RU2781306C1 - Method for determining the wound-healing activity of samples of products of supercritical extraction of plant raw materials for use as wound-healing drugs in vitro - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, pharmacology. A method is proposed for determining the wound-healing activity of samples of supercritical extraction of plant raw materials in vitro, including the cultivation of mouse fibroblast cells of the NIH/3T3 line on a modified F-12 medium, after achieving 70% confluence at the bottom of the well of a 24-well tablet, a scratch in the form of a cross is applied to 100 mcl using a sterile dispenser tip, and the solution of the samples in the nutrient medium is added to each well in a semi-maximal effective concentration previously calculated using a tetrazolium test, after incubation for 48 hours using an inverted microscope, the scratch area is calculated in comparison with the control: a nutrient medium without adding samples.

EFFECT: invention provides an expansion of the arsenal of methods for testing the wound-healing activity of products of supercritical extraction of plant raw materials without the use of laboratory animals.

1 cl, 1 ex

Description

The invention relates to pharmacology, and in particular to methods for the qualitative and quantitative determination of cytotoxicity and wound healing activity of products of supercritical extraction of plant materials in vitro. The invention consists in developing a method for rapid assessment of cytotoxicity and wound healing activity of supercritical plant extracts in vitro without the use of laboratory animals.

Currently, in connection with the general increase in the life expectancy of people on earth, a particularly relevant area of research is the creation and study of drugs that preserve the quality of life of people over 35 years old. For example, such drugs include antibiotics and drugs that allow you to deal with the phenomena of multi-resistance (bacteria or tumor cells). All these characteristics are possessed by natural substances isolated from natural raw materials. For example, terpene glycosides and flavonoids are very effective in combating multidrug resistance of bacteria and fungal infections to antibiotics. Many natural polysaccharides have antimicrobial and antiviral properties (while not being antibiotics). Glycosaminoglycans are very effective probiotics, and their use is often more effective than the use of antibiotics [1]. Sulfated polysaccharides contribute to the thinning of human blood, which in general has a very positive effect on health.

However, it should be noted that the extraction of such compounds is associated with many unsolved technical problems. For example, with classical extraction methods, the yield of the finished product may be insufficient. In this regard, it is more promising to use supercritical fluid extraction methods for these purposes. The supercritical extraction industry is now developing rapidly and is of great interest, since it is distinguished by the highest yields of finished extracts and a gentle extraction regime. New approaches in this direction can give competitive advantages in the isolation of new substances and in the development of technological processes for the production and research of innovative drugs and other substances of this type [2].

The Siberian Federal District has a unique flora, a large number of rare plants and endemics of the region. The use of supercritical fluid extraction technologies will allow the isolation and study of minor compounds that were not possible to detect with previous extraction methodologies.

Vegetable raw materials are a rich source of vitamins, essential amino acids and polyunsaturated fatty acids. Also in Russia, as well as in Europe, the direction of functional nutrition, which came from Asia, is actively developing as part of personalized medicine. This trend is gradually replacing the use of dietary supplements. The production of functional food allows to reduce the time between the development and entry of goods to the market, simplifies the registration of product exports, and most importantly, contributes to the development of a food culture of the population. In addition, the complex consumption of wholesome foods has a synergistic effect and is often more effective than the consumption of active ingredients separately.

The use of products of supercritical extraction of plant raw materials will solve a number of environmental problems caused by the massive use of chemical plant protection products. This determines the high relevance of the search for alternative means of increasing plant resistance to adverse factors (including plant pathogens) based on the action of triterpene acids in extracts from needles, spruce flavonoids, and brassinosteroids. Cheap sources of such compounds can be plant extracts obtained using supercritical fluid technologies. Therefore, the study of biologically active compounds of plants is an important and urgent task. The solution of this problem is important for fundamental science and for the development of formulations of high-quality natural functional food products.

Based on the foregoing, the study and development of methods for assessing the activity of secondary metabolites and plant polysaccharides is very important for the development of modern medicine and the fight against socially significant and alimentary-dependent diseases, which is proved by the availability of relevant patents [3; four].

When new compounds are obtained by supercritical extraction from plant materials, the problem of assessing their biological activity arises. It should be noted that at present there are no patents directly on methods for studying the biocompatibility of such substances and evaluating their effect on biological processes, in particular, on cell division. Our task was to develop a method for studying the wound healing activity and cytotoxicity of the products of supercritical extraction of plant materials in vitro.

