RU2549494C1 - Method for producing silymarin and nanoselenium agent having inhibitory action of tumour cell growth - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for producing an agent inhibitory the tumour cell growth, involving preparing a mixture of aqueous solution of selenious acid and PEG 400; that is followed by preparing a mixture of aqueous solution of hydrazine hydrochloride and PEG 400; the produced mixtures are combined; the solution is put to dialyse against distilled water; surplus of water is driven off in a rotary evaporator; the produced solution is added with silymarin dissolved in Solufor with dialysis against distilled water; pH is reduced to 7.2-7.4; the method is implemented in certain circumstances.

EFFECT: agent produced by the given method possesses high inhibitory action on the tumour cell growth.

4 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of pharmacology and medicine, and in particular to a method for producing an agent representing a colloidal aqueous solution of nasalen and silymarin to inhibit the growth of tumor cells.

Silymarin is a complex of flavonolignans (the main biologically active substances of milk thistle Silybum marianum), which includes: silibinin, or silibin (it accounts for 60-70%), silicristin (20%), silidianin (10%) and isosilibine (5%) .

A large amount of experimental in vitro and in vivo data has been accumulated on experimental cancer models that show that edible and medicinal plants contain cancer-preventing compounds. Among these plant-derived agents, indole-3-carbinol (13C) and silibinin are antitumor agents. 13C is glucobrassicin (C16H19N2O9S2). This derivative is widely represented in consumed cruciferous vegetables such as cabbage, cauliflower, broccoli and Brussels sprouts. It is secreted from cruciferous vegetables by the action of myrosinase, an enzyme that is present in these vegetables, but is secreted only when plant tissue is damaged by chewing or maceration (1). 13C inhibits tumorigenesis in various animal models, including cancer of the breast, uterus, stomach, colon, lung, and liver, by modulating the carcinogen with metabolism, spreading inhibition of tumor cells, inducing apoptosis, inhibiting tumor angiogenesis and invasion (2-6). In the acidic environment of the stomach, 13C undergoes a condensation reaction capable of giving rise to several products, the predominant of which is 3,3-diindolylmethane (7). 3,3-Diindolylmethane is believed to be responsible for many physiological effects of 13C in vivo (8).

Silibinin is a polyphenolic flavonoid isolated from milk thistle (Milk Thistle L. Gaertn) (9). The antitumor activity of silibinin in animal models for oncology of the skin, prostate, lung, liver and colon was revealed through inhibition of the cell cycle, suppression of antiapoptotic gene products, inhibition of cell enzymes, inflammatory transcription factor, angiogenesis and metastasis (10-12; 23-26) .

Silibinin inhibits several cytokine-induced signaling pathways that regulate the expression of nitric oxide synthase (iNOS) in the tumor line of A549 cells and inhibits their growth in vivo, and a decrease in systemic toxicity of doxorubicin is observed in these studies (13, 14). Dietary silibinin reduces cell proliferation and angiogenesis by binding (iNOS) in a urethane-induced lung cancer model (15). It has also been shown in chemotherapy for urethane-induced lung adenocarcinoma, where silibinin also inhibits angiogenesis (16). Silibinin also reduces the spread of human non-small cell lung carcinoma (NSCLC) H1299, H460, and H322 cells by cell cycle orientation (17).

The antiangiogenic efficacy of silibinin can be mediated through the inhibition of nitric oxide (NO), which plays an important role in tumor angiogenesis. NO is a free radical that regulates a variety of physiological and pathological processes (18). NO is synthesized in the three main isoforms from NO synthase (iNOS), endothelial cells (eNOS), neurons (nNOS) NOS and induced NOS (iNOS). Of these isoenzymes, iNOS produces the largest amount of NO (19). Greater iNOS activity is observed in lung tumors than in surrounding normal tissues, and iNOS levels also increase in alveolar and tumor-associated macrophages, pulmonary endothelium, and airway epithelium (20). INOS expression / activity correlates with angiogenic status and metastatic potential in a wide range of tumors. iNOS inhibition by natural and synthetic compounds was effective in the study of cancer chemoprevention (21, 22).

It is known to use selenium as a therapeutic agent, i.e. a drug capable of participating in redox processes of body cells (see, for example, RF patents No. 2394583, No. 2426444, sites http://fitoapteka.com/read/ru; http://www.naturoprof.ru. http: //bezvreda.com/selen-v-pitanii/). Selenium normalizes the exchange of proteins and nucleic acids; regulates specific and nonspecific immunity by activating the function of neutrophils, proliferation of T-, B-lymphocytes, generating production of antibodies, lymphokines; improves body adaptation; reduces the toxic effect of substances, salts of heavy metals, drugs, other various antibiotics; prevents the development of the oxidative process, free radical diseases, including atherosclerosis and its complications, liver necrosis, pancreatitis, multiple sclerosis, parkinsonism, and other diseases.

