INTRODUCTION:

Currently, the interest to the use of herbal medicines determines the prospects for pharmaceutical production, focused on plant substances. It has been proven by centuries of practice that phytopreparations for the treatment of a number of diseases are not inferior in efficiency to synthetic analogs, which is confirmed by the absence of side effects, less toxicity and mild action. The study of the experience of traditional medicine in the use of medicinal plants undoubtedly contributes to an increase in the number of pharmacotherapeutic agents in the arsenal of practical medicine and veterinary medicine^1-3.

Wild medicinal plants serve as a source of more than 650 drugs, providing up to 55% of the total supply of medicinal plant raw materials for the needs of health care and chemical-pharmaceutical industry^4,5.

Phytopreparations take their rightful place in the prevention and treatment of many diseases. In our country, there are more than 800 species of various plants that have medicinal properties. Of these, only up to 10% are used in clinical veterinary practice. A significant proportion of medicines are purchased from foreign countries. There is an acute issue of developing its own full-cycle pharmaceutical production: from raw materials and substances to finished dosage forms. It should be noted that in many countries, up to 50% of drugs are obtained from natural raw materials, mainly from medicinal plants^6-8.

In connection with the deterioration of the ecological situation, it is very important to create new phytopreparations, which, in comparison with synthetic ones, have not only symptomatic, but also more effective pathogenetic effects. The use of antibacterial agents and chemotherapy drugs often leads to the development of various side reactions: allergies, addiction of pathogenic microflora to them, suppression of immune reactivity and a decrease in the effectiveness of treatment. In contrast to them, phytopreparations have a wider range of pharmacotherapeutic action, are non-toxic or low-toxic, which makes it possible to use them for a longer time and without the risk of any pronounced side effects^9-11.

With a rational combination of herbal medicines with synthetic drugs, their therapeutic capabilities are significantly expanded. Phytotherapy can be used especially effectively for the prevention and treatment of chronic, recurrent diseases, especially in pathology of the kidneys, respiratory and digestive organs, skin, and in functional disorders of the neuroendocrine system^12-14.

The search for alternative treatments for bacterial infections immediately highlighted herbal medicine. Now more and more in the practice of treatment includes the use of complex or combined phytopreparations containing two or more active components. The main advantage of these preparations is that the effect of each ingredient is taken into account when creating them. This makes it possible to simulate natural processes in the body, to purposefully control them by changing the ratio of active substances^15,16.

Based on the above, it follows that the widespread use of phytopreparations from plant raw materials is of particular importance for veterinary medicine, since such drugs have a wider range of action, fewer side effects and fewer risks of interaction with other drugs, are much cheaper than synthetic ones, are environmentally safe and can successfully replace^17-19.

Therefore, the search for new phytopreparations from local plant raw materials, the study of their pharmacological, toxicological, immunological properties, their widespread introduction into clinical veterinary practice is currently of great scientific and practical importance all over the world, especially the richness of the flora of the Republic of Kazakhstan opens up great prospects in this direction.

In this regard, the study of this issue is undoubtedly an urgent problem at the present stage of development of veterinary science and practice.

The aim of this work was to manufacture dosage forms (infusion, extracts) from local plant materials and study their toxicity in an experimental mode.

MATERIALS AND METHODS:

To resolve this aim, we used 20kg of local plant materials, 70 white mice, 20 rats, 10 guinea pigs, 20 calves, culture test Str.pyogenes, St.aureus, E.coli.

For the purpose of making dosage forms from local raw materials and studying their toxicity, we used the following local plant materials: licorice root (Glyceryhiza glabra), plantain leaves (Plantago glabra), foalfoot herb (Tussilago farfara), oregano herb (Origanum vulgare), horsetail herb (Equisetum arvense).

The chemical composition of these plants is extremely rich in various biological active substances that have pronounced pharmacotherapeutic properties in various respiratory and gastrointestinal diseases of humans and animals.

When collecting medicinal plants, it should be remembered that the value and good quality of plant materials largely depends on their correct drying. Raw materials were dried immediately after harvesting, drying of raw materials was carried out with natural (air-solar) heat on the shelves. The average drying time was within 5-7 days.

