RU2843363C1 - Method of producing biologically active food additive with anticancer activity - Google Patents

Abstract

FIELD: foods or foodstuffs.

SUBSTANCE: invention relates to nutriciology, in particular, to methods for production of biologically active food additives possessing anticancer effect. Biologically active food additive production method is as follows: aboveground biomass of Phlojodicarpus sibiricus and Inonotus obliquus are used. Method comprises extraction of dried and milled vegetal components separately with ratio of raw material-extractant 1:10 by alternate sequence three times with 70 % aqueous solution of ethanol and twice with 96 % aqueous solution of ethanol at temperature 20-24 °C for 24 hours at each stage. Ethanol extracts are evaporated and then lyophilically dried to obtain a dry extract. Produced dry extracts are mixed in equal proportions.

EFFECT: invention allows to expand the range of biologically active food additives possessing antineoplastic activity.

1 cl, 1 tbl

Description

The invention relates to the field of nutritionology, namely methods for producing biologically active food supplements that have an antitumor effect.

Among the means for the prevention of oncological diseases based on natural raw materials, there are known means, for example, based on tincture of the herb of spiny milk thistle (see RU No. 2356566, class A61K 36/28, A61P 35/00, A61P 37/02, published on 27.05.2009); plant compositions of membranous milk thistle, roots of baicalensis skullcap, rhizomes and roots of Siberian inflated carpel (see RU No. 2603465, class A61K 36/539, A61K 36/23, A61K 36/481, B01D 11/02, A61P 9/10, A61P 39/06, published on 27.11.2016); plant compositions from the leaves of the water sedge, water burdock, alder flowers, dill seeds, celandine herb, nettle and sage in equal parts (see RU No. 2011151961, class A61K 36/00, published on 27.06.2013); plant compositions of wild rosemary, calamus, burnet, calendula, horse sorrel, black currant, sandy immortelle, rose hips, chicory, knotweed, plantain, black chokeberry, bilberry, field horsetail, black mulberry, thyme, wormwood, licorice, St. John's wort, blackthorn (see RU No. 2080867, cl. A61K 35/78, A23C 3/00, published 10.06.1997).

In addition, there are known agents for the prevention and treatment of oncological diseases (see RU No. 2118166, class A61K 35/78, published on 27.08.1998; RU No. 2208446, class A61K 35/78, A61P 35/00, A61P 39/06, published on 20.07.2003; RU No. 2118536, class A61K 35/78, published on 10.09.1998), which include extracts from geranium, plantain, calendula, shrubby cinquefoil (Kuril tea) and angelica (angelica). The objects of testing the claimed agents were cell cultures of the A-549 lung carcinoma line and BDF1 mouse line with transplanted tumors: lymphocytic leukemia P-388, myelomonocytic leukemia L-1210 and Ehrlich tumor.

At the same time, the known solutions do not contain the results of studies of the microbiological contamination of the preparations, which are necessary for assessing their effectiveness. In addition, the multi-component composition of the products leads to an increase in their cost price, due to the collection of bio-raw materials growing in various conditions, and the costs of manufacturing bio-preparations associated with conducting numerous analyses.

In folk medicine, the use of Siberian swollen fruit fungus and chaga fungus is known as a remedy against many pathological conditions.