It is generally recognized that the trace element selenium (Se) is a necessary nutrient for the normal functioning of the human body and animals, as it is part of most hormones and enzymes, actively participating in the metabolism. It performs catalytic, structural and regulatory functions in the body; interacts with vitamins, enzymes and biological membranes; participates in redox processes, cellular respiration, metabolism of fats, proteins and carbohydrates. The role of selenium in the body is largely determined by its inclusion in the composition of one of the most important enzymes - glutathione peroxidase, which protects cells from peroxidation products. Thus, selenium and its compounds exhibit significant antioxidant activity. This element is also a part of other enzymes, participates in the detoxification of xenobiotics, regulates the functions of the thyroid and pancreas, exhibits a hepatoprotective effect, stimulates the body's antitoxic defense, positively affects the reproductive system, and has a radioprotective effect.

However, the use of an agent based on colloidal selenium and silymarin obtained by the proposed method as an agent that effectively inhibits the growth of tumor cells is still unknown.

The objective of the invention is to obtain an effective agent having an inhibitory effect on the growth of tumor cells.

The technical result is to obtain funds by the proposed method, which has a high inhibitory effect on the growth of tumor cells.

The technical result is achieved by the proposed method of obtaining funds, including the preparation of the mixture (1) by adding 250 μl of a 0.5 M aqueous solution of selenic acid to 8 ml of polyethylene glycol 400 (hereinafter PEG 400), intensive stirring on a magnetic stirrer at at least 750 rpm, The pH of this mixture is 7.55, then the mixture (2) is prepared by adding 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, intensively stirred on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0 , 68, with vigorous stirring All (1) mixture (2) is added dropwise, PEG 400 and hydrazine hydrochloride are removed by dialysis of the resulting mixture against distilled water, the excess water is distilled off on a rotary evaporator, silymarin, previously dissolved in the solufor, is added to the resulting solution, the solufor is removed by dialysis, the pH is adjusted to 7, 2-7,4, while the components are mixed in an amount that ensures their content in the tool, in wt.%:

Silymarin 1.7 Selenium 0,062 Distilled water up to 100

The advantage of the agent obtained by the proposed method is that, using selenium-based nanoparticles, silymarin is delivered to the cell. In this case, the biodynamics of silymarin, combined with colloidal selenium, occurs by the lymphogenous route and is less subjected to enzymatic degradation and neutralization by the liver. The effects of silymarin and selenium in relation to the inhibition of tumor cell growth.

Also, a certain advantage is that selenium itself is part of the metabolic chain of the body and is able to be absorbed in the intracellular space. This eliminates the undesirable consequences associated with the "disposal" of the carrier itself.

The foregoing allows us to conclude that there is a criterion of "inventive step" in the claimed invention.

Bringing the pH to 7.2-7.4 is due to the need to create a stable system, since acidity values above or below the specified limits will lead to the destruction of the colloidal solution.

The selection of the conditions of the technological stages in the implementation of the method is due to the need to match the problem being solved. The values of the concentrations of the components ensures uniformity and stability of the obtained funds.

Example 1

- A mixture of selenic acid with PEG 400 (mixture 1) is prepared by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, intensively stirred on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55 .

- Prepare a mixture of hydrazine hydrochloride with PEG 400 (mixture 2) by adding 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, mix intensively on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68 .

- With vigorous stirring, the mixture (2) is added dropwise to the mixture (1). The resulting solution is put on dialysis against distilled water, while PEG 400 and hydrochloric acid hydrazine are removed from the solution.

- The excess water is distilled off (in an amount equal to the volume of the further silymarin added).

- Silymarin, previously dissolved in a solufor, is added to the resulting solution to a final concentration of 1.7%, the solufor is removed by dialysis.

- Adjust the pH to 7.2-7.4.

Total in this tool we get:

Silymarin 0.017 g / ml Selenium 0,00062 g / ml Distilled water rest

As a percentage of the total volume, the composition of the product will be as follows, in wt.%:

Silymarin 1.7 Selenium 0,062 Distilled water up to 100

The product obtained by the proposed method is a clear solution, the color of which varies from brick red to orange. The product is stored at a temperature of +2 to + 20 ° C in a dark place.

The agent obtained by the proposed method is a colloidal solution of selenium nanoparticles with silymarin particles adsorbed on its surface.

Nanoparticles in the preparation were detected by electron microscopy. Figure 1 shows a snapshot of a drug based on colloidal selenium with silymarin. Figure 1 shows nanoparticles with sizes from 40 to 100 nm.

The concentration of silymarin in the tool was determined chromatographically. Figure 2 shows the chromatogram of a sample of silymarin. It was found that the concentration of silymarin in the samples corresponded to that introduced.

