RESEARCH OF ANTIMICROBIAL ACTIVITY OF ANTHELMINTHIC MIXTURE FOR NEMATODOSIS TREATMENT

Parasitic infections are one of the most acute problems of the modern society as this group of diseases is met in all countries of the world. According to the World Health Organization around 1.2 billion people are infected with diverse parasitic infections each year. Helminthiases of digestive system, primarily nematodosis, constitute a group of special risk, since a particularly sensitive audience for them are children of younger and middle age. Phytotherapy, which is based on the centuries-old experience of folk medicine, promises to be a solution to this problem, enabling to create effective complex medicines for nematodosis prevention and treatment. Thus, the herbal mixture was prepared on the basis of the offered composition of medicinal plant raw material. The research of antimicrobial activity of the studied herbal mixture was conducted in vitro by the method of diffusion into agar at the Biotechnology Department of the National Pharmaceutical University under the direction of Doctor of Pharmaceutical Sciences, prof. Strelets O. P. The obtained results of the carried out research of the antimicrobial properties of the herbal mixture samples allowed examining the level of antimicrobial activity. The further research of the investigated herbal mixture and the offered phytocomposition is promising for creation of new effective medicines with the complex action including anthelminthic (specific to nematodes), antiparasitic, anti-inflammatory, antibacterial, antiseptic, hepatoprotective, nephroprotective, laxative, antiallergic, antispasmodic, analgesic, sedative activities.


Introduction
Helminths in humans are classified into round (nematodes), tape (cestodes) and flukes (trematodes).The toxic effect of the products of vital activity and disintegration of helminths parasitizing in the human body causes damage to the nervous system, dyspeptic disorders, suppression of the secretory and motor functions of the digestive system.Often there are allergic reactions, expressed by fever, rash, itching, eosinophilia or asthmatic attacks.The mechanical effect of helminths and their larvae vital activities lead to trauma caused by worms' suckers, crests and teeth.For example, ascaride conglomerates can clog the bile ducts and cause intestinal insufficiency [1,2].
The centuries-old heritage of folk medicine in the treatment of digestive system helminthiases contains many recipes for monoand polycomponent remedies.The analysis of this experience, as well as drugs offered by phytomedicine, made it possible to identify a number of medicinal plant raw material, which is promising for the creation of complex herbal medicines with polyvalent pharmacological activity [6,7].
On the basis of the conducted research [8,9] the promising medicinal plant raw materials for the development of a new phytomedicines with complex anthelminthic action (specific to nematodes) are: -Tansy flowers (Flores Tanaceti vulgaris, Tanacetum vulgare L., Asteraceae); -Flax seeds (Semina Lini, Linum usitatissimum L., Linaceae); -Horsetail herb Based on the offered composition the herbal mixture containing aqueous and water-glycerine extracts of the said medicinal plant raw material was prepared.Such composition provides anthelminthic (specific to nematodosis), antiparasitic, anti-inflammatory, antibacterial, antiseptic, hepatoprotective, nephroprotective, laxative, antiallergic, antispasmodic, analgesic and sedative activities.
Abstract: Parasitic infections are one of the most acute problems of the modern society as this group of diseases is met in all countries of the world.According to the World Health Organization around 1.2 billion people are infected with diverse parasitic infections each year.Helminthiases of digestive system, primarily nematodosis, constitute a group of special risk, since a particularly sensitive audience for them are children of younger and middle age.Phytotherapy, which is based on the centuries-old experience of folk medicine, promises to be a solution to this problem, enabling to create effective complex medicines for nematodosis prevention and treatment.Thus, the herbal mixture was prepared on the basis of the offered composition of medicinal plant raw material.The research of antimicrobial activity of the studied herbal mixture was conducted in vitro by the method of diffusion into agar at the Biotechnology Department of the National Pharmaceutical University under the direction of Doctor of Pharmaceutical Sciences, prof.Strelets O. P. The obtained results of the carried out research of the antimicrobial properties of the herbal mixture samples allowed examining the level of antimicrobial activity.The further research of the investigated herbal mixture and the offered phytocomposition is promising for creation of new effective medicines with the complex action including anthelminthic (specific to nematodes), antiparasitic, anti-inflammatory, antibacterial, antiseptic, hepatoprotective, nephroprotective, laxative, antiallergic, antispasmodic, analgesic, sedative activities.Keywords: nematodosis, helminthiases, medicines of natural origin, medicinal plant raw material, antimicrobial activity, parasitology, digestive system, phytotherapy, pharmacotherapy.Thus, the aim of our research was to evaluate the antimicrobial activity of the proposed herbal mixture in order to determine the prospects of the further specified studies and development of new medicine with complex anthelminthic activity (specific to nematodosis).

Methods
The research of the antimicrobial activity was carried out at the Biotechnology Department of the National Pharmaceutical University, under the direction of Doctor of Pharmaceutical Sciences, prof.Strelets O.P. in May-June 2017.
The antimicrobial activity of the studied herbal mixture was investigated in vitro by the method of diffusion into agar (the method of "wells") [10,11].This method is based on the ability of the active substances to diffuse into the agar medium, previously inoculated with microorganisms' cultures.The results of the research characterize both the antimicrobial activity of the medicine and the release of antimicrobial substances from the base as zones of microorganisms' growth inhibition are formed as a result of the diffusion of these substances into a dense nutrient medium.
The following pure cultures were used as the test cultures: gram-positive microorganisms Staphylococcus aureus ATCC 25293, spore culture Bacillus subtilis ATCC 6633, gram-negative cultures Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.Fungicidal activity was examined towards yeast fungi of the genus Candida albicans ATCC 885-653.
During the studies, one-day suspensions of bacterial microorganisms in physiological saline and a two-day culture of yeast fungi were used.The microbial load was 