RU2038086C1 - Method for extracting biologically active product from wax moth larvae - Google Patents

Abstract

FIELD: medicine; biology; pharmacology. SUBSTANCE: method involves treating grown up moth larvae having 150 g in weight with 40% ethanol solution during 20 days to achieve complete extraction of the end product. Moth larvae are cultured in darkness on dark wax soil at 20-25 C. EFFECT: improved biostimulating activity, enhanced effectiveness in promoting adaptation and preventing heart diseases. 3 dwg, 6 tbl

Description

 The invention relates to biology and pharmacology and can be used to obtain a biologically active product from the larvae of the large wax moth.

 The large wax moth of Galleria mellonella L. (Lepidoptera, Pyralidae, hereafter wax moth) is a ubiquitous parasite of the bee family. Wax moth is bred and used for various purposes: as a laboratory host for the cultivation of entomophage insects used for biological plant protection in agriculture; as a test object for assessing the activity and quality of bacterial preparations; as well as an object of biochemical and physiological research in entomology.

 For growing wax moth in laboratory conditions, bee honeycombs, flower pollen, pollen with the addition of beeswax, and artificial nutrient media of various compositions are used as feed. The most convenient and widely used for mass insect cultivation is an artificial environment with the addition of beeswax and honey, developed by Marston et al and modified by Yu.I. Kuznetsova. In laboratory conditions, wax moth is grown in Petri dishes, glass jars equipped with lids with a fine meshed soldered mesh. For mass breeding of insects use special containers.

 There is a method of isolating a preparation of lipases from wax moths, which are insect digestive enzymes that have an antimicrobial effect against Mycobacterium fuberculosis. To do this, wax moth larvae collected in a bee hive are ground and dried by the Willstatter method, then lipases are extracted from the obtained powder with an emulsifying mixture containing 50% glycerol and 1.5% carboxel, then the lipases are precipitated with acetone and lyophilized.

 The disadvantage of this method is the limited spectrum of biological activity of the resulting drug.

A known method of obtaining an extract from a wax moth that has a growth-promoting effect on lymphoblastoid cells of the Raj ⁱ line in culture. According to the method, wax moth larvae collected in a beehive, subjected to extraction with 40% ethanol at 20-25 ^{° C} in the dark for 20 days.

 The disadvantage of this method is the impossibility of obtaining significant quantities of the drug due to the economic inexpediency of breeding wax moth larvae in a bee hive, which leads to a weakening and death of the bee family. In addition, the method does not provide a reproducible biological activity of the product due to the uncontrolled growing conditions of the larvae and the food absorbed by them.

 Other methods for producing biologically active preparations from wax moth larvae are not known from the literature.

 The purpose of the present invention is to increase the yield and stabilize the biological activity of the product, as well as having adaptogenic, cardioprotective effect.

 For this, the method, including the extraction of wax moth larvae, involves growing larvae outside the hive, and using dark wax sushi containing the products of many years of activity of the bee family as feed.

 The invention is based on the first discovered and studied biological properties of dark wax land, which is the product of many years of activity of the bee family. Dark wax land is formed from bee honeycombs after growing in the last several generations of bee brood for 3-4 years. In chemical composition, it differs significantly from bee honeycombs, light wax drying and refined wax, traditionally used to grow wax moths. Dark wax sushi contains a smaller amount of wax compared to light sushi (40-55% instead of 70% with a moisture content of 2.2-3.8% instead of 01, -0.5%). It has been established that, in addition to wax, it contains vital products of bees (the remains of royal jelly, larval membranes, bee brood integument, carbohydrates, peptides, nitrogen-containing substances, serotonin-like compounds), as well as flavonoids and aromatic acids of plant origin. The presence of these components makes dark wax dry as a full-fledged nutrient medium for wax moth larvae and allows you to do without the use of pollen, honey and other expensive food additives. It has also been established that dark wax land, which does not in itself have significant biological activity, undergoes metabolic transformation by wax moth larvae and turns into a biologically active product with a wide spectrum of action.

 The product obtained by the proposed method contains several biologically active components. Its chemical composition differs from the composition of the extract obtained in a similar way from wax moth larvae grown on artificial feed, as well as from the composition of the extract of dark wax land.

 Studies conducted on various biological models have shown that the extract of wax moth larvae obtained by the proposed method has a wide spectrum of biological activity, adaptogenic, growth-promoting and cardioprotective effects, and also stimulates the oxidative metabolism of cardiac and vascular tissues.

 At the same time, the extract obtained from wax moth larvae grown on artificial feed, as well as the extract of dark wax land, do not exhibit biological activity.

