RU2767908C1 - Lipid-correcting agent of seaweed - Google Patents

Abstract

FIELD: medicine; pharmaceutics.SUBSTANCE: invention relates to the field of medicine and pharmaceutics, namely to the use of the lipid fraction of Anfeltia Tobuchinsky thallus extract - Ahnfeltia tobuchiensis (Kanno et Matsubara) Makijenko, department Rodophyta, order Ahnfeltiales, family Ahnfeltiaceae, as a lipid-correcting agent in alimentary dyslipidemia.EFFECT: reduced content of triacylglycerols, total cholesterol in blood, low-density lipoprotein cholesterol and atherogenicity index with increase of content of high-density lipoprotein cholesterol in blood.1 cl, 4 tbl, 1 ex

Description

The invention relates to the use of plant extracts containing a composition comprising a complex of lipids as a lipid-correcting agent for the prevention or treatment of cardiovascular pathologies, and also as an agent that increases the body's resistance to fat and cholesterol loads that occur in the process of eating disorders leading to the development of dyslipidemia (DLP), causing the development of atherosclerosis.

It is known that most cases of cardiovascular diseases, which are one of the main causes of death, are initiated by risk factors, among which dyslipidemias are not the last, which can be controlled and treated. According to the literature, 65% of men and 62% of women suffer from various forms of DLP [Bockeria L.A., Oganov R.G. All about cholesterol: a national report. Moscow, 2010. Izd. A.N. Bakuleva RAMS], [Novgorodtseva T.P., Somova L.M., T.A. Gvozdenko, Yu.K. Karaman, N.V. Bivalkevich. Alimentary dyslipidemia: experimental and morphological aspects. - Vladivostok, 2011. Dalnevost Publishing House. Feder. Unta], that is, the correction of DLP remains an urgent problem of modern medicine.

There are various medications for DLP based on synthetic lipid-lowering drugs. One of these basic means for correcting lipid metabolism disorders are statins, which are synthetic inhibitors of the key enzyme of cholesterol biosynthesis, HMG-CoA reductase [Oganov R.G. Statins are first-line drugs in the prevention and treatment of atherosclerosis // Medical Alphabet. 2013. Vol. 1, N 1. R. 12-15]. Also known lipid-lowering drugs belonging to the group of substances that reduce the reabsorption of bile acids, which is one of the most important factors in the regulation of plasma cholesterol homeostasis [Kramer W., Glombik H. Bile acid reabsorption inhibitors (BARI): Novel hypolipidemic drugs // Curr. Med. Chem. 2006. V. 13. No. 9. P. 997-1016]. Fibrates are also widely used to correct lipid metabolism, and in particular to reduce the level of triglycerols and increase the concentration of HDL (high density lipoproteins) [Gomaraschi M., Adorni M.R., Banach M., Bernini F., Franceschini G., Calabresi L. Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass Distribution, and Function // High Density Lipoproteins: From Biological Understanding to Clinical Exploitation. - Berlin: Springer - Verlag Berlin, 2015. P. 593-615 (Handbook of Experimental Pharmacology; Vol.224)].

The main disadvantage of effective medicinal lipid-lowering agents is their high cost and the presence of contraindications in some cases. The action of synthetic drugs is narrowly focused and does not affect all parts of the pathogenetic process. In addition, with their long-term use, as well as when used in high doses, they can cause a number of quite serious adverse side effects, so these drugs are contraindicated for certain groups of patients [Dyadyk A.I., Kugler T.E., Suliman Yu.V. ., Zborovsky S.R., Zdikhovskaya I.I. Side effects of statins: mechanisms of development, diagnosis, prevention and treatment // Archives of Internal Medicine. 2018 Vol. 42, No. 4. R. 266-276].

The search for alternative, safer non-drug lipid-lowering agents based on natural raw materials is topical. Unlike synthetic drugs, natural drugs can combine the total biological effects mediated by the diverse composition of their components.

Promising in this regard are biologically active complexes from raw materials of marine origin. These include lipid complexes from marine hydrobionts containing omega-3 polyunsaturated fatty acids (PUFAs). It is known that omega-3 PUFAs are considered the fundamental building material for the structural components of cells, tissues and organs, as well as a precursor for the synthesis of a number of biologically active substances.

