RU2154270C1 - Method of assay of biological activity of preparation - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to method of estimation of quality and biological activity of preparation prepared of celandine (Chelidonium majus L.). Method involves preparing preparation samples, culturing cells to be tested in nutrient medium, counting living cells and comparison with control in cultures without addition of preparation. Cells are grown in medium α--MEM with 10% embryonic calf serum up to monolayer formation. Then culturing medium is removed, 0.25-% trypsin solution is added and incubated for 3-10 s. Culture is immersed in 0.02% Versene solution and incubated for 10 min at 37 C. Detached cells are collected, washed out from Versene solution twice by centrifugation and incorporation level of ³H-thymidine by proliferating cells is determined. Preparation is considered to be tested if degree of inhibition of total incorporation of ³H-thymidine and/or adhesion is 50% (not less) of control level in cultures without addition of preparation. Proposed method ensures to examine some indices simultaneously and provides the quantitative characterization of preparation. EFFECT: simplified method, decreased cost. 3 dwg

Description

 The invention relates to the field of agriculture and can be used in the biotechnological and food industries to assess the quality and biological activity of preparations made from plants, mainly large celandine (Chelidonium majus L.).

 A known method for determining the biological activity of plant cells under the influence of adverse factors [1].

 However, this method does not allow to determine the antitumor activity of the drug, requires special equipment.

 There is also known a method of testing a biologically active drug [2], which includes the preparation of samples of the drug, growing test cells in a nutrient medium, counting living cells and comparing with control in cultures without adding the drug.

However, this assessment method is very laborious and often requires a number of tests: determining the total number of cells and the number of living cells, mitotic and oxidase activity, and the level of ³ H-thymidine incorporation by proliferating cells.

 The objective of the invention is to determine the biological activity of the drug from the celandine of large (Cheltdonium majus L.), in particular "Blastophagus" "Ramifara" and others in a simpler and cheaper way and gives a quantitative characteristic of the drug, independent of the experimenter, which can be used in biotechnology industry in the production and testing of the drug.

 As a result of the use of the present invention, cost reduction is achieved when testing the biological activity of a preparation from celandine, in particular the antitumor activity of a blastophage.

The specified technical result is achieved in that the determination of the biological activity of the drug, mainly "Blastophagus", prepared from plants of large celandine (Chelidomum majus L.), includes the preparation of samples of the drug, growing test cells on a nutrient medium, counting living cells and comparing with control in cultures without the addition of the drug, the cells are grown on αMEM medium with 10% fetal calf serum until a monolayer is obtained, then the culture medium is removed and a 0.25% trypsin solution is added and the they are shaken for 3-10 s, after which they are immersed in culture in a 0.02% Versene solution and incubated at 37 ^° C for 10 minutes, then the detached cells are harvested and washed 2 times from Versene solution by centrifugation and the number of living cells is counted and determine the level of inclusion of ³ N-thymidine by proliferating cells, the drug is considered tested if the degree of suppression of the total inclusion of ³ N-thymidine and / or adhesion is at least 50% of the control level in cultures without adding the drug.

 Figure 1 shows the profiles of the suppression of adhesion in 5 variants of the drug.

 In FIG. 2 shows the dependence of the sensitivity of cell lines to the blastophage.

 Figure 3 presents the total activity of each test blastophage series in relation to all 20 lines.

 An example of a specific implementation.

Testing is proposed to be conducted by the level of ³ H-thymidine incorporation by proliferating cells, as it gives a high degree of correlation with other studied methods, is easy to implement, and allows analysis of several parameters at once (adhesion and the total number of proliferating cells in the culture) and quantitatively characterize the drug.

 The test was carried out on cells of the CHO line, as one of the most sensitive and unpretentious cell lines.

 The testing methodology includes 6 stages.

1. Cells are grown on αMEM medium with 10% fetal calf serum until a monolayer is obtained, then the culture medium is removed and a 0.25% trypsin solution is added and exposed for several seconds (3-10 s), after which it is immersed in 0 , 02% Versen’s solution and incubated at 37 ^o C for 10 min, then the detached cells are collected and washed 2 times from Versen’s solution by centrifugation.

