Method of obtaining cosmetic and medical-cosmetic products

Abstract

The present invention relates to the field of cosmetics, namely, to a method of obtaining cosmetic and medical-cosmetic products by sequential repetitive vibration treatment of the initial substance – biological molecules of cells, cellular structures, fluid media (blood, lymph), and water, located mainly in the skin, hair, nails, and oral mucosa or included in the tissue fluid of the skin or having a regulatory effect on functional and metabolic processes in the skin, hair, and nails, and oral mucosa, as well as the biological products, which during preparation acquire the ability to exert an effect similar to the properties of the initial substance, thereby exerting cosmetic or medical-cosmetic action on the skin, hair, nails, or oral mucosa.

Description

[0001] Method of obtaining cosmetic and medical-cosmetic products

[0002] The present invention relates to the field of cosmetics, namely, to a method of obtaining cosmetic and medical-cosmetic products by sequential repetitive vibration treatment of the initial substance - biological molecules of cells, cellular structures, fluid media (blood, lymph), and water, located mainly in the skin, hair, nails, and oral mucosa or included in the tissue fluid of the skin or having a regulatory effect on functional and metabolic processes in the skin, hair, and nails, and oral mucosa, as well as the biological products, which during preparation acquire the ability to exert an effect similar to the properties of the initial substance, thereby exerting cosmetic or medical-cosmetic action on the skin, hair, nails, or oral mucosa.

[0003] It is known from the prior art that certain long-lasting structural changes can appear in water after any vibration treatment - both mechanical (Gudkov S.V. et al., 2019; Astashev M.E. et al., 2023; Demangeat JL., 2022; Duval E. et al., 2012) and electromagnetic (D'Emilia E. et al., 2017; Sronsri C. et al., 2021; Wu T. et al., 2020).

[0004] It is also known from the prior art that when solutions of an initial substance are prepared by multiple dilutions using at each dilution cycle an external mechanical action in the form of vertical shaking (vibration) - up to the level of ultra-high dilutions, the latter acquire a number of new physical properties that determine their activity even at speculatively small amounts in dilutions of the initial substance.

[0005] Such properties include the ability of high dilutions obtained by applying multiply repeated vibration treatment to exert a non-contact (distant) effect on other substances or solvents [Jerman, I.; Ruzic, R.; Krasovec, R.; Skarja, M.; Mogilnicki, L.; Ruzic, R.; Krasovec, R.; Skarja, M.; Mogilnicki, L. "Electrical Transfer of Molecule Information into Water, Its Storage, and Bioeffects on Plants and Bacteria", Electromagnetic Biology and Medicine 2005, 24 (3), 341-353. https: / / doi.org / 10.1080 / 15368370500381620; Ruzic, R., Jerman, I., Skarja, M., Leskovar, R., Mogilnicki, L., "Electromagnetic Transference of Molecular Information in Garden Cress Germination", Int J High Dilution Res 2008; 7(24): 122-131 2008, 7, 122-131; Igor Jerman, Linda Ogrizek, Vesna Pericek Krapez and Luka Jan, Physicochemical study of the molecular signal transfer of ultra-high diluted antibodies to interferon gamma. Int. J. Mol. Sci. 2023, 24; N. Penkov, N. Penkova. Analysis of Emission Infrared Spectra of Protein Solutions in Low Concentrations. Front. Phys., December 18, 2020 Sec. Interdisciplinary Physics, Volume 8 - 2020 | https: / / doi.org / 10.3389 / fphy.2020.624779; Penkov N., 2021; Novikov V.V. and Yablokova E.V., 2022)], as well as the ability of dilutions to have a modifying effect on the initial substance [WO2012017324, US7,229,648, US4,311,897, RU2192888, 2577137, 2577136, 2536230, 2519695, 2778521, 2509572, 2531048, 2517085, 2505312, 2577299] Modifying effects are manifested in changes in the structure of the molecules of the initial substance, their hydrate shell, which leads to modulation of biological activity of the initial substance [Tarasov SA, Gorbunov EA, Don ES, Emelyanova AG, Kovalchuk AL, Yanamala N, Schleker ASS, Klein-Seetharaman J, Groenestein R, Tafani JP, van der Meide P, Epstein OI. Insights into the Mechanism of Action of Highly Diluted Biologies. J Immunol. 2020;205(5): 1345-1354. doi: 10.4049 / jimmunol.2000098; Woods KN. Modeling of protein hydration dynamics is supported by THz spectroscopy of highly diluted solutions. Front Chem. 2023; 11 : 1131935. doi: 10.3389 / fchem.2023.1131935] and determines the presence of biological (pharmacological) activity in high dilutions.

[0006] The modifying effect underlying the specific pharmacological activity of highly diluted drugs is quite pronounced, which allowed the evaluation and the standardization of high dilutions of biological substances using generally accepted analytical methods (see State Pharmacopoeia of the Russian Federation XV, General Pharmacopoeia Monography (GPM) 1.7.0001). The technology of solution production in the form of sequential dilution of biological substances and vibration treatment of dilutions after the release of GPM received the official name ‘gradual’ technology. Currently, products based on gradual technology, gradualized drugs, are produced mainly from antibodies and therefore belongs to the category of biological products [Mkrtumyan A, Ametov A, Demidova T, Volkova A, Dudinskaya E, Vertkin A, Vorobiev S. A New Approach to Overcome Insulin Resistance in Patients with Impaired Glucose Tolerance: The Results of a Multicenter, Double-Blind, Placebo-Controlled, Randomized Clinical Trial of Efficacy and Safety of Subetta. J Clin Med. 2022; 11(5): 1390. doi: 10.3390 / jcml 1051390; Geppe NA, Blokhin BM, Shamsheva OV, Abdrakhmanova ST, Alikhanova KA, Myrzabekova GT. Efficacy and Safety of Ergoferon in Children from 6 Months to 6 Years Old with Acute Respiratory Viral Infections in Contemporary Outpatient Practice: A Multicenter, Double-Blind, Placebo-Controlled Randomized Trial. Can Respir J. 2021; 2021 :5570178. doi: 10.1155 / 2021 / 5570178; Lashch NU, Kamchatnov PR, Fedorova TN, Muzychuk OA, Khacheva KK, Pizova NV, Malygin AU, Shavlovskaya OA, Fateeva VV, Nikulina KV, Abrosimov AV, Gerasimova YA, Glushkov KS, Lebedeva AV. Efficacy and Safety of Divaza for the Correction of Oxidative Disturbances in Patients with Cerebral Atherosclerosis: A Randomized Controlled Trial. Cerebrovasc Dis. 2021; 50(4):472-482. doi: 10.1159 / 000515233; Avdeev SN, Vizel AA, Abrosimov VN, Zaicev AA, Ignatova GL, Khamitov RF, Mikhaylusova MP, Shapovalova JS, Pavlysh EF, Trofimov BI, Emelyanov AV, Martynenko TI, Martynenko VA, Kostina NE, Chizhov DA, Chizhova OY, Kuzubova NA, Makova EV, Makarova EV. Management of Cough in Patients with Chronic Obstructive Pulmonary Disease: Results of the Multicenter Randomized Placebo-Controlled Clinical Trial. Int J Chron Obstruct Pulmon Dis. 2021; 16: 1243-1253. doi: 10.2147 / COPD.S292109; Parfenov VA, Zhivolupov SA, Poverennova IE, Nesterova MV, Ushakova SE, Zhukova NG, Glazunov AB, Nikulina KV, Alexandrov MV, Lapatukhin VG, Zhestikova MG. Treatment of Cognitive Impairment and the Role of Demographic Factors in Disease Progression: The Final Results of the Russian Observational Program "DIAMANT". Eur Neurol. 2020; 83(6):591-601. doi: 10.1159 / 000508184; Ivashkin VT, Poluektova EA, Glazunov AB, Putilovskiy MA, Epstein OI. Pathogenetic approach to the treatment of functional disorders of the gastrointestinal tract and their intersection: results of the Russian observation retrospective program COMFORT. BMC Gastroenterol. 2019; 20(l):2. doi: 10.1186 / sl2876-019-l 143-5; Parfenov VA, Ostroumova OD, Ostroumova TM, Kochetkov Al, Fateeva VV, Khacheva KK, Khakimova GR, Epstein OI. Vascular cognitive impairment: pathophysiological mechanisms, insights into structural basis, and perspectives in specific treatments. Neuropsychiatr Dis Treat. 2019; 15: 1381-1402. doi: 10.2147 / NDT.S197032; Pushkar D, Vinarov A, Spivak L, Kolontarev K, Putilovskiy M, Andrianova E, Epstein O. Efficacy and safety of Afalaza in men with symptomatic benign prostatic hyperplasia at risk of progression: a multicenter, double-blind, placebo-controlled, randomized clinical trial. Cent European J Urol. 2018; 71(4):427-435. doi: 10.5173 / ceju.2018.1803; Mkrtumyan A, Romantsova T, Vorobiev S, Volkova A, Vorokhobina N, Tarasov S, Putilovskiy M, Andrianova E, Epstein O. Efficacy and safety of Subetta add-on therapy in type 1 diabetes mellitus: The results of a multicenter, double-blind, placebo- controlled, randomized clinical trial. Diabetes Res Clin Pract. 2018; 142: 1-9. doi: 10.1016 / j.diabres.2018.04.044; Rafalsky V, Averyanov A, Bart B, Minina E, Putilovskiy M, Andrianova E, Epstein O. Efficacy and safety of Ergoferon versus oseltamivir in adult outpatients with seasonal influenza virus infection: a multicenter, open-label, randomized trial. Int J Infect Dis. 2016; 51 :47-55. doi: 10.1016 / j .ijid.2016.09.002; Don E, van der Meide N, Egorov V, Putilovskiy M, Tarasov S. The level of natural autoantibodies to IFN-gamma in varicella infection treated with antiviral drug Anaferon for children: A pilot study. Immunol Lett. 2020; 222:90-94. doi: 10.1016 / j.imlet.2019.10.015; Tarasov SA, Gorbunov EA, Don ES, Emelyanova AG, Kovalchuk AL, Yanamala N, Schleker ASS, Klein-Seetharaman J, Groenestein R, Tafani JP, van der Meide P, Epstein OI. Insights into the Mechanism of Action of Highly Diluted Biologies. J Immunol. 2020; 205(5): 1345-1354. doi: 10.4049 / jimmunol.2000098; Woods KN. Modeling of protein hydration dynamics is supported by THz spectroscopy of highly diluted solutions. Front Chem. 2023; 11 : 1131935. doi: 10.3389 / fchem.2023.1131935; Alinkina E, Don E, Gizitdinova O, Samsonova L, Petrova A, Stepanov G, Tarasov S. A novel technique for studying the effects of technologically processed antibodies by evaluating the rate of oxidation of ascorbic acid during the reduction of the green-blue ABTS + radical. Spectrochim Acta A Mol Biomol Spectrosc. 2024; 304: 123323. doi: 10.1016 / j.saa.2023.123323; Emelianova AG, Petrova NV, Fremez C, Fontanie M, Tarasov SA, Epstein OI. Therapeutic potential of highly diluted antibodies in antibiotic-resistant infection. Eur J Pharm Sci. 2022; 173: 106161. doi: 10.1016 / j.ejps.2022.106161; Petrova NV, Tarasov SA, Epstein OI, Dubroca C, Sulpice T. Highly Diluted Antibodies to eNOS Restore Endothelium Function in Aortic Rings From Hypertensive Rats. Dose Response. 2022; 20(2): 15593258221099281. doi: 10.1177 / 15593258221099281; Don ES, Bobrovnik SA, Sherriff G, Myslivets AA, Tarasov SA, Epstein OI. Advanced approach to activity evaluation for released-active forms of antibodies to interferon-gamma by enzyme-linked immunoassay. J Immunoassay Immunochem. 2019;40(3):250-268. doi: 10.1080 / 15321819.2019.1567536; Kardash EV, Ertuzun IA, Khakimova GR, Kolyadin AN, Tarasov SA, Wagner S, Andriambeloson E, Ivashkin VT, Epstein OI. Dose-Response Effect of Antibodies to SI 00 Protein and Cannabinoid Receptor Type 1 in Released-Active Form in the Light-Dark Test in Mice. Dose Response. 2018; 16(2):1559325818779752. doi: 10.1177 / 1559325818779752; Don ES, Emelyanova AG, Yakovleva NN, Petrova NV, Nikiforova MV, Gorbunov EA, Tarasov SA, Morozov SG, Epstein OI. Dose-dependent antiviral activity of released-active form of antibodies to interferon-gamma against influenza A / California / 07 / 09(H1N1) in murine model. J Med Virol. 2017; 89(5):759- 766. doi: 10.1002 / jmv.24717; Don E, Farafonova O, Pokhil S, Barykina D, Nikiforova M, Shulga D, Borshcheva A, Tarasov S, Ermolaeva T, Epstein O. Use of Piezoelectric Immunosensors for Detection of Interferon-Gamma Interaction with Specific Antibodies in the Presence of Released-Active Forms of Antibodies to Interferon-Gamma. Sensors (Basel). 2016;16(l):96. doi: 10.3390 / sl6010096; Gorbunov EA, Nicoll J, Kachaeva EV, Tarasov SA, Epstein OI. Subetta increases phosphorylation of insulin receptor P-subunit alone and in the presence of insulin. Nutr Diabetes. 2015; 5(7):el69. doi: 10.1038 / nutd.2015.20; Gorbunov EA, Ertuzun IA, Kachaeva EV, Tarasov SA, Epstein OI. In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to SI 00 protein in released- active form. Neuropsychiatr Dis Treat. 2015; 11 :2837-46. doi: 10.2147 / NDT.S92456; Bailbe D, Philippe E, Gorbunov E, Tarasov S, Epstein O, Portha B. The novel oral drug Subetta exerts an antidiabetic effect in the diabetic Goto-Kakizaki rat: comparison with rosiglitazone. J Diabetes Res. 2013; 2013:763125. doi: 10.1155 / 2013 / 763125; Nicoll J, Gorbunov EA, Tarasov SA, Epstein 01. Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro. Int J Endocrinol. 2013; 2013:925874. doi: 10.1155 / 2013 / 925874; Andrianov VV, Epstein OI, Gainutdinova TKh, Shtark MB, Timoshenko AKh, Gainutdinov KL. Antibodies to calcium- binding SIOOB protein block the conditioning of long-term sensitization in the terrestrial snail. Pharmacol Biochem Behav. 2009; 94(l):37-42. doi: 10.1016 / j.pbb.2009.07.003; Castagne V, Lemaire M, Kheyfets I, Dugina JL, Sergeeva SA, Epstein OI. Antibodies to SI 00 proteins have anxiolytic-like activity at ultra-low doses in the adult rat. J Pharm Pharmacol. 2008; 60(3):309- 16. doi: 10.121 l / jpp.60.3.0005; Chu X, Zhavbert ES, Dugina JL, Kheyfets IA, Sergeeva SA, Epstein OI, Agmo A. Sildenafil and a compound stimulating endothelial NO synthase modify sexual incentive motivation and copulatory behavior in male Wistar and Fisher 344 rats. J Sex Med. 2008; 5(9):2085-99. doi: 10.1111 / j.1743-6109.2008.00937.x; Chu X, Agmo A.

