RU2747147C1 - Pharmaceutical composition exhibiting cytotoxicity towards human colon carcinoma cells - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to a pharmaceutical composition exhibiting cytotoxicity towards human colon carcinoma cells, characterized by being an oily solution containing alpha-pinene, beta-pinene, beta-myrcene, 1,8-cineole, sabinene, abietic acid, ginsenosides Re, Rb1, Rg2, phloroglucinol, usnic acid and a vegetable oil as a pharmaceutically acceptable solvent with the following content of components, mg/l: alpha-pinene 430 to 530; beta pinene 400 to 500; beta myrcene 300 to 400; 1,8-cineole 380 to 480; sabinene 330 to 430; abietic acid 280 to 380; ginsenoside Re 300 to 400; ginsenoside Rb1 350 to 450; ginsenoside Rg2 300 to 400; phloroglucinol 150 to 250; usnic acid 100 to 200; the vegetable oil - the rest.EFFECT: pharmaceutical composition exhibiting cytotoxicity towards human colon carcinoma cells.1 cl, 2 dwg, 6 tbl, 4 ex

Description

The invention relates to the pharmaceutical industry and medicine, in particular oncology, concerns the creation of a pharmaceutical composition based on active substances isolated from plants, which can be used in patients with colorectal cancer with peritoneal carcinomatosis.

Colorectal cancer occupies one of the leading places in Russia and in the world. More than 1 million new cases are diagnosed worldwide every year. Of these, approximately 50% of patients die annually from this disease. More than 25% of patients at the time of diagnosis have stage IV disease (Malignant neoplasms in Russia in 2015. Morbidity and mortality / edited by A.D. Kaprin, V.V. Starinskiy, G.V. Petrova. P. A. Herzen - branch of the Federal State Budgetary Institution "NMIRTS" of the Ministry of Health of Russia, 2017).

Plants are a valuable source of biologically active compounds with diverse therapeutic potential (Fridlender, M .; Kapulnik, Y .; Koltai, H. Plant derived substances with anti-cancer activity: From folklore to practice. Front. Plant Sci. 2015, 6,799.) ...

According to researchers, more than 60% of drugs with anti-cancer activity are either plant extracts or substances obtained from plants (Fridlender M, Kapulnik Y, Koltai H. Plant derived substances with anti-cancer activity: From folklore to practice. Front Plant Sci. 2015; 6: 799; Ijaz, S .; Akhtar, N .; Khan, MS; Hameed, A .; Irfan, M .; Arshad, MA; Ali, S .; Asrar, M. Plant derived anticancer agents: A green approach towards skin cancers, Biomed. Pharmacother., 2018, 103, 1643-1651.).

Most of the species of medicinal plants of interest for use in oncology include such groups of substances of secondary metabolism as phenolic compounds (simple phenols, flavonoids, phenolic acids, tannins, etc.), terpenoids (mono-, diterpenoids, triterpene saponins, etc.) etc.), nitrogen / sulfur-containing compounds (alkaloids, etc.) (Shin SA, Moon SY, Kim WY, Paek SM, Park HH, Lee CS Structure-based classification and anti-cancer effects of plant metabolites. // Int J Mol Sci 2018; 19 (9): 2651-2685.). They have pronounced antitumor, antioxidant, immunomodulatory, anti-inflammatory, antibacterial properties (Gali-Muhtasib, N .; Hmadi, R .; Kareh, M .; Tohme, R .; Darwiche, N. Cell death mechanisms of plant-derived anticancer drugs: Beyond apoptosis. 2015,20,1531-1562.).

Combined herbal products have a more pronounced effect.

For example, from the prior art patent RU 2693378 C1, publ. 07/02/2019, which discloses a pharmaceutical composition exhibiting a cytotoxic effect on human urinary bladder cancer cells, which is an oily phyto extract of the fruits of common juniper, St. John's wort, flowers of tansy, rose hips, rhizomes of Potentilla erectus, rhizomes and roots of Rhodiola rosea, ginseng roots, licorice roots, peppermint leaves, rhizomes and roots of Valerian officinalis, oregano herb, eucalyptus leaves, pine buds, birch buds, taken in certain proportions.

Medicines that are phytoextracts, including those disclosed in the cited source, are usually well tolerated and have less severe side effects. However, the complexes of biologically active substances included in their composition, especially multicomponent ones, are difficult to standardize, and can differ significantly in the quantitative composition and biologically active properties, the presence of ballast substances, due to both the peculiarities of plant materials (geographical origin, growing conditions) and extraction technology ...