Experimental studies have demonstrated that a high omega-3 diet plays a central role in the prevention of atherosclerosis [Torres N.M. Guevara-Cruz L.A., Velazquez-Villegas, Tovar A.R. // Nutrition and Atherosclerosis. Archives of Medical Research. 2015. Vol. 46, No. 5. P. 408-426]. At the same time, unlike statins, omega-3 PUFAs can affect several key cellular processes associated with the development of atherosclerosis, including a decrease in the intensity of monocyte migration and their adhesion to endothelial cells, inhibition of the uptake of modified LDL (low density lipoprotein) by macrophages, and suppression of migration smooth muscle cells, changing the ratio of omega-6 / omega 3 fatty acids in membrane phospholipids in the direction of its reduction [Ramji DP Polyunsaturated Fatty Acids and Atherosclerosis: Insights from Pre-Clinical Studies // European Journal of Lipid Science and Technology. 2019 Vol. 121, No. 1].

These properties will allow the use of complexes of marine hydrobionts rich in omega-3 PUFAs for the prevention and treatment of various dyslipidemias.

However, the main part of the well-known dietary supplements (BAA) uses fat of deep-sea fish and crustaceans of cold-water seas and its concentrates as a basis (Wild Norwegian Cod Liver Oil, Source Naturals, Arctic Pure, Wild Alskian Salmon Oil, Rimfrost Antarctic krill, etc.). as well as ethyl ester concentrate of omega 3 PUFAs (Ultra Omega, DHA-500, Omega-3, Triple Omega-3, etc.) and omega 3 PUFAs in the form of triglycerides (Omega-3 Triglyceride Fish Oil, CGN Omega-3 Premium Fish Oil, etc.).

There is also known an agent isolated from the liver of the Commander squid, which has a lipid-correcting and immunological effect (RF patent No. 2259824 C2, publ. 09/10/2005), containing 10% PUFA, as well as an agent isolated from the liver of the king crab, which has lipid-corrective, hypocoagulant and antioxidant properties (RF patent No. 2302248 C2, publ. 10.07.2007).

It should be noted that the main source of raw materials for obtaining dietary supplements containing omega-3 PUFAs and their complexes are nektonic and periphytonic forms of hydrobionts, which are actively exploited by humans and their reserves tend to be depleted, which stimulates research aimed at finding and studying new sources of biologically active substances. . In this regard, benthic forms of hydrobionts are of great interest, namely, various types of seaweeds, which are the primary producers of omega-3 PUFAs in the food chain of marine organisms. Algae serve as a food source for periphyton forms of hydrobionts, which, in turn, form the food base for nekton forms of hydrobionts.

At the same time, insufficient attention is currently paid to the study of their biological properties and therapeutic use in conditions of a hypercholesterol diet. Experimental studies are known that studied the lipid-lowering effect of lipid extracts from brown algae Sargassum pallidum, green Ulva fenestrata and sea grass Zostera marina under conditions of experimental hyperlipidemia caused by a single administration of Triton-1339 (Tyloxapol) [Krivoshapko O.N., Popov AM, Artyukov A.A., Kostetsky E.Ya. Features of the corrective action of polar lipids from marine hydrobionts in violations of lipid and carbohydrate metabolism // Biomedical Chemistry. 2012. V. 58. Issue. 2. S. 189-198]. This model is characterized by a sharp increase in the content of triglycerols and total cholesterol in the blood serum, which corresponds to a greater extent to toxic hyperlipidemia and, with large assumptions, can be extrapolated to hyperlipidemia that develops under real conditions of a hypercholesterol diet.

It is known that an extract obtained by alcoholic extraction of crushed dry brown algae of Japanese kelp (Laminaria japonica) has a lipid-correcting property, containing 80-90% lipids, including 26.7-38.7 polyunsaturated fatty acids from the total fatty acids (US Pat. RF No. 2309763, published 11/10/2007).

Thus, there is a problem of expanding the arsenal of biological lipid-correcting agents from seaweed.

The problem is solved by using the lipid fraction of the extract of anfeltia tobuchiensis thallus - Ahnfeltia tobuchiensis (Kanno et Matsubara) Makijenko, Rhodophyta department, Ahnfeltiales order, Ahnfeltiaceae family, in alimentary dyslipidemia as a lipid-correcting agent.

