WO2023149795A1 - Method for producing an entomological compound with a keratolytic effect - Google Patents

Abstract

The invention relates to the field of biotechnology and can be used in cosmetology and pharmaceutics for treating keratoses of various origins and in cosmetic exfoliating procedures. A method for producing an active entomological compound with a high degree of keratolytic activity consists in raising insects until the seventh larval instar (21-23 days after hatching) on a first nutrient medium having a specific composition, and subsequently transferring them to a second nutrient medium which differs from the first in that it contains a larger quantity of wax and a smaller quantity of honey and further contains zinc acetate as a protein synthesis stimulator. The insects are grown for 180 hours, after which the larvae are harvested, frozen at -18°С, washed and ground at a temperature of 20°С, lyophilized, re-ground at an extremely low temperature of -55°С, homogenized, packaged and stored in a freezer compartment at -18°С. The invention makes it possible to obtain a larger quantity of larval biomass and to increase the keratolytic activity of the target product.

Description

METHOD FOR PRODUCING ENTOMOLOGICAL COMPLEX C

KERATOLYTIC ACTION

The invention relates to the field of cosmetology and pharmaceuticals, namely to preparations with a keratolytic effect, effective in the treatment of keratoses of various etiologies and gentle exfoliation as part of cosmetic procedures.

There are two groups of drugs with keratolytic action - obtained by chemical synthesis and natural origin. Among the most well-known keratolytic drugs obtained by chemical synthesis, ■ - resorcinol (Benzene-1, 3-diol; C6H4 (OH) 2). This substance at a concentration of 5-10% has a keratolytic and cauterizing effect on the stratum corneum [D]. The disadvantage of this drug is that the skin actively absorbs the substance, and application to large surfaces or the use of high concentrations can cause pronounced side effects: , tinnitus, sweating, shortness of breath, increased heart rate, convulsions, cyanosis, etc. Also, resorcinol gives a pronounced reddish tint to fair hair, and in case of insufficient resorcinol purification, the skin at the site of application acquires a purple hue.

There are several preparations of natural origin with keratolytic action. For example, a skin care composition is known that acts on the basis of three proteolytic enzymes that ensure the elimination of dead cells and the renewal of the epidermis. This composition is obtained by combining three proteolytic enzymes - thiol protease, primary serine protease obtained as a result of the metabolic processes of Aspergillus melleus, and secondary serine protease. In addition, the composition of the drug also includes a plant-derived hydroxy-acid obtained by extracting plants, as well as an antioxidant, which can also be of plant or synthetic origin [2].

The disadvantage of this drug is the high price of the finished product, which is determined by the production of three types of proteolytic enzymes with a high degree of purity, as well as the high irritating potential of the drug, determined by its composition, especially the presence of alpha hydroxy acids. Also, this composition does not contain components that facilitate the process of skin restoration in places subjected to keratoses.

Also known is a biological product that removes moles, nevi, polyps, keloids, carcinomas and other skin formations [3]. The biological product consists of 51.7 - 52.0% of a specific protein, which contains 10% of biologically active substances in terms of amino acids: Glycine. Asparazine, Leucine, Pyr. Phenylalanine, Lysine, as well as lanolin - 25%, glycerin - 25%, glycerin - 25% milk powder, walnut extract 10%, nipagin 0.12%, nipazole 0.08%, bidistilled water 4.8%, percentages are expressed by weight. To obtain the drug, pupae of Lymantria dispar are collected from the affected forests. The disadvantage of this drug is that the biological material is obtained by collecting from nature, thus it is impossible to control the composition of the active substance. In addition, the collection of biological material from nature does not allow planning a certain amount of production, depending on environmental conditions and fluctuations in the abundance of the species Lymaniria dispar.