The use of pure, isolated bioactive phytocomponents allows for a higher level of therapeutic efficacy, easier assimilation by the body and storage of drugs for a long period. Therefore, the identification and study of natural complexes of biologically active substances in accordance with their structure, non-toxic for normal cells and a healthy organism, but capable of inhibiting the development of tumor processes, reducing the side effects of anticancer drugs, seems to be very relevant for researchers.

Currently, a number of combinations based on such active ingredients are known as anticancer therapy agents.

In particular, there is a known composition containing apigenin, arbutin, hyperoside, glycyrrhizic acid, quercetin, luteolin, naringenin, alanine, aspartic acid, arginine, glutamic acid, proline, tyrosine, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb2, ginsenoside Rb2 , ginsenoside Rq1, aralozide A, aralozide B, aralozide C, eleutheroside A, eleutheroside B, eleutheroside C, eleutheroside E, salidroside, rosavin, rosiridin, rhodionin, schizandrin, schizanterin, 2477ubl. ). This composition is aimed at preventing carcinogenesis and can be used in clinical practice for the prevention and treatment of cancer patients.

Known compositions for the treatment of cancers, such as colon cancer, which are drugs enriched with at least two pure phytochemicals selected from the group consisting of isoflavones, lignans, saponins, catechins and phenolic acids (US patent 6395279 B1 , publ. 2002-05-28). According to the source, when creating the compositions, specific isoflavones and photochemicals are selected, adapted to the needs of specific diseases.

Known pharmaceutical composition for the treatment of colon cancer, which includes ginsenoside Rh4 and irinotecan (CN 109045052 A, publ. 2018.12.21). The composition exhibits side effects on the hematopoietic system and may cause nausea and vomiting.

The listed compositions known from the prior art are not sufficiently effective in the metastasis of colon cancer.

Moreover, it is known from the prior art that metastasis to the peritoneum is a fatal variant of the progression of colorectal cancer. Metachronous peritoneal carcinomatosis occurs in 4.2% of radically operated patients. Recurrence of peritoneal carcinomatosis after complete cytoreductive surgery is observed in more than 50% of patients (Segelman J., Akte O., Gustafsson UO, Bottai M., Martling A. External validation of models predicting the individual risk of metachronous peritoneal carcinomatosis from colon and rectal cancer. // Colorectal Dis. - 2016. - Vol. 18 (4): 378-385).

The classical approach, including cytoreductive surgery followed by hyperthermic intra-abdominal chemotherapy, made it possible to improve survival in the contingent of patients with limited peritoneal carcinomatosis (PCL <5). At the same time, the median relapse-free survival was 13.3 months (Sushkov O.I., Shelygin Yu.A., Achkasov S.I., Ponomarenko A.A., etc. Factors for predicting the survival of patients operated on for peritoneal carcinomatosis in colorectal cancer intestines // Surgery. Journal named after N.I. Pirogov 2019, No. 8, issue 2, pp. 16-23).

Only complete cytoreduction can allow patients to survive the five-year milestone (Gelli M., Huguenin J., de Baere T. at al. Peritoneal and extraperitoneal relapse after previous curative treatment of peritoneal metastases from colorectal cancer: What survival can we expect? // Eur J Cancer. 2018 Sep; 100: 94-103; Elias D., Mariani A., Cloutier AS, Blot F. et al. Modified selection criteria for complete cytoreductive surgery plus HIPEC based on peritoneal cancer index and small bowel involvement for peritoneal carcinomatosis of colorectal origin. // Eur J Surg Oncol. 2014 Nov; 40 (11): 1467-73).

Consequently, today the problem of increasing the effectiveness of treatment of patients with colorectal cancer with peritoneal carcinomatosis is urgent. The use of drugs that have not only cytostatic, but immunomodulatory and antioxidant effects when used independently after cytoreductive surgeries, as well as in combination with cytoreductive surgeries followed by intra-abdominal hyperthermic chemotherapy, could increase the effect of eliminating residual tumor cells in the abdominal cavity.

From the prior art, a pharmaceutical composition is known that has an antitumor effect, the ability to induce a system of apoptosis in cells, to exert an anti-relapse and antimetastatic effect, exhibiting immunomodulatory, antibacterial, wound healing, anti-inflammatory, analgesic effects, containing a capsule extract of coniferous trees, additionally containing sesquiterpenoids, neutral diterpenoids, diterpenic acids, triterpenic acids, unsaturated and saturated fatty acids, phenolic compounds and monoterpenoids, as well as vegetable oil (patent EA 017659 B1. publ. 2013.02.28). This solution can be indicated as the closest analogue of the prototype.