EFFECT: reduced content of triacylglycerols, total blood cholesterol, low-density lipoprotein cholesterol and atherogenic index while increasing high-density lipoprotein cholesterol content in blood.

The hepatoprotective property of this lipid fraction of the extract of anfeltia tobuchensis thallus - Ahnfeltia tobuchensis, is known (p. RF No. 2667472 C1). It is shown that the effect of this fraction of the extract as a hepatoprotective agent, like all similar hepatoprotectors, is based on its ability to restore the lipid composition and the spatial matrix of hepatocyte membranes, which is disturbed when exposed to toxins on the liver, like all similar hepatoprotectors.

However, experimental studies of the effect of this fraction on blood parameters in alimentary dyslipidemia conducted by the authors showed that this fraction has the ability to restore the disturbed ratio of low and high density lipoprotein cholesterol, as well as triacylglycerols and total cholesterol in blood plasma. This property of this extract was not previously known, which allows us to speak about its use as a lipid-correcting agent for lipid metabolism disorders in conditions of alimentary dyslipidemia (metabolic syndrome, obesity).

The thallus of anfeltia tobuchiensis (Kanno et Matsubara) Makijenko is used as a raw material for obtaining the lipid complex. It is distributed in Russia in Peter the Great Bay (Sea of Japan), Sakhalin, Kunashir, Shikotan islands. It is an important commercial object and is used as food It should also be noted that in Ahnfeltia tobuchiensis, the content of the most important eicosapentaenoic acid for the manifestation of the pharmacological effect is more than 3 times higher than that in Japanese kelp (Titlyanov E.A., Cherbadgy LL, Chapman DJ A review of the biology, productivity and economic potential of the agar-containing red alga, Ahnfeltia tobuchiensis (Kanno et Matsubara) Makijenko, in the seas of the Far East of Russia // Int J Algae 1999 Vol 1 N 4 pp. 28-67).

The thallus is the part of the plant that reaches the largest size and is characterized by the maximum growth rate, which requires constant biosynthesis of secondary metabolites necessary for the construction of newly formed cells. This determines the highest lipid content in the thallus during the growing season.

It is known that the composition of the complex of polyunsaturated fatty acids obtained from algae depends on the type of raw material used for extraction, so it was impossible to assume that the lipid fraction from Ahnfeltia tobuchiensis had a lipid-correcting property.

Isolation of the lipid complex can be carried out by conventional methods for the isolation of lipids from plant materials, for example, as described in paragraph RF No. 2667472 C1 or the article (Bligh EG, Dyer WJ A rapid method of total lipid extraction and purification // Canadian Journal of Biochemistry and Physiology 1959 Vol 37 No 8 P 911-917) adapted to our conditions.

Below is a method for isolating the lipid complex used further for the study.

Samples of the algae Ahnfeltia tobuchiensis were collected in August - September in the Peter the Great Bay of the Sea of Japan (Stark Strait, Popov Island). Harvested algae were thoroughly cleaned of contaminants from other algae, small invertebrates and particulate matter. The algae were washed first in sea water, then in fresh water, then squeezed out and immersed in boiling water for 2 min to inactivate the enzymes. The algae were dried to a dry state, which leads to destructive processes, especially of the PUFA fraction and a change in its native composition, as well as a decrease in the total yield of the lipid fraction. It was experimentally found that when drying algae to a residual moisture content of 40%, the yield of the lipid fraction obtained during extraction increases by 25% compared to the extraction of dry-air raw materials. Therefore, after enzyme inactivation, the algae were dried to a residual moisture content of ~40% and stored until the extraction stage at a temperature of -18°C. Next, thallus was placed in a freezer at -80°C for 2 hours to increase brittleness, then crushed with a laboratory mill to a particle size of 0.5-1 mm and extracted with a mixture of chloroform:ethanol (1:2). One kilogram of raw material was poured into 1.5 liters of the extraction mixture, thoroughly mixed and left for 2 hours, then 0.5 liters of chloroform was added, and after thorough mixing, 0.5 liters of water was added. The resulting extract was separated on a Buchner funnel under vacuum and left overnight in a refrigerator for phase separation. After settling, the upper phase containing water and ethanol was removed using a water jet pump. The lower phase containing lipids was transferred to a separating funnel and washed with 0.2 volumes of 0.73% sodium chloride solution. After phase separation, the lower chloroform layer containing lipids was transferred into a round-bottomed flask and evaporated on a rotary evaporator (T<37°C) until the odor of chloroform was absent. The resulting lipid extract is a green-brown oily mass. Re-extraction of a portion of raw materials showed that more than 90% of lipids were extracted with a single process, which made it possible to make a decision to use a single extraction procedure. The output of the lipid complex was 2.2% by weight of the algae in terms of dry-air raw materials. Table 1 shows the chemical composition of the obtained lipid complex of anfeltia, corresponding to the known composition according to paragraph RF No. 2667472 C1.

