RU2818479C1 - Method of producing a biopreparation of phlojodicarpus sibiricus, having antitumour activity - Google Patents

Abstract

FIELD: biochemistry; pharmaceutics.

SUBSTANCE: invention relates to biochemistry and pharmaceutical industry, specifically to a method for producing a preparation based on biologically active substances of a dry solution of Phlojodicarpus sibiricus having an anti-tumour effect. Method of producing an extract of aboveground biomass of Phlojodicarpus sibiricus characterized by the fact that aboveground biomass of Phlojodicarpus sibiricus, collected during flowering, is used as raw material, for which preliminary dried and crushed raw material with ratio of raw material-extractant of 1:10 is successively extracted three times with 70% aqueous solution of ethanol and twice with 90% aqueous solution of ethanol at temperature of 23–25 °C for 22–24 hours at each stage, obtained ethanol extracts are combined and evaporated in an evaporator to obtain an aqueous fraction, which is subjected to lyophilic drying to obtain a dry extract. Use of the aboveground biomass extract of Phlojodicarpus sibiricus, obtained by the method according to cl. 1, as a biopreparation having anti-tumour action.

EFFECT: above-described method enables to obtain an extract by using the aboveground part of the Phlojodicarpus sibiricus biomass, which does not pose a threat of reducing the population of the perennial plant, the obtained extract expands the range of biopreparations having anticancer activity.

3 cl, 4 dwg, 1 tbl

Description

The invention relates to the field of biochemistry and the pharmaceutical industry, namely to a method for producing a drug based on biologically active substances of a dry solution of Siberian spleen (lat. Phlojodicarpus sibiricus ), which has an antitumor effect.

It is known that polyphenols of natural origin have a protective effect against the development of tumors of the oral cavity, stomach, duodenum, liver, lungs, skin, ovaries, cervix, mammary 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; 17 (8): 1352. DOI: 10.3390/ijms 17081352; compounds. J. Clin. 2009; 27 (16): 2712-2725. DOI: 10.1200/jco.20.6235; Zakl. Hig. 2014; 65 (2): 79-85; cells in vitro and in vivo. Asian Pac. Prev. 2015; 16 (7): 3035-3042. Plant polyphenols as dietary antioxidants in human illness and disease. Oxid. Med. Cell. Long. 2009; 2 (5): 270-278. DOI: 10.4161/oxim.2.5.9498; Pratheeshkumar P., Sreekala C., Zhang Z., Budhraja A., Ding S., Son Y.O., Wang X., Hitron A., Kim H.J., Wang L., Lee J.C., Shi X. Cancer prevention with promising natural products : Mechanisms of action and molecular targets. Anticancer Agents Med. Chem. 2012; 12 (10):1159-1184. DOI: 10.2174/187152012803833035; Romano B., Pagano E., Montanaro V., Fortunato A.L., Milic N., Borrelli F. Novel insights into the pharmacology of flavonoids. Phytother. Res. 2013; 27 (11): 1588-1596. DOI: 10.1002/ptr. 5023; Sak K. Cytotoxicity of dietary flavonoids on different human cancer types. Pharmacogn. Rev. 2014; 8 (16): 122-146. DOI: 10.4103/0973-7847.134247, 18. Johnson I.T., Williamson G., Musk S.R.R. Anticarcinogenic factors in plant foods: A new class of nutrients. Nutr. Res. Rev. 1994; 7 (1): 175-204. DOI: 10.1079/nrr19940011; 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; 8(8):E515. DOI: 10.3390/nu 8080515).