However, testing samples of this kind is associated with many methodological difficulties. Usually, various cell lines with different properties are used to study the wound healing activity and cytotoxicity of prototypes of functional materials for biomedical purposes.

The prototype of the developed method for assessing cytotoxicity is the standard method described in GOST R ISO 10993-5-2009 [5]. However, the standard method has many variations, and one of the main difficulties is the discrepancy between critical experimental parameters in existing methods. Such parameters are types of cell lines, methods of sample preparation and incubation. The standard method is universal and does not take into account the important parameters necessary for the study of cytotoxicity and wound healing activity of the samples. At the same time, each researcher offers his own variants of the methodology, which leads to divergence of research results, complicating the reproducibility of experiments and making meta-analyses difficult. In this regard, it seems particularly relevant to develop a method for conducting experiments on testing the cytotoxicity of plant extracts.

The technical result is the ability to quickly assess the cytotoxicity and wound healing activity of supercritical plant extracts in vitro without the use of laboratory animals.

The problem is solved by the fact that the method of qualitative and quantitative determination of cytotoxicity and wound healing activity of products of supercritical extraction of plant materials for use as wound healing drugs in vitro, including sample preparation, cell line cultivation and direct tetrazolium test, differs from the prototype in that on the first stage in the claimed method assess the overall cytotoxicity of the samples and select the least toxic of them using tetrazolium test, then at the second stage evaluate the wound healing activity by the method of scratch healing.

NIH/3T3 mouse fibroblasts are used to assess overall cytotoxicity.

For cell line cultivation, a modified F-12 medium containing inactivated fetal calf serum, L-glutamine, gentamicin solution and a mixture of penicillin-streptomycin is used.

To assess wound healing activity, after reaching 70% confluence at the bottom of the well of a 24-well plate, a scratch in the form of a cross is applied using a sterile 100 μl dispenser tip, then a solution of samples in a complete nutrient medium is added to each well in a half-maximal effective concentration, as a control, use a complete nutrient medium without adding samples, after incubation using an inverted microscope, the scratch area is calculated compared to the control.

The developed method includes:

1. Selection of cell lines that have medical significance and respond satisfactorily to exposure to samples.

2. Type and composition of the nutrient medium most suitable for cell culture growth.

3. Sample preparation parameters most suitable for samples of plant extracts.

4. Culture parameters most suitable for a given cell line.

The claimed method for assessing the cytotoxicity and wound healing activity of plant extracts in vitro was implemented in the course of a scientific study devoted to testing these samples. It should be noted that the assessment of cytotoxicity and wound healing activity of plant extracts in vitro is carried out in two stages. At the first stage, the overall cytotoxicity of the preparations submitted for the study and the selection of the least toxic ones are evaluated, at the second - their wound healing activity. The implementation of this method was as follows.

The NIH/3T3 cell line was used to study the cytotoxicity of the samples. This line represents dividing mouse fibroblasts. Fibroblasts secrete collagen proteins that are used to maintain the structural basis of many tissues. Dermal fibroblasts produce connective tissue and extracellular matrix components that influence epidermal regeneration and promote wound healing. Therefore, cells of this line are often used for biotesting of wound healing drugs. In this study, the cytotoxicity of the sample was studied by the MTT test. This is a method for determining the viability of cell cultures. Its action is based on the ability of living cells to convert soluble yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide (MTT) into insoluble purple-blue intracellular crystals of MTT-formazan (MTT-f). Non-viable cells do not have this ability. The intensity of MTT conversion to MTT-f reflects the general level of dehydrogenase activity of the studied cells and is modulated by the activity of coupled enzyme systems, for example, the respiratory electron transport chain, etc. The microtetrazolium test is widely used in assessing the effects of toxicants, pharmacological preparations, and adverse environmental factors on cells, making it possible to assess the toxicity of the studied preparations in vitro [6].

Culture medium preparation:

For cultivation, a modified nutrient medium F-12 was used. The modification of the nutrient medium consisted in adding to 450 ml of the prepared medium 50 ml of previously inactivated fetal calf serum, 300 mg of L-glutamine, 50,000 IU of a mixture of penicillin-streptomycin, and a solution of gentamicin in a ratio of 1 μl/1 ml of the prepared medium. This composition of the nutrient medium ensures stable growth of the cell line and prevents contamination of the culture. Inactivation of fetal calf serum was carried out by keeping it in a water bath at 57°C for 20 min, followed by filtration through a membrane-type bacterial filter with a pore diameter of 0.22 μm.