The object of the invention is to provide an effective pharmaceutical composition exhibiting a cytotoxic effect on human colon carcinoma cells.

The problem is solved by the fact that the proposed pharmaceutical composition with immunomodulatory and antioxidant components (hereinafter FC), exhibiting cytotoxicity against human colon carcinoma cells, characterized in that it is an oil solution containing alpha-pinene, beta-pinene, beta myrcene 1,8-cineole, sabinene, abietic acid, ginsenosides Re, Rb1, Rg2, phloroglucinol, usnic acid with the following content of components, mg / l:

alpha pinene 430-530 beta pinene 400-500 beta myrcene 300-400 1,8-cineole 380-480 sabinen 330-430 abietic acid 280-38 ginsenoside Re 300-400 ginsenoside Rb1 350-450 ginsenoside Rg2 300-400 phloroglucinol 150-250 usnic acid 100-200

vegetable oil the rest

The technical result of the invention is to increase the efficiency of elimination of residual tumor cells in the abdominal cavity in patients with colorectal cancer with peritoneal carcinomatosis.

The active substances that make up the pharmaceutical composition can be isolated from plant raw materials by known physicochemical methods.

Various parts of representatives of the Pinaceae family (pine, fir), fruits of common juniper (Juniperus communis), coriander (Coriandrum sativum), leaves of eucalyptus (Eucalyptus viminalis), common thyme grass (Thymus vulgaris), buds silver birch (Betulapendula), male fern rhizome (Dryopteris filix-mas), lichens of the Cladoniaceae family, real ginseng root (Panax ginseng) and other plants containing active compounds included in the claims.

Table 1 shows the list and characteristics of the chemical substances that make up the pharmaceutical composition.

The vegetable oil can be any oil suitable as a pharmaceutically acceptable solvent, such as soybean oil, avocado oil, linseed oil, sesame oil, olive oil, rapeseed oil, corn oil, sunflower oil. The most preferred is the use of linseed, corn and rapeseed oil.

Biologically active compounds that are part of the claimed pharmaceutical composition have versatile pharmacological action.

For example, alpha / beta-pinenes, beta-myrcene, 1,8-cineole, sabinene, abietic acid, ginsenosides Re, Rb1, Rg2 block the signaling pathways for the activation of the nuclear transcription factor NF-kB (Jain N., Dhingra N., Narsinghani T ., Sharma R. Insights into the mechanism of natural terpenoids as NF-κВ inhibitors: an overview on their anticancer potential. // Exp. Oncol. 2016; 38 (3): 158-68; Lee CW, Song KH, Kim YS , Kim HP Ginsenosides from Korean Red Ginseng ameliorate lung inflammatory responses: inhibition of the MAPKs / NF-κB / c-Fos pathways. // J Ginseng Res. 2018; 42 (4): 476-484).

Alpha-pinene, 1,8-cineole, ginsenosides Re, Rb1, Rg2 have cytostatic and proapoptotic effects along with an increase in the activity of effector caspase-3 (Hou J., Zhang Y., Zhu Y., Zhou B. et al. Α- Pinene Induces Apoptotic Cell Death via Caspase Activation in Human Ovarian Cancer Cells. // Med Sci Monit. 2019; 25: 6631-6638; Abdalla AN, Shaheen U., Abdallah Q., Flamini G. et al. Proapoptotic Activity of Achillea membranacea Essential Oil and Its Major Constituent 1,8-Cineole against A2780 Ovarian Cancer Cells. // Molecules. 2020 Mar 30; 25 (7): 1582-1594; Xiao H., Xue Q., Zhang Q. et al. How Ginsenosides Trigger Apoptosis in Human Lung Adenocarcinoma Cells. // Am J Chin Med. 2019; 47 (8): 1737-1754)

Phloroglucinol has anti-inflammatory properties (Ashour R., Okba MM, Menze ET, El Gedaily RA Eucalyptus Sideroxylon Bark Anti-inflammatory Potential, Its UPLC-PDA-ESI-qTOF-MS Profiling, and Isolation of a New Phloroglucinol. // J Chromatogr Sci . 2019 Jul 1; 57 (6): 565-574).