 In contrast to the known preparation of lipases from wax moth larvae (analogue), the product obtained by the proposed method does not have antimicrobial activity in relation to Mycobacterium tubereulasis and other microorganisms.

 The following is a specific example of the proposed method. Biological material for the cultivation of wax moths (egg laying, larvae, cocoons, butterflies) was collected in apiaries in the summer. The determination of the insect was carried out according to the directory of diseases and pests of bees. Subsequently, continuous cultivation of wax moths was carried out in laboratory production conditions.

To grow wax moth larvae, a container 30x30x40 cm in size was used, equipped with a lid and a ventilation gap tightened by a fine-mesh metal mesh with openings smaller than 1 mm. 500 g of dark wax sushi (dark brown and black, non-translucent in bottoms, containing 40-55% of wax with a moisture content of 2.2-3.8%) and a laying of eggs of a wax moth (about 500 eggs) were placed in a container. Cultivation larvae was carried out at 20-25 ^{° C} and 80% relative humidity for 35 days. For the extraction of biologically active substances, larvae were selected that reached the last stage of development before pupation, weighing 150 mg or more. The selected larvae (100 g) were placed in a wide-necked vessel, the cobweb and the remnants of wax land were removed. Then the larvae were transferred into a 600 ml extraction vessel with a ground glass stopper and 450 ml of 40% rectified ethyl alcohol were added. Extraction was carried out at 20-25 ^{° C} in the dark with occasional stirring for 21 days until complete extraction of the product. Then the extract was filtered through a paper filter. Received 420 ml of extract. The resulting product is a reddish-yellow clear liquid containing about 1% dry matter and exhibiting characteristic fluorescence (440 nm at an excitation light wavelength of 350 nm). The extract was stored in the dark at ⁴ ° C, the biological activity is maintained throughout the year.

 Study of the chemical composition of the resulting product. The chemical composition of the obtained product was studied by various biochemical methods using thin layer chromatography, gas chromatography, disk electrophoresis, amino acid and elemental analysis, NMR, UV and luminescent spectroscopy. Fractionation of the product was carried out on a column with Sephadex LH-20 (figure 1).

The following components were found in the composition of the product:
proteins and peptides (2% dry matter), including acidic peptides containing a linked aromatic component, as well as alkaline serine protease (fraction 1);
free amino acids (50-60% dry matter), (fractions 2-5, table 1);
low molecular weight growth-stimulating factor (fraction 3);
low molecular weight aromatic compounds (fraction 7);
lipids, including fatty acids (table 2);
carbohydrates.

 At the same time, an extract of larvae grown on a medium developed by Marston et al. And modified by Yu.I. Kuznetsova was prepared in the same way. The medium contained the following components (g / kg): bran 100, wheat flour 110, corn flour 140, yeast 130, milk powder 130, glycerin 210, honey 100, wax 80. A product was obtained, which was a clear liquid of light yellow color containing about 1% dry matter and not fluorescence. The chemical composition of the extract was investigated by the same methods. Fractionation of the extract was carried out on a column with Sephadex IH-20 (figure 2).

The following extracts were found in the extract of wax moth larvae grown on artificial feed:
small amounts of protein (fraction 1);
free amino acids glycine, alanine, serine, threonine, glutamate, aspartate, valine, histidine, proline, etc. (fractions 2-5);
lipids;
carbohydrates.

 Dark waxy sushi extract was also prepared. Dark wax dry (100 g) was ground, placed in an extraction vessel and 450 ml of 40% rectified ethyl alcohol was added. The extraction was carried out under the same conditions for 21 days, then the extract was filtered through a paper filter. A product was obtained, which was a dark brown transparent liquid containing 2.5% dry matter and possessing characteristic fluorescence (440-460 nm at an excitation light wavelength of 350 nm). The chemical composition of the extract was investigated by the same methods. Fractionation of the extract was carried out on a column with Sephadex LH-20 (figure 3).

In the extract of dark wax sushi found:
aromatic acids;
flavonoids;
carbohydrates;
lipids.

From FIG. 1-3 shows that the chromatographic elution profiles of extracts of wax moth larvae grown on dark wax land and artificial nutrient medium, as well as extract of dark wax land, were different. Differences were observed both in composition and in the relative content of chromatographic fractions. The results of the analyzes indicate a difference in the chemical composition of the studied samples. A biologically active product from wax moth larvae grown on dark wax land contains active ingredients that are not present in the extract of wax moth larvae grown on artificial feed and dark wax land extract:
acidic peptides containing an associated aromatic component (fraction 1);
low molecular weight growth-stimulating factor (fraction 3);
low molecular weight aromatic compounds (fraction 7).