Izvest is another way to obtain an entomological complex in propylene glycol with a keratolytic effect. Entomological complex in propylene glycol with keratollonic action, obtained from insects of the order Lepidoptera, family Pyralidae. genera Galleria,. Asopia or JgZo&w, contains subtilisin, chitosan tin polysaccharides, amino acids and jarapylene glycol, exhibits bird keratol activity, activity 46.5 - 51.0 U / ml, The procedure for obtaining this entomological complex consists in growing insects from the order Lepidoptera, family Pyralidae, genera Galleria, Asopia or on various artificial nutrient media, then the last instar larvae are collected, washed, disinfected with UV rays and crushed. The resulting biomass is lyophilized and extracted with bidisgillated water for 2-8 hours, then the extract is separated by decantation, concentrated, jaropylene glycol is added and the water is evaporated; The active complex is used for the manufacture of drugs with keratollic action in the form of ointments and gels and cosmetics with keratolnetic action in the form of cream, tonic, cleansing lotion and exfoliating gel. [4] .

The disadvantage of this method is the use of very complex nutrient media, consisting of more than 30 components. For example, for the growth of insects of the genus Galleria, the medium contains yellow corn extract, red corn extract, glucose, fructose, sucrose, maltose, casein, retiol, tocopherol acetate, vitamin C, riboflavin, calcium pantothenate, thiamine, pyridoxine, biotin, niacin, pinobanksin, pinocembrin, glycerin, NaNOy, KH2PO, NaCl, CaCl, MgSOrTHjO, 2JJSO*7H2O, MnSO^SHjO, CUSC SHSO, ECL6H2O, EDTA, yellow wax, purified water. Also, the biomass of larvae is collected immediately after molting, a period when the amount of enzymes with keratoltic action decreases as a result of their use in the process of removing the old external skeleton.

The closest solution to the problem of this invention is a method for obtaining biomass of Galleria melloaella larvae with high biological activity, one of the variants of which is to obtain entomological biomass with high keratolytic activity. The method consists in growing the larvae of Galleria meliopia at a temperature of 27°C and a humidity of 75% for 19 days with reseeding of the larvae at least 2 times on a fresh nutrient medium consisting of wheat flour; corn flour, wheat bran, yeast extract, milk powder, honey, glycerin, wax powder, pinobanksin, ninotsembrin, purified water. [5].

The disadvantage of this technique is that the larvae of the 6th larval age are used, which is the penultimate one in the normal life cycle of the insect, the weight of the larvae is below the maximum possible and, therefore, the keratolytic activity is also below the maximum possible level. The problem that the present invention solves is to develop a method for obtaining an entomological complex from the larvae of Galleria mellonella, which provides a greater amount of biomass with a higher keratolytic activity through the use of larvae of VII age grown in special conditions that contribute to the accumulation of biomass and increase its biological activity.

The essence of the invention lies in the fact that a method is proposed for obtaining an active entomological complex with high keratolytic activity, which consists in growing insects until reaching the 7th - larval age (21-23 days after hatching), on nutrient medium 1 from the nearest solution [5 {; transferring them to another nutrient medium - nutrient medium 2, which differs from the first in a large amount of wax powder and less bee honey, as well as in the presence of a protein synthesis stimulator - zinc acetate, followed by the composition (per kg of medium):

Wheat flour 185 - 200

Corn flour 180 - 190

Wheat bran 110 - 130

Yeast extract 15 - 20

Powdered milk 120 - 140

Bee honey 40-50

Glycerin 80 - 90

Wax powder 65-85

Pinobanksin 0.02 - 0.03

Piyocembrin 0.02 - 0.03

Zinc acetate dihydrate (ZnCCHjCO ^HjO) 0.01-0.02

Purified water up to 1000g, their cultivation for 180 hours, after which the larvae are collected, frozen at -18°C; washed and crushed at a temperature of 20 ° C - thus obtaining the active complex Keratolysis Plus, lyophilized; re-grind at extremely low temperature (-55°C); homogenized, vaccinated and stored in a freezer (-18°C).