The Siberian swollen carp (Phlojodicarpus sibiricus) was used among the peoples of Siberia and Yakutia for obesity, pulmonary tuberculosis, diseases of the thyroid gland, stomach and heart, rheumatism, atherosclerosis, in the treatment of wounds and tumors of the stomach and esophagus (see Batorova S.M., Yakovlev G.P., Aseeva T.A. Directory of medicinal plants traditional Tibetan medicine / Batorova S.M., Yakovlev G.P., Aseeva T.A., Novosibirsk: Nauka, 2013; Kuznetsova L.V., Mikhaleva L.G., Zakharova V.I. Atlas of medicinal plants of Yakutia / Kuznetsova L.V., Mikhaleva L.G., Zakharova V.I., ed. Ivanov, Yakutsk: Publishing house YANTS SO RAS, 2003. 224 pp.; Barnaulov, O.D.; Belenovskaya, L.M.; Medvedeva, L.I. Plant Resources of USSR: Flowering Plants, Chemical Components, Uses-Family Rutaceae-Elaegnaceae / Barnaulov, O.D.; Belenovskaya, L.M.; Medvedeva, L.I., Leningrad: Nauka, 1988. 357 p.). It was actively used in pharmaceuticals in the production of dimidine, flowerin and safinor, which was suspended due to a decrease in population numbers and, accordingly, a lack of raw materials (see Vasilyeva O.D. Siberian inflated fruit Phlojodicarpus Sibiricus (Steph. ex Spreng.) K.- Pol. in Yakutia: Biology, introduction, conservation. Dissertation ... candidate of biological sciences. Yakutsk, 2005. 170 p.; Gromakova A.I., Isaykina A.P., Krivut B.A., Vandyshev V.V. Study of the content of visnadine and dihydrosamidine in the raw material of Siberian inflated fruit // Khim.-pharm. zhurn. 1982. Vol. 16. No. 4. pp. 60-66; Pimenov M.G., Babilev F.V., Nikonov G.K. Phlojodicarpus Turcz. and Libanotis L. As sources of coumarins with spasmolytic activity // Plant resources. 1968. Vol. 4. No. 4. Pp. 486-491). It is known that polyphenols of natural origin have a protective effect on the development of tumors of the oral cavity, stomach, duodenum, liver, lungs, skin, ovaries, cervix, mammary glands and prostate glands (see Amararathna M., Johnston M.R., Rupasinghe H.P.V. Plant polyphenols as chemopreventive agents for lung cancer // Int. J. Mol. Sci. 2016. No. 8 (17). P. 1352; Amin A.R.M.R., Kucuk O., Khuri F.R., Shin D.M. Perspectives for cancer prevention with natural compounds // J. Clin. Oncol. 2009. No. 16 (27). P. 2712–2725; Kozlowska A. Szostak-Wegierek D. Flavonoids – food sources and health benefits // Rocz. Panstw. Zakl. Hig. 2014. No. 2 (65). pp. 79–85; Li Q., Ren F.Q., Yang C.L., Zhou L.M., Liu Y.Y., Xiao J., Zhu L., Wang Z.G. Anti-proliferation effects of isorhamnetin on lung cancer cells in vitro and in vivo // Asian Pac. J. Cancer Prev. 2015. No. 7 (16). pp. 3035–3042; Pandey K.B., Rizvi S.I. Plant polyphenols as dietary antioxidants in human illness and disease // Oxid. Med. Cell. Long. 2009. No. 5 (2). pp. 270–278; Romano B., Pagano E., Montanaro V., Fortunato A.L., Milic N., Borrelli F. Novel insights into the pharmacology of flavonoids. // Phytother. Res. 2013. No. 11 (27). pp. 1588–1596; Sak K. Cytotoxicity of dietary flavonoids on different human cancer types // Pharmacogn. Rev. 2014. No. 16 (8). pp. 122–146; Zhou Y., Zheng J., Li Y., Xu D.P., Li S., Chen Y.M., Li H.B. Natural polyphenols for prevention and treatment of cancer // Nutrients. 2016. No. 8 (8)).