In particular, flavonoids are able to inhibit three stages of cancer cell development: initiation - influence on oxidative stress and inactivation of carcinogens; activation (promotion) - inhibition of cell proliferation and proliferation - 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; 5 (9): 1-12. DOI: 10.4103/0973-7847.79093; Kandaswami C., Lee P.P., Hwang J.J., Huang Y.T., Lee M.T. The antitumor activities of flavonoids. (5): 895-909, Sak K. Cytotoxicity of dietary flavonoids on different human cancer types. Pharmacogn. Rev. 2014; 8 (16): 122-146. Frachet V., Bhouri W., Ghedira K., Chekir-Ghedira L., Ronot X. Flavones inhibit the proliferation of human tumor cell lines by inducing apoptosis. Chem. 2012; 10. DOI: 10.3109/01480545.2011.564180; Kozlowska A., Szostak-Wegierek D. Flavonoids - food sources and health benefits. Li A.N., Li S., Zhang Y.J., Xu X.R., Chen Y.M., Li H.B. Resources and biological activities of natural polyphenols. Nutrients. 2014; 6 (12): 6020-6047. DOI: 10.3390/nu6126020; Li F., Li S., Li H.B., Deng G.F., Ling W.H., Wu S., Xu X.R., Chen F. Antiproliferative activity of peels, pulps and seeds of 61 fruits. J. Function. Foods. 2013; 5 (3): 1298-1309. DOI: 10.1016/j.jff.2013.04.016; Li F., Li S., Li H.B., Deng G.F., Ling W.H., Xu X.R. Antiproliferative activities of tea and herbal infusions. Food Function. 2013; 4 (4): 530-538. DOI: 10.1039/c2fo30252g; Majewski G., Lubecka-Pietruszewska K., Kaufman-Szymczak A., Fabianowska-Majewska K. Anticarcinogenic capabilities of plant polyphenols: Flavonoids and stilbene. Pol. J. Public Health. 2012; 122 (4): 434-439. DOI: 10.12923/j.0044-2011/122-4/a.19; Mantena S.K. Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells. Carcinogenesis. 2005; 27 (8):1682-1691. DOI: 10.1093/carcin/bgl030; Middleton E.Jr., Kandaswami C., Theoharidis T.C. The effects of plant flavonoids on mammalian biology: Implications for inflammations, heart disease and cancer. Pharmacol. Rev. 2000; 52 (4): 673-751; Pandey K.B., Rizvi S.I. Plant polyphenols as dietary antioxidants in human illness and disease. Oxid. Med. Cell. Long. 2009; 2 (5): 270-278. DOI: 10.4161/oxim.2.5.9498).

It is known that 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. Oxidative damage results from endogenous and exogenous factors producing reactive oxygen species. 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. 53(3).

Excessive production of free radicals causes inhibition of natural cellular antioxidant defenses and leads to deterioration of the body's functionality (see Poljsak B., Milisav I. Aging, oxidative stress and antioxidants. In: J.A. Morales-González (Ed.), Oxidative Stress and Chronic Degenerative Diseases . A Role for Antioxidants, Intech Open Access Publisher, Croatia 2013. R. 331-356 doi: 10.5772/51609).

One of the most important strategies for reducing oxidative stress is the consumption of exogenous antioxidants, usually of plant origin (see Ames B.N., Shigenaga M.K., Hagen T.M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA. 1993, 90 7915-7922; Packer L. Oxidative stress, antioxidants, aging and disease. In: Cutler R. G., Packer L., Mori A. (eds) Molecular and Cell Biology Updates. Basel. 1995. R. 1-14. doi.org/10.1007/978-3-0348-7337-6_1).

Thus, a remedy for the treatment of oncological diseases is known (see RU No. 2118166, class A61K 35/78, published on August 27, 1998), containing aqueous extracts of plants of the genera Geranium, Plantago and plants of the species Calendula officinalis, obtained in the following ratio of plants and water, g: Geranium - 10-60 (dry.) or 300-360 (fresh.), Plantago - 10-60 (dry.), Calendula officinalis - 10-60 (dry.), water - 3000. The product can additionally contain an extract of a plant of the species Pentaphylloides fruticosa, obtained from 10-60 g (dry.), as well as an extract of a plant of the genus Angelica, obtained from 10-60 g (dry.). The method for obtaining aqueous extracts involves soaking in water, followed by filtering and centrifugation.

The disadvantages of the known solution is that the extract is obtained by maceration without the use of aseptic conditions, which leads to a decrease in shelf life and an increase in the microbiological load of the body during use.