The keratolytic activity of the entomological complex with keratolytic activity was determined using a spectrophotometric test using keratin azure [6], which is carried out as follows: 5 mg of keratin azure powder is suspended in 1 ml of Tris buffer - 50 mM HC! (pH 8.0). The reaction mixture contains 1 ml of keratin-azure suspension and 1 mg of a complex with keratolytic activity. The reactions are carried out at 50°C in a water bath with constant stirring at 200 rpm for 30 min. After incubation, the reaction is stopped by adding 2 ml of 0.4 M trichloroacetic acid, followed by centrifugation at 3000 g for 20 minutes. The absorbance of the supernatant is measured at 595 nm. In the same way, a control is prepared, while replacing the complex with keratolytic activity with keratinase. Keratolytic activity, expressed in units of activity per mg of substance (U/mg), indicates the amount an enzyme that causes a 0.01 increase in absorbance between sample and control at 595 nm under specified conditions.

The results obtained are presented in table 1

The data in table I prove that the application of the proposed method provides an increase in the amount of biomass obtained per inch of feed used by 23.7% compared with the prototype. The keratolytic activity of the active complex also increases by 69.1%.

The technical result of the invention in comparison with the nearest solution is that the process of obtaining an active complex with keratolytic activity involves the use of two nutrient media for growing larvae. - one before reaching the 7th larval age, and the other within 180 hours For larvae of the 7th instar before they pupate. The use of 7th instar larvae provides a higher biomass of larvae, and a medium with low honey content and high wax content, as well as zinc acetate content, stimulates the elimination of proteases with keratolytic activity.

The keratolytic active complex obtained by this method can be used as an active substance in cosmetic and pharmaceutical products.

For the preparation of cosmetics, the active complex will be used in an amount of 0.3-2.0%, preferably 1.0-1.5% of the total weight of the composition.

Also cosmetic compositions will include solvents, preferably purified water, which depending on the chosen physical form may vary from 3% to 87% by weight of the composition; or alternatively glycerol, benzyl alcohol, etc.

In addition, cosmetic products (in the form of a lotion, cream, gel or serum) will include one or more emulsifiers, such as organic alcohols with a chain of 14-22 carbon atoms (C14-22 alcohols), C 14-20 alkyl glycosides, alkyl sulfate, alkyl phosphates, acyl isothionate, cetyl alcohol, stearic alcohol, glyceryl stearate, etc., substances with these properties or combinations of these substances. The mass of components with emulsifying properties will be from G% to 12% of the total mass of the composition.

As emulsion stabilizers, cosmetic products will contain C 14-22 organic alcohols, esters of glycerol such as glycerol stearate; synthetic polymers or other components with emulsion stabilizer properties, including in various combinations.

Cosmetic compositions will necessarily contain emollients, such as: one or more fats or oils, hydrogenated or non-hydrogenated, their esters, or various combinations of emollients that are acceptable for this type of product. The content of this type of components in the formulations will be from 0.5 to 10% of the total weight of the composition.

Cosmetic formulas will also contain humectants capable of retaining water in the skin tissues. As such substances, urea, pyroglutamic acid, amino acids or other substances with hygroscopic properties accepted for this type of product, as well as combinations of these substances, can be used. The amount of substances with hygroscopic properties in the product formulations will be from 0.1% to 10.0% by weight of the composition.

Cosmetic products will contain preservatives such as phenoxyethanol, EDTA, EDTA-conjugated fatty acids, isothioazolin, parabens such as methylparaben and propylparaben, propylene glycol, sorbates, urea derivatives such as diazolidinyl urea or imidazolidinyl urea, and other substances with preservatives. accepted for such products or combinations thereof. The amount of preservatives in the composition of the products will be 0.3% - 2.0% by weight of the final composition.

Cosmetic products will also include chelating agents such as EDTA derivatives or combinations thereof. The amount of these components in cosmetic products will be 0.01% - 0.2% by weight of the final composition.

Cosmetics will also include buffer compounds to help maintain the correct pH level. As such substances, adipic acid, glycine, citric acid, calcium hydroxide, triethanolamine or other substances with similar properties adopted for such products and combinations thereof can be used.