The presence of radicals in the cells of the body is associated with vital processes and plays an important role in the prooxidant-antioxidant balance. In this case, oxidative damage is the result of endogenous and exogenous factors in the formation of active oxygen species. The internal sources of oxidants include the mitochondrial electron transport chain and the reaction of nitric oxide synthase synthesis, as well as non-mitochondrial reactions (see Gilca M., Stoian I., Atanasiu V., Virgolici B. The oxidative hypothesis of senescence // J. Postgrad. Med. 2007. No. 3 (53). P. 207–213). Excessive formation of free radicals causes suppression of natural cellular antioxidant protection and leads to deterioration of the body's functionality (see Poljsak B., Milisav I. Aging, oxidative stress and antioxidants edited by Morales-González J.A., Croatia: Intech Open Access Publisher, 2013. Pp. 331–356). One of the most important strategies for reducing oxidative stress is the consumption of exogenous antioxidants, usually of plant origin (see Packer L. Oxidative stress, antioxidants, aging and disease edited by Cutler R.G., Packer L., Bertram J., Mori A., 1995.P. 1–14; Ames B.N., Shigenaga M.K., Hagen T.M. Oxidants, antioxidants, and the degenerative diseases of aging // Proc Natl Acad Sci USA. 1993. (90). P. 7915–7922). In particular, flavonoids are capable of inhibiting three stages of cancer cell development: initiation – influence on oxidative stress and inactivation of carcinogens; activation (promotion) – inhibition of cell proliferation and spread - activation of apoptosis, inhibition of angiogenesis and suppression of tumor metastasis (see Chachar M.K., Sharma N., Dobhal M.P., Joshi Y.C. Flavonoids: A versatile source of anticancer drugs // Pharmacogn. Rev. 2011. No. 9 (5). P. 1–12; Li F., Li S., Li H.B., Deng G.F., Ling W.H., Xu X.R. Antiproliferative activities of tea and herbal infusions // Food Funct. 2013. No. 4 (4). P. 530–538 Li Q., Ren F.Q., Yang C.L., Zhou L.M., Liu Y.Y., Xiao J., Zhu L., Wang Z.G. Anti-proliferation effects of isorhamnetin on lung cancer cells in vitro and in vivo // Asian Pac. J. Cancer Prev. 2015. No. 7 (16). pp. 3035–3042; Majewski G., Lubecka-Pietruszewska K., Kaufman-Szymczak A., Fabianowska-Majewska K. Anticarcinogenic capabilities of plant polyphenols: Flavonoids and stilbene // Pol. J. Public Health. No. 4 (122). pp. 434–439; Mantena S.K. Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells // Carcinogenesis. 2005. No. 8 (27). C. 1682–1691; Middleton E.Jr., Kandaswami C., Theoharidis T.C. The effects of plant flavonoids on mammalian biology: Implications for inflammation, heart disease and cancer // Pharmacol. Rev. 2000. No. 4 (52). pp. 673–751).

It is known that the extract of the leaves of the sibiricus plant growing in Yakutia contains: essential oil 2.11 mg / g dry weight, coumarins 56.28 mg / g dry weight, caffeoylquinic acids 18.62 mg / g dry weight, water-soluble polysaccharides 3.74, pectin 2.67, triacylglycerol 755.1 μg / mg protein. The component composition of the leaves of P.sibiricus includes phenylpropanoids: 1-O-caffeoyl glucose, 1-O-caffeoyl quinic acid, 6-O-caffeoyl glucose and 5-O-caffeoyl quinic acid; flavonoids: diosmetin-7-O-glucoside and chrysoeriol-7-O-glucoside; helactones and its derivatives: helactone 4'-O-methyl ester, helactone 4'-O-acetyl ester, helactone 3'-O-acetyl-4'-O-isobutyroyl ester (huganin D), helactone 3'-O-isovaleroyl-4'-O-acetyl ester (dihydrosamidine), helactone-3'-O-glucoside (praeroside II); coumarins: umbelliferone-O-deoxyhexosyl-O-hexoside, umbelliferone-7-O-(6”-apiosil)-glucoside (6”-apiosilskimmin), pecedanol-3'-O-glucoside (see Olennikov DN, Fedorov IA, Kashchenko NI, Chirikova NK Cecile Vennos. Khellactone Derivatives and Other Phenolics of Phlojodicarpus sibiricus(Apiaceae): HPLC-DAD-ESI-QQQ-MS/MS and HPLC-UV Profile, and Antiobesity Potential of Dihydrosamidin // Molecules. No. 12 (24). P. 2286). In addition, it includes hellactone, hellactone 4′-O-methyl ether, hughanin D, hellactone 3′-O acetyl-4′-O-(2-methylbutyroyl) ether, 6′-apiosyl-skimmin, visnadin, suksdorfin, and isoimperatorin (see Gantimur D., Syrchina AI, Semenov AA Isoimperatorin from Phlojodicarpus sibiricus. // Chem. Nat. Comp. 1986. (22). pp. 103–104; Gantimur D., Syrchina AI, Semenov AA Khellactone derivatives from Phlojodicarpus sibiricus. // Chem. Nat. Comp. 1986. (22). pp. 103–104; Nikonov GK, Vandyshev VV Visnadin—A new component of the plant genus Phlojodicarpus. // Chem. Nat. Comp. 1969. (5). pp. 101–102).