The lipid complex for pharmacological testing was previously dissolved in a ratio of 1:2 in vaseline oil of the “medical” category. Vaseline oil was chosen as a diluent because it is not absorbed during digestion in the gastrointestinal tract, has an unchanged chemical composition, does not contain substances toxic to the body, does not accumulate in the body itself, and, therefore, cannot affect the received in experiment results.

When studying the composition of the resulting lipid complex (table 1), it was determined that 90% are total lipids, of which 80% are metabolically active components important for the regulation of lipid metabolism. As a metabolically active fraction, we took the sum of essential phospholipids (26%), glycolipids (30%) and the total fraction of free fatty acids and triacylglycerols (25%), which are the main source of polyunsaturated fatty acids, which are an important component of the regulation of lipid metabolism in conditions of hyperlipidemia and hypercholesterolemia (alimentary dyslipidemia) [Taratukhin E.O. Atherosclerosis and fatty acids: an important relationship and a new direction in therapy // Russian Journal of Cardiology. 2011. No. 5. S. 77-80]. The total content of SFA from the total amount of fatty acids in the lipid complex is not less than 66%, and the amount of PUFA of the n-3 family, mainly eicosapentaenoic acid, represents the "active principle" and exceeds 21%, which is two times higher than that of the prototype and corresponds to the composition of the lipid complex according to clause RF No. 2667472 C1.

Low toxicity (LD50 is more than 5000 mg/kg) and safety with long-term administration in the stomach and parenterally allowed for experimental studies.

The use of the obtained agent in an experiment on rats under conditions of a hypercholesterol diet with a high-fat load (hyper-fat diet) gave a result that exceeded the expectations expected from its use and consisted in a lipid-correcting effect, namely, restoring the disturbed ratio of low-density and high-density lipoprotein cholesterol, as well as triacylglycerols and total plasma cholesterol.

The experiment was carried out on outbred white male rats weighing 146 ± 3 g, obtained from the nursery of the Stolbovaya branch of the Federal State Budgetary Institution Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency of Russia. Animal studies were performed in accordance with the order of the Ministry of Health and Social Development of Russia dated 01.04.2016 No. 199n "On Approval of the Rules of Laboratory Practice" and the requirements of GOST R 53434-2009 "Principles of Good Laboratory Practice". The animals were adapted to the vivarium for 7 days before the start of the experiment. During this period, a daily examination of the external condition of the rats was carried out, and animals without signs of abnormal health were taken into the experiment. Animals were kept 1 in individual plastic cages on sawdust bedding at 20-22°C and 12/12 hour lighting. Animals received drinking water without restrictions and food daily at the same time in free access mode. After passing the quarantine, the rats were randomly divided into intact (control), who consumed the standard general diet throughout the experiment, and rats, subjected to simulation of alimentary dyslipidemia. The most popular is the high fat diet model, which is a hypercholesterol diet with a high fat load induced by feeding animals a diet with excess cholesterol and saturated fatty acids. According to the studies of Lutsenko M.T., Ryzhavsky B.Ya., Chertov A.D., Lutsenko N.V. Adaptation of the body to high cholesterol / ed. M.T. Lutsenko. Blagoveshchensk, 1973. 143 pp., when exposed to hypercholesterol load for 30 days in experimental animals, an organism reaction is formed, characterized by alimentary dyslipidemia. In this regard, the development of alimentary dyslipidemia in our experiment was carried out by feeding animals with a high-fat diet for 30 days, consisting of a basic standard diet with the addition of 2% cholesterol and 20% beef lard of the total diet [Guidelines for conducting preclinical studies of drugs. Part one / ed. A.N. Mironova M.: Grif and K, 2012]. Another group of rats in a high-fat diet was added to the lipid complex of anfeltia at a dose of 1 g/kg of animal weight [Novgorodtseva T.P., Gvozdenko T.A., Kasyanov S.P., Knyshova V.V., Karaman Yu.K. The use of biologically active food supplements based on lipids of marine aquatic organisms in an experiment on rats // Problems of nutrition. 2010. V. 79, No. 2. S. 24-27]. In the group with the reference drug, which is the lipid preparation Omega-3 (Now Foods, USA), this lipid complex was introduced into the diet at the same dose. The Omega-3 preparation included a natural complex isolated from fish oil, which included 18% eicosapentaenoic (EPA) and 12% docosahexaenoic (DHA) acids. The dose of the lipid complex introduced into the diet was equivalent to the daily norm of polyunsaturated fatty acids (PUFAs) developed for humans. The choice of the lipid complex Omega-3 as a reference drug is associated with a similar chemical composition of the PUFAs of the n-3 family in the studied lipid complex of anfeltia and, accordingly, the proposed mechanism of action. It is known that fatty acids belonging to the n-3 family have great potential for maintaining human health due to their high efficiency in the treatment of various pathologies [Jump D.B., Depner C.M., Tripathy S., Lytle K.A. Potential for Dietary omega-3 Fatty Acids to Prevent Nonalcoholic Fatty Liver Disease and Reduce the Risk of Primary Liver Cancer // Adv. Nutr. 2015. Vol. 6, No. 6. P. 694-702].

