RU2005485C1 - Method for production of medicine against animal skin diseases - Google Patents

Abstract

FIELD: veterinary medicine. SUBSTANCE: method involves extraction (two times) of air-dry ground algae Laminaria with 96-% ethanol at 65-75 C. The extract is kept at ambient air temperature for at least 16 h, filtered, sediment from ethanol is re-crystallized, the filtrate is boiled down dry. The residue is extracted with 96-% ethanol and dried by boiling down. EFFECT: higher medicine quality.

Description

 The invention relates to veterinary medicine, in particular to methods for producing medicines for animals.

 Diseases of the skin of animals caused by injuries, burns, pathogenic fungi and other factors are very common and lead to the culling of a large part of livestock and sports horses, and also cause the death of domestic dogs, cats and other animals. Injuries to the skin are especially common in sports horses, and injury occurs not only during competitions and training, but also in the process of constant transport of animals. Along with synthetic drugs, drugs obtained from natural sources are widely used to treat such diseases.

Known methods for producing drugs from various algae, including methods for producing drugs of lipid nature. The methods consist in preparing pastes or aqueous extracts from seaweed, or in extracting lipids and other lipophilic substances from cultured unicellular algae. The drugs obtained by these methods can be used in veterinary medicine, however, the cited sources do not contain specific data on their effectiveness in the treatment of skin diseases. In addition, the above methods have disadvantages. When extracted with aqueous solutions, highly active carotenoids and polyunsaturated lipids are not extracted. In the preparations, which are the products of mechanical processing of algae (pastes, creams, etc.), the concentration of active components is very low, and the use of such agents for treating animals is limited, since it is associated with dressings. Moreover, the use of algae in its natural form is uneconomical, since they contain a number of substances that do not have a therapeutic effect (and sometimes have a negative effect), but are widely used in other areas and are classified as scarce. As for lipid preparations from unicellular algae, the process of obtaining them is complicated. It includes a long cultivation operation requiring special equipment, and the desired product extraction is carried out at a reduced temperature (0 to 5 ^{° C)} using a highly toxic and extremely undesirable methanol chloroform (MACs - 5mg / ^m3 of air), which is also extremely difficult to completely remove from lipids.

 Closest to the technical nature of the proposed method of obtaining funds for the treatment of skin diseases in humans and animals from marine brown algae of the genus Laminaria, which consists in drying the algae in air and subsequent grinding. The use of the drug is carried out by treating it with hot water and applying chilled pulp to the affected areas of the skin.

 However, in the resulting preparation, the concentration of substances that have a therapeutic effect is negligible, as a result of which its effectiveness is low. The use of the drug obtained by this method is complicated by the fact that in most cases it requires a bandage, which is not always possible in the treatment of animals. In addition, the method does not provide for the extraction of their algae with high-value D-mannitol, which does not have a therapeutic effect when treating damaged skin, but is contained in a very large amount in kelp.

 The purpose of the invention is to increase the therapeutic effectiveness of an agent for treating skin diseases in animals obtained from algae of the genus Laminaria, and the simultaneous production of D-mannitol.

The object is achieved in that the air-dry milled algae obtained by a known method, was extracted twice with 96% ethanol at a temperature of 65-75 ^{° C,} the extracts are combined and maintained at ambient temperature for at least 16 hours, the precipitate was filtered off D-mannitol, and recrystallized from aqueous ethanol, the filtrate was evaporated to dryness, the residue was extracted with 96% ethanol, and the extract was evaporated to dryness.

 The use of raw or insufficiently dried algae entails the need to increase the extraction ratio, which leads to a significant increase in solvent consumption, and most importantly, leads to contamination of the target product with ballast and undesirable substances. The use of 96% ethanol as an extractant is due to the fact that it is low toxic, it dissolves the lipophilic bioactive components of algae fairly well, satisfactorily extracts D-mannitol, but slightly dissolves inorganic salts present in large quantities in the feed. With a single extraction, these bioactive compounds are recovered no more than 65%, with a double extraction the degree of extraction is not less than 92%. Although the degree of lipid extraction increases somewhat during further extraction, the extract is contaminated with inorganic salts and unidentified organic substances that are not removed during subsequent re-extraction; the presence of these substances in the target product causes negative effects during its use - skin irritation, itching, etc.