The presence of caffeoylquinic acids in the extract of P. sibiricus leaves may cause hypoglycemic, hypocholesterolemic, hepatoprotective, and antitumor effects on the human body (see Giamperi L., Bucchini A., Cara P., Fraternale D., Ricci D., Genovese S., Curini M., Epifano F. Composition and antioxidant activity of Nepeta foliosa essential oil from Sardinia (Italy) // Chemistry of Natural Compounds. 2009. (45). P. 554–556). It was found that the extract of the herb P. sibiricus has a cytotoxic effect on diffuse large B-cell lymphoma, causing a decrease in the expression of anti-apoptotic members of the Bcl-2, Bcl-xL and Mcl-1 family, inducing the collapse of the mitochondrial membrane potential, which causes apoptosis (see Kim Jisu, Kim Dong, Nam Jehyun, Jeon Byeol, Okhlopkova Zhanna, Zulfugarov Ismayil, Kim Sang-Woo Anti-lymphoma Activities of Phlojodicarpus sibiricus and Artemisia kruhsiana Besser Extracts // Journal of Life Science. 2020. No. 4 (30). P. 379–385). Special attention of researchers is also paid to hellactone esters with their high efficiency as antiadipogenic agents; it has been established that dihydropyranocoumarins of the hellactone group can have an inhibitory effect on adipogenesis (see Nugara, RN; Inafuku, M.; Takara, K.; Iwasaki, H.; Oku, H. Pteryxin: A coumarin in Peucedanum japonicum Thunb leaves exerts antiobesity activity through modulation of adipogenic gene network. // Nutrition. 2014. (30). P. 1177–1184). Thus, the plant components contained in P. sibiricus extracts can influence the processes of preparation and dissemination of cells from the primary focus during lymphohematogenous metastasis.

In addition, the prior art discloses the use of P. sibiricus as a raw material with immunomodulatory activity (see RU No. 2496510, cl. A61K 36/738, A61K 36/23, A61K 36/28, A61K 36/484, A61K 36/533, A61K 36/55, A61K 36/57, A61K 36/704, B01D 11/02, A61P 37/02, published 27.10.2013), a source of macro- and microelements (see RU No. 2263511, cl. A61K 35/78, A61P 39/00, published 10.11.2005), a source of pyranocoumarins widely used in pharmaceuticals (see RU No. 2666920, class A01H 4/00, published on 13.09.2018), a source of dihydrosamidine and visnadine, which became the basis of the drug "Floverin" (see SU No. 876136, class A61K 35/78, published on 30.10.1981), a component of herbal tea (see RU No. 2746625, class A23F 3/34, published on 19.04.2021). In the method for obtaining a biopreparation based on Siberian inflated carp according to patent RU No. 2818479 (class A61K 36/23, B01D 11/02, A61P 35/00, published 02.05.2024), the aboveground biomass of Siberian inflated carp collected during the flowering period, namely leaves, stems and flowers, is used as raw material, while the pre-dried and crushed raw material is extracted with aqueous ethanol solutions.

Chaga ( Inonotus Obliquus , beveled tinder fungus, black birch mushroom, ash conk, clinker polypore, cinder conk, birch polyp, beveled inonotus, chaga, etc.) is an ancient folk remedy that has long been used by the population of Russia, northwestern Europe, Siberia, Korea, Canada to treat gastrointestinal diseases, inflammatory processes and cancer. It has been established that chaga and preparations based on it have a beneficial effect on the diseased organism and may be promising for use in therapeutic and preventive oncology. Due to the total complex of biologically active substances (humic-like polyphenolic compounds, flavonoids, lectins, micro- and macroelements), chaga preparations have a multifunctional pharmacological effect, including anti-inflammatory and general tonic. They activate metabolism in brain tissue and increase the bioelectrical activity of the cerebral cortex, regulate metabolic processes and increase the body's defense reactions, relieve pain, dyspeptic symptoms and normalize bowel function, improve the general condition and quality of life even in patients with stage IV cancer (see Shashkina M.Ya., Shashkin P.N. Chaga in oncology // Russian Biotherapeutic Journal. 2005. No. 4). It was also found that the aqueous extract of chaga significantly inhibited the activity of bladder cancer organoids (see Abugomaa A, Elbadawy M, Ishihara Y, Yamamoto H, Kaneda M, Yamawaki H, Shinohara Y, Usui T, Sasaki K. Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids. // Front Pharmacol. 2023. (19)), while the isolated fractions of triterpenoid acids have high antiproliferative activity in relation to the cancer cell lines HT-29, AGS, MCF-7 and PC3 (see Kim J, Yang SC, Hwang AY, Cho H, Hwang KT. Composition of Triterpenoids in Inonotus obliquus and Their Anti-Proliferative Activity on Cancer Cell Lines // Molecules. No. 18 (25). P. 4066).