The animals were divided into the following groups of 10 rats each: group 1 - control (intact, standard basic diet), group 2 - high-fat diet, group 3 - high-fat diet + ahnfeltia lipid complex, group 4 - high-fat diet + Omega 3 lipid complex Animals were taken out of the experiment by decapitation under light ether anesthesia in compliance with the Rules and international recommendations of the European Convention for the Protection of Vertebrate Animals used for experiments or for other scientific purposes (Strasbourg, 1986). The design of the experiment was approved by the Ethics Committee of the Pacific Ocean Institute. IN AND. Ilyichev FEB RAS.

The therapeutic efficacy of the studied lipid fractions was evaluated by their effect on weight characteristics (body weight, liver weight) and biochemical parameters of the liver and blood plasma. The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triacylglycerols (TAG) were determined in blood plasma using biochemical reagent kits from Olvex Diagnosticum (Russia). The atherogenic index was also calculated using the formula TC - HDL-C/HDL-C, where TC is total cholesterol, HDL-C is high-density lipoprotein cholesterol, etc. [Klimov A.N., Nikulcheva N.G. Metabolism of lipids and lipoproteins and its disorders. SPb: Peter Kom, 1999. 512 pp.], as the main criterion for analyzing the effectiveness of the studied lipid-correcting drugs.

The state of the antioxidant system was assessed by the level of antiradical activity (ARA) against alkyl-peroxyl radicals [Bartosz G., Janaszewska A., Ertel D., Bartosz M. Simple determination of peroxyl radical-trapping capacity // Biochemistry and molecular biology international. 1998 Vol. 46, no. 3. P. 519-528], the level of malondialdehyde (MDA) [Buege JA, Aust SD. Microsomal lipid peroxidation // Methods Enzymol. 1978;52:302-310], superoxide dismutase (SOD) activity [Paoletti F., Aldinucci D., Mocali A. et al. A sensitive spectrophotometric method for the determination of superoxide-dismutase activity in tissue-extracts // Anal, biochem. - 1986. - vol. 154.-n 2.-p. 536-541] and enzymes of the glutathione link - glutathione reductase (GR) [D.M. Goldberg, R.J. Spoone., Methods of enzymatic analysis, H.U. Bergmeyer (ed. in-chive), Weinheim; Deerfield Beach, Florida; Basel, 3, (1983), 258-265] and glutathione peroxidase (GP) [R.F. Burk, R.A. Lawrence, J.M. lane. Liver necrosis and lipid peroxidation in the rat as the result of paraquat and diquat administration. Effect of selenium deficiency // J. Clin. Invest., 65(5), 1024-1031. 1980] in blood plasma, as well as by the level of reduced glutathione (G-SH) in the liver tissue [Novgorodtseva T.P., Endakova E.A., Yankova V.I. Guidance on methods for studying the parameters of the system "Lipid peroxidation - antioxidant protection" in biological fluids. Vladivostok, 2003, 80 p.]