[0007] Sexual incentive motivation in old male rats: the effects of sildenafil and a compound (Impaza) stimulating endothelial NO synthase. Pharmacol Biochem Behav. 2008; 89(2):209-17. doi: 10.1016 / j.pbb.2007.12.012; Epstein OI, Zapara TA, Simonova OG, Ratushnyak AS, Shtark MB. Plasticity of neuronal responses induced by low concentrations of exogenous ligands affecting cellular calcium stores. Front Biosci. 2004; 9:809-15. doi: 10.2741 / 1244; Epstein OI, Beregovoy A, Sorokina NS, Starostina MV, Shtark MB, Gainutdinov KhL, Gainutdinova TKh, Muhamedshina DI. Membrane and synaptic effects of anti-S-100 are prevented by the same antibodies in low concentrations. Front Biosci. 2003; 8:a79-84. doi: 10.2741 / 1025],

[0008] Biological substances - biological molecules, cells and cellular structures of auto-, allo-, or heterogeneous origin, including those from fetal or embryonic biological material have a great potential for introduction as cosmetic and medical-cosmetic agents, as unlike synthetic drugs have a wide range of activity and gentle, maximally physiological character of action. However, the use of molecules not subjected to gradualization procedure of biological molecules has a number of limitations in the form of adverse events, such as toxicity for monoclonal antibodies, which is unacceptable for cosmetics.

[0009] Currently, individual therapy of various diseases using various biological objects, primarily fetal tissues containing stem cells and used in the form of cell transplants, has seen substantial progress. This therapy has advantages, first of all, in the form of its ability to have a regenerative effect, which makes it attractive for use in cosmetics. However, the use of fetal tissue products in cosmetics has a number of limitations, one of the primary ones being the limited availability of biomaterial. Fetal tissues are difficult to standardize and store for long periods of time, which makes it difficult to use them widely in the form of cosmetics. The use of fetal and embryonic materials may be accompanied by immunologic rejection reactions, viral or prion contamination. In this regard, the use of gradualized biological substances subjected to multiple dilutions accompanied by vibration treatment is a priori a promising approach in cosmetics. However, obtaining high dilutions of a biological substance is a difficult task, since during dilution of preliminary homogenizated biological substance this substance loses part of its initial activity.

[0010] The applicant found that even without dilution of the initial substance, successive vibration treatment of a test tube containing various molecules, including biological ones, i.e., antibodies, together with a closely located test tube containing a solvent, results in the solvent changing its physical-chemical properties and turning into an artificial material object called iteration. It was found that iterations were distributed by physical and chemical properties into fractions, each of which reproduced one or another biological and chemical effects of the used molecules.

[0011] The technical task of the present invention was to create a method of obtaining from the initial biological substance (biological molecules, cell biological objects, tissue fluid and water) products for use in cosmetology, having a similar physiological action to the initial substance, directed in the body mainly on the skin, hair, nails and oral mucosa.

[0012] The applicant has discovered a way of obtaining a cosmetic product, reproducing the properties of the biological substance, affecting in the body on molecular or cellular targets, physiological and metabolic processes localized mainly in the skin, hair, nails and oral mucosa, which is a biological product - iterations of a biological substance, prepared using external rhythmic effect (Fig. 1).

[0013] There are articles in the prior art relating to high dilutions. For example, the article 'Interaction between highly diluted samples, protein solutions and water in a controlled magnetic field' by V. V. Novikov was published in Applied Sciences (2022), 'Modifying distant effect of high dilutions of inorganic and biological substances' by E. S. Alinkina in the Bulletin of Experimental Biology and Medicine (Vol. 175, 2023); Jerman I. et al., 'Molecular signal transfer of highly diluted antibodies to interferon-gamma regarding kind, time, and distance of exposition', International Journal of Molecular Sciences (2024) and 'Antibodies processed using high dilution technology distantly change the structural properties of an IFN gamma aqueous solution', by N. Penkov, published in Pharmaceutics (2021). These articles describe a stock solution of a substance that is diluted multiple times (HD), and an experiment on the distant action of HD IFNg on IFNg. However, this is not relevant to the present invention, as the application discloses iterations that are prepared from a neutral carrier in the presence of a biological product. Another known article is “The Supramolecular Matrix Concept” by O. Epstein, Symmetry, 2023, describes ultrahigh dilutions (i.e. preparations obtained by successive reduction of concentration and shaking of solutions of various substances) and the probable mechanism of action of such preparations. It describes the so-called distant effect, which implies that ultrahigh dilutions can affect their targets from a distance. However, the above article does not contain information on the preparation and use of iterations of cosmetics objects.

[0014] The article by Stepanov, G. O. et al., 'The effect of aqueous solutions processed using gradual technology on the emission of a carbohydrate (lactose) in the RF range', published in Physics of Wave Phenomena (2024), reports on changes in the EMI intensity of lactose treated with HD antibodies to interferon gamma or HD antibodies to eNOs, compared with the control group. The mechanism of HD's remote action is investigated. However, the article does not provide information on the preparation of iterations for cosmetic purposes.

[0015] The article 'Specific features of the concentration dependence of impurities in condensed media' by I. A. Shcherbakov, published in Physics of Wave Phenomena (Allerton Press, Heidelberg, Vol. 28, 2020), investigates the relationship between impurity content in water and dilution degree during serial dilution. The publication also reports that shaking dilutions leads to structural changes at the molecular level. The article does not provide information on the preparation of iterations, much less for cosmetic purposes.

[0016] The article by Novikov, V. V., titled 'The Role of Water in the Effect of Weak Combined Magnetic Fields on the Production of Reactive Oxygen Species (ROS) by Neutrophils', published in Applied Science (Vol. 10, 2020), investigates how physical factors influence the modification of water properties to enhance ROS release by neutrophils. The article also indicates that using irradiated water dilutions leads to an increase in ROS release by neutrophils. However, the article does not contain information on the preparation of iterations derived from biological objects, especially for cosmetic purposes.