Example 2

Biological studies of funds based on selenium with silymarin obtained in example 1

The pig embryonic kidney cell cultures carrying oncoviruses A and C (SPEV-2) and human cancer cells HeLa used in the work, hamster fibroblasts were obtained from the cryobank of the cell culture collection of the Virology Laboratory of the Research Veterinary Station of the Russian Academy of Sciences (Saratov). Cell cultures were cultured in plastic bottles in complete RPMI medium (10%) fetal serum, gentamicin, ampicillin, amphotericin) at 37 ° C. Dissociation of monolayer culture cells was achieved by washing the monolayer with trypsin solution for 10 min.

Animal studies were performed using the transplantable rat kidney cancer line. Before the introduction, the tumor was crushed in a Hanks solution from 0.001% trypsin to single cells. The suspension was centrifuged at 450g for 15 minutes. Oncological cells from the supernatant were injected subcutaneously between the shoulder blades. After 2 weeks, subcutaneous tumor formation was observed.

The dose of silymarin administered to rats intraperitoneally is 100 μg / kg. The drug was administered three times a day for 30 days. The dose of selenium is 3.6 μg / kg.

To accomplish the tasks used 4 groups of animals (rats) of 5 animals each. The general experimental design is presented in table 1.

When studying the possibility of using selenium spherical nanoparticles for intracellular delivery of silymarin, the optimal number of cells was determined. Cell counting was carried out using a Goryaev camera.

The MTT test was carried out according to the following procedure: a pure cell culture and cells with added preparations were incubated in 500 μl in eppendorf tubes at 37 ° C for 48 hours. Each tube with cell suspensions at the end of incubation was centrifuged for 10 min at 1000 g. Re-dissolve the resulting pellet in 500 μl of MTT solution and incubate for one hour. After incubation, the cells were redissolved in 500 μl of DMSO, 200 μl of suspension was taken from each tube and placed in the wells of a 96-well flat-bottom plate. The optical density readings were read on a reader.

The biological properties of the compound of colloidal selenium with silymarin were studied on HeLa cell lines.

When incubating HeLa cells in depleted and enriched media for seven days, it was determined that in both cases their number is approximately the same (5 × 10 ⁴ pcs / ml), respiration rate indicators are approximately equal, which indicates that there is no effect of the composition of the nutrient medium on properties of the cell population at low exposure.

In this case, we took the optical density of the sample with the maximum amount of cellular material for 100% of the respiratory activity of the cells when measured with a biochemical analyzer at a wavelength of 492 nm.

The concentration of the introduced active substances:

1. Colloidal selenium with silymarin - 0.001 M silymarin.

2. Colloidal selenium - 0.001 M selenium.

Silymarin 1 ng / ml was introduced into the cell population; selenium 5 ng / ml.

MTT test. The studied points: selenium silymarin; selenium; the control.

The cytostatic activity of the solution of silymarin with selenium was tested using the HeLa cell line (see Figure 3).

During the study, it was found that selenium reduces the respiratory activity of cells by 47%, the agent obtained by the proposed method based on silymarin with selenium by 68%, and silymarin by 40% (as a control, a solution of selenium in complete RPMI medium was used) .

Selenium with silymarin is absorbed by HeLa cells at a concentration of approximately 2 mg / ml.

The influence of the agent obtained by the proposed method on the inhibition of tumor development was carried out using an transplantable tumor of a rat kidney cancer line of RA.

The agent was administered three times with an interval of 7 days, after which the animals were euthanized and the tumor was weighed and its volume changed. The data are presented in table 2

The degree of inhibition of the tumor is calculated by the formula:

TPO% = (V control - V experience) / V control × 100

As a result of the studies, the following results were obtained: selenium inhibits tumor development by 54%, silymarin - by 60%, and the agent obtained by the proposed method based on selenium with silymarin - by 91%.

The agent obtained by the proposed method has an increased inhibitory effect on the growth of tumor cells.

In parallel, we conducted a study on normal non-cancer cells. The cell line used was a hamster fibroblast cell line.

During the study, it was found that selenium increases the respiratory activity of normal cells by 34%, silymarin with selenium - by 16%, and silymarin does not affect healthy cells (see Figure 4). A selenium solution in complete RPMI medium was used as a control.

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Claims (1)

A method of obtaining an agent that inhibits the growth of tumor cells, including the preparation of a mixture of 1 by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, intensive stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55 then mix 2 is prepared by adding 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, stirring vigorously with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring is added to the mixture 1 mixture 2 dropwise, the resulting solution becoming It is used for dialysis against distilled water by removing PEG 400 and hydrazine hydrochloride, the excess water is distilled off on a rotary evaporator, silymarin, previously dissolved in the solufor, is introduced into the resulting solution, the solufor is removed by dialysis, the pH is adjusted to 7.2-7.4, while the components are mixed in the amount providing their content in the tool, wt.%:
Silymarin 1.7 Selenium 0,062 Distilled water up to 100

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