In Siberia and Yakutia, the use of the Siberian bloat carp is known as a food and medicinal plant, which has a wide therapeutic effect: a decoction of the roots of the Siberian bloat carp was used 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 Atlas of medicinal plants of Yakutia / Compiled by L.V. Kuznetsova, L.G. Mikhaleva, V.I. Zakharova and others; Editor-in-chief B.I. Ivanov. - Yakutsk: Publishing House YSC SB RAS , 2003. - 224 p.; Batorova S.M., Yakovlev G.P., Aseeva T.A. Directory of medicinal plants of traditional Tibetan medicine. Novosibirsk: Nauka, 2013. P. 127-129; L.M.; Medvedeva, L.I. (Eds.) Plant Resources of USSR: Flowering Plants, Chemical Components, Uses-Family Rutaceae-Elaegnaceae; Nauka: Leningrad, Russia, pp. 150-152. chemical composition, use; Family Rutaceae - Elaegnaceae L., 1988. 357 pp.).

Previously, the Siberian swollen carp was used in pharmaceuticals in the production of the drugs Dimidin, Flowerin and Safinor, which was suspended due to a decrease in population numbers and, accordingly, lack of raw materials (see Vasilyeva O.D. Siberian swollen carp Phlojodicarpus Sibiricus (Steph. ex Spreng.) K.- Pol. in Yakutia: Biology, introduction, protection. Dissertation of candidate of biological sciences, Yakutsk, 2005. 170 pp.; Isaikina A.P., Krivut B.A., Vandyshev V.V. Study of the content of visnadin and dihydrosamidine in the raw material of the Siberian puffball // Khim.-pharm. 1982. T. 16. No. 4. P. 60-66; Phlojodicarpus Turcz. and Libanotis L. As sources of coumarins with antispasmodic activity // Plant resources 1968. Vol. 4. 486-491).

At the same time, solutions for obtaining known drugs are characterized by the following disadvantages:

- there are no results of studies of microbiological contamination of drugs;

- the multicomponent composition of drugs makes it difficult to standardize them during phytochemical studies, which can lead to higher costs when collecting biological raw materials and a significant increase in costs in the manufacture of biological products due to analysis.

Some approaches to overcome the current situation in the absence of raw materials for the preparation of biological products may be the creation of medicinal plant nurseries and the use of above-ground biomass raw materials .

The problem to be solved by the claimed invention is expressed in the production of a biological product based on the Siberian pustule, which has antitumor activity.

The technical effect obtained by solving the problem is expressed in the production of a biological product based on the Siberian pustule by using the above-ground part of the biomass, which does not pose a threat to the reduction of the population of the perennial plant.

To solve this problem, the method for obtaining an extract of above-ground biomass of the Siberian bloat carp (Phlojodicarpus sibiricus) is characterized by the fact that the above-ground biomass of the Siberian bloat carp (Phlojodicarpus sibiricus), collected during the flowering period, is used as a raw material, namely, leaves, stems and flowers, and pre-dried and crushed raw materials with a raw material-extractant ratio of 1:10 are sequentially extracted with aqueous solutions of ethanol at a temperature of 23-25 ° C for 22-24 hours at each stage according to the following scheme: first with a 70% solution, then with a 90% solution, then, again with a 70% solution, after which, with a 90% solution, and finally again with a 70% solution, the resulting ethanol extracts are combined and evaporated in an evaporator at a temperature of 50±2°C to obtain an aqueous fraction, the aqueous concentrate is subjected to freeze-drying in a lyophilizer until complete removing the solvent and obtaining a dry residue. In addition, distillation from water-ethanol extraction is reused to extract plant materials. The resulting dry extract is stored in the refrigerator in sealed packaging. In addition, the extract of the above-ground biomass of the Siberian swollen carp (Phlojodicarpus sibiricus), obtained by the above method, is used as a biological product with an antitumor effect.