Cosmetic products will also contain antioxidant substances such as ascorbic acid, tocopherol, plant extracts such as moringa seed extract, grapevine extract, or any other substance with such properties, or combinations thereof.

Also, all cosmetic products based on the active entomological complex may contain fragrances, dyes, UV-A and UV-B protection factors, natural plant extracts or other organic raw materials.

Thus, it is possible to choose a wide variety of formulas that will ensure maximum activity of the active complex without changing its keratolytic effect.

Cosmetic compositions may also include pharmaceutical plant extracts; .Car a papaya, Ficus carted,: Ananas sp. _t Actmidia. deliciosa etc. or/'and other substances such as purified proteolytic enzymes with similar and / or complementary action that does not change the action of the active components of the entomological complex.

The physical form of a cosmetic product cannot be limited to homogeneous mixtures, but can also be represented by separated phases.

Pharmaceutical preparations containing an entomological complex may be presented in the form of an ointment, gel, serum, spray, or other physical forms accepted for pharmaceutical products of this type. Pharmaceutical compositions may include any suitable active ingredients and components suitable for such formulations.

All pharmaceutical products according to this patent will contain an entomological complex in an amount of 1.0-4.0%, preferably 1.5-2.0% of the total weight of the composition.

Pharmaceutical compositions based on an active complex of entomological origin in cropylene glycol will include solvents (in the case of this patent, purified water is preferred, which, depending on the chosen physical form, can vary from 3% to 87% by weight of the drug), compositions or alternatively - glycerol, benzyl alcohol etc.).

Likewise, pharmaceutical products (in the form of a spray, ointment, gel or serum) will include one or more emulsifiers such as C14-22 chain organic alcohols (C14-22 alcohols), C14-20 alkyl glycosides, alkyl sulfate , alkyl phosphates, acyl isothionate, cetyl alcohol, stearyl alcohol, glyceryl stearate and other substances with similar properties or combinations of these substances. The mass of components with emulsifying properties will be from 1% to 12% of the total mass of the composition.

Pharmaceutical compositions necessarily contain emollients, such as: one or more fats or oils, hydrogenated or non-hydrogenated, their esters, paraffins, petroleum jelly, or various combinations of emollients accepted for this type of product. The content of this kind of components in pharmaceutical formulations will be from 0.5 to 10% of the total weight of the composition.

The pharmaceutical formulations of this patent will also contain humectants capable of retaining water in skin tissues. As such substances, glycerin, urea, pyroglutamic acid, amino acids or other substances with hygroscopic properties adopted for this type of product, as well as combinations of these substances, can be used. The amount of substances with hygroscopic properties in the formulations of pharmaceutical products according to this patent will be from 0.1% to 10.0% by weight of the composition.

Pharmaceutical products will contain preservatives such as sorbates, phenoxyethanol, ED 1A, EDTA-conjugated fatty acids, isothioazolin, parabens such as methylparabe and propylparaben, propylene glycol, urea derivatives such as diazolidipylurea or imidazoyaidinylurea, and other substances with preservatives accepted for such products or combinations thereof. The amount of preservatives in the composition of pharmaceutical products according to this patent will be 0.3% ~ 2.0% by weight of the final composition.

Pharmaceutical products will also contain antioxidant substances such as butylated hydroxyanisole, ascorbic acid, tocopherol, plant extracts such as moringa seed extract, grapevine extract, or any other substances with similar properties, or combinations thereof.

Thus, it is possible to choose a wide variety of recipes that will ensure the maximum activity of the entomological complex without changing its keratolytic action.

Pharmaceutical compositions may also include pharmaceutical extracts of plants: Calendula officinalis, Aloe vera, Salvia officinalis, etc. D- or / 'and other substances: allantoin, vitamin E, antibiotic substances, etc. having complementary therapeutic effects: anti-inflammatory, healing, antibiotic.

The physical form of a pharmaceutical product cannot be limited to homogeneous mixtures, but can also be represented by separated phases.