The good quality of herbal medicinal raw materials depends on many factors and is determined by the correctness and timeliness of its procurement, the content of biologically active substances, and the level of moisture, the absence of mold, impurities and pests. Medicinal raw materials are not allowed to be used without quality analyzes. Determined the good quality by special research methods, which include the acceptance of raw materials, sampling and their analysis. The authenticity of medicinal plant materials was determined by four analyzes: macroscopic, microscopic, chemical, and luminescent^20,21.

In the process of work, collections from medicinal plants (the size of the crushed parts of plants does not exceed 4 - 6mm) were made, before use, they were brewed or infused with water in a ratio of 1:10 (1 part plant, 10 parts water), as well as the preparation of an alcohol extract (dry extract).

The study of the toxicity of the prepared extracts from the collection of medicinal plants was carried out according to the method developed by the department. The indicators of acute toxicity were carried out by subcutaneous administration on 10 white mice with an average toxic dose (LD₂₀), cumulative properties - on 40 white mice. The study of the allergenic activity of the extracts was carried out on guinea pigs weighing 200-250g using the "histamine shock test".

The study of antimicrobial activity was carried out against pathogenic microorganisms test culture Str.pyogenes, St.aureus, E.coli. by the method of serial dilutions in a liquid nutrient medium²².

The measurements of the absolute body weight and internal organs were carried out in 10 rats of the experimental and control groups.

The study of the effect of dosage forms on clinical indicators (temperature, pulse, respiration) was carried out in a farm on one-month-old calves of the Alatau breed of cattle. Calves were selected taking into account their general condition, age, body weight and were kept in the same conditions of housing and feeding. The animals were regularly subjected to veterinary control and routine preventive treatments. During the period of the experiments, the farms were safe for infectious and invasive diseases.

Biochemical and morphological parameters of blood were determined using Reader StatFax 2100EIA devices (USA); EIA automatic analyzer Immulight 1000 (USA), general protein - refractometric; protein fractions - by vertical electrophoresis on agar gel. Clinical and laboratory blood tests were performed in the clinical diagnostic laboratory of the “Sana” Medical Center (Almaty).

The obtained digital data were processed by the constant method of variation statistics with the calculation of arithmetic mean values and their statistical errors (M±m), the reliability (P) of the compared indicators was determined by the Student's test. For calculations, we used the package of statistical analysis Microsoft Excel.

RESULTS:

Each new pharmacological preparation, before being recommended for use, must be subjected to a comprehensive pharmacotoxicological study. Since, only a multilateral assessment of an organism as a single biological system can make a reliable judgment about the harmfulness or harmlessness of one or another level of chemical exposure.

Based on this aim, the first stage of research was to obtain water and alcohol extracts from the above plant species. For the preparation of extracts, they take dried and thoroughly cleaned plant material from mechanical impurities. Raw materials for water extract were crushed, roots up to 5mm, leaves and grass up to 3mm.

The extraction was carried out as follows: 100g of the prepared raw material was poured into an enamel dish and filled with distilled water at the rate of 1 part of the raw material and 9 parts or 900ml of distilled water. Leaves and grass were brought to a boil (roots were boiled for 30 minutes over low heat), and left for two hours, after which they were wrung out and filtered. It was evaporated in a water bath at a temperature of +60°C to dry extract and ground to a powdery state. The extract was weighed on a laboratory scales and stored in dark tightly closed vials in a refrigerator at a temperature from +2 to +4⁰C. Also, 70% ethyl alcohol was used as an extractant. Medicinal plants were crushed on a homogenizer, then crushed raw materials were poured into glass jars and alcohol was added at a ratio of 1:10. The extraction was carried out for 14 days at room temperature, after which it was squeezed out and filtered. Evaporation and storage were carried out in the same way as in water extraction.

Table 1 shows that the yield of dry matter from medicinal plants when using distilled water as an extractant increased in plantain leaves by 15%, foalfoot herbs - by 16.3% and oregano herbs - by 13.8%. When 70% ethyl alcohol was used as an extractant, the yield of dry matter increased in licorice root by 19.9%, and in horsetail herbs - by 15.2%.