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 combination of essential features provides a solution to the stated technical problem, namely, expanding the range of biological products with antitumor activity and methods for their production. In this case, a lyophilized extract is obtained that can suppress the growth of HepG2 cancer cells, which can be used to suppress tumor growth, both separately and together with other drugs in complex treatment.

The essence of the claimed invention is that for the preparation of a biological product (working name “Flodis”), above-ground biomass of the Siberian swollen carp, collected during the flowering period, is used. For the experiments, we used a plant grown in a nursery on the territory of the Botanical Garden of the Institute of Biological Problems of the Cryolithozone SB RAS (IBPK, Republic of Sakha (Yakutia), Yakutsk). Drying of above-ground biomass was carried out in room conditions without access to light. Extraction was carried out sequentially with water-ethanol 70% (three times) and 96% (twice) solutions. The extracts were combined and evaporated on a rotary evaporator to an aqueous fraction, which was dried in a lyophilizer. The distilled ethanol was used to extract the next batch of raw materials. The resulting dry extract was stored in vacuum packaging at room conditions without access to light. The lyophilisate of Siberian spleen was tested for contamination with spores of microorganisms and fungal conidia on an agar medium. Microbiological studies of the lyophilisate were carried out according to the TRTS 021/2011 standard. The test results confirmed the absence of microbiological contamination and the presence of aseptic conditions for obtaining the biological product (see table).

Testing of the resulting preparation for the iron-reducing ability of antioxidants corresponded to 0.13 mg of quercetin equivalent/g of preparation. For comparison, an analysis of the antioxidant activity of ascorbic acid was carried out, which was 1.25 mg quercetin equivalent/g. The data obtained suggest a possible weak effect of the new biological drug on oxidative stress, which affects the initiation of cancer cells.

From literary sources, it is known that the extract of the leaves of the Siberian swollen carp, 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 mass, water-soluble polysaccharides 3.74, pectin 2.67, triacylglycerol 755.1 μg/mg protein. The component composition of the leaves of the Siberian spleen includes phenylpropanoids: 1-O-caffeoyl-glucose, 1-O-caffeoylquinic acid, 6-O-caffeoyl-glucose and 5-O-caffeoylquinic acid; flavonoids: diosmetin-7-O-glucoside and chrysoeriol-7-O-glucoside; chelactones and its derivatives: hellactone 4'-O-methyl ether, hellactone 4'-O-acetyl ester, hellactone 3'-o-acetyl-4'-o-isobutyroyl ether (huganin D), hellactone 3'-O-isovaleroyl -4′-O-acetyl ester (dihydrosamidine), hellactone-3'-O-glucoside (praeroside II); coumarins: umbelliferone-O-deoxyhexosyl-O-hexoside, umbelliferone-7-O-(6”-apiosil)-glucoside (6”-apiosilskimmine), pecedanol-3'-O-glucoside [11]. Hellactone, hellactone 4'-O-methyl ester, huganin D, hellactone 3'-O acetyl-4'-O-(2-methylbutyroyl) ester, 6'-apiosyl-skimine, visnadine, suxdorphin and isoimperatorin (see Gantimur, D.; Syrchina, A.A. Isoimperatorin. Chem. Nat. Comp. 1986, 22, 104-105. Syrchina, A.A. Khellactone derivatives. Chem. Nat. Comp. 1986, 103-104. Visnadin-A new component of the plant genus Chem. Nat. Comp. 1969, 5, 101-102.

The presence of caffeoylquinic acids in the leaf extract can cause hypoglycemic, hypocholesterolemic, hepatoprotective, 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. - R. 554-556., Olthof M.R., Hollman P.C., Katan M.B. caffeic acid are absorbed in humans // J. Nutr. - 2001. - V. 131 (1.) - P. 66-71).

The herb extract of the herb Siberian carp has been found to have a cytotoxic effect on diffuse large B-cell lymphoma, causing a decrease in the expression of anti-apoptotic family members Bcl-2, Bcl-xL and Mcl-1, inducing 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 2020 Vol. 4. 379~385. ). Currently, special attention of researchers is paid to hellactone esters with their high efficiency as anti-adipogenic agents; it has been established that dihydropyranocoumarins of the hellactone group can have an inhibitory effect on adipogenesis (see Nugara, R.N.; 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, 1177-1184. .015). This aspect is very important due to the fact that the plant components contained in the extracts Siberian bloat, can influence the processes of preparation and spread of cells from the primary focus during lymphohematogenous metastasis.