Cell line preparation:

1. Cells were thawed by placing the ampoules in a water bath at 37°C for 1–2 min. After that, the entire contents of the ampoule were pipetted into a sterile tube with a capacity of 15 ml. Then 1 ml of pre-prepared nutrient medium warmed up to 37°C was slowly added.

2. Resuspend the cell pellet in complete nutrient medium to achieve a cell concentration of 5×10 ³ cells/cm ² . After that, the cells were placed in culture vessels with an area of 75 cm ² with 15 ml of medium, 500 thousand cells each.

3. Cells were incubated in an incubator with humidity 37°C, 5% CO ₂ . When the cells adhered to the bottom of the vessel (within 48 hours), the supernatant was discarded and replaced with fresh, prewarmed (37°C) medium. At the same time, at least two passages were performed before using the cells in the tests.

4. Cells were cultured in an incubator until a confluence of 60-70% was reached. At the same time, complete confluence of cells was not allowed.

5. The medium was aspirated from the culture vessels and the cells were washed with warm 1X PBS.

6. Added 5 ml trypsin-EDTA solution and incubated at 37°C for 2 minutes. To avoid clumping, the cells were not shaken by hitting or shaking the flask until they separated. Once the cells detached from the substrate, the cell suspension was transferred to a vessel with 6-8 ml of complete growth medium to inactivate trypsin.

7. Cells were centrifuged at 1200 rpm for 3 minutes (4°C) and cells were resuspended in appropriate volumes of medium. Appropriate aliquots of the cell suspension were added to the culture flasks (approximately 5×10 ³ cells/cm ² ). The medium was changed approximately every 3 days.

8. The culture medium was changed to fresh one the day before subculture in 24-well plates. Then, on the day of seeding, a cell suspension was prepared in the plate in complete nutrient medium. 100,000 cells in complete medium were added to each well.

9. Cells were incubated for 24 hours ± 2 hours (37 °C ± 1 °C, humidity 90% ± 10%, 5.0% ± 1.0% CO ₂ / air) so that the cells formed less than half (< 50 %) confluent monolayer. This incubation period ensures cell recovery, adherence and transition to the exponential growth phase.

Preparation of MTT solution:

MTT powder was dissolved in 1X PBS to a concentration of 5 mg/ml, aliquoted and stored at -20°C as a working solution.

Conducting an in vitro cytotoxicity experiment:

1. Gently aspirate all medium from the plate. Then, 1 ml of a solution of the corresponding sample in a complete nutrient medium was added to each well. The concentration of the samples was selected experimentally and amounted to 10; one; 0.1 and 0.01 mg/ml. The concentration value could shift in one direction or another depending on the detected activity of each specific substance. As a control, cell growth in a complete nutrient medium without the addition of samples was used.

2. Place the cell plate in the incubator for 48 hours.

3. Aspirate all media from the plate and wash each well once with 500 µl PBS.

4. Aspirate the PBS solution and add 500 µl of MTT solution to each well, then place the plates in the incubator for 4 hours (37°C ± 1°C, 90% ± 10% humidity, 5.0% ± 1.0% CO ₂ /air).

5. Remove the MTT solution by gentle aspiration.

6. Add 500 µl of DMSO to each well and place the plate on a shaker at 500 rpm for 10 minutes to dissolve the formazan crystals.

7. Transfer 100 µl of DMSO solution from each well to 5 wells of a 96-well plate.

8. Measured absorbance at 620 nm using a Tecan Infinite F50 ELISA reader. The lower the absorbance value compared to the control, the fewer cells survived after contact with the corresponding sample, therefore, the cytotoxicity of the sample is greater. Based on the absorbance data, a calibration plot was built and the half maximum effective concentration for each sample (EC ₅₀ ) was calculated.

Conducting an experiment to assess wound healing activity in vitro:

At the second stage of the study, the wound healing activity of the samples that showed the least cytotoxicity was studied. For this, the scratch method was used.