[0017] In his article 'Effect of Mechanical Shaking on the Physicochemical Properties of Aqueous Solutions', published in the International Journal of Molecular Sciences (Vol. 21, 2020), Gudkov S.V. investigates the chemiluminescence of water when it is shaken. The publication reports that physical action on water and aqueous solutions is a complex process leading to changes in the physicochemical properties of water. The article does not provide information on the preparation of iterations derived from biological objects, much less for cosmetic purposes. Thus, this source, taken alone, does not violate the requirement of novelty or inventive step. In their article, 'New Perspectives in Cell Communication: Bioelectromagnetic Interactions', published in Seminars in Cancer Biology (Vol. 21, 2011), Rossi et al. report the results of their study of cell signalling, including electromagnetic signalling. The research method involved morphometry of cells separated by a barrier that transmits electromagnetic interference (EMI). The paper reports on the importance of research of these properties of cells in the composition of tissues, including in the study of the mechanisms of oncology. The paper does not contain information on the preparation of iterations derived from biological objects, especially for cosmetic purposes.

[0018] M. E. Astashev et al., 'Influence of the vibration impact mode on the spontaneous chemiluminescence of aqueous protein solutions', Physics of Wave Phenomena, Vol. 31, 2023. This publication investigates the chemiluminescence of water and protein solutions when shaken or subjected to a magnetic field. The researchers report that modes with a frequency of 10 Hz and an amplitude of 12 mm, as well as with a frequency of 30 Hz and an amplitude of 2.3 mm cause the most pronounced changes in the chemiluminescence intensity of water. For protein solutions, the most pronounced effects were observed for modes with a frequency of 30 Hz and duration of 60 s. The dependence of the effect of alternating magnetic field on its frequency and on protein concentration was revealed. The most pronounced effects were found for a protein solution with a concentration of 1 mg / mL exposed to a field with a frequency of 50 Hz and an inductance of 50 pTL for 5 min. The results obtained should allow a better understanding of the fundamental mechanisms of the mutual influence of mechanical stress and magnetic field on the physical properties of water and dissolved proteins. From the practical point of view, the obtained results can be used to optimize the processes of obtaining biologically active substances for household needs. The article does not contain information on the preparation of iterations derived from biological objects, much less for cosmetic purposes.

[0019] Iteration preparation technology is fundamentally different from the technology used to prepare high dilutions. High dilutions involve a sequential reduction in the concentration of the starting substance by physical action on the solution. In contrast, iteration technology involves sequential vibratory action on a neutral carrier in the presence of a solution of the substance. These are two distinct technologies.

[0020] Therefore, the articles cited above are not relevant to the present application. Some articles provide information on the distance effects of products obtained using the high dilution preparation technique. However, this information relates to the properties of such preparations, whereas the presence of the substance in close proximity to a neutral carrier within the iterative preparation technology is a condition for its realization, which is in no way disclosed in the articles. Thus, the average person skilled in the art, having knowledge of the high dilution technology or the properties thereof, would not be able to implement the iteration technology.

[0021] The use of iterations from biological substances, liquid media or water allows to obtain accessible cosmetic and medical-cosmetic products with safe and physiological action.

[0022] A distinctive feature of the claimed method of obtaining biological products is the possibility of using biological molecules, integral cells and their structures (cell culture, tissues, organs) as an initial biological substance without diluting this biological substance. It is necessary in order to transfer into iterations the properties of biological molecules that retain the original conformational characteristics of cellular and biological objects that retain their morpho- functional integrity.

[0023] The iterations obtained in the course of successive vibration treatment of biological molecules or cellular objects have a biological (pharmacological) effect that partially or completely reproduces the biological effects of a biological substance. The range, direction and extent of action of different fractions of iterations of biological substances are different and can be established experimentally.

[0024] Furthermore, an obligatory part of the technical solution is the separation of the obtained iterations into fractions by evaluating their physical-chemical properties using generally accepted analytical methods and further determination of their biological (pharmacological) properties using also generally accepted methods for experimental biology

[0025] In this case, the method includes obtaining at the initial stage of ‘iteration zero’ by vibration treatment of tubes with the initial biological substance and solvent, which can be carried out both simultaneously and alternately, leading to the transformation of the solvent into a material object with new physical, chemical and biological properties different from both the properties of the initial substance and the solvent, and further sequential vibratory treatment of the previous iteration and the solvent to obtain subsequent iterations 1, 2, 3, etc. from the solvent.

[0026] By biological substance within the scope of the present invention is meant a biological molecule, cell, cell, groups of cells, cell associations or cell structures, fluid media located predominantly in the skin, hair, nails and oral mucosa or forming part of the tissue fluid of the skin or having a regulatory effect on functional and metabolic processes in the skin, hair, nails and oral mucosa - such as blood, lymph, water. Thus, molecules predominantly found in the skin and determining its structure and functional activity, or molecules determining specific functional, metabolic or immunological processes in the whole organism, including the skin, may be used as the initial biological substance used for obtaining the drug by the claimed method. Examples of molecules used as initial substances are the following: - Filaggrin. Iterations derived from it will affect both the protein itself in the body and have humectant effects: moisture saturation and retention in the skin, structuring the cytoskeleton of horny cells and stimulating epidermal lipid production, antimicrobial action; effect on the functional activity of enzymes involved in ceramide metabolism; modulate the activity of the serine protease cascade required for coordinated epidermal differentiation and keratinized cell envelope formation; photoprotective effect on the skin. As a consequence, filaggrin-based iterations have a therapeutic and preventive effect on atopic dermatitis.

[0027] - Involucrin (IVL). The iterations derived from it will provide mechanical strength of the epidermal barrier; influence the formation of a waterproof insoluble shell of epidermal stratum corneum cells; and affect type 1 transglutaminase (TGM1), which catalyzes the formation of cross-links between lysine residues in structural proteins of the epidermis.

[0028] - Elastin. Its derived iterations have an effect on the elasticity (ability to stretch and return to its original position) of the skin.

[0029] - Elastase. The iterations derived from it have an effect on wound healing.

[0030] - Collagen. The iterations derived from it have an effect on the tightening and firmness skin.

[0031] - Collagenase. Its derived iterations have an effect on the breakdown of collagen fibrils; affect wrinkles; have a therapeutic effect on festering wounds, scabs and break down dead tissue.

[0032] - Hyaluronidase. Iterations obtained on its basis have an effect on, cleavage of hyaluronic acid into monosaccharides; affect the thickness of the epidermis, architectonics of collagen fibers, reduction of synthesis of sulfated glycosaminoglycans by skin cells, water-ionic homeostasis.

[0033] - Squalene. Iterations obtained on its basis have antioxidant or pro-oxidant effect; protect the skin surface from lipid peroxidation due to UV radiation, ozone, etc.; protect linoleic acid, linolenic acid, docosahexaenoic acid and eicosapentaenoic acid from self-oxidation; moisturize the skin.

[0034] - Main Histocompatibility complex (MHC) class I and MHC class II. Iterations derived from them have protective, anti-inflammatory effects.

[0035] - Vanilloid receptor type 1 (TRPV1). Its derived iterations have antipruritic and analgesic effects, as well as reduction of neurogenic inflammation; they have therapeutic effects on the treatment of dermatitis.

[0036] - Insulin receptor. Iterations derived from it have an effect on skin repair during wound healing, an effect on keratinocyte survival, regulating inflammatory reactions (reducing, in particular, the number and activity of macrophages); resistance to sepsis and protein synthesis in the area of skin damage.

[0037] - Cannabinoid receptors type 1 (CB1R). Iterations derived from it have antipruritic and analgesic effects.

[0038] - Bradykinin. Its derived iterations have an effect on skin redness in rosacea; on microcirculation.

[0039] - Erythropoietin. Iterations obtained on its basis have a stimulating and anti-apoptotic effect in wound healing; increases proliferative and functional activity of human skin cells.

[0040] - Granulocyte colony-stimulating factor (G-CSF). Iterations derived from it have antiinflammatory effect, accelerates wound healing; affects collagen in the dermis adjacent to the wound, inflammatory responses and increase the expression of epidermal growth factor (EGF) and Vascular endothelial growth factor (VEGF) genes that promote wound healing.

[0041] Both a homogeneous set of cells and a combination thereof may be used as an initial biological substance for obtaining the claimed product. For example, both individually derived stem cells (undifferentiated cells capable of transforming into any of the cell types in the body, derived from embryos, fetuses, uterine fetuses, umbilical cord blood, bone marrow, induced pluripotent stem cells) and stem cells contained in fetal tissues commonly used as cell transplants for personalized biological regenerative medicine can be used.

[0042] Fetal or embryonic cells containing regional blast cells in addition to stem cells along with differentiated cells, specialized cells and bioactive substances can be used as a biological object. Due to this iteration of such cells (or tissues), can be used both as a cosmetic and medical-cosmetic products for the treatment of a wide range of diseases associated with the skin [RU2160112],

[0043] The cosmetic effect produced by the products obtained by the claimed method is an effect on the initial substance or their molecular or cellular targets in the body, as well as on functional and metabolic processes regulated by the initial substance.

[0044] Within the scope of the present invention, the cosmetic action provided by the resulting active ingredient may comprise a caring action (to maintain and improve the appearance of the skin - i.e. moisturizing, nourishing, repairing, and to delay signs of aging), a protective action (protection from harmful environmental effects such as sunlight, low or high temperatures, antioxidant action, prevention of photo-aging, irritation and other problems associated with negative environmental effects on the skin; therapeutic and preventive action (care and prevention or reduction of cosmetic skin defects such as acne, pigmentation, increased sensitivity, dryness or oiliness of the skin). The claimed product can be used as an active ingredient for creams, masks, serums, tonics and other forms of cosmetic skin care, therapeutic, preventive and protective products.

[0045] Since in some cases iterations of the same substance have multidirectional effects, it is possible to experimentally select the iterations that have the most pronounced cosmetic effect.

[0046] Biological molecules or biological objects with known biological properties are selected depending on which functional and metabolic processes of skin, hair, nails or which target molecules are supposed to be affected. This approach allows to use a wide range of experimental and clinical data on the activity of biological objects, which facilitates the development of new cosmetic products.

[0047] Cells in the form of an isolated cell or a group of cells may be used as biological objects, wherein the claimed product will affect the same functions performed by these cells in skin, hair, nails.

[0048] Both structurally normal cells and cell cultures can be used as a biological object. In some cases in order to weaken the activity of a biological object its homogenates or supernatants can be used.

[0049] Cell cultures grown outside the body may be used as the biological object.