Abietic acid, alpha / beta-pinenes, beta-myrcene, ginsenosides Re, Rb1, Rg2 have high antibacterial activity (Ghaffari T., Kafil HS, Asnaashari S. et al. Chemical Composition and Antimicrobial Activity of Essential Oils from the Aerial Parts of Pinus eldarica Grown in Northwestern Iran. // Molecules. 2019; 24 (17): 3203-3213; Wang L., Huang Y., Yin G., Wang J. et al. Antimicrobial activities of Asian ginseng, American ginseng, and notoginseng Phytother Res. 2019 Dec 29.doi: 10.1002 / ptr.6605).

Usnic acid is a molecule with diverse biological properties, including antibacterial, antiviral, antitumor, antioxidant activity (Patent RU No. 2536873; Luzina O.A., Salakhutdinov N.F. Biological activity of usnic acid and its derivatives. Part 2 The action of usnic acid and its derivatives on higher organisms, molecular and physicochemical aspects of biological activity. // Bioorganic chemistry 2016; volume 42, No. 3, pp. 276-300).

Substances included in the claims, such as myrcene, cineole, ginsenosides Rb1 and Rg2, exhibit antioxidant activity, suppressing the accumulation of malondialdehyde and increasing the levels of glutathione, catalase and glutathione peroxidase (Ciftci O., Ozdemir I., Tanyildizi S., Yildizi , Oguzturk H. Antioxidative effects of curcumin, β-myrcene and 1,8-cineole against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced oxidative stress in rats liver Toxicol Ind Health 2011; 27 (5): 447-53; Shaukat A., Yang C, Yang Y., Guo YF et al. Ginsenoside Rb 1: A novel therapeutic agent in Staphylococcusaureus-induced Acute Lung Injury with special reference to Oxidative stress and Apoptosis. // Microb Pathog. 2020 12; 143: 104109; Chen CF, Chiou WF, Zhang JT Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium. // Acta Pharmacol. Sin. 2008 Sep; 29 (9): 1103-8).

The combination of components with immunomodulatory, antioxidant and cytotoxic properties selected according to the invention unexpectedly made it possible to obtain a unique complex of biologically active components providing the above technical result.

Further, to confirm the possibility of carrying out the invention, examples of the compositions of the claimed pharmaceutical composition exhibiting a cytotoxic effect on human colon carcinoma cells are presented.

Example 1.

FC1 - oil solution containing beta-myrcene, alpha-pinene, beta-pinene, 1,8-cineole, sabinene, phloroglucinol, ginsenoside Re, ginsenoside Rb1, ginsenoside Rg2, abietic acid, usnic acid at the following content of components (mg / l ):

alpha pinene 430 beta pinene 400 beta myrcene 300 1,8-cineole 380 sabinen 330 abietic acid 280 ginsenoside Re 300 ginsenoside Rb1 350 ginsenoside Rg2 300 phloroglucinol 150 usnic acid 100

vegetable oil the rest

The substances included in the composition of FC1 in the above amount are dissolved in 1 liter of vegetable oil. Linseed, corn, rapeseed oil can be used as a solvent.

Example 2.

FC2 - oil solution containing beta-myrcene, alpha-pinene, beta-pinene, 1,8-cineole, sabinene, phloroglucinol, ginsenoside Re, ginsenoside Rb1, ginsenoside Rg2, abietic acid, usnic acid at the following content of components (mg / l ):

alpha pinene 480 beta pinene 450 beta myrcene 350 1,8-cineole 430 sabinen 380 abietic acid 330 ginsenoside Re 350 ginsenoside Rb1 400 ginsenoside Rg2 350 phloroglucinol 200 usnic acid 150 vegetable oil rest

The substances included in the composition of FC2 in the amount indicated above are dissolved in 1 liter of vegetable oil. Linseed, corn, rapeseed oil can be used as a solvent.

Example 3.

FC3 - oil solution containing beta-myrcene, alpha-pinene, beta-pinene, 1,8-cineole, sabinene, phloroglucinol, ginsenoside Re, ginsenoside Rb1, ginsenoside Rg2, abietic acid, usnic acid at the following content of components (mg / l ):

alpha pinene 530 beta pinene 500 beta myrcene 400 1,8-cineole 480 sabinen 430 abietic acid 380 ginsenoside Re 400 ginsenoside Rb1 450 ginsenoside Rg2 400 phloroglucinol 250 usnic acid 200 vegetable oil rest

The substances included in the composition of FC3 in the above amount are dissolved in 1 liter of vegetable oil. Linseed, corn, rapeseed oil can be used as a solvent.