Fig. 1: Dynamics of morphological parameters of blood with intramuscular injection of extracts in the experimental and control groups

Study of the allergenic activity of extracts. The experiments were carried out on guinea pigs weighing 200-250g using the "histamine shock test". The evaluation criterion was the time from the moment of administration of the drug to the moment the animals assumed the lateral position. So, when administered at a dose of 100mg/kg of body weight of animals, histamine shock occurred, on average, after 19.80±0.32, and at a dose of 300mg/kg - after minutes. Consequently, the phytopreparation in the indicated doses does not potentiate the effect of histamine, which indicates the absence of allergenic properties in them.

Upon completion of the experiments, five animals from each group were slaughtered and pathomorphological studies were performed. In this case, only necrotic areas of the muscles were found in the area of administration of the extract due to injury from the syringe needle.

The prepared extracts from the collection of medicinal plants do not have a pronounced toxic effect on the body of white mice, as evidenced by the absence of obvious negative effects on the clinical and morphological parameters of the blood of laboratory animals.

The indicators of acute toxicity were carried out by subcutaneous administration in 10 white mice with an average toxic dose (LD₂₀), cumulative properties in 40 white mice.

For the study, 50% ethanol and lipid extracts were taken. The research data are shown in Table 4 and Fig. 2.

The data indicated in the table and diagram indicate that the introduction of 50% ethanol extract at a dose of 0.1-0.3 ml and lipid extract at a dose of 0.1-0.5 ml do not cause significant changes in the clinical state of white mice, and the introduction of 50% ethanol extract at a dose of 0.32-0.35 ml and a lipid extract at a dose of 0.5-0.55 ml cause minor clinical changes and these changes cover only a short time (2-3 hours after administration), after which come to their original physiological state.

Table 4: Indicators of acute toxicity of extracts made from the collection of medicinal plants, (M±m; n = 10)

No.	Indicators	50% ethanol extract	Lipid extract
1	LD₀ml/kg	15,1 ± 0,13	24,1 ± 0,17
2	LD ₅₀ml/kg	19,3 ± 0,21	29,2 ± 0,12
3	LD ₁₀₀ml/kg	25,2 ± 0,18	34,1 ± 0,15
4	LD₁₆ ml/kg	27,8± 0,28	25,3 ± 0,19
5	LD₈₄ ml/kg	29,4 ± 0,23	32,4 ± 0,21
Variable lethal dose rate		1,2 ± 0,05	1,3 ± 0,03

Fig. 2: Dynamics of indicators of acute toxicity of manufactured extracts from the collection of medicinal plants, ml/kg

10-15 minutes after the injection of doses exceeding moderate toxic doses of extracts LD₅₀(50% ethanol extract 0.45 ml, lipid extract-0.65 ml), laboratory animals show significant clinical signs: acute agitation, salivation, ruffled coat, fast heart rate, choking. After 20-30 minutes, the excitement changes to the stage of weakening, the mice hide at one angle, the coordination of movements is inhibited, they do not respond to external stimuli, remain chained in one place and ends in death. As a result of the immediate slaughter of dead mice, their death is attributed to acute cardiac arrest.

Thus, it was found that the maximum permissible dose (MPD) of 50% ethanol extract was (LD₀) -15.1±0.13; average toxic dose (LD₅₀) -19.3±0.21; absolute lethal dose (LD₁₀₀) -25.2 ± 0.18 ml/kg. The LD₀of the lipid extract was 24.1±0.17; LD₅₀-29.2±0.12; LD₁₀₀- 34.1±0.15 ml/kg.

In general, the danger of phytopreparations is limited not only by their moderate toxic doses, but also determined by their physicochemical and cumulative properties, resistance in the external environment, metabolic nature in the body, species sensitivity of animals. From this point of view, an objective assessment of the poisoning should be given. Therefore, a variable lethal dose coefficient was introduced in the ratio of LD₈₄and LD₁₆. The variable coefficient of lethal doses for oral administration of extracts from the above plants to white mice was determined as follows: for 50% ethanol extract, 1.2±0.05, and for lipid extract, 1.3±0.03 ml/kg.

