHO Expert Committee on Food Additives. World Health Organization. Series of technical reports No. 488, Geneva, 1974, p.13.

Claims (1)

A method of obtaining a natural melanoid antioxidant, including extraction of unmilled sunflower husk with water during boiling for 30-55 min, with a husk to water ratio of 1:10, filtering and evaporating to a gel-like state, characterized in that before extraction the husk is washed with water and dried at a temperature 90-110 ° C, and before evaporation is treated with food adsorbent KSM No. 6C.