The claimed technical solution is illustrated in the drawing, where figure 1 shows a diagram of the reaction of the conversion of MTT into formazan, figure 2 shows the cytotoxic effect of various concentrations of Siberian pustule, figure 3 shows the cytotoxic effect of various concentrations of Siberian puffin compared to the control and doxorubicin, figure 4 - cytotoxic effect of various concentrations of Siberian spleen compared to control and doxorubicin.

The method of obtaining a biological product is implemented as follows.

The above-ground biomass of the Siberian swollen carp (Phlojodicarpus sibiricus), collected during the flowering period, was used as a raw material, while the leaves, stems and flowers were pre-dried and crushed to particle sizes, for example, 1-2 mm, by known methods.

The prepared raw materials were sent for extraction with an aqueous solution of ethanol, for which 70 and 90% ethanol solutions were used. At the same time, in order to obtain the maximum content of the extract and antibacterial treatment, an alternating extraction scheme was used, consisting of five stages: starting with a 70% ethanol solution, then again with a 90% ethanol solution, then again with a 70% ethanol solution, then with a 90% ethanol solution and finally with a 70% ethanol solution. Thus, extraction of bioraw materials includes alternate three-fold extraction with a 70% ethanol solution and two-fold extraction with a 90% ethanol solution.

Each extraction stage was carried out at a raw material-extractant ratio of 1:10 at a temperature of 23-25°C for 22-24 hours. In this case, the distillation from water-ethanol extraction was reused for the extraction of plant materials.

The resulting ethanol extracts were combined and evaporated in an evaporator at a temperature of 50±2°C to obtain an aqueous fraction. The aqueous concentrate was subjected to freeze-drying in a lyophilizer until the solvent was completely removed and a dry residue was obtained. The dry extract was stored in the refrigerator in sealed bags.

The developed method for obtaining a biological product from the Siberian stupor for the treatment of oncological diseases can help reduce the degree of metastasis during the lymphohematogenous spread and improve long-term treatment results and, compared with known solutions, has the following main advantages:

- the possibility of using renewable, environmentally friendly and economically profitable raw materials to obtain a biological product, namely, the above-ground biomass of the Siberian swollen carp, including with the plantation method of growing the plant in conditions of a sharply continental climate in the permafrost zone of Yakutia;

- a preparation containing biologically active substances of plant origin, has antitumor activity and is an aseptic dry extract of long-term storage of one type of plant, ready for use and easily mixed when encapsulating/tableting.

The effect of the invention is achieved by the fact that the introduction of the drug in concentrations from 0.025 g/ml (1:4) (0.027 (0.022; 0.032)) to 0.017 g/ml (1:6) (0.028(0.026; 0.037)) provides a decrease in viability and death of cancer cells (see Fig. 4).

The proposed drug for reducing the proliferative activity and number of cancer cells is characterized by the following list of qualities:

• natural raw materials are used, which have a complex of biologically active properties and have not been subjected to chemical treatment;

• has antioxidant activity, which can lead to a decrease in the level of oxidative stress in the body;

• in the studied doses gives the same effect as the drug doxorubicin;

• technological preparation of the drug component is traditional for biopharmaceuticals;

• dry extract of Siberian spleen is convenient in biotechnology, is well stored and easily transported.

The biological product can be used as follows.

Option 1. The use of Siberian bloat as a biological product with antitumor activity at a dose of 0.02 g/ml (1:5), when used, a decrease in the number of HepG2 cancer cells was observed relative to control values by 52.6% and achieved the same cytotoxicity indicators relative to the use drug doxorubicin.