[0050] Both a homogeneous set of cells and a combination thereof may be used as an initial substance for obtaining the claimed product.

[0051] The claimed invention provides a method for producing a biological product comprising the following steps:

[0052] 1) preparation of an artificial material object - ‘iteration zero’ obtained by external vibration treatment of the solvent in the presence of the initial biological molecule or cell biological object (with their further exposure - incubation);

[0053] 2) obtaining the first iteration by vibration treatment the intact solvent in the presence of ‘iteration zero’;

[0054] 3) obtaining a series of iterations, where each successive iteration is obtained by externally vibration treatment of the solvent in the presence of the previous iteration.

[0055] In the first step, a ‘iteration zero’ is obtained, into which the solvent is transformed by joint vibration treatment of the solvent and the initial artificial substance (with their further exposure - incubation). Then, by vibration treatment of the preceding iteration and the solvent from the solvent, subsequent iterations, the first, second, third, etc., are obtained.

[0056] The process of obtaining iterations may differ in terms of the time during which the external vibration treatment is applied, or incubation (exposure) - post-vibration contact of the substance (or iterations) with the solvent; the type of external rhythmic physical effect - horizontal or vertical mechanical treatment (shaking, rotation on a vortex), ultrasound, electromagnetic field, etc., as well as variants of external rhythmic influence in frequency and amplitude. Various solvents, such as water or a water-alcohol mixture, may also be used. The series of iterations may be derived from a single biological substance, or a combination thereof, such as an auto-, allo- or heterogeneous cell transplant containing different cell types.

[0057] The time of external vibration treatment and incubation (co-incubation of test tubes with intact solvent and iteration or initial biological substance at room temperature) depends on the amount of initial biological raw material - molecules or biological object, as well as solvent and is selected experimentally for each specific case. Test tubes or vials are placed side by side close to each other or at a distance, mostly 1-3 cm. Separate vibration treatment of substance (or iterations in the case of the preparing subsequent iterations) and solvent with their further incubation is also possible, when tubes with substance (or iterations in the case of the preparing subsequent iterations) and solvent are placed side by side for a time of 1 sec or more.

[0058] Iterations obtained by successive external vibration treatment on the test tubes with solvent and preceding iterations or initial substance (to obtain ‘iteration zero’) represent a biological product, as they are obtained from biological raw materials and their mechanisms of action are biological. They are aimed at biological targets in the body or in case of water iteration the drug exhibits non-specific action.

[0059] In contrast to individually applied biological products, such as cell transplants, biological cosmetic products obtained by the claimed method have a number of advantages: they can be characterized by relatively simple physical-chemical criteria and validated using generally available analytical methods. They can be stored for a long time, do not cause rejection reactions and other side effects, for example, there is no risk of viral or prion contamination.

[0060] It has been experimentally established that fractions of iterations with common physical, first of all spectral properties have common biological properties; therefore, the separation of iterations into fractions is a necessary technological step in the preparation of cosmetic products of this type. Further experimental determination of their activity should be done.

[0061] The method of separation into fractions according to the present invention, includes the following steps:

[0062] 1. For each iteration obtained, its physical and chemical properties are evaluated by known analytical methods, e.g. specific electrical conductivity, radiometry, pH, the amount of dissolved oxygen, dynamic light scattering, high-resolution thermography, immunoassay (ELISA) etc . 2. Depending on the evaluation, iterations are separated into fractions exhibiting their own physicochemical properties different from those of the initial neutral solvent;

[0063] 3. For each fraction, the intrinsic biological activity, which is similar to the activity of the biological object or biological molecules used as the initial substance, is determined experimentally.

[0064] Thus, knowing the properties of a biological substance, it is possible to anticipate what properties the obtained iterations (technological product) may exhibit and to study them using generally accepted methods.

[0065] The external vibration treatment used to obtain iterations means horizontal, vertical mechanical shaking or a combination thereof, as well as an external influence exerted by acoustic and microfluidics methods [RU2724254], electromagnetic, ultrasonic influence or other external rhythmic influence. The test tubes with solvent and substance (or the preceding iteration) may be subjected to joint (simultaneous) vibration treatment. Each of the test tubes could be independently of the other undergoes vibration treatment and in this case different types of external rhythmic influence may also be used.

[0066] Intrinsic physical-chemical properties mean the appearance of long-lasting physicalchemical properties in iterations that are quantitatively or qualitatively different from the physical -chemi cal properties of the initial substance or neutral solvent.

[0067] The neutral carrier can be either a solvent, whose transformation results in iterations: water, aqueous-alcoholic solution, or solids - lactose or any pharmaceutically acceptable solvent or excipient.

[0068] An intact carrier / solvent is understood to be a neutral carrier / solvent, until the moment of vibration treatment.

[0069] To obtain iterations, the initial biological substance may be used in a conditionally solid unaltered form or in the form of homogenate, lyophilizate, supernatant, solution or high dilution of homogenate in a specially selected solvent, in fresh or frozen form. The concentration of the initial biological substance in the tube can be from 0.1 pg / mL to 10 mg / mL and for each particular case the concentration is selected experimentally.

[0070] Iteration-based cosmetics are more commonly used in the traditional form of an ointment or cream, as well as parenteral and oral products, as well as spray, lotion, tonic, emulsion, serum, masks and other cosmetic forms.

[0071] Thus, for example, obtaining a cream using as an active ingredient a product obtained by the claimed method comprises preparing an iteration based on the desired biological substance in the form of an aqueous-alcoholic solution; preparing an oil phase comprising known excipients such as stearic acid, glycerol monostearate, vaseline oil, petroleum jelly, vaseline, cetearate-20 and the like and then emulsifying the above prepared aqueous and oil phases by active stirring followed by the addition of known preservatives and stabilizers, as well as, if necessary, fragrances and packaging.

[0072] Thus, for example, a product obtained by the claimed method can be used in a solid dosage form and contains a technologically necessary (effective) amount of neutral carrier saturated with iterations and pharmaceutically acceptable additives, which include, for example, lactose, microcrystalline cellulose, magnesium stearate and others. To obtain a solid oral form of the claimed medicinal product in a fluidized bed unit (e.g., of the ‘Huttlin Pilotlab’ type manufactured by Huttlin GmbH), irrigation is performed until the granules of a neutral substance, lactose (milk sugar), introduced into the fluidized-boiling bed are saturated with a previously obtained aqueous or aqueous alcohol solution of iterations with simultaneous drying in the flow of heated air supplied under the grate at a temperature not exceeding 40 °C. The resulting tablet mass is evenly mixed and tableted by direct dry pressing (e.g., in tablet press Korsch XL 400) [W02007105981(A1), 20.09.2007], After tableting, tablets weighing 300 mg and impregnated with aqueous or aqueous-alcoholic solution of iterations are obtained.

[0073] The figures below, together with the drawings attached, are offered to illustrate the present invention:

[0074] Fig. l. The scheme of the technology for obtaining iterations.

[0075] The production of an artificial product is hereinafter described in more detail with reference to the enclosed Figure 1, which shows the scheme of the technology for obtaining iterations:

[0076] The production of an artificial product (iterations) consists of several stages:

[0077] 1. A test tube with the initial substance and a test tube with a neutral carrier (water) are placed close together and subjected to joint vibrational treatment using a vortex.

[0078] 2. The test tubes are then incubated at room temperature. Empirically, a minimum incubation time of 1 minute has been established as sufficient for obtaining iterations. In some cases, the incubation time can be reduced or skipped if the transformation of the neutral solvent into a material object (iteration) has been experimentally confirmed.

[0079] After stages 1 and 2, a change occurs in the physical properties of the solvent in the first test tube compared to the intact solvent.

[0080] The test tube with the neutral carrier (solvent), altered by the first vibrational treatment is called the ‘iteration zero’ of the substance (10). 3. Then, 10 is placed close to a test tube with water (a new portion), and both test tubes are subjected to vibrational treatment and incubation at room temperature. As a result, the water in the second test tube also changes its properties and transforms into the first iteration of the substance (II).

[0081] 4. The described vibrational treatment procedure is repeated to obtain subsequent iterations from the previous ones — 12, 13,..., In iterations of the substance.

[0082] 5. Afterward, the stage of selecting the obtained iterations, which have separated into fractions after vibrational treatment.

[0083] Fractions that are potential candidates for cosmetic and medical-cosmetic products after vibrational treatment should differ in physical properties from the solvent. For practical purposes, it is sufficient to select candidates after obtaining a series of six iterations and choose two main groups that differ the most in physical properties, especially in their influence on the spectral characteristics of the initial substance. It has been empirically established that these fractions also differ the most in biological and chemical properties, allowing for the selection of the most suitable iteration for a specific task. It is sufficient to use only one iteration from the entire series of obtained iterations or to mix several iterations of one fraction together.

[0084] Given the methods we use, the first six iterations (11-16) can only be divided into four fractions based on their physical characteristics, the ratio of which may vary for each initial substance, or one of the fractions may be absent. Technical terms proposed for the fractions are native, seminative, semi-active, and active.

[0085] 1. Native - Iterations that, according to conductometry (C) and radiometry (GHz) results, do not have significant changes in physicochemical properties compared to the intact neutral carrier (water) and, according to terahertz (Thz) spectroscopy (effect on initial substance) and immunoassay (ELISA), do not possess modifying activity towards the initial substance, i.e., C-, GHz-, THz-, ELISA.

[0086] 2. Semi-native - Iterations in which statistically significant changes in physicochemical properties were found compared to the intact neutral carrier (water), but like natives, do not possess modifying activity towards the initial substance, i.e., C+ and / or GHz+, THz-, ELISA-.

[0087] 3. Semi-active - Iterations that do not have significant changes in physicochemical properties compared to the intact neutral carrier (water), but unlike natives, possess modifying activity towards the initial substance, i.e., C-, GHz-, THz+ and / or ELISA+. 4. Active - Iterations in which statistically significant differences in physicochemical properties were found compared to the intact neutral carrier (water) and which possess modifying activity towards the initial substance, i.e., C+ and / or GHz+, THz+ and / or ELISA+.

[0088] Usually, the choice is made between the semi-native and active fractions, which are tested on the same biological or pharmacological experimental models to determine their activity, which in varying degrees of different fractions of iterations reproduces the effects of the initial substance.

[0089] Example 1. Iterations obtained using elastin and water as a neutral carrier.