Example 4 Study of pharmacological activity.

The determination of the cytotoxic activity of pharmaceutical activity (FC1-FC3) was carried out on the HCT 116 cell line of human colon carcinoma by the colorimetric method using the MTT test (Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. // J Immunol Methods 1983 Vol. 65 (1-2): 55-63).

Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine (PanEco, Russia) and penicillin-streptomycin (PanEco, Russia) at 37 ° C under 5% CO _2.

Tumor cells were seeded in 96-well flat-bottomed plates (Costar, USA). The concentration of cells in each well is 1.7 × 10 ⁴ cells / ml of complete culture medium RPMI-1640. A day later, flaxseed oil, stock FC1-FC3 oil solutions, as well as their dilutions with a 2-fold step (1: 2; 1: 4; 1: 8; 1:16; 1:32). Each concentration was examined in triplets. Control wells with cells were filled with 20 μl of medium, which was used to prepare dilutions of solutions. Plates with cells treated FK1-FK3 in various dilutions, incubated at 37 ° C under 5% CO ₂ for 24 hours and then added to wells of 20 .mu.l of MTT solution [3- (4,5-dimetiltiazolin-2) - 2.5 diphenyltetrazolium bromide] at a concentration of 1 mg / ml and incubated for 4 h at 37 ° C in 5% CO ₂ . After the formation of formazan, the supernatant was removed, the precipitate was dissolved in 150 μl of dimethisulfoxide (DMSO). Then the plates were placed in a thermostat at 37 ° C for 10 minutes, then shaken on a shaker for 10 minutes, after which the color intensity of the medium was measured on a Multiskan EX photometric enzyme immunoassay analyzer (Thermo Labsystems) at λ = 540 nm. The absorption rate is directly proportional to the number of surviving cells.

Cytotoxicity, C (in%) was determined by the formula:

Ts = (1- (Oo / Ok)) * 100%,

where OK is the optical density in the control wells, Oo is the optical density in the experimental wells.

Tumor cells were considered sensitive to the studied compositions of the pharmaceutical composition if the number of dead cells in the culture was at least 50%.

The results are shown in Tables 2-4.

Table 2 shows the results of the MTT test to determine the cytotoxic effect of the FC1 pharmaceutical composition on HCT-116 cells. Linseed oil was used as a solvent.

Table 3 shows the results of the MTT test to determine the cytotoxic effect of the FC2 pharmaceutical composition on HCT-116 cells. Linseed oil was used as a solvent.

Table 4 shows the results of the MTT test to determine the cytotoxic effect of the FC3 pharmaceutical composition on HCT-116 cells. Linseed oil was used as a solvent.

As follows from the results, HCT 116 cells appeared to be sensitive to the cytotoxic effects of the proposed examples of PK. FC1-FC3 both in the original form and in dilutions from 1: 2 to 1:16 showed a high cytotoxic effect against HCT 116 cells. Their death ranged from 71 to 84.3%. The highest cytotoxicity of FC1-FC3 was found at a dilution of 1:16 (80.9 ± 3.4; 81.9 ± 4.0; 84.3 ± 7.5%, respectively). At higher dilutions, the cytotoxic activity of the pharmaceutical compositions decreased (20.6 ± 7.7; 21.2 ± 5.2; 24.8 ± 3.2%, respectively). No cytotoxicity was found in flaxseed oil.

Thus, the proposed FC variants both in their original form and in dilutions from 1: 2 to 1:16 showed high cytotoxicity towards HCT 116 cells.

The determination of the mechanism of cell death induced by PK was carried out using the example of PK3 at a dilution of 1:16, which showed the greatest cytotoxicity against tumor cells.

The type of cell death (apoptosis / necrosis), i.e. detection of apoptotic or necrotic cells, as well as the number of living / dead cells (in%) was determined by the method of double staining with Annexin V-FITC (AnnV-FITC) and propidium iodide (PI).

HCT 116 cells were plated into 6-well plates at 200 thousand / well in 4 ml of complete RPMI-1640 medium with 10% fetal bovine serum. One day later, FC3 was added to the wells with cells at the cytotoxic concentration previously determined in the MTT test (1:16 dilution). In control wells, cells were incubated without preparations. After 24 h of incubation, the cells were washed in 500 μl of Versene's solution. FITC Annexin V Apoptosis Detection Kit I (BD, 556547) was used to assess cell death. Cells were placed in flow cytometry tubes in 100 μl of annexin-binding buffer, 5 μl of AnnV-FITC and 5 μl of PL were added. Samples were incubated in the dark at room temperature for 15 minutes. Next, 400 μL of annexin-binding buffer was added to the samples and analyzed on a FACSCanto II flow cytomofluorometer (Becton Dickinson).