The content of total lipids in anfeltia was determined by the gravimetric method. A suspension of silica gel brand "KSK" and plates 6x6 cm in size for two-dimensional microthin-layer chromatography of phospholipids and neutral lipids were prepared according to the method [Svetashev V.I., Vaskovsky V.E. A simplified technique for thinlayer chromatography of lipids // J. Chromatogr. 1972 Vol. 67. P. 376-378]. For fractional separation of phospholipids, the solvent systems described by Roser were used [Rouser G., Kritchevsky G., Yamamoto A. Column chromatographic and associated procedures for separation and determination of phosphatides and glicolipids // Lipid chromatogr. Anal. N.Y.: Dekker. 1967 Vol. 1. P. 99-162]. To detect choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine, sphingomyelin), Dragendorf's reagent was used [Wagner H., Horhammer L., Wolff F. Thin-layer chromatography of phosphatides and glycolipides // Biochem. Z. 1961. Bd. 334. S. 175-184] - lipids appeared as orange spots on a yellow background. To detect phospholipids containing an amino group (phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine), the plates were sprayed with a 5% solution of ninhydrin in acetone [Rouser G., Kritchevsky G., Yamamoto A. Column chromatographic and associated procedures for separation and determination of phosphatides and glicolipids / / Lipid chromatogr. Anal. - N.Y.: Dekker, 1967. Vol. 1. P. 99-162] followed by heating for 2-3 minutes over water vapor until pink spots appear on a white background. Phospholipids containing hydroxyl groups (phosphatidylinositol) were detected using the periodate Schiff reagent [Kates M. Lipidology Technique. M.: Mir. 1975. 221 pp.], lipid spots had a pink-lilac color. For the manifestation of all phospholipid fractions, a molybdate reagent was used [Vaskovsky V.E., Kostetsky E.Y., Vasendin I.M. Anuniversal reagent for phospholipid analysis // J. Chromatogr. 1975 Vol. 114, N 1. P. 129-141] and a reagent based on malachite green [Vaskovsky V.E., Latyshev N.A. Modified Jungnickel’s reagent for detecting phospholipids and other phosphorus compounds on thin-layer chromatograms // J. Chromatogr. 1975 Vol. 115, No. 1. P. 246-249]. The lipids appeared as blue or green spots on a white background. Fractions were quantified using a 10% solution of sulfuric acid in methanol followed by heating. The number of individual fractions of phospholipids and the values of total phospholipids were calculated according to the method [Vaskovsky V.E., Kostetsky E.Y., Vasenden I.M. A universal reagent for phospholid analysis // J. Chromatography. 1975 Vol. 114, no 1. P. 129-141] with the accepted algorithm for their quantitative determination. Fractional separation of neutral lipids was carried out by one-dimensional microthin-layer chromatography on silica gel [Amenta J.S. A rapid chemical method for quantification of lipids separated by thin-layer chromatography // J. LipidRes. 1964 Vol. 5. P. 270-272]. The content of individual fractions was expressed as a percentage of the total amount of phospholipids and neutral lipids, respectively.

Quantitative data were processed using the statistical package Instat 3.0 (GraphPad. Softwarelnc. USA, 2005) with a built-in procedure for checking the compliance of the sample with the normal distribution law. To determine the statistical significance of differences depending on the distribution parameters, the parametric Student's t-test or the non-parametric Mann-Whitney U-test were used.

A high-fat diet for 30 days was accompanied by an increase in animal weight by 29% (p<0.001), and the specific mass of the liver (g/100 g of body weight) increased by 72% (p<0.001) in relation to control indicators (Table 2) characteristics of rats under the influence of a high-fat diet and the introduction of the lipid complex of anfeltia and Omega3 (M ± m)).