Antitumor activity is achieved either by suppression of multiple oncogenic signals, including, but not limited to, NF-κB and FAK activation, as well as RhoA/MMP-9 expression via the ERK1/2 and PI3K/Akt signaling pathway. Antitumor activity can also be achieved by inhibiting tyrosinase activity via the PAK1-dependent signaling pathway, or by altering lysosomal membrane permeability by blocking tubulin polymerization and/or disrupting energy metabolism via the LKB1/AMPK pathway. In addition, I. obliquus metabolites also have potential against the molecular mechanisms of drug resistance either through selective inhibition of P-gp/ABCB1 or MRP1/ABCC1 proteins or through induction of G2/M checkpoint arrest in tumor cells of chemoresistant phenotypes mediated by the Nox/ROS/NF-kB/STAT3 signaling pathway (see Zhao Y, Zheng W. Deciphering the antitumoral potential of the bioactive metabolites from medicinal mushroom Inonotus obliquus // J Ethnopharmacol. 2021. (265)). Inhibition of cancer cell growth in vitro and in vivo is mediated by macrophages due to high production of IL-12p70 triggered by polysaccharides of I. obliquus (see Wold CW, Christopoulos PF, Arias MA, Dzovor DE, Oynebraten I, Corthay A, Inngjerdingen KT Fungal polysaccharides from Inonotus obliquus are agonists for Toll-like receptors and induce macrophage anti-cancer activity. // Commun Biol. 2024. No. 1 (7). P. 222).

In addition, the prior art contains solutions related to the use of birch chaga (see RU No. 2438685, cl. A61K 36/06, B01D 11/02, published on 10.01.2012; RU No. 2548767, cl. A61K 36/06, B01D 11/02, published on 20.04.2015; RU No. 2406515, cl. A61K 36/06, A61P 35/00, published on 20.12.2010; RU No. 2597160, cl. A61K 36/07, B01D 11/02, published 10.09.2016). The technologies are mainly aimed at extracting biologically active molecules of chaga for subsequent use in the pharmaceutical industry. Oncoprotective, immunomodulatory, antioxidant activities are repeatedly mentioned.

The problem, which the claimed invention is aimed at solving, is expressed in obtaining a biologically active food supplement based on Siberian bloater and oblique tinder fungus, which has antitumor activity.

The technical result obtained by solving the set task is expressed in obtaining an environmentally friendly and safe biopreparation based on the above-ground part of the Siberian swollen fruit fungus and the oblique tinder fungus.

To solve the set problem, the method for obtaining a biologically active food supplement based on Siberian inflated fruit (Phlojodicarpus sibiricus) and oblique tinder fungus (Inonotus Obliquus) is characterized by using the aboveground biomass of Siberian inflated fruit (Phlojodicarpus sibiricus), extracting the dried and crushed plant components separately at a raw material-extractant ratio of 1:10 in an alternate sequence three times with a 70% aqueous ethanol solution and twice with a 96% aqueous ethanol solution at a temperature of 20-24°C for 24 hours at each stage, then evaporating the ethanol extracts with subsequent lyophilic drying until a dry extract is obtained, the resulting dry extracts are combined in equal proportions.

A comparative analysis of the features of the claimed solution with the features of analogues indicates that the claimed solution meets the “novelty” criterion.

The set of essential features ensures the solution of the stated technical problem, namely, the expansion of the range of biopreparations with antitumor activity and methods for their production. In this case, a lyophilized extract is obtained that is capable of suppressing the growth of HepG2, MCF-7, HeLa, HEp-2 cancer cells, which can be used to suppress tumor growth both separately and together with other drugs in complex treatment. In addition, the simplicity of performing technological operations to obtain a lyophilized extract contributes to its use in prevention and adjuvant therapy to suppress tumor growth.

Thus, the biologically active food supplement is a composition of extracts of two natural components: the above-ground part of Siberian inflated fruit and chaga. Given the antitumor effect on cancer cells, encouraging results of in vivo studies and numerous empirical references in folk medicine about the effectiveness of various products based on Siberian inflated fruit and chaga, it is assumed that the combination of these plant components will contribute to the prevention of cancer and adjuvant cancer therapy.

To ensure the release of the widest spectrum of the biochemical composition of biologically active substances from the intracellular environment into the extractant, water-alcohol extraction technology is used. Biologically active compounds obtained by extraction with water-ethanol mixtures are environmentally friendly and toxically safe (see Vasiltsova I.V., Bokova T.I. Possibility of using plant extracts as biologically active additives // Innovations and food security. 2015. (3). P. 5–10; Nechiporenko I.A. Extraction of biologically active substances from finely ground plant materials: dissertation of candidate of technical sciences, 1984; Ponomarev V.D. Extraction of plant raw materials. / Ponomarev V.D., Moscow: Meditsina, 1976. 202 p.).

The claimed technical solution includes a multi-stage separate water-ethanol extraction of Siberian bloatberry and birch chaga, followed by evaporation, lyophilization and mixing of the dry residue masses in equal proportions.

Thus, the essence of the declared solution is that for the preparation of the biopreparation, the above-ground biomass of Siberian bloatberry, grown, for example, in plantations on open ground, and birch chaga, growing in natural conditions, is used.

For the experiments, the drying of the above-ground biomass of Siberian inflated fruit and the purified finely chopped biomass of chaga was carried out in room conditions without access to light and in different rooms.

The dried components were ground mechanically to a powder state, after which the powders were extracted successively with water-ethanol 70% (three times) and 96% (two times) solutions in a raw material-extractant ratio of 1:10 for 24 hours at each stage at a temperature of 20-24°C. The extracts were combined, evaporated on a rotary evaporator to obtain an aqueous fraction, which was dried in a lyophilization unit. The resulting dry extract was stored in vacuum packaging under room conditions without access to light. The lyophilisate was tested for contamination according to TR/TS 021/2011.

For the experiments, the resulting mixture was studied in the laboratory to identify antitumor activity.

Microbiological studies. The mixture was tested for the absence of microbiological agents in accordance with the requirements for raw materials for biologically active food supplements according to the TRTS 021/2011 standard. The results of the study confirm the absence of such agents in the mixture (see table).

Determination of cytotoxic activity. The tests were conducted to assess the inhibitory effect of the samples on cancer cell lines in vitro. The following cell lines were used: HepG2 (hepatocellular carcinoma), MCF-7 (adenocarcinoma of the mammary gland ducts), HEp-2 (epidermoid carcinoma of the larynx), HeLa (epithelioid carcinoma of the cervix). Healthy somatic cells - human dermal fibroblasts, cultured under the same conditions - were used as a control. Doxorubicin, a drug used to treat leukemia, lymphoma, and solid tumors, was also used for comparison with known drugs with antitumor activity. Doxorubicin inhibits DNA and RNA synthesis: it intercalates into the DNA double helix between pairs of nitrogenous bases (in this case, the matrix is disrupted and the spatial structure changes) and causes DNA cleavage due to the formation of free radicals. In addition, the antitumor effect may be due to changes in cellular functions as a result of binding to lipids of cell membranes and interaction with topoisomerase II (see US No. 3590028, cl. C07C 47/18, C07C 95/04, published 06/29/1971).

The cells were cultured at 37°C with 5% _CO2 in a DMEM medium supplemented with fetal bovine serum to 10% of the volume, 100 mM sodium pyruvate solution to 1% of the volume, an antibiotic-antimycotic solution containing 10,000 U/ml penicillin, and 10 mg/ml streptomycin to 1% of the volume. Upon reaching a monolayer, the culture was detached to obtain a cell suspension using the enzyme trypsin (0.25% solution) followed by its inactivation with a nutrient medium.

A 96-well plate was used to perform the test. From 2500 to 2800 cells in 200 μl of the culture medium were added to each well. The plates with the cells were then incubated for 24 hours in a CO ₂ incubator for cell adhesion at a temperature of 37°C. After 24 hours, a serial dilution of the sample in the nutrient medium was added to the wells under study. The dilution was performed in the range of 1:10–1:10000 (0.01 g/ml (1:10); 0.001 g/ml (1:100); 0.0001 g/ml (1:1000) and 0.00001 g/ml (1:10000), based on the initial concentration of 0.1 g/ml (1:1)). Doxorubicin was added at a concentration of 0.5 mg/ml. After adding the test substance, the cells were incubated in a _CO2 incubator under standard conditions for 72 hours. After the specified time, the culture medium with the test substances was removed using a vacuum aspirator.

The antitumor activity was measured using the MTT method. The MTT test is an indicator of mitochondrial function in viable cells based on tetrazolium reduction. The method was chosen because MTT has a positive charge and easily penetrates living eukaryotic cells. (3-[4,5-dimethylthiazolyl-2-el]-2,5-iphenyltetrazolium bromide) – the MTT reagent under the action of mitochondrial dehydrogenases of viable cells is converted into a water-insoluble formazan having a violet color.

For one 96-well culture plate, a solution of 9 ml of the culture medium with the addition of 1 ml of the MTT reagent (5 mg / ml in Hanks' solution) was used, for which 100 μl were added to each well of the plate and incubated in a CO ₂ incubator for 3.5 hours. After the incubation time, the nutrient medium with the substances under study was carefully removed using an aspirator and 100 μl of dimethyl sulfoxide (DMSO) were added to each well, then incubated for 10 min. After which, violet coloration was observed in the wells of the plate. Detection of violet coloration was carried out on a plate reader at a wavelength of 650 nm. During cell lysis, crystals formed in living cells easily pass into the DMSO (dimethyl sulfoxide) solution. The activity of mitochondrial dehydrogenases and, accordingly, the viability of the cells were determined by the optical density of the formazan solution. The obtained optical density data in the studied wells were entered into a database, which was then processed using the IBM SPSS Statistic 23 software package.

Comparative analysis revealed statistically significant differences indicating the most effective cytotoxicity towards cancer cells at concentrations of 0.01 g/ml (1:10) and 0.02 g/ml (1:5) compared to other lower concentrations and the control group (p=0.000). Each concentration was presented in 16 wells of the plate.

The results of statistical analysis indicate that the concentration of the sample has a pronounced cytotoxic effect. 0.02 g/ml (1:5), which turned out to be close to the indicators of the drug Doxorubicin. Also, the absence of cytotoxic effect of the samples on the cultures of dermal fibroblasts was revealed.

Thus, the conducted experimental study indicates the presence of inhibitory activity of the lyophilisate of the water-ethanol extract of Siberian bloatberry and birch chaga in relation to cultured cancer cells HepG2, MCF-7, HeLa, HEp-2.

The proposed method for obtaining a biopreparation has the following advantages compared to known analogues:

- consists of two plant components - Siberian inflated fruit and birch chaga, the antitumor activity of each of which has been proven experimentally, which ensures high efficiency in the treatment and prevention of oncological diseases;

- plant components are obtained from renewable, environmentally friendly and cost-effective raw materials, using the above-ground part of the biomass of Siberian inflated fruit, grown, for example, by the plantation method, and the fleshy base of birch chaga, which grows in the conditions of a sharply continental northern climate;

- is an aseptic dry extract with a long shelf life, ready to use and easy to mix during encapsulation/tabletting.

Table

Results of microbiological studies of the herbal mixture

№№
p/p The indicators under study Acceptable levels Results 1 The number of mesophilic aerobic and facultative anaerobic microorganisms (QMAFAnM), CFU/g, no more than 5*10 ⁵ Not found 2 Bacteria of the coliform group (coliforms), not allowed in the product mass, g 0.01 Not found 3 E. Coli, not allowed in product mass, g 0,1 Not found 4 S. aureus, not allowed in product mass, g 1.0 Not found 5 Mold, CFU/g, no more than 10 ³ Not found 6 Yeast, CFU/g, no more than 100 Not found

Claims (1)

A method for obtaining a biologically active food supplement based on Phlojodicarpus sibiricus and Inonotus Obliquus, characterized by using the aboveground biomass of Phlojodicarpus sibiricus, extracting the dried and crushed plant components separately at a raw material-extractant ratio of 1:10 in an alternate sequence three times with a 70% aqueous ethanol solution and twice with a 96% aqueous ethanol solution at a temperature of 20-24°C for 24 hours at each stage, then evaporating the ethanol extracts with subsequent lyophilic drying until a dry extract is obtained, the resulting dry extracts are combined in equal proportions.