[0090] The process of obtaining iterations included several stages. A vial with a capacity of 60 ml with a freshly prepared elastin solution (5 mg / ml) as initial substance in 50 mM Tris-HCl buffer in a volume of 50 ml and a vial with a capacity of 250 ml with purified water (neutral carrier) in a volume of 180 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0091] Next, a 250 ml vial with Io (180 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0092] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0093] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0094] Each of the obtained iterations also has its own physical and chemical properties, determined in the following ways:

[0095] 1. Conductivity. To evaluate the physicochemical properties of a series of iterations, the conductometry method was used. To measure specific electrical conductivity (SEC), 10 ml of sample was taken into 15 ml vials and an electrode (Mettler Toledo, USA) was positioned so that the electrode membrane was completely immersed in the solution, after which the SEC was measured. In total, 16 iterations were obtained. Using the conductometry method, it was revealed that iterations No. 1, 2, 3, 6, 9, 10, 12, 16 have significant differences from the neutral carrier.

[0096] 2. Radiometry. The assessment of the iterations' intrinsic electromagnetic radiation was carried out using the radiometry method on a special installation, which consisted of a Faraday cage - an aluminum frame covered with a copper mesh. Inside the cage there was placed a tripod with a claw for attaching a detector, a thermal shaker (BIOSAN PST- 60HL-4) for heating the samples, and a TES-92 electromagnetic radiation (EMR) detector (TES Electrical Electronic Corp., Taiwan), which makes it possible to detect EMR flux density in the range from 1 pW / m2to 30.93 W / m2and frequency range from 50 MHz to 3.5 GHz. The measurement mode used was maximum average value (MAX AVG). The test sample (10 ml) was placed in a Petri dish, then the Petri dish with the sample was placed on a thermal shaker and the sample was heated to a temperature of 37±1°C. After this, the detector was placed above the sample at a distance of 0.5 cm, and the lid of the Petri dish was removed. The measurements were carried out with a closed Faraday cage. The measurement time was 10 minutes. After the time had elapsed, the maximum average value of the EMR flux density from the device display was recorded. Using the radiometric method, it was revealed that iterations No. 1, 2, 3, 6, 9, 10, 12, 16 have significant differences from neutral carrier.

[0097] 3. Terahertz spectroscopy. Analysis of changes in spectral characteristics in the terahertz region of the target molecule after adding iterations was carried out using a TeraView TeraPulse Lx terahertz spectrometer (England). To do this, 1 part (5 pL) of the iterations or control was added to 99 parts (495 pl) of a solution of the substance, which is a specific target for the iterations under study, and the refractive and transmission spectra were recorded. To analyze the data, the coefficients of the equation of the well-known Debye function that describes the resulting spectra (Penkov N., Fesenko E. Development of terahertz time-domain spectroscopy for properties analysis of highly diluted antibodies. Appl. Sci. 2020; 10:7736. doi: 10.3390 / appl0217736.) and characterizes the dielectric constant in relaxation regions was used. Using terahertz spectroscopy, it was revealed that iterations No. 4, 5, 6, 9, 12, 13, 15 have significant differences from the neutral carrier in the value of the coefficients Asl (amplitude of the relaxation process Rl) or As2 (amplitude of the relaxation process R2).

[0098] 4. Enzyme-linked immunosorbent assay (ELISA). Also, the modifying properties of iterations were studied using ELISA. To do this, the studied iterations were added to the antigen - the target molecule, and the modifying activity was indirectly assessed by changes in the degree of its binding to monoclonal antibodies depending on their concentration. Using the ELISA method, it was revealed that iterations No. 4, 5, 6, 9, 12 have significant differences from the neutral carrier in terms of the curve of dependence of optical density on the concentration of monoclonal antibodies to elastin after their interaction with elastin (1 pg / ml) in the presence of samples.

[0099] Thus, using the above methods, the obtained iterations with numbers from 1 to 16 (Ii- Iie) were divided into the following fractions: having altered physicochemical properties and exhibiting a modifying effect (E, I9, I12); having altered physicochemical properties and not exhibiting a modifying effect (Ii, b, I3, I10, lie); not having altered physicochemical properties, but exhibiting a modifying effect (I4, I5, I13, I15); not having altered physicochemical properties and not exhibiting a modifying effect (I7, Is, In, I14).

[0100] Since it is known that elastin is a protein produced by fibroblasts that is responsible for the elasticity (the ability to stretch and return to its original position) of the skin, the resulting iterations may exhibit specific activity in relation to the biomechanical parameters of the skin. The assessment of the specific activity is currently being carried out on an in vitro human skin model.

[0101] Example 2. Iterations obtained using fibroblast cells and water as a neutral carrier.

[0102] With aging, the number of skin fibroblasts in which active synthesis of type I collagen occurs decreases. Iterations may exhibit specific activity in relation to the expression of markers of aging of human skin fibroblasts, in particular, type I collagen and sirtuin-6 protein (SIRT6), which is a critical regulator of transcription, genome stability, telomere length, DNA repair, and cellular homeostasis.

[0103] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (suspension of fibroblast cells, 5xl04cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0104] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named h. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0105] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0106] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0107] In this study In (iteration No. 11) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0108] Fibroblasts were isolated from the skin of the parotid region obtained during circumferential facelift surgery in a woman (bom 1987). The skin sample was treated under sterile conditions with a solution of dispase II (2.4 U / ml) for 18 hours at 4 °C. Then the epidermis was mechanically separated from the dermis. To obtain a cell suspension, the dermis was cut with scissors into 2-3 mm pieces and placed in type I collagenase solution in M199 medium. Cells were pelleted at 1000 rpm for 5 min and resuspended in complete cell culture medium (M199 medium, 10% fetal bovine serum, 1% L-glutamine, 1.5% Hepes buffer, penicillin and streptomycin). After 5 days, the monolayer of primary cells was formed; the cells were trypsinized and subcultured at a ratio of 1 :3. The cell concentration for the zero passage was 50,000 cells per 1 ml of complete medium in one well of a 24-well plate (sterile, with an adhesive surface, CellATTACH, Jet Biofil). Passaging was carried out after 3 days on the 4th, when the culture reached a monolayer. Cells of the 3rd and 14th passages were divided into three groups: 1st - control (intact cells); 2nd - cultures with the addition of Sample 2 (control, purified water) in a volume of 100 pl; 3rd - cultures with the addition of Sample 1 (iterations of fibroblast) in a volume of 100 pl. In accordance with the recommendation of the International Association for Cell Culture Research (San Francisco, 2007), the 3rd passage culture was regarded as “young”, and the 14th as “old”.

[0109] Immunofluorescent staining of the cells was performed using primary monoclonal antibodies to type I collagen (1 : 100, Abeam, USA) and sirtuin-6 (1 :200, Abeam, USA). Confocal microscopy was carried out using an Olympus Fluoview CM FV300-IX70 inverted confocal microscope with a 488 nm argon laser. Cell nuclei were counterstained with Hoechst 33258 (Sigma). Secondary antibodies were conjugated with Alexa Fluor 488 (1 : 1000; Abeam). For image analysis, VideoTest Morphology v. 5.2 software was used. In each case, five fields of view were analyzed at a magnification of 200x. The expression area was calculated as the ratio of the area occupied by immunopositive cells to the total area of cells in the field of view and expressed as a percentage. This parameter characterizes the number of cells in which the marker under study is expressed. Also, the fluorescence intensity of staining, reflecting aging marker expression level in one cell, was assessed in arbitrary units (a.u.).

[0110] Statistical analysis was performed using Statistica 6.0 software. The Shapiro-Wilk test was used to analyze the type of distribution. To test the statistical homogeneity of several samples, the Kruskal -Wallis one-way ANOVA nonparametric test was used. In cases where analysis of variance revealed statistically significant heterogeneity in several samples, multiple comparison procedures using the Mann-Whitney U test were used to subsequently identify heterogeneous groups (by pairwise comparisons). Differences between groups were considered statistically significant at p<0.05.

[0111] The results of the study are presented in Tables 1 and 2. Table 1. Effect of the test samples on expression area of aging markers in skin fibroblasts (M±m)

[0112] Note: * - differences are significant compared to Sample 2 (control, purified water) in the corresponding culture, # - differences are significant compared to intact cell group in “young” cultures

[0113] Table 2. Effect of the test samples on fluorescent intensity of staining of aging markers in skin fibroblasts (M±m)

[0114] Note: * - differences are significant compared to Sample 2 (control, purified water) in the corresponding culture, # - differences are significant compared to intact cell group in “young” cultures

[0115] It was shown that the area of expression of type I collagen in “old” fibroblast cultures was 3 times smaller than in “young” cultures. At the same time, the fluorescent intensity of type I collagen staining in “young” and “old” cultures of skin fibroblasts did not differ significantly. The data indicate that with aging, the number of skin fibroblasts, in which active synthesis of type I collagen occurs, decreases. Sample 1 significantly increased the area of type I collagen expression in “old” fibroblast cultures, but did not affect this indicator in “young” cultures. At the same time, Sample 1 increased the fluorescent intensity of type 1 collagen staining in “young” and “old” fibroblast cultures. The area of sirtuin-6 expression in “old” fibroblast cultures was 3.2 times smaller than in “young” cultures. The same trend was observed for the fluorescence intensity. The fluorescence intensity of sirtuin-6 staining in “old” cultures of skin fibroblasts was 2.8 times lower than in “young” cells. The data indicate that with aging, the number of skin fibroblasts in which sirtuin-6 is synthesized decreased. Sample 1 statistically significantly increased the area and the level of sirtuin-6 expression in “young” and “old” fibroblast cultures.

[0116] Thus, it has been proven that Sample 1 (iterations of fibroblast) exhibits specific activity regarding the functional activity of skin fibroblasts and slows down their aging.

[0117] Example 3. Iterations obtained using skin explants and water as a neutral carrier.

[0118] When any damaging factors act on the skin, the blood supply to its various layers is disrupted, starting from the epidermis, and this, in turn, leads to the development of oxidative stress in the skin tissues, aggravating the pathological process. Iterations can exhibit specific activity in relation to indicators of the skin's antioxidant system.

[0119] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (skin explants of dorsal skin of outbred female mice, 20 mg) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 2 minutes at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0120] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 20 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2 and No. 3) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0121] For each study, iterations were prepared by one operator on one day. Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used. Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0122] In this study E (iteration No. 3) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0123] The study was conducted on an excisional skin wound model in outbred female mice of two age groups: “young” (2.5 months old, weighing 17-19 g, n=30), and “old” (18 months old, weighing 25-28 g, n=30). Each age group of animals was divided into 3 subgroups of 10 animals each. One subgroup (subgroup 1) remained intact. In the rest of the animals, wounds were inflicted on the dorsal skin, followed by treatment either with Sample 1 (iterations of skin explants) (subgroup 2) or Sample 2 (control, purified water) (subgroup 3). To inflict excisional wounds, symmetrical areas of the dorsal skin with an area of 1 cm2were shaved. The animals were anesthetized with ketamine at a dose of 0.5 mg / kg. Surface damage was applied to the shaved areas using a scarifier. After damage, Sample 1 or Sample 2 was applied to areas located on the right side of the animal’s body, once daily for 7 days. The areas on the left side were used as controls and healed naturally. 2 weeks after mechanical damage was applied, the animals were decapitated, and pieces of damaged skin weighing 200-300 mg were harvested and stored at - 80°C until used for further analysis.