The main indicator of the toxicological assessment of pharmacological preparations is the determination of their cumulative properties.

We carried out the cumulative properties of the extracts on 40 white mice. They were divided into 2 groups of 20 animals each. In the experimental group of mice, extracts were orally administered in the following amounts: during the first 4 days, 50% ethanol extract at a dose of 1.62 ml/kg, lipid extract - 2.5 ml/kg (LD₀ 1/10 part), then through every 4 days the doses of extracts are increased 1.5 times. At the end of the experiment, the daily dose of 50% for the ethanol extract was -18.4; and the lipid extract is 28.0 mg/kg. The extracts were injected subcutaneously with a syringe in a warm state (T 37.5⁰C). The control was carried out for 28 days.

The data obtained indicate that for 50% ethanol extract with a daily increase, the non-lethal dose was 30.5 ml/kg, and for the lipid extract - 81.0 ml/kg.

The total number of all doses leading to the death of mice from 50% ethanol extract begins to appear within 84.9 ml/kg of the total number of doses used, and from lipid extract - 131.0 ml/kg, and a further increase in their doses is characterized by an increasing probability death of laboratory animals.

The data obtained in the course of the general study indicate that the death of laboratory animals as a result of exposure to extracts does not lead to a mass process. In the group using 50% ethanol extract, the mortality rate was 3 heads, and 2 mice died from the lipid extract. That is, it was found that the average degree of toxicity (LD₅₀) after repeated administration from 50% ethanol extract was 84.9 ml/kg, and from the lipid extract - 131.0 ml/kg. The cumulative index (coefficient) for 50% ethanol extract was 4.13, and for the lipid extract - 4.21.

Thus, the data obtained in the course of the study can be formulated as characteristic weak cumulative properties of the prepared extracts from the collection of medicinal plants.

The results of measuring the absolute weight of the body and internal organs in animals of the experimental and control groups showed that under the influence of herbal dosage forms, their growth was stimulated, at the same time, the relative expression of the indicators did not have significant differences, as evidenced by the absence of a pronounced harmful organotropic effect of the prepared extracts.

Biochemical studies of blood serum in animals treated with phytopreparations revealed shifts in the quantitative content of protein, glucose, urea and aminotransferase activity. These indicators were within the physiological norm and did not significantly differ from those in the control group rats (Table 5).

So, for example, on the first day after giving the extract at a dose of 0.08 ml/kg, the protein concentration increases by 5.7%, glucose - by 13.7%, AAT - by 8.5%, creatinine decreases - by 9.5 %, and on the 30th day, protein decreases by 13.1%, urea - by 20%, glucose increases - by 3.2%. These changes in physiological parameters are not particularly large and do not reflect side effects on the general condition of animals.

Table 5: Biochemical parameters of rat blood serum after plant extract administration and withdrawal (M±m; n = 10)

Indicators	Control animals	Extract injection
Indicators	Control animals	0,08 ml/kg	0,8 ml/kg
on the 1^st day of experience
Protein (g/l)	75,0±0,33	75,0±0,17	79,3±0,37
Glucose (mmol/l)	5,84±0,38	6,64±0,62	6,87±0,39
Urea (mmol/l)	7,45±0,27	6,83±0,54	7,64±0,72
Creatinine (mmol/l)	117,3±3,4	112,2±13,58	119,32±4,43
Alanine aminotransferase (μmol ml/h)	0,94±0,07	1,02±0,05	0,77±0,09
Aspartate aminotransferase (μmol ml/hour)	0,85±0,05	0,93±0,18	1,07±0,25
on the 30^th day of experience
Protein (g/l)	78,0±0,42	67,8±0,44	65,2±0,18
Glucose (mmol/l)	5,88±0,26	6,07±0,42	6,48±0,86
Urea (mmol/l)	7,32±0,38	5,85±0,65	6,25±0,54
Creatinine (mmol/l)	118,30±2,20	122,00±2,52	112,34±2,30
Alanine aminotransferase (μmol ml/h)	0,76±0,03	0,89±0,07	0,95±0,06
Aspartate aminotransferase (μmol ml/hour)	1,16±0,44	1,25±0,08	1,37±0,67

We conducted research on the clinical state of healthy and sick with catarrhal bronchitis calves, also after giving herbal remedies.