Table 3 shows the biochemical parameters of the blood plasma of rats under the action of a hyperfat diet and the introduction of the lipid complex of anfeltia and Omega 3 (M±m, mmol/l). It can be seen that the level of triacylglycerols in the blood plasma increased by almost 2.5 times (p<0.001) and total cholesterol by 55% (p<0.001) in relation to the control indicators. At the same time, the level of atherogenic cholesterol (LDL-C) was increased by 13% while reducing HDL-C by 14% (p<0.05). That is, under the influence of a high-fat diet for 30 days, a pronounced picture of dyslipidemia was formed in rats. It is known that LDL deliver lipids (mainly cholesterol) from the liver, where they are formed, to peripheral tissues. At the same time, the amount of HDL, which remove cholesterol from cell membranes to the liver, decreases. In this regard, the calculated atherogenic index in these animals was significantly higher than the control values by 2.5 times. This indicates a violation of lipid metabolism in blood plasma under the influence of a hyperfat diet and is an unfavorable risk factor for metabolic syndrome and cardiovascular diseases.

It is known from the literature that lipid metabolism disorders due to a high-fat diet (alimentary dyslipidemia, metabolic syndrome) are accompanied by the development of oxidative stress [Avelar T.M.T., Storch A.S., Castro L.A., Azevedo G.V.M.M., Ferraz L., Lopes P.F. Oxidative stress in the pathophysiology of metabolic syndrome: which mechanisms are involved? // Jornal Brasileiro de Patologia e Medicina Laboratorial. 2015. V. 51. No. 4. P. 231-239], which is expressed in the imbalance of the system lipid peroxidation - antioxidant defense of the body (LPO-AOD). In this regard, we studied the effect of the lipid complex of anfeltia tobuchinskaya on the main indicators of this biochemical system.

An analysis of the state of AOP under the action of a high-fat load revealed the accumulation of secondary products of lipid peroxidation in the blood. The level of MDA increased by 2 times (p<0.001) with a simultaneous decrease in ARA by 16% (p<0.05) and SOD (superoxide dismutase) activity by 55% (p<0.001) relative to the control group (Table 4). SOD is a key enzyme that acts as the first line of defense against free radicals and, as a result, this leads to their rapid consumption and depletion. At the same time, the activity of glutathione peroxidase (GP), which catalyzes the reduction of hydrogen peroxide (H2O2) and organic peroxides in the presence of reduced glutathione (G-SH), increased by 17% (p<0.001). The high activity of glutathione peroxidase at a low content of reduced glutathione indicates the presence of an excess amount of LPO products (lipid peroxidation), (Kulinsky V.I., Kolisnichenko V.S. Glutathione system. I. Synthesis, transport of glutathione transferase, glutathione peroxidase // Biomed. chemistry. 2009. V. 53, No. 3. S. 255-277), which is confirmed by a significant increase in the level of MDA and a decrease in the content of reduced glutathione in the blood plasma by 41% (p<0.001) on the 30th day of the experiment. All this points to the exhaustion of the reserve of adaptive capabilities of the body. The activity of another enzyme of the glutathione link, glutathione reductase (GR), decreased by 13% (p<0.05), which is quite natural, since this enzyme is actively consumed for the synthesis of reduced glutathione in the presence of NADPH.

Based on the above, it follows that the experimental model of a hypercholesterol diet with a high-fat diet for a period of 30 days was accompanied by the development of alimentary dyslipidemia with impaired lipid metabolism. It showed up:

• an increase in the mass of the liver due to fatty infiltration of hepatocytes;

• in an increase in the level of triacylglycerols, total cholesterol, LDL cholesterol and a decrease in HDL cholesterol in blood plasma;

• Mismatch of the main parameters of the endogenous system of antioxidant protection, leading to a decrease in the overall antiradical activity of blood plasma.

Studies on the use of the lipid complex of anfeltia and the reference drug Omega-3 when introduced into a high-fat diet showed a clear direction towards the normalization of the studied parameters compared to those in group 2 (high-fat diet). It should be noted a pronounced tendency to restore the weight characteristics of animals (Table 2): a decrease in body weight relative to group 2 by 16% (p<0.001) with the introduction of anfeltia extract and by 9% (p<0.01) with the introduction of Omega-3. However, in relation to the control parameters, the specific mass of the liver in animals of the 3rd group (anfeltia) remained slightly increased (by 11%; p<0.05), while in animals of the 4th group (Omega-3) it was 1.5 higher than the control times (p<0.01).