Option 2. Using the drug at a dose of 0.01 g/ml (1:10), with the use of which a decrease in the number of HepG2 cancer cells was observed relative to control values by 47.4%, which can be used for therapeutic purposes.

Moreover, the conditions for culturing cancer cells in the control group and experimental groups were the same and were used without adding aqueous solutions of the drug.

Cytotoxicity results were processed using SPSS. The results of statistical processing are presented in the form: Me (median) (25th and 75th percentile), i.e. Me (25;75).

Thus, the above data indicate that the drug of plant origin (lyophilized extract of the aerial part of a wild plant - Siberian bloat) helps reduce the proliferation of cancer cells and reduce their survival. The antioxidant activity of the components of the biological product can affect oxidative stress in the body.

Laboratory test results

Type of study. The studies were carried out under sterile conditions in vitro, and the effectiveness of the drug was tested on HepG2 cell lines. HepG2 is a human hepatocellular carcinoma cell line, this cell line was derived from liver carcinoma of a 15-year-old male (see Nature, 1979, 282, 615; Science, 1980, 209, 497).

Research methodology. Measurement of antitumor activity is based on the MTT method. The MTT test is an indicator of mitochondrial function in viable cells, based on tetrazolium reduction.

To perform this test, HepG2 cancer cells were cultivated at 37°C with an atmosphere containing 5% CO ₂ in DMEM medium with the addition of 10% fetal bovine serum until a monolayer was formed. To obtain a cell suspension, the resulting cell monolayer was trypsinized, followed by inactivation of trypsin with the addition of a nutrient medium.

A 96-well plate was used to carry out the test. From 2500 to 2800 cells were added to each well in 200 μl of culture medium. Next, the plates with cells were incubated for 24 hours in a CO ₂ incubator for cell adhesion at a temperature of 37°C. After the proliferation and adhesion of HepG2 cells, a pre-prepared suspension of lyophilisate of Siberian bloat was added, 22 μl per well, except for control wells. After adding the test substance, the cells were incubated in a CO ₂ incubator under standard conditions for 72 hours. After the specified time, the culture medium with the test substances was removed using a vacuum aspirator.

Separately, in a bath for a multichannel dispenser, a solution was prepared from 9 ml of culture medium + 1 ml of MTT reagent (5 mg/ml in Hanks' solution) for one 96-well culture plate. The prepared solution was added to each well of the plate in 100 μl and incubated in a CO ₂ incubator for 3.5 hours. After the incubation time, the nutrient medium with the test substances was removed using an aspirator and 100 μl of DMSO was added to each well, after which it was re-incubated for 10 min. After the last incubation, a purple color appears in the wells. Detection of violet coloring was carried out on a plate reader (Bio-Rad iMark) at a wavelength of 650 nm.

Preparation of MTT solution. The choice of technique is due to the fact that MTT has a positive charge and easily penetrates into living eukaryotic cells. (3-[4,5-dimethylthiazolyl-2-el]-2,5-ifenyltetrazolium bromide) - MTT reagent, under the action of mitochondrial dehydrogenases of viable cells, is converted into a water-insoluble formazan with a purple color (see Fig. 1).

During cell lysis, crystals formed in living cells are easily transferred to a DMSO (dimethyl sulfoxide) solution. The optical density of the formazan solution was used to determine the activity of mitochondrial dehydrogenases and, accordingly, cell viability.

The obtained optical density data in the test wells were entered into a database, which were further processed by the IBM SPSS Statistic 23 software package.

Doxorubicin was chosen as a sample of a drug with proven antitumor activity. Doxorubicin, described in US Pat. No. 3,590,028, has a wide range of uses in the treatment of cancer and is used in the treatment of leukemia, lymphoma and solid tumors. It suppresses DNA and RNA synthesis: it intercalates into the DNA double helix between pairs of nitrogenous bases (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 cell membrane lipids and interaction with topoisomerase II.

The resulting lyophilisate was compared with the drug Doxorubicin.