 2. Count the number of living cells. To do this, mix 50 μl of the cell suspension with 50 μl of a 0.1% trypan blue solution and calculate the number of unpainted cells in the Goryaev chamber. The suspension is diluted in a culture medium to a concentration of 250 thousand / ml and poured into the wells of a 96-well flat-bottomed tablet of 0.1 ml. Add 0.1 ml of the test drug in different concentrations.

3. The tablets are placed in a CO ₂ incubator and cultured for 48 hours at 37 ^° C, 90% humidity and 5% CO ₂ . After incubation, 0.5 μCi ³ N-thymidine (specific activity 37 GBq / mM) was added to each well and incubated for another 16 hours.

 4. Using a harvester, non-adherent cells are transferred to fiberglass filters, washing each well with warm αMEM medium (1 wash). 50 μl of trypsin solution and 150 μl of Versen's solution are added to each well of the plate. The plates are incubated for 10 min in an incubator and again washed on the harvester with distilled water and ethyl alcohol, transferring the detached cells to the filters.

 5. The filters are dried for several hours and placed in scintillation vials with 5 ml of scintillation fluid (toluene with 3 g / l PPO and 0.1 g / l POROP). Radioactivity is calculated on a β-spectrometer. Separately determine the radioactivity deposited on the filters after 1 and 2 washes. To determine the total inclusion of isotopes, the results of 1 and 2 swabs for each well are summarized.

6. The drug is considered tested if the degree of suppression of the total incorporation of ³ H-thymidine (2 rinses) and / or adhesion is at least 50% of the control level in cultures without adding the drug. The concentration of the blastophage should be no higher than 5 μl / well (25 μl / ml).

 Using this test, activity profiles of 5 investigated blastophage series at a dose of 5 μl / well on 20 lines were obtained (Fig. 1). Cell lines are arranged in order of increasing sensitivity to the drug. It can be seen that the most active drug was PF2, which in no case caused a stimulating effect. At the same time, it was this drug that had the maximum stimulating activity against intact spleen lymphocytes.

 Three cell lines turned out to be almost insensitive to the bastophage, and 4 highly sensitive (Fig. 2). The least sensitive was the MDCK line (dog kidney cells). Then two leukemia lines follow - MT4 and Daudi. Highly sensitive lines include breast tumor cells (McF-7wt), Chinese hamster ovary (CHO), human fetal lung (L41), and hamster embryonic fibroblasts (HETR).

 The ambiguous effect was also given by different series of the blastophage (Fig. 3). PF2 generally inhibited tumor cell adhesion by 78.7%. Drugs 3 and 7 were less active - the degree of suppression of 60 and 60.2%, respectively. The 1D and 8 preparations turned out to be weaker - 48.9 and 42.1%, respectively.

 The proposed method for determining the biological activity of the drug in comparison with the known one gives a high degree of correlation with other studied methods, is simple to implement, and allows analysis of several parameters at once (adhesion and the total number of proliferating cells in the culture) and allows to obtain a quantitative characteristic of the drug, does not contain expensive ingredients and is more effective in assessing the therapeutic properties of drugs, for example, the antitumor activity of the blastophage, and can be used in otehnologicheskoy industry.

Sources of information:
1. Patent of the Russian Federation N 1722313, M.cl. A 01 G 7/06, publ. B.I. N 12, 1992.

 2. Requirements for the preclinical study of the general toxic effects of new pharmacological substances (temporary guidelines) Moscow, Ministry of Health of the USSR, 1985

Claims (1)

A method for determining the biological activity of a preparation, including preparation of preparation samples, growing test cells on a nutrient medium, counting live cells and comparing their number with the number in the control in cultures without adding a preparation, characterized in that the cells are grown on α MEM medium with fetal calf serum up to a monolayer, then the culture medium is removed and trypsin solution is added, after which Versen solution is added to the culture and incubated at 37 ^° C for 10 min, then the detached cells are they shave and wash 2 times from Versen's solution by centrifugation and count the number of living cells, after which the level of ³ H-thymidine incorporation by proliferating cells is determined, and the drug is considered to have biological activity if the degree of suppression of the total incorporation of ³ H-thymidine and / or adhesion is at least 50% of the control level in cultures without the addition of the drug.

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