[0124] For biochemical analysis, skin tissue was homogenized in 50 mM potassium phosphate buffer, pH 7.4. The content of TBA-active products (TBCAP), reduced glutathione (GSH), activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and catalase (CAT)) were determined. When determining TBCAP, a mixture of 0.375% thiobarbituric acid (TBA) and 15% trichloroacetic acid at a ratio of 9: 1 was added to the samples. The mixture was heated at 95°C and, after cooling, centrifuged for 10 min at 13,000 rpm. TBCAP content was determined spectrophotometrically at 335 nm. To determine the glutathione content, 2 ml of a solution containing Ellman's reagent (5,5'-dithiobis- 2 nitrobenzoic acid) and 0.8 M Tris buffer, pH 8.9. Samples were analyzed using spectrophotometry after 4 min at 412 nm. SOD activity was measured using an indirect spectrophotometric method based on the generation of superoxide radicals in a mixture consisting of reduced nicotinamide adenine nucleotide (NADH), phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). To do this, 0.45 ml of H2O, 0.125 ml of chloroform, and 30 mg of dry K2HPO4 were added to 0.05 ml of the homogenate and, after thorough mixing, the samples were centrifuged for 30 min at 4000 rpm. Then, 2 ml of 50 mM K+-phosphate buffer, 0.1 mM EDTA, 60 pM NBT and 100 pM NADH was added to 0.01 ml of the resulting homogenate. The reaction was started by adding FMS (30 pM), after 5 min absorbance at 560 nm was measured, and enzyme activity was expressed in U / g of protein. Glutathione peroxidase activity was determined by the modified Tappel method. The reaction mixture consisted of 0.39 mM glutathione, 0.19 mM NADH, 1.55 U / ml glutathione reductase and a buffer containing 50 mM Tris pH 7.6 and 0.1 mM EDTA. Then 0.01 ml of supernatant used to determine SOD activity was added to the sample, and the reaction was started by adding 0.1% H2O2. Enzyme activity was determined spectrophotometrically at 340 nm and expressed in mU / g of protein. Protein concentration was determined according to Lowry.

[0125] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0126] For statistical analysis, a one-way analysis of variance with Tukey's post-hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0127] The results of the study are presented in Tables 3 and 4.

[0128] Table 3. Effects of the test samples on indicators of the antioxidant system activity after mechanical damage to the skin in young animals (M±m).

[0129] Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to intact animal group and Sample 1. Table 4. Effects of the test samples on indicators of the antioxidant system activity after mechanical damage to the skin in old animals (M±m)

[0130] Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to intact animal group and Sample 1.

[0131] The data demonstrates that skin damage causes oxidative stress due to increased peroxidation processes. Application of Sample 1 to both young and old animals restored the activity of antioxidant enzymes (SOD and GTP) and the level of reduced glutathione (GSH) in the skin. Also, Sample 1 restored the activity of glutathione-S-transferase (GST) and reduced the intensity of lipid peroxidation, which is indicated by the normalization of the level of TBA- active products (TBCAP). Thus, the activity of Sample 1 indicates the normalization of the process of eliminating toxic products of free radical oxidation in the skin.

[0132] Thus, it can be concluded that Sample 1 (iterations of skin explants) exhibits specific activity in relation to the indicators of the antioxidant system of the skin. This is expressed by the ability of Sample 1 to prevent the development of oxidative stress when the skin is exposed to damaging factors.

[0133] Example 4. Iterations obtained using involucrin and water as a neutral carrier.

[0134] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (involucrin, 5 mg) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0135] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 3 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0136] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0137] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0138] In this study mixture of I? (iteration No. 7) and I23 (iteration No. 23) (hereinafter referred to as Sample 1) was used. The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0139] Since involucrin is known to be one of the skin barrier proteins synthesized by keratinocytes, the resulting iterations may exhibit specific activity in relation to biomechanical skin properties as well as the integrity of the skin barrier function.

[0140] The study was conducted on hairless SKH-1 male mice (4 weeks old, n=30), following exposure to lewisite vapors for 21 days (model of lewisite-induced skin damage). Animals were divided into 3 groups (n=10 each). One group remained intact. In the rest of the animals, lewisite-induced skin damage was modeled, followed by treatment either with Sample 1 (mixture of iterations of involucrin) or Sample 2 (control, purified water).

[0141] Prior to lewisite exposure, the mice were anesthetized with a mixture of ketamine hydrochloride (33.3 mg / kg, i.p.) and diazepam (37.5 mg / kg, i.p.) and for pain control were subcutaneously injected with buprenorphine (0.05 mg / kg). The occluded vapor cup technique 1 was used to expose the skin (0.5 cm2) to lewisite- saturated vapors (10 pL of neat liquid L for a period of 8 min) and to achieve deep dermal burns. On the dorsal skin surface on each animal, three sites were exposed to lewisite and the remaining site served as a control.

[0142] Biomechanical skin properties were estimated by assessment of the skin elasticity at 0, 14, 17, and 21 days of the experiment with a suction extensometer (Cutometer SEM 580, Courage + Khazaka, Cologne, Germany). The following parameters were measured: Ua / Uf (R2, gross elasticity), Ur / Ue (R5, elastic function), Uv / Ue (R6, visco-elastic ratio), and Ur / Uf (R7, elastic recovery). The skin barrier integrity was assessed by the measurement of transepidermal water loss (TEWL in g / m2 / h) using an evaporimeter (Aquaflux, Biox System, London, United Kingdom) at the same days of the experiment (DO, D14, D17, and D21).

[0143] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0144] For statistical analysis, a two-way repeated measures ANOVA with Tukey's post-hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0145] The results of the study are presented in Tables 5 and 6.

[0146] Table 5. The effect of test samples on the skin biomechanical properties in SKH-1 hairless mice following exposure to lewisite vapors (M±SD) Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to intact animal group and Sample 1

[0147] Table 6. The effect of test samples on the transepidermal water loss (TEWL, g / m2 / h) in SKH-1 hairless mice following exposure to lewisite vapors (M±SD)

[0148] Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to intact animal group and Sample 1

[0149] The data demonstrates that before the lewisite challenge (DO), the values for each R parameter were similar in all the skin sites. Following lewisite exposure, in Sample 2 group the values for three elasticity parameters (R2, R5, and R7) differed from the control sites. The elastic function (R5) and the biological elasticity (R7) values showed a marked increase at D14, whereas the gross elasticity levels (R2) were significantly higher at D14 and D17. By D21, all three ratios had returned to basal values. The measurements of TEWL values revealed that in Sample 2 group loss went up from DO to D14 after lewisite exposure, suggesting a skin barrier disruption during skin necrosis. TEWL values remained stable up to D21. The activity of Sample 1 indicates the normalization of all estimated parameters.

[0150] Thus, it can be concluded that Sample 1 (mixture of iterations of involucrin) exhibits specific activity in relation to the indicators of biomechanical skin properties and the integrity of the skin barrier function.

[0151] Example 5. Iterations obtained using collagen and water as a neutral carrier.

[0152] The iterations were produced using the technology for obtaining iterations. The distribution of the obtained iterations into fractions was made as described in the Example 1. The regenerative properties of iteration of collagen (iteration No. 2) were studied in a prospective, randomized, double-blind, placebo-controlled, multicenter study in which patients were observed after elective surgical interventions. Thus, in the study group, patients in the postoperative period with a well-established skin suture were prescribed a cream containing the iteration of collagen component on days 4-5. The cream was applied in a thin layer, once a day, and recovery results were assessed on days 5, 20, and 40. In the control group, some patients did not use anti-scarring drugs, and some used creams and ointments containing both enzymatic (hyaluronidase, lidase, and ronidase) and non-enzymatic active components, such as allantoin and niacinamide. According to the results of observations, patients from the study group (iteration of collagen) showed a clear tendency towards better and faster postoperative skin recovery.

[0153] Researchers have noted positive clinical effects at the stages of inflammation, proliferation and epithelization during the healing of postoperative wounds. Significant results include a decrease in the intensity and duration of local inflammatory signs, rapid and painless resolution of the scab, complete and uniform scarring of the postoperative wound, and early epithelization. There were no cases of excessive growth of granulation tissue and healing by secondary intention during the observation period in the study group. In the control group, 35% of patients showed excessive growth of granulation tissue, and in 28%, healing of postoperative wounds occurred by secondary intention. On the fortieth day of observation, 100% of patients in the study group and 67% of patients in the control group showed complete wound healing. The improvement in this parameter in the study group (iteration of collagen) was 49.2 relative percent. During observation, no adverse reactions or other complaints from the patient about the treatment were noted in both groups. The researchers concluded that the use of cream containing the iteration of collagen in the postoperative recovery protocol significantly improved clinical outcomes and patient satisfaction with the overall treatment.

[0154] The improvement in the healing of postoperative wounds in the study group (iteration of collagen) are explained by the ability to stimulate cellular regeneration, suppress the formation of keloid fibroblasts, prevent the proliferation of granulation tissue, and immunomodulatory effects.

[0155] Example 6. Evaluation of human dermal fibroblasts survival in culture after shortterm exposure to UV in the presence of vibrational iterations of UV-treated water

[0156] Materials and methods

[0157] Initial substance

[0158] The initial substance is ultrapure water treated with UV radiation. UV cabinet (LEDVANCE TIBERA UVC 15W G13, 100-280 nm) was used for UV treatment (duration of treatment: 2 minutes).

[0159] Preparation of vibrational iterations

[0160] The initial substance and intact ultrapure water (neutral carrier) were used for preparing vibrational iterations according technology described in Example 1. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier in a volume of 200 mL in close contact with the bottle made of transparent borosilicate glass (250 mL) with the initial substance (200 mL) was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200 mL of vibrational iteration zero was obtained from neutral carrier (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 17) were obtained from each previous one. Samples from 10 to 17 comprised a row of vibrational iterations. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (Type 1) with a resistivity of 18.2 MQ- cm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of the vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0161] In this work, vibrational iterations were analyzed using intact water as a control, classified into 4 groups (fractions) by their unique physico-chemical properties, as well as by their ability to influence physico-chemical properties of intact water (the so-called "modifying effect") (Table 7). These 4 types of fractions were named "Active", "Native", "Semi-native", and "Semi-active".