Determination of biological activity
1. The study of adaptogenic properties. To determine the adaptogenic properties of the extracts, a test for the limiting duration of swimming was used. The experiments were performed on white outbred male rats weighing 180-200 g. Two groups of animals received an extract of wax moth larvae grown on dark wax land and artificial nutrient medium, subcutaneously at a dose of 0.2 ml / 200 g of body weight daily for 30 days . Two other groups of animals received an extract of wax moth larvae grown on dark wax land, orally at a dose of 0.1 ml / 200 g body weight daily for the same period of time. Control animals received an equivalent amount of 40% ethanol. On days 1, 5, 10, 15, 20, 25, and 30, the duration of limiting swimming of rats with a load of 20 g was determined. The results are presented in Tables 3 and 4.

 From these tables it can be seen that the extract of wax moth larvae grown on dark wax land has a pronounced adaptogenic effect, significantly lengthening the swimming time of rats, both subcutaneously and when administered orally. The extract of wax moth larvae grown on an artificial nutrient medium, as well as the extract of dark wax land, did not show pronounced aptogenic properties.

 2. The effect on oxidative tissue metabolism. The study of the biological activity of the extract was carried out on 24 white outbred male rats weighing 180-200 g. Two groups of animals received an extract of wax moth larvae grown on dark wax land, orally for 30 days in doses of 0.01 and 0.1 ml / day / 200 g body weight. The control group of rats received an equivalent amount of 40% ethanol. Two hours after the last administration of the extract, the main indicators of the oxidative metabolism of animal tissues were determined: the content of lactate, pyruvate and the NAD / NAD ˙ N ratio (pyridine nucleotide reduction coefficient).

 The results are presented in table. 5.

 The experimental results indicate that the introduction of the animal extract causes a significant increase in the content of pyruvate in the tissues of the heart, aorta and liver while reducing the level of metabolite in the blood. The lactate content decreased slightly (heart, aorta) or remained close to control. The ratio NAD / NAD ˙ N changed in the direction of increasing oxidation-aerobization.

 The results indicate a marked increase in oxidative metabolism and energy processes in the cardiac and vascular tissue under the action of an extract of wax moth larvae grown on dark wax land.

 3. The study of cardioprotective properties. The study of the biological activity of the extract was carried out on 34 frogs weighing 30 g. Two groups of animals received an extract of wax moth larvae grown on dark wax land, orally at a dose of 0.05 ml / 30 g body weight for 7 and 14 days. Control animals received an equivalent amount of 40% ethanol. Two hours after the last injection, the resistance of frog heart muscle to strophanthin was determined by standard methods. The results obtained are presented in table.6.

 The experimental results indicate that the extract of wax moth larvae grown on dark wax land has a cardioprotective effect, increasing the resistance of the frog’s heart muscle to the toxic effect of strophanthin.

 Thus, the above examples show that the proposed method allows to obtain a biologically active product from wax moth larvae using low activity products of the bee family as the initial substrate. The resulting product has a wide range of biostimulating effects.

 The proposed method in comparison with the known has the following advantages: the simplicity of growing wax moths in laboratory production conditions and its high fecundity make it possible to obtain a biologically active product on an industrial scale; the ability of wax moths to reproduce year-round provides the possibility of obtaining a biologically active product, regardless of the time of year; the use of standard nutritious raw materials for growing larvae (dark brown and black wax drier of the 3rd grade which is not transparent at the bottoms) under controlled laboratory and production conditions allows us to obtain a standardized product.

 Due to the cheapness of the nutrient substrate used, which belongs to the wax raw materials of the lower category, a low cost of production of the product is achieved (about 25 rubles / l).

 The raw material needs of large-scale production of the product can be met by existing production volumes of low-grade wax raw materials. For the production of the product does not require the development of special equipment.

Claims (1)

METHOD FOR PRODUCING A BIOLOGICALLY ACTIVE PRODUCT FROM LARGE Wax MOTH LARVES by extraction of Galleria mellonella L moth larvae with 40% ethyl alcohol at 20 25 ^o С in the dark, characterized in that, in order to increase the yield and stabilize the biological activity of the product, which also has an adapt cardioprotective effect, as a raw material to produce a product using larvae grown at the expense of the hive is feeding wax sushyu dark at 20 25 ^o C, the final product contains free amino acids 50% monosaccharides and 60 disaha 2 4.7% ides the nucleotides and nucleosides of 1.5% high molecular compound 1 2% fatty acid 0.1% minerals, 7.1% 9

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