All animals were diagnosed with catarrhal bronchitis on the basis of clinical and serological studies. The calves had rapid breathing, coughing, a positive tracheal pressure reflex, moist wheezing in the lungs and trachea.

In the first days of observation, the body temperature in some animals exceeded the physiological norm, however, after the 5th day of treatment, it did not go beyond the physiological norm and did not undergo serious changes (on average, temperature fluctuations were in the range of 38.7-40.5°C). Indicators of heart contraction were on different days either within the physiological norm, or exceeded it, and averaged 90-123 beats per minute. The presence of some tachycardia in calves is explained by our individual reaction to the study and by the hot weather in the days of the experiment. In the first week of the disease, the calves had rapid breathing, the respiratory rate exceeded the physiological norm by almost 2 times. By the end of the second week of observation, this indicator in all animals slightly exceeded the norm, which, apparently, is associated with an increased temperature in the calf barn.

After giving the phytopreparation by the 15th day, all the calves completely disappeared coughing and wheezing in the lungs and trachea, i.e. recovery has come.

DISCUSSION:

The healing properties of plants are due to the presence in them of a variety of chemicals and compounds, the quantity and quality of which depends on the type of plant, its growing conditions, collection time, drying methods and storage conditions. Any medicinal plant contains, in the presence of certain conditions, to manifest certain properties in human and animal health. More often they concentrate only in certain of its organs, and therefore, for medical needs, they use either the entire plant as a whole, or only parts of it that contain active principles. The number of active principles contained in a medicinal plant in different periods of growth and development of the latter is not the same and fluctuates, therefore, the time of collection of medicinal plants is a fundamentally important issue for the preparation of a phytopreparation.

Each new pharmacological preparation, before being recommended for use, must be subjected to a comprehensive pharmacotoxicological study. Only a comprehensive assessment of the organism as a unified biological system can make a reliable judgment about the harmfulness or harmlessness of one or another level of chemical exposure.

Based on the results of the experiment, it was found that in order to obtain a larger amount of concentrated extract, it is better to use distilled water as an extractant for plantain leaves, foalfoot and oregano herbs, and 70% ethyl alcohol for licorice root and horsetail herb.

Establishing the mechanism of action of certain biologically active medicinal substances is extremely important both from a theoretical and practical point of view. A clear understanding of this allows a more rational use of drugs, makes it possible to outline the path to the creation of new, more perfect drugs.

The danger of phytopreparations is limited only by their moderate toxic doses, and is also determined by their physicochemical and cumulative properties, resistance in the external environment, metabolic nature in the body, species sensitivity of animals.

The results of the experiment showed that the prepared extracts from the collection of medicinal plants did not have a pronounced toxic effect on the organism of white mice and rats, as evidenced by the absence of obvious negative effects on the clinical and morphological parameters of the blood of laboratory animals.

The results of measuring the absolute weight of the body and internal organs in rats of the experimental and control groups showed that under the influence of phytopreparations, their growth was stimulated, as evidenced by the absence of the harmful organotropic action of the prepared extracts.

Biochemical studies of the blood serum of rats treated with phytopreparations revealed shifts in the quantitative content of protein, glucose, urea and aminotransferase activity. These indicators were within the physiological norm and did not significantly differ from those in the control group.

The main question concerning the action of a biologically active and medicinal substance on microbes-causative agents of infectious diseases is the question of changes in the metabolic processes of a microbial cell under the influence of the substance under study. The presence of changes on the part of the main metabolic reactions of the microbe will indicate the active action of the medicinal substance. Hence, it should be noted that the pronounced bactericidal and bacteriostatic effect of approved medicinal plants is apparently due to the presence of various components in the chemical composition of these plants, in particular, essential oils, glycosides, alkaloids, tannins and ascorbic acid.