[0162] Table 7.

[0163] Note: the test result was considered positive (“+”) if it met the acceptance criteria. Otherwise, the result was taken as negative The acceptance criteria: the values obtained for vibrational iterations should statistically significantly (p < 0.05) differ from those of intact water by ±5% or more (by conductometry and THz spectroscopy) and by +10% or more (by radiometry). The values obtained for intact water were taken as 100%.

[0164] The following vibrational iterations belonging to different fractions were selected as test samples (Table 8).

[0165] Table 8

[0166] Cell lines

[0167] Immortalized human dermal fibroblasts (hTERT-HDFa-d220) were obtained from the Institute of Biological Research of the Russian Academy of Sciences. Method of colony formation: 2D cell culture as a monolayer.

[0168] Scheme of the experiment

[0169] To evaluate the specific activity of the test samples 01-04, cells were seeded into 96- well culture plates and incubated for 24 hours at 37 °C in a humidified atmosphere containing 5% CO2. Following incubation, the test samples are added to the wells. The final sample-to- medium ratio in each well was 1 :5 (v / v), the total volume per well was 100 pl, and 6 wells were used for each sample. The cells are incubated with the samples for 48 hours. Next, UV exposure was carried out on cells incubated with the test samples (with the lid open, duration of treatment: 2 minutes). Untreated cells served as the negative control. Cell survival was assessed using a colorimetric MTT assay.

[0170] Conducting MTT test

[0171] MTT solution (10 pl) with a concentration of 5 mg / mL was added into each well (except edge wells), after which the plates were incubated for 2 h at 37 °C, 5% CO2. Next, the culture medium was carefully removed from the wells, and 100 pl of DMSO was added to dissolve the formed formazan crystals, after which the plates were incubated at room temperature for 10 min. After shaking, plate for 2 min, the absorbance values were measured using Multiscan GO at wavelengths of 554 nm and 700 nm.

[0172] Statistical analysis

[0173] For statistical processing of the data, the RStudio 2024.12.1+563 software was used. The Shapiro-Wilk test was used to assess the normality of the distribution of data obtained in repetitions. The Student t-test was used to compare data between groups. Differences between groups were considered statistically significant at p < 0.01.

[0174] Results

[0175] The effect of test samples 01-04 on the survival of fibroblasts after exposure to UV for 2 min is presented in Table 9.

[0176] Table 9

[0177] * Statistically significant difference from intact water (Sample 01) (p < 0.01).

[0178] &Fibroblast’s survival relative to UV untreated fibroblasts, %.

[0179] In the presence of Sample 03 (Active fraction of vibrational iterations of UV-treated water (15)), the survival rate of fibroblasts after UV exposure was statistically significantly increased by 25% relative to the survival rate of fibroblasts after UV exposure in the presence of Sample 01 (Intact water, control).

[0180] Conclusion

[0181] Vibrational iterations of UV-treated water have the property of increasing the survival of fibroblasts when the cells are exposed to UV.

[0182] Example 7. Evaluating of the efficacy and safety of the claimed cosmetic product for skin care.

[0183] The tested cream was a formulation containing, an Native fraction of artificial material object - iterations, produced by sequential vibration treatment of a neutral carrier - purified water in the presence of a biological substance - water (13, 17, Il l - selected after determination physical -chemi cal and specific biological properties using methods described in Example 1) and, as excipients, water (technologically processed aqueous matrix), carbomer, glycerin, stearic acid, glyceryl monostearate, petrolatum, triethanolamine, ceteareth-20, Euxyl K 9010, and ethanol.

[0184] A total of 544 women aged 18 years and older (see Table 10) with various skin types (oily, combination (mixed), normal, dry) were included in the study. The distribution of participants by skin type is presented in Table 11.

[0185] Table 10. The distribution of participants by age.

[0186] Table 11. The distribution of participants by skin type. Participants applied the cream twice daily for one month. The cream was primarily applied to the face (95% of participants), hands (47.8%), body (7.7%), and neck (3.1%). Less than 1% of participants applied the cream to the decollete and lips.

[0187] The effect was assessed after 4 weeks based on a questionnaire in which participants answered “yes / no” to the following questions:

[0188] 1. Has the skin become more hydrated?

[0189] 2. Has the skin become smoother?

[0190] 3. Has the skin become firmer (more elastic)?

[0191] 4. Does the skin look better cared for, improved in appearance?

[0192] 5. Does the cream effectively restore (regenerate) the skin?

[0193] After four weeks of treatment, the majority of participants (96.5%) reported a moisturizing effect of the cream. 88.8% and 78.7% of participants noted increased smoothness and firmness of the skin, respectively. 80.1% observed a restoring (regenerating) effect of the cream-fluid (see Table 12). Nearly all participants (93.2%) confirmed a visible improvement in the skin, stating that “the skin looks better and more cared for.”

[0194] Table 12. Results of using the cream

[0195] No adverse events were recorded during the 4-week application period. Only 3 out of 544 participants (0.55%) reported no effect from using the cream. Thus, the study demonstrated the efficacy of the cream for the care of various skin types. The moisturizing, restoring, regenerating, and rejuvenating effects of the cream were confirmed.

[0196] Example 8. The treatment of atopic dermatitis.

[0197] The tested cream was a formulation containing, an Native fraction of artificial material object - iterations, produced by sequential vibration treatment of a neutral carrier - purified water in the presence of a biological substance - water (II, 15, 17 - selected after determination physical -chemi cal and specific biological properties using methods described in Example 1) and, as excipients: carbomer, glycerin, stearic acid, glyceryl monostearate, petrolatum, triethanolamine, ceteareth-20, Euxyl® PE 9010, and ethanol.

[0198] The aim of the study was to evaluate the efficacy and safety of claimed cosmetic product in the form of a cream in young children with AD.

[0199] Materials and Methods

[0200] Design: Double-blind, placebo-controlled, randomized study.

[0201] The study included children aged 6 months to 12 years with a confirmed diagnosis of atopic dermatitis (ICD-10 codes: L20. Atopic dermatitis; L20.8 — Other atopic dermatitis; L20.9 — Atopic dermatitis, unspecified), with mild to moderate disease severity.

[0202] Patients with any infectious complications of the underlying diagnosis, immunodeficiency conditions (Wiskott-Aldrich syndrome, hyperimmunoglobulinemia E syndrome, etc.), or those receiving hormonal drug therapy were excluded from the study.

[0203] Enrolled patients were randomized into two groups:

[0204] Cream group - 40 subjects.

[0205] Placebo group - 20 subjects.

[0206] Patients in the first group received therapy with the cream containing iterations, while patients in the second group received a cream containing distilled water. The organoleptic properties of the cream and the composition of excipients were identical in both groups. Neither the patients nor the physicians knew which preparation was being administered.

[0207] No additional drug therapy was provided in either group. In case of condition deterioration, the patient was to be withdrawn from the study and transferred to standard therapy.

[0208] The treatment duration was 14 days. During this period, the patient had five visits to the physician on days 1, 3, 5, 7, and 14. At each visit, the physician conducted an objective examination, assessed general and local symptoms of the main disease, and evaluated the safety of the cream. The following parameters were used for objective assessment of symptoms: intensity of itching / pain (very severe, severe, moderate, mild, none), severity of skin lesions (qualitatively: bright, moderate, pale, none), sleep disturbances (severe, moderate, none), and overall assessment of dermatitis course (negative dynamics, no dynamics, positive dynamics).

[0209] The efficacy of treatment was compared between the main and placebo groups by the degree of reduction in clinical manifestations of the disease, improvement in patients’ quality of life, speed of skin recovery, and number of infectious complications.

[0210] Results

[0211] A total of 60 children were enrolled in the study: 40 in the main group and 20 in the placebo group. The number of boys and girls did not differ between groups — 19 (47.5%) / 21 (52.5%) in the main group, and 11 (55.0%) / 9 (45.0%) in the placebo group. The age distribution of study participants is presented in Table 13.

[0212] Table 13. Age distribution of study participants

[0213] During therapy, the intensity of itching / pain decreased in both groups. More pronounced dynamics were observed in the Cream group. By day 14, the differences between the groups reached marginal statistical significance (P -value = 0.0531) (Table 14).

[0214] Table 14. Dynamics of itching / pain intensity. Post-visit comparisons

[0215] On days 3 and 14, a statistically significant predominance of regression in the severity of skin lesions was observed in the Cream group. By day 14, skin lesions were absent in more than half of the children in the Cream group, compared to only one child (5.9%) in the placebo group (Table 15).

[0216] Table 15. Severity of skin lesions. Post-visit comparisons

[0217] By day 7, the severity of sleep disturbances was significantly lower in the Cream group — 10.0% vs. 41.2%.

[0218] The physician’s assessment of dermatitis progression showed a predominance of positive dynamics over negative dynamics in the Cream group. The differences compared to the placebo group were statistically significant. By day 14, almost all patients (94.9%) in the Cream group were assessed by the physician as having positive dynamics, compared to only 58.8% in the placebo group. The remaining patients were classified as having no change or negative dynamics (Table 16).

[0219] Table 16. Overall assessment of dermatitis course. Post-visit comparisons

[0220] Adverse events, including those associated with worsening of atopic dermatitis, predominated in the placebo group. Overall, adverse events were reported in 15% of patients in the placebo group (infectious complications, worsening of AD), and in 2.5% of patients in the Cream group (worsening of AD).

[0221] Thus, the study demonstrated the efficacy and safety of the Cream for the treatment of atopic dermatitis in children.

[0222] Example 9. In vitro study of the effect of vibrational iterations of ultrapure water on release and permeation kinetics of caffeine from emulsion

[0223] Materials and methods

[0224] Materials

[0225] Normal rabbit skin was provided by the Shumakov National Medical Research Center of Transplantology and Artificial Organs. The test was performed using a Copley’s compact HDT 1000 test system.

[0226] Preparation of vibrational iterations

[0227] The vibrational iterations were prepared using the technology described in the Example 1. As the initial substance was used ultrapure water and as the neutral carrier - ultrapure water. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier (ultrapure water) in a volume of 200 mL in close contact with the bottle made of transparent borosilicate glass (250 mL) with the initial substance was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200 mL of vibrational iteration zero was obtained from neutral carrier (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 17) were obtained from each previous one. Samples from 10 to 17 comprised a row of vibrational iterations. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (type 1) with a resistivity of 18.2 MQx -cm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0228] In this study, two samples were examined (Table 17).

[0229] Table 17.