It is important to note that in the blood plasma of rats treated with anfeltia lipid complex, there were no significant differences in the content of total cholesterol, HDL cholesterol and LDL cholesterol from the control. Accordingly, the atherogenic index in these animals remained within the normal range. At the same time, in animals treated with Omega 3, the content of total cholesterol was 18% higher than the control (p<0.001), and HDL cholesterol was lower by 10% (p<0.001). At the same time, the atherogenic index was increased by 55% (p<0.001) relative to the control.

The state of the antioxidant system in animals of the 3rd and 4th groups treated with lipid complexes was characterized by positive dynamics and a tendency to restore the studied parameters against the background of a high-fat load (Table 4), which indicates the prevention of the development of lipid peroxidation processes. Confirmation of the inhibition of lipid peroxidation processes is a decrease in the content of MDA with a simultaneous increase in the level of ARA in the blood plasma compared with the 2nd group. In animals treated with the lipid complex from ahnfeltia (group 3), there were no significant differences in the activity of antioxidant enzymes (SOD, GR and GP) from the control, in contrast to animals of the 4th group (omega), in which SOD activity was significantly below.

A significant increase in the level of G-SH in the liver of animals of the 3rd group (by 40%; P<0.001), which received the lipid complex from anfeltia, was noted in comparison with the 2nd group. It should also be noted that the indicators of the antioxidant system in animals with the introduction of the Omega complex into the diet still significantly differed from the control values (MDA, SOD, G-SH), in contrast to the corresponding indicators in the anfeltia group, which may indicate a more pronounced effect of PUFAs. n-3, which are in the native form of the natural lipid complex, compared with the purified Omega-3 preparation.

Thus, the introduction of the lipid complex of both anfeltia and the Omega-3 preparation contributed to the correction of disorders caused by a high-fat diet for 30 days, however, the restoration of the studied parameters under the action of the lipid complex of anfeltia turned out to be more effective. Apparently, the main reason for the observed difference is that the biological activity of PUFAs of the n-3 family (eicosapentaenoic and docosahexaenoic acids), which are part of the Omega-3 preparation, is opposed by the multicomponent native lipid complex of anfeltia. Greater efficiency, according to the applicant, is due to the presence in its composition of all known classes of phospholipids of marine origin, as well as a complex of glycolipids, which are an important source of PUFAs. The fatty acid composition of the lipid complex of anfeltia is characterized by a wider range of fatty acids, which, in addition to PUFA n-3, includes monoenoic fatty acids and PUFA n-6.

Thus, the lipid fraction of the extract of anfeltia tobuchiensis Ahnfeltia tobuchiensis has a pronounced lipid-correcting effect in alimentary dyslipidemia. The mechanism of the lipid-correcting action of the lipid complex of anfeltia is due to the restoration of the metabolic reactions of the liver disturbed by the alimentary load:

• reduces the level of triacylglycerols, total cholesterol, LDL cholesterol and increases the level of HDL cholesterol in blood plasma;

• activates the body's endogenous antioxidant defense system and reduces the accumulation of toxic lipid peroxidation products.

According to the studied indicators, the ahnfeltia lipid complex is more effective in restoring lipid metabolism.

The use of the lipid complex of Ahnfeltia tobuchiensis (Kanno et Matsubara) Makijenko under the conditions of a hypercholesterol and high-fat diet surpasses the omega-3 reference drug in terms of its therapeutic efficacy in terms of its ability to restore body weight and liver of animals to control values and in terms of the effectiveness of reducing triglycerols in the blood, total cholesterol and atherogenicity index against the background of an increase in the content of HDL cholesterol.

Thus, the previously unknown property of the lipid fraction of the extract of the red seaweed Ahnfeltia tobuchiensis (Kanno et Matsub.) Makijenko to restore the disturbed ratio of low and high density lipoprotein cholesterol, as well as triacylglycerols and total cholesterol in blood plasma, allows expanding the range of lipid correcting agents with using new natural sources - seaweed Ahnfeltia tobuchiensis.

Claims (1)

The use of the lipid fraction of the extract of anfeltia tobuchinskaya thallus - Ahnfeltia tobuchiensis (Kanno et Matsubara) Makijenko, department of Rodophyta, order Ahnfeltiales, family Ahnfeltiaceae, as a lipid-correcting agent for alimentary dyslipidemia.