Carrying out the extraction at elevated temperature increases the degree of extraction of the active compounds. However, at temperatures above 75 ^{° C} becomes very noticeable oxidation and decomposition of carotenoids and polyunsaturated lipids. At temperatures below 65 ^about With the extraction efficiency decreases sharply and there is a need to increase the extraction ratio.

 Maintaining the extract at ambient temperature aims to precipitate the bulk of the recovered D-mannitol. Maximum precipitation is achieved in at least 16 hours. With a longer exposure, the amount of sediment does not increase. Precipitation of D-mannitol at a low temperature is impractical because some active lipid components precipitate from the solution. To further concentrate the active substances, the filtrate obtained after separation of the crude mannitol precipitate was evaporated to dryness, and the residue was extracted with 96% ethanol. After complete distillation of ethanol from the extract, the expected product is obtained. As a result of recrystallization of "raw" mannitol from aqueous ethanol, a D-mannitol preparation is obtained with a basic substance content of 98.8-99.3%.

 In the above-described manner, from 1.0 kg of air-dried algae L. saccharina or L. digitata, 20.0-22.5 g of the target product and 37-42 g of D-mannitol are obtained. The target product obtained from both types of kelp is almost the same in terms of physico-chemical and medicinal properties. The product is a greenish-brown ointment in a thin layer with a characteristic odor of water, it consists of lipids (82-86% of the total mass), carotenoids (3.8-4.2%), amino acids and dipeptides (3.5-4 5%), porphyrin derivatives (3-4%), inorganic salts (1.2-1.4%), flavonoids (0.09-0.12%). The composition of lipids includes neutral glycerides (60-64%), phospho- and glycolipids (27-29%).

The target product is characterized by the following physico-chemical parameters:
density of a 50% solution in 96% ethanol 0.909 ± 0.001 g / cm ³ at ²⁰ ° C;
solidification temperature 12.5 ± ± 0.2 ^о С;
the relative viscosity of a 50% solution in 96% ethanol at ²⁰ ° C 6.0 ± 0.1 with respect to 96% ethanol;
the refractive index of a 50% solution in α-bromonaphthalene n _D ²⁰ 1,6802 ± 0,0004;
optical absorption of a 0.1% solution in 96% ethanol λ _max. (E): 407 (1.43 ± 0.02), 505 (0.40 ± 0.02), 538 (0.33 ± 0.01), 607 (0.27 ± 0.02), 666 (0.91 ± 0.02) nm;
mass fraction of bound carbohydrates (no free carbohydrates) 0.3 ± ± 0.1%;
mass fraction of lipid phosphorus 0.85 ± 0.15%;
ash content 1.6 ± 0.2%;
acid number 18.1 ± 0.5 mg sodium hydroxide (0.452 ± 0.012 mEq) per 1.0 g of the drug;
saponification number 86.5 ± 0.6 mg of potassium hydroxide (1.545 ± 0.011 mEq) per 1.0 g of the drug;
iodine number 542 ± 10 mg of iodine bound 1.0 g of the drug.

Each of the above parameters was determined on at least 10 samples taken from various batches of the target product. The D-mannitol preparation obtained simultaneously contains 98.8-99.3% of the basic substance, has a melting point of 167-168 ^о С and specific optical rotation [α] _D ²⁰ - 0.55 ± 0.15 ^о (water; from 6) .

Despite the high degree of unsaturation, the drug is stable at room temperature and stored without appreciable change of physico-chemical and therapeutic properties at least 1 year at a temperature of 15-25 ° ^C. This is due to the presence therein of natural antioxidants - diosmetin and luteolin.