The results of our studies on the toxicity of medicinal raw materials are reliably confirmed by the scientific works of a number of research scientists^23-36.

CONCLUSION:

Thus, the analysis of the presented data allows us to conclude that the local herbal preparations used by us from local medicinal raw materials do not have pronounced toxic properties, as evidenced by the absence of obvious side effects on the organism of laboratory animals. Consequently, the manufactured dosage forms can be successfully used in the future to treat diseases of the respiratory system of young agricultural animals.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 19.11.2021 Modified on 30.12.2021

Research J. Pharm. and Tech. 2022; 15(8):3540-3548.

DOI: 10.52711/0974-360X.2022.00594

Medicinal raw material	Ratio of raw material and extractant		Dry matter yield		Dry extract yield, in%
Medicinal raw material	water	alcohol	water	alcohol	water	alcohol
Licorice root (Glyceryhiza glabra)	1/10	1/10	14,2±0,06	19,9±0,07	14,2	19,9
Plantain leaves (Plantago glabra)	1/10	1/10	15,0±0,07	11,2±0,08	15	11,2
Foalfoot herb (Tussilago farfara)	1/10	1/10	16,3±0,05	13,2±0,04	16,3	13,2
Oregano herb (Origanum Vulgare)	1/10	1/10	13,8±0,04	10,3±0,06	13,8	10,3
Horsetail herb (Equisetum arvense)	1/10	1/10	11,5±0,04	15,2±0,09	11,5	15,2

S. No.	Extracts of medicinal plants	Test culture
		*St. pyogenes*		*St.aureus*		*E.coli*
		B/cid. properties	B/stat. properties	B/cid. properties	B/stat. properties	B/cid. properties	B/stat. properties
1	Licorice root	1:160	1:320	1:80	1:320	1:40	1:80
2	Plantain leaves	1:160	1:320	1:80	1:160	1:40	1:80
3	Foalfoot herb	1:80	1:160	1:80	1:160	1:40	1:80
4	Oregano herb	1:80	1:320	1:80	1:160	1:80	1:160
5	Marshmallow rhizomes	1:160	1:320	1:80	1:320	1:40	1:80
6	Trinity violet leaves	1:80	1:320	1:80	1:160	1:80	1:160

Indicators	Before experience		After experience
Indicators	Control group	Experimental group	Control group	Experimental group
Erythrocytes, 10¹²/l	7,48 ± 0,62^х	7,21 ± 0,22^хх	7,24 ± 0,52^хх	5,63 ± 0,32^ххх
Hemoglobin, gr %	16,1 ± 0,12^х	15,5 ± 0,15^х	13,5 ± 0,61^ххх	11,75 ± 0,30^хх
Leukocytes, 10⁹/l	7,25 ± 0,48^хх	7,60 ± 0,21^хх	9,5 ± 0,61^х	13,3 ± 0,16^ххх
Stab, in %	4,0 ± 0,12^х	5,1 ± 0,23^хх	5,5 ± 0,18^хх	2,5 ± 0,19^ххх
Segmented, in %	17,5 ± 0,25^хх	23,5 ± 0,65^х	21,5 ± 0,81^ххх	23,0 ± 0,62^ххх
Eosinophils, in %	8,0 ± 0,13^х	8,5 ± 0,19^х	2,5 ± 0,17^хх	4,0 ± 0,16^ххх
Monocytes, in %	3,0 ± 0,18^х	4,0 ± 0,13^х	4,0 ± 0,28^х	5,5 ± 0,29^хх
Lymphocytes, in %	67,5 ± 2,15^х	59,1 ± 2,69^х	66,5 ± 2,83^хх	65,2 ± 2,52^хх
Body temperature, ⁰С	36,4 ± 0,39^х	36,6 ± 0,45^х	36,7 ± 0,56^хх	36,5 ± 0,60^х
Body weight, in gr.	32,6 ± 0,55^х	39,3 ± 0,82^хх	34,4 ± 0,66^хх	41,5 ± 1,02^ххх