Research results. At the initial stage of the study, experiments were carried out with different concentrations of the test substance - a suspension of lyophilisate of Siberian puffball: 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 an initial concentration of 0.1 g/ml (1:1). A comparative analysis revealed statistically significant differences, indicating the most effective cytotoxicity against HepG2 cancer cells at a concentration of 0.01 g/ml (1:10) compared to other lower concentrations (see Fig. 2) and the control group (p= 0.000). Each concentration was presented in 16 wells of the plate.

At the second stage of the study, in addition to the control group (only cancer cells without the addition of Siberian puffball), a group with the drug Doxorubicin was added to the experimental groups. The concentration of the tablet form of the drug was adjusted to 0.5 mg/ml. Taking into account the previous results obtained, indicating a pronounced degree of cytotoxicity at a concentration of 0.05 g/ml (1:10), at this stage higher concentrations of Siberian puffball were determined:0.02 g/ml (1:5), 0.01 g/ml (1:10) and 0.006 g/ml (1:15). Each comparison group had 16 wells. The results of statistical analysis indicate that a pronounced cytotoxic effect has a concentration of Siberian bloat carp of 0.02 g/ml (1:5), which turned out to be close to the indicators of the drug (see Fig. 3). Comparative analysis did not reveal statistically significant differences in optical density between the values of the suspension with Siberian puffball with a concentration of 0.02 g/ml (1:5) (0.008 (0.005; 0.012)) and doxorubicin (0.007 (0.003; 0.0107)). Compared to the control, statistically significant differences in optical densities are due to the drug and concentrations of 0.02 g/ml (1:5) and 0.01 g/ml (1:10).

At the third stage of the study, the following concentrations of Siberian puffball were considered : 0.08 g/ml (1:1.25); 0.025 g/ml (1:4); 0.017 g/ml (1:6) and 0.014 g/ml (1:7.5) compared to drug and control (see Fig. 4). The results indicate that the more effective concentration of Siberian carp is in the range of 0.025 g/ml (1:4) (0.027 (0.022; 0.032)) to 0.017 g/ml (1:6) (0.028(0.026; 0.037)), the median of which are as close as possible to the cytotoxicity value of the drug (0.025 (0.020; 0.036)).

Thus, the experimental study indicates the antitumor activity against cultured HepG2 cancer cells of a suspension of lyophilisate of the Siberian bloat with its most effective concentration of 0.025 g/ml (1:4) to 0.017 g/ml (1:6).

Table. Results of a microbiological study of lyophilisate of the Siberian bloat carp, performed according to the TRTS standard 021/2011

No. Indicators studied Acceptable levels Result 1 Number of mesophilic aerobic and facultative anaerobic microorganisms (QMAFAnM), CFU/g, no more 5*10 ⁵ Not detected 2 Bacteria of the group of coliforms (coliforms), not allowed in the product mass, (g) 0.01 Not detected 3 E. Coli, not allowed in the product mass, (g) 0.1 Not detected 4 S. aureus, not allowed in the product mass, (g) 1.0 Not detected 5 Mold CFU/g, no more 10 ³ Not detected 6 Yeast CFU/g, no more 100 Not detected

Claims (3)

1. A method for obtaining an extract of above-ground biomass of the Siberian bloat carp (Phlojodicarpus sibiricus), characterized by the fact that the above-ground biomass of the Siberian bloat carp ( Phlojodicarpus sibiricus ) collected during the flowering period is used as a raw material, for which pre-dried and crushed raw materials at a raw material-extractant ratio of 1: 10 are extracted in an alternate sequence three times with a 70% aqueous solution of ethanol and twice with a 90% aqueous solution of ethanol at a temperature of 23-25°C for 22-24 hours at each stage, the resulting ethanol extracts are combined and evaporated in an evaporator to obtain an aqueous fraction, which is subjected to lyophilization drying to obtain a dry extract. 2. The method for obtaining the extract according to claim 1, characterized in that distillation from water-ethanol extraction is reused to extract plant raw materials. 3. The use of an extract of above-ground biomass of the Siberian swollen carp ( Phlojodicarpus sibiricus ), obtained by the method according to claim 1, as a biological product with an antitumor effect.