PRI me R 1. In a glass reactor with a capacity of 15 l load 1.0 kg of air-dry (residual moisture 25%) and powdered algae L. saccharina collected on the White Sea, and 8.0 l 96% ethanol. The mixture was stirred for 2 hours at 65.degree ^. C. and immediately filtered without cooling through a glass porous filter (hereinafter, filter No. 2) with suction. The filtered biomaterial is loaded into the same reactor and extracted with 96% ethanol under the indicated conditions. The extracts were combined and incubated 16 hours at 20 ^C. The white crystalline precipitate consisting mainly of D-mannitol, filtered, and the filtrate was evaporated to dryness (hereinafter ethanol distillation is carried out on a rotary evaporator at a bath temperature of 35-40 ^about C and a residual pressure of 20-25 mm RT. Art.). To the residue (54 g) was added 600 ml of 96% ethanol and the mixture was stirred for 1 hour at ²⁰ ° C, then filtered and the filtrate evaporated to dryness. The residue was dried for 4 hours ^at 40 ° C and residual pressure of 0.2 mm Hg. Art. 20.0 g of the expected product are obtained.

The filtered precipitate of "raw" mannitol (dry weight 42.8 g) was poured into 300 ml of 80% ethanol, the mixture was stirred at moderate boiling until the precipitate had stopped dissolving (10 min), after which it was filtered while hot. The filtrate was allowed to stand for 2 hours at 20 ^{° C} and 6 hours at 10 ° ^C. The crystalline precipitate was filtered off and dried for 4 hours ^at 50 ° C and a pressure of 20 mm Hg. Art. Obtain 37.1 g of D-mannitol with a basic substance content of 99.3% according to gas chromatography of the corresponding trimethylsilyl derivative.

PRI me R 2. Air-dried and powdered algae L. saccharina (1.0 kg) is twice extracted with 96% ethanol in the conditions of example 1, except that the extraction is carried out at 75 ^about C. processing of the extract is carried out as described in example 1. Obtain 22.1 g of the target product and 41.9 g of D-mannitol with a basic substance content of 98.8%.

PRI me R 3. Air-dried and powdered algae L. saccharina (1.0 kg) is twice extracted with 96% ethanol in the conditions of example 1, except that the extraction is carried out at 71 ^about C. Further processing of the extract is carried out as described in example 1. Obtain 22.0 g of the target product and 42.0 g of D-mannitol with a basic substance content of 99.3%.

PRI me R 4. Air-dried and powdered algae L. digitata (1.0 kg) is twice extracted with 96% ethanol in the conditions of example 1, except that the extraction is carried out at 72 ^about C. Processing the extract is carried out as described in example 1. Receive 20.2 g of the target product and 37.4 g of D-mannitol with a basic substance content of 99.1%.

The above method is a fundamentally new process, which has the following main advantages compared to the known method:
the target product obtained by the proposed method is much more enriched with biologically active substances of lipid nature (carotenoids, lipids, including polyunsaturated fatty acids, and others), which dramatically increases the therapeutic effect of the drug;
the target product is a greasy substance that is well retained on the surface of the skin, which allows it to be successfully used to treat animals;
the method allows to simultaneously obtain a high-purity D-mannitol preparation; D-mannitol is widely used in the pharmaceutical and food industries, in the production of surfactants, resins, varnishes, and in addition, it is used in scientific research.

Toxicological studies of the target product, which is named "Algalipin", were carried out on 256 non-linear white mice. Doses of the drug for experimental animals were calculated per 1 kg of body weight and were administered intragastrically. Algalipin was tested, starting with a dose that does not cause a reaction in any of the animals, to a dose that causes 100% death. Clinical observations of experimental animals were conducted for seven days. The LD ₅₀ value was calculated by the Kerber method. For Algalipin, LD ₅₀ is 27.0-34.0 ml / kg, and for its 50% solution in 60% ethanol 19.5-21.0 ml / kg. According to GOST 12.1.07.76, the drug belongs to low-toxic substances.