Examples of the implementation of the invention

Example 1;

Obtaining an entomological complex with keratolytic activity from the larvae of Galleria mellonella

The pupae of Galleria mellonella are removed from the refrigerator, where they are kept at a temperature of 3-5°C, and left at room temperature for 2 hours. After this time, the pupae are transplanted into containers covered with perforated lids, which contain a small amount of nutrient medium (1/10 of the container volume) for further development of the pupae, their transition to the imago phase and laying eggs. In order to start the hatching of the larvae from the eggs, the containers are kept at a temperature of 27°C and a relative humidity of 75% in the dark. 48 hours after placing the pupae in containers, the first instar larvae appear. The process is controlled until reaching 80% yield, but not more than 6 hours.

Nutrient medium 1, the following composition, g/kg:

Wheat flour 185

Corn flour 180 Wheat bran 110

Yeast extract 15 Milk powder 120 Bee honey 75

Glycerin, 80

Wax powder 40

Pinobanksin 0.02

Pinocembrin 0.02

Purified water rest

Prepare new containers with nutrient medium. After 6 hours from the start of hatching, the first instar larvae are removed with tweezers from the containers in which they appeared and placed in the prepared containers.

The growth of larvae continues until the larvae of the seventh age are obtained, but not more than 23 days. The temperature in the containers is maintained at 27° C. and the relative humidity is 75% under dark conditions. During this time, two or three transfers of larvae are carried out in containers with fresh nutrient medium.

Larvae of VII age are transplanted onto nutrient medium 2 of the following composition, g/kg:

Wheat flour 185

Cornmeal 180

Wheat bran 110

Yeast extract 15

Powdered milk 120

Bee honey 45

Glycerin!, 80

Wax powder 80

Pinobanksin 0.02

Pinocembrin 0.02

Zinc acetate dihydrate (ZnfCHjCO^a'SHiO) 0.015

Purified water rest

180 hours after transfer, the larvae are harvested with tongs and transferred to special freezer containers. Larvae are frozen at ~18°C. Frozen raw materials can be stored for 24 months and used depending on the need.

Before use, the larvae are washed twice with four volumes of 0.9% saline and two more times with four volumes of purified water.

Next, the biomass is subjected to grinding in a grinder with stainless steel knives so that the size of the resulting particles does not exceed 2 mm. The resulting active complex is then subjected to a lyophilization process.

The last stage consists in grinding the lyophilized active complex in a cryogenic mill at an extremely low temperature, which ensures the production of dispersed particles up to 200 microns in size.

The active complex obtained as described above is sealed in aluminum bags and stored until use (up to 24 months) in a freezer (t ~ -18±3 ^Q C). The keratolytic activity of the entomological complex obtained according to this example is 6.48 units/mg of dry matter.

Example 2

Recipes for the manufacture of pharmaceutical preparations based on an entomological complex with a keratolytic effect

* Pharmaceutical product in the form of a cosmetic cream based on an entomological complex with keratolytic action, containing:

1 . Entomological complex with keratolytic action 1.0%

2. Poyaigyaceryl-3-methylglucose distearate 5.0%.

3. Glycerin 3.0%

4. Isopropyl palmitate 2.0%

5. Grape seed oil 3.0%

6. Kadryl/capric triglycerides 1.0%

7. Butyrospermum Parkii 2.0%

8. Cyclopentasiloxane 0.5%

9. Polysorbate 80 2.0%

10. Glyceryl stearate 2.5%

1 1. Soy protein clay 1.0%

12. Diazy Lidinyl urea 0.6%

13. Methylparaben 0.3%

14. Proylparaben 0.10%

15. Disodium salt EDTA 0.05%

16. Perfume 0.20%

17. Water up to 100%

• Pharmaceutical product in the form of a tonic and cleansing lotion. the basis of an entomological complex with keratolytic action, which contains:

1 Entomological complex with keratolytic action 1.2%

2. Glycerin 5.0%

3. Pyatylene glycol 3.0%

4. D-panthenoia 0.5%

5. .Allantoin 0.10%

6. Sodium laurate sulfate 0.10%

7. Diazolidinylurea 0.40%

8. Methylparaben 0.30%

9. Propyl paraben 0.10%

10. Water up to 100% • Pharmaceutical product in the form of an exfoliating gel based on an active complex of entomological origin, which contains:

1. Active entomological complex 1.5%

2. Carbomer 0.8%

3. Triethanolamine 0.8%

4. Sodium hyaluronate 0.2%

5. Bisabolol 0.1%

6. Diazolidinylurea 0.4%

7. Mstilparaben 0.30%

8. Proyliaraben 0.10%

9. Water up to 100%

• Pharmaceutical product in the form of an ointment based on an entomological complex with keratolytic action, which it contains:

1 . Entomological complex with keratolytic action 2.0%

2. Butium hydroxyanierl 0.1%

3. Cetyl alcohol 4.0%

4. Polysorbate 80 3.0%

5. Potassium sorbate 0.50%

6. piperine 4.0%

7. Liquid paraffin 3.0%

8. White Vaseline 3.0%

9. Purified water up to 100%

• A drug in the form of a gel based on an entomological complex with a keratolytic effect, containing:

1. Entomological complex with keratolytic action 2.0%

2. Carbon 940 2.0%

3. Glycerin 6.0%

4. Sodium hydroxide solution 15% 1.0%

5. Propyl englycol 6.0%

6. methyl p-hydroxybenzoate 0.10%

7. Propyl p-hydroxybenzoate 0.20%

8. Purified water up to 100%

Bibliography:

1. Marian A. Barna M.V. Proceedu de fabrivare a rezorcinei. Brevet de invenjie RO

117694 Bl. BOPl nr.6, 2002 .

2. Kusmirek K. Skincare composition comprising a trienzyme activity. UK Patent GB 2440117 B , Ciuhrii M. Preparat biologic pentru lizarea alunijelor, nevilor, polipilor, keloizilor, earcinoamelor §1 a altor farmapuni cutanate, procedeu de oblinere utilizare a acestuia. Brevet de inventie, RO 1 19113 Bl . BOPI nr.4, 2004. Ciuhrii V. Complex de origine entomologies In propylenglycol cu actiune cheratoiitica, procedeu de oblinere a acestuia, produse farmaceutice §i cosmetics pe baza lui (variants). Brevet de invenpe MD 4198, BOPI nr. 2/2013, Ciuhrii C. Procedeu de obfinere a biomasei de larve de Galleria mellonella cu activitate biologies inalta. Brevet de inventie MD 4646, BOPI, nr.9/2019 . Cai, C., Chen, J,, Qi, J., Yin, Y,, Zheng, X, Purification and characterization of keratinase from a new Bacillus subtilis strain. Journal of Zhejiang University SCIENCE B, 2008, 9(9), 713-720.

Claims

FORMULA OF THE INVENTION of an active entomological complex with high keratolytic activity, which consists in growing insects until reaching the 7th - larval age (21-23 days after hatching), on a nutrient medium 1 from the nearest solution [5]; transferring them to another nutrient medium - nutrient medium 2, which differs from the first in a large amount of wax powder and less bee honey, as well as in the presence of a protein synthesis stimulator - zinc acetate, followed by the composition (per kg of medium): Wheat flour 185 - 200 Corn flour 1.80 - 190 Wheat bran 110 - 130 Yeast extract 15 - 20 Powdered milk 120 - 140 Bee honey 40-50 Glycerin 80 - 90 Wax powder 65-85 Pinobanksin 0.02 - 0.03 Pinocembrin 0.02 - 0.03 Pink acetate dihydrate Water purified to 100 g nx cultivation for 180 hours, after which the larvae are collected, frozen at -18 "C; washed and crushed at a temperature of 20 ° C, lyophilized; re-crushed at an extremely low temperature (-55 ° C); homogenized, packed and stored in a freezer (-18°C).