[0230] Methods of analysis

[0231] A drug release study was performed using the Franz diffusion cell technique. Freshly excised normal rabbit skin was employed as a membrane, representing a biological barrier between donor and receptor chambers. The emulsion-loaded chamber was placed on top of the receptor chamber. The receptor chamber and membrane were removed after a certain time period, and the concentration of caffeine was evaluated using HPLC quantitative analysis. Multiple time endpoints were implemented to allow evaluation of release kinetics (15, 30, 60 minutes, 3 hours, and 6 hours).

[0232] Intact water served as a negative control. The chemical skin permeation enhancer (dimethyl sulfoxide (DMSO)) served as a positive control.

[0233] Permeation aid (PA) were calculated using the following formula:

[0234] PA (%) = [Cs - Cv / Cv] x 100%

[0235] - Cs is concentration of caffeine released by emulsion containing vibrational iteration sample or positive control

[0236] - Cv is concentration of caffeine released by emulsion containing negative control

[0237] Statistical analysis

[0238] Statistical data processing was carried out in the R Studio program (2023.09.1 Build 494 © 2009-2023, R Foundation for Statistical Computing, Vienna, Austria) using the R package version 4.2.2. The data were tested for normality of the distribution using the Shapiro-Wilk test and for homogeneity of the variances by the Bartlett test. The groups were compared using the Student / Welch t-test with Holm’s correction for multiple comparisons. The differences between the groups were considered significant at p < 0.05.

[0239] Results

[0240] The results of the study are presented in the table below.

[0241] Table 18.

[0242] * Statistically significant differences compared to the negative control (p<0.05)

[0243] Conclusions

[0244] Studied vibrational iterations of ultrapure water enhance the permeation of caffeine through skin. The effect of the Native fraction of vibrational iterations of ultrapure water (14) was the most pronounced. The use of this sample led to a statistically significant (compared to the negative control) enhancement of the permeation of caffeine.

[0245] Example 10. Hydration effects of the vibrational iterations of water on skin microstructure.

[0246] Purpose of the study

[0247] The aim of the study was to investigate the effect of cream with vibrational iterations of water as an active ingredient on human skin explants ex vivo compared to reference cream.

[0248] Materials and methods

[0249] The following parameters were evaluated during the experiment:

[0250] Cell viability;

[0251] Genomic changes on days 3 and 8 of the experiment;

[0252] Expression of key skin proteins: filaggrin, involucrin, S100A7, CK2, IL-1 beta, and dermcidin.

[0253] Preparation of vibrational iterations

[0254] The vibrational iterations were prepared using the technology described in the Example 1. As the initial substance was used ultrapure water and as the neutral carrier - ultrapure water. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier in a volume of 200 mL in close contact with the bottle made of transparent borosilicate glass (250 mL) with the initial substance was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200 mL of vibrational iteration zero was obtained from neutral carrier (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 17) were obtained from each previous one. Samples from 10 to 17 comprised a row of vibrational iterations. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (type 1) with a resistivity of 18.2 MQx-cm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0255] Vibrational iterations were analyzed using intact ultrapure water as a control. Then they were classified into four groups (fractions) by their unique physico-chemical properties and modifying effect (Table 19). These four types of fractions were named "Active", "Native", "Semi-Native", and "Semi- Active".

[0256] Table 19.

[0257] Note: the test result was considered positive (“+”) if it met the acceptance criteria. Otherwise, the result was taken as negative (“-”).

[0258] In this study, the following vibrational iteration was examined (Table 20).

[0259] Table 20.

[0260] Studied samples

[0261] Sample 1 is the control sample, which is a reference cream with intact water (placebo):

[0262] Intact water, water, acrylates / cosporyl alkyl acrylate C10-30, glycerin, stearic acid, glyceryl stearate SE, liquid paraffin, petroleum jelly, triethanolamine, dimethicone, cetearate-20, phenoxyethanol and ethylhexylglycerin, ethyl alcohol.

[0263] Sample 2 is the study sample, which is a reference cream formulation with the addition of the Native fraction of vibrational iterations of water (16) as an active ingredient:

[0264] Native fraction of vibrational iterations of water (16), water, acrylates / crosspolymer of alkyl acrylate Cl 0-30, glycerin, stearic acid, glyceryl stearate SE, liquid paraffin, petroleum jelly, triethanolamine, dimethicone, cetearate-20, phenoxyethanol and ethylhexyl glycerin.

[0265] Ex vivo model

[0266] Ex vivo living human skin explants were used as a model.

[0267] Topical treatment (~2 mg / cm2) was applied with sample 1 or 2 on day 0 (DO), DI, D2, D3, D6, and D7.

[0268] Skin explant sampling was performed on DO, D3, and D8.

[0269] RNA extraction from all explants was performed on DO, D3, and D8.

[0270] Parameters analyzed: cell viability and genomic analysis using 8*60K human whole genome chip and immunostaining for filaggrin, involucrin, S100A7, cytokeratin 2, interleukin- 1 beta and dermcidin with semi-quantification by image analysis.

[0271] Genomic analysis

[0272] 1. RNA isolation and validation

[0273] 2. Amplification, cRNA labeling

[0274] 3. Whole human genome 8*60K on-chip hybridization (Agilent)

[0275] 4. Quantitative and qualitative chip validation

[0276] 5. Data normalization Selection of modulated genes

[0277] Assay level III: Determination of biological activity through functional correlation between genes and biological processes in response to sample 2 after comparison with sample 1.

[0278] Statistical analysis

[0279] The microarray data were quantified according to the protocol of the quantification software (Feature Extraction 12.2.0.7). In order to compare samples, the raw data were normalized by Quantile Method (inter-and intra-array normalization) including a batch effect correction (array background) using several packages in R statistical software. In order to visualize the similarities between samples, Principal Component Analysis (PCA) was performed, using moal package (vl.1.9), on normalized sample intensity, after removal of potential batch effect due to the microarray slides.

[0280] Results

[0281] Histologic analysis

[0282] The results of the study are summarized in Table 21.

[0283] Table 21.

[0284] The tested samples are well tolerated by the skin after 3 days of treatment. After 8 days of treatment, sample 2 causes an improvement in epidermal viability compared to placebo sample 1. Moreover, hyperkeratinization of the granular layer (the last of the living layers of the epidermis) indicates impaired final differentiation on day 8.

[0285] Sample 2 induces decreased expression of keratinocyte differentiation markers (filaggrin, profilaggrin, involucrin, S100A7, CK2), indicating impaired differentiation, compared to placebo sample 1. In addition, sample 2 induces decreased expression of an antimicrobial marker (dermci din) compared to placebo sample 1.

[0286] Genomic analysis

[0287] Compared to placebo sample 1, the use of sample 2 induces an inflammatory state after D3, and in response to this inflammation, an increase in keratinocyte proliferation is initiated. In addition, after 8 days of sample 2 use, suppression of keratinocyte differentiation marker genes indicates impaired differentiation.

[0288] Conclusions

[0289] The active ingredient in sample 2 is safe and does not cause pathologic changes. It has a stimulating effect on epidermal renewal and may influence keratinocyte differentiation. It may be promising for use in anti-aging cosmetics, hyperkeratosis therapy, and post-procedural care. Thus, sample 2 has potential applications in various fields of cosmetology and dermatology.

Claims

1. Claims1. A method for obtaining a cosmetic product, which is an artificial material object - iteration, produced by sequential vibration treatment of a neutral carrier in the presence of a biological substance selected from biological molecule, cell, group of cells or structures composed of cells, fluid media having a regulatory effect on functional and metabolic processes in skin, hair, nails and oral mucosa.

2. The method of claim 1, wherein the biological substance is represented by individual cells, groups of cells, cell associations, or cellular structures located predominantly in skin, hair, nails, and oral mucosa or contained within tissue fluid.

3. The method according to claim 1, wherein the biological substance is represented by biological molecules predominantly found in the skin that determine its structure and functional activity, or molecules that have a regulatory effect on metabolic, functional or immunologic processes throughout the body, including skin.

4. The method of claim 3, wherein the biological molecules are enzymes, receptors, proteins, factors.

5. The method of claim 1, wherein the fluid media, are blood, lymph, tissue fluids or components of tissue fluid, including water.

6. The method according to claim 1, wherein the vibration treatment means the process comprising: a) external vibration treatment of test tubes containing biological substance and neutral carrier to obtain from neutral carrier the primary artificial object - ‘iteration zero’; b) external vibration treatment of the previous iteration and neutral carrier to obtain from neutral carrier the subsequent iteration.

7. The method of claims 1, 6, wherein neutral carrier is water or an aqueous- alcoholic solution.

8. The method of claim 6, wherein the vibration treatment may be horizontal or vertical mechanical shaking.

9. The method of claim 6, wherein the vibration treatment is accomplished by electromagnetic, ultrasonic, acoustic or other rhythmic physical action.

10. The method according to claim 1, wherein the cosmetic action is a caring, protective, therapeutic and preventive action on the skin.

11. The method according to claim 1, wherein the cosmetic action comprises activating or inhibiting the biological substance or their molecular or its cellular targets in theorganism as well as impact on the functional and metabolic processes regulated by the biological substance.

12. A Product having a cosmetic effect obtained by the method according to claim 113. A method of distribution into fractions a product obtained according to claim 1, comprising: a) preliminary distribution of the obtained iterations into fractions depending on the physical -chemi cal properties they acquire after vibration treatment, which are different from the properties of neutral carrier, b) determination of specific biological or pharmacological activity for each fraction using standard methods.

14. The method of claim 13, wherein the physicochemical properties of the substance are determined using generally accepted analytical methods.

15. The method of claim 14, wherein as an analytical method is selected measurement of specific electrical conductivity (SEC), radiometry, terahertz spectroscopy, immunoassay (ELISA), pH measurement, determination of the amount of dissolved oxygen, dynamic light scattering, and high resolution thermography.

16. The method according to claim 13 wherein the separation into fractions is based on the presence of altered physicochemical properties compared to neutral carrier.

17. The method according to claim 13 wherein the determination of specific activity is accomplished by determining the biological and / or chemical activity of the fraction using methods generally accepted in experimental biology and chemistry.

18. A method according to claim 13, wherein iterations for further experimental determination of the biological (pharmacological) action are selected from two fractions that markedly differ in their physico-chemical properties both from the neutral carrier and from each other - in the presence or absence of a modifying effect on the physical characteristics of the initial biological substance.

19. A fraction obtained by the method of claim 13, comprising an artificial substance - iteration having a cosmetic effect.

20. A cosmetic product comprising as an active ingredient a product according to claim 12 or a fraction according to claim 19.

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