AnnV-FITC and PI stained cells were evaluated according to the following criteria: live cells negative for AnnV-FITC and PI (AnnV-FITC ^- / PI ^- ), early apoptotic cells positive for AnnV-FITC and negative for PI (AnnV-FITC ⁺ / PI ^- ), late apoptotic cells positive for AnnV-FITC and PI (AnnV-FITC ⁺ / PI ⁺ ) and necrotic cells negative for AnnV-FITC and positive for PI (AnnV-FITC ^- / PI ⁺ ).

The results of double staining of HCT 116 cells with AnnV-FITC, and PI are presented in Figure 1 and Table 5. Figure 1 shows the results of double staining of HCT 116 cells of human colon carcinoma line using AnnV-FITC, and PI (A - control ( intact cells); B - cells treated with FC3 at a 1:16 dilution).

Table 5 shows the results of the effect of FC3 on the death of human colon carcinoma cells of the HCT 116 line. Table 5 shows that when exposed to FC3 at a dilution of 1:16, 27% of living tumor cells were detected, which is consistent with the results of the MTT test: FC3 at a dilution 1:16 caused the death of more than 80% of HCT 116 cells. At the same time, 47.6% of cells at the stage of early apoptosis and 24.3% of cells at the stage of late apoptosis were identified. In other words, about 72% of tumor cells are found at the stage of apoptosis. The necrotic mechanism of death was observed in 1.1% of tumor cells.

The FITC Active Caspase-3 Apoptosis Kit (BD Pharmingen) was used to determine the activation of caspase-3, an effector enzyme involved in apoptotic cell transformation.

HCT 116 cells were plated into 6-well plates at 200 thousand / well in 4 ml of complete RPMI-1640 medium with 10% fetal bovine serum. One day later, FC3 was added to the wells with cells at a 1:16 dilution. In control wells, cells were incubated without preparations. After 24 h of incubation, the cells were washed in 500 μl of Versene's solution. Then the cells were washed twice with cold PBS, resuspended in cold Cytofix / Cytoperm buffer at a concentration of 1 * 10 ⁶ cells / 0.5 ml of buffer. Incubation was carried out on ice for 20 minutes. Then they were washed from Cytofix / Cytoperm twice in Perm / wash buffer (0.5 ml of buffer for 1 * 10 ⁶ cells) at room temperature. Antibodies to active caspase-3 were diluted in Perm / wash buffer (for one tube 20 μl of antibodies in 100 μl of buffer). The cells were resuspended in the resulting solution with antibodies and incubated for 30 minutes at room temperature in the dark. Then each sample was washed in 1 ml Perm / wash buffer, resuspended in 0.5 ml Perm / wash buffer and analyzed on a FACS Canto II flow-through pyrometer (Becton Dickinson).

Table 6 shows the results of the effect of FC3 on the activation of caspase-3. Figure 2 illustrates the activation of caspase-3 by FC3 (A - control, intact cells; B - cells treated with FC3 at a 1:16 dilution). The study showed that pharmaceutical compositions (for example, a FC3) activate a pro-apoptotic enzyme, effector caspase-3, which suggests cell death as a programmed death (apoptosis mechanism).

Thus, the data obtained indicate the ability of the proposed FC variants to provide high cytotoxicity against human colon carcinoma cells, causing the death of tumor cells by the apoptosis mechanism.

Claims (2)

A pharmaceutical composition exhibiting cytotoxicity against human colon carcinoma cells, characterized in that it is an oily solution containing alpha-pinene, beta-pinene, beta-myrcene, 1,8-cineole, sabinene, abietic acid, ginsenosides Re, Rb1, Rg2, phloroglucinol, usnic acid and vegetable oil as a pharmaceutically acceptable solvent with the following content of components, mg / l: alpha pinene 430-530 beta pinene 400-500 beta myrcene 300-400 1,8-cineole 380-480 sabinen 330-430 abietic acid 280-380 ginsenoside Re 300-400 ginsenoside Rb1 350-450 ginsenoside Rg2 300-400 phloroglucinol 150-250 usnic acid 100-200 vegetable oil rest

Images