 The irritant effect of Algalipin was studied on 21 rabbits of the chinchilla breed with a body weight of 2-3 kg. 2 drops of Algalipin were introduced into the conjunctival sac of the right eye of each animal with an eye dropper; the state of the left eye served as a control. For comparison, a separate group of rabbits was injected in the same way with Vishnevsky balsamic liniment. A visual assessment of the irritating effect of the drugs was performed after 1, 2, 3, 4, and 24 hours, noting changes in the blood supply of the conjunctiva, the presence of lacrimation, and the condition of the cornea. Signs of eye irritation after administration of Algalipin completely disappeared after 1 h, which indicates, rather, a mechanical cause of irritation. The liniment of Vishnevsky had exactly the same effect. The conjunctival irritation with the above alcoholic solution of Algalipin was more prolonged (2 hours). Thus, Algalipin gives a mild irritant effect.

 The irritating effect of Algalipin on the skin was evaluated in white mice, rats, rabbits and guinea pigs. On the back of the animal, a section was shaved: 1 x 1 cm in mice, 2 x 2 cm in rats and guinea pigs, 4 x 5 cm in rabbits, and Algalipin or its alcohol solution in the amount of 0.2 ml per day was rubbed 1 kg of animal weight for 5 minutes Each batch of the drug (a total of 6 batches) was tested on 10 mice, 5 rats, 3 guinea pigs and 3 rabbits. The control groups of animals (of the same number) were rubbed with Vishnevsky liniment. It was established that rubbing the preparations for 7 days once a day does not cause changes in the behavior of animals, redness of the skin, scratching, and thickening of the skin fold were not observed in the areas of application of the preparations.

 Thus, the complex of tests indicates that Algalipin and its alcohol 50% solution are low toxic and do not cause side effects with repeated use. The reparative properties of Algalipin and its alcohol solution were studied on 12 Romanov sheep at the age of 1 year and on 9 dogs. For comparison, the balsamic liniment of Vishnevsky was used. Before the experiment, 24 hours after wounding, as well as on the 3rd, 5th, 10th, 15th, 20th, and 23rd days, clinical and hematological parameters (hemoglobin concentration, erythrocyte count, and leukocyte count in blood, protein content in blood serum), histological studies were performed and the level of ATP was determined.

 A clear anti-inflammatory and anti-edematous effect of Algalipin and its alcohol solution, as well as its ability to stimulate regenerative processes, has been ascertained. So, three days after the start of treatment, mononuclear forms predominated in the leukocyte infiltrate, and signs of fibroplasia appeared in the deeper layers with more mature granulation tissue. In the case of using Vishnevsky liniment, a similar histological picture was observed, mainly, only on day 5-6, in untreated animals - on day 7-10, and when using the kelp preparation obtained in accordance with the prototype method, on day 7-8 . Algalipin and its alcohol solution alkalize the wound medium in the first phase of the wound process, which contributes to the growth and development of full-fledged granulation tissue. Liniment Vishnevsky stimulates regeneration in the second phase of the wound healing process. Complete healing of wounds under the influence of Algalipin occurs in animals faster than when using the Vishnevsky liniment, and much faster than when using the preparation of dry kelp. (56) Kizevetter I.V. et al. Processing of algae and other commercial aquatic plants. M.: Food Industry, 1967.

Claims (1)

METHOD FOR PRODUCING A MEDICINE FOR TREATING ANIMAL SKIN DISEASES, including air drying and subsequent grinding of algae of the Laminaria genus, characterized in that, in order to increase the therapeutic effectiveness of the agent and simultaneously produce D-mannitol, air-dried ground algae are twice extracted with 96% - ethanol at 65 - 75 ^o C, the extracts are combined and kept at ambient temperature for at least 16 hours, the precipitated precipitate of D-mannitol is filtered off and recrystallized from aqueous ethanol, filter the atom is evaporated to dryness, the residue is extracted with 96% ethanol, and drying is carried out by evaporation of the extract to dryness.