The preparation of nutrient media and cell lines in this method was carried out similarly to the previous one. The difference was that after reaching 70% confluence, a cross-scratch was created at the bottom of the well of a 24-well plate using a sterile 100 µl pipette tip. Cells were then washed with 1X PBS/PBS to remove debris. After that, a solution of samples in complete nutrient medium at a concentration of EC ₅₀ was added to each well. A complete culture medium without the addition of samples was used as a control. Incubation was carried out for 48 hours (37°C ± 1°C, humidity 90% ± 10%, 5.0% ± 1.0% CO ₂ /air). After incubation using an inverted microscope in phase contrast mode, the area of the scratch was read in comparison with the control. The resulting photographs were processed using the ImageJ software product.

An example of a specific implementation of the invention.

Using the developed method, the cytotoxicity and wound-healing activity of the prototype dosage form, which is a thin hydrophilic film made by pouring into molds with subsequent evaporation of the solvent, was studied. The components of the prototype dosage form were betulin, miramistin and inert excipients. Betulin (3β, 28-dihydroxy-20 (29) - lupene or loop-20 (29) - en-3β, 28-diol) is a natural pentacyclic triterpenoid of the lupan series. This substance is found in birch bark, hazel bark, calendula and other medicinal plants. A sample of the same composition, but without the addition of miramistin, was used as a positive control. A complete nutrient medium standard for this cell line was used as a negative control. For the experiment, sample solutions were used in a complete nutrient medium with concentrations of 7 mg/ml; 0.7 mg/ml; 0.07 mg/ml; 0.007 mg/ml; 0.0007 mg/ml; 0.00007 mg/ml and 0.000007 mg/ml. The duration of cell incubation with the sample was 48, 72, and 120 h.

As a result of the experiment, it was revealed that the sample of the dosage form prototype presented for the study has cytotoxicity. It was found that the level of its cytotoxicity is higher than that of the complete nutrient medium (p < 0.05). The effect was observed only at a sample concentration of 7 mg/mL. If we take the number of live cells in the negative control sample as 100%, then the sample reduced their number by 50%.

On the contrary, the positive control sample at the working concentration showed not only the absence of any cytotoxicity, but also a significant increase in fibroblast proliferation, which can be determined by a 90% increase in optical density. This was confirmed statistically (p < 0.05). It should be noted that the effect was also observed only at a sample concentration of 7 mg/ml, while at other concentrations, the increase in proliferation did not differ from the control.

It was also found that the greatest effects of both samples were recorded at an incubation duration of 48 hours. At an incubation duration of 72 hours, many dead cells were found in all cultures, and at an incubation duration of 120 hours, all results did not differ from the negative control (p > 0.05).

As a result of the first stage of the experiment, it was revealed that a sample of the prototype dosage form has cytotoxicity against fibroblasts. At the same time, a sample of a similar composition, on the contrary, accelerates the proliferation of fibroblasts.

At the second stage of the study, the wound healing activity of the samples was studied by the scratch method. For the experiment, a solution of samples with a concentration of 7 mg/ml was used. The duration of incubation was 48 hours. A complete culture medium was used as a negative control without the addition of samples.

As a result of the second stage of the experiment, it was revealed that under the influence of the positive control sample, the scratch area decreased by 62% compared to the negative control (p < 0.05). It was also found that the sample with the addition of miramistin, on the contrary, slowed down the healing of the scratch compared to the negative control by 25% (p < 0.05).

Literature

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2. Maksudov R.N., Tremasov E.N., Egorov A.G., Mazo A.B. Supercritical extraction of oilseeds // Bulletin of the Kazan Technological University. 2010. №2.

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5. GOST R ISO 10993-5-2011 Medical products. Evaluation of the biological effect of medical devices. Part 5. Cytotoxicity studies: Methods. 01/01/2013.

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Claims (1)

A method for determining the wound healing activity of samples of products of supercritical extraction of plant materials in vitro, including the cultivation of mouse fibroblast cells of the NIH / 3T3 line on a modified F-12 medium containing inactivated fetal calf serum, L-glutamine, a solution of gentamicin and a mixture of penicillin-streptomycin, after reaching 70 % confluence at the bottom of the well of a 24-well plate is scratched in the form of a cross using a sterile 100 µl dispenser tip, then a solution of samples in the indicated nutrient medium is added to each well at a half-maximal effective concentration previously calculated using a tetrazolium test, nutrient is used as a control environment without adding samples; after incubation for 48 hours, using an inverted microscope, the scratch area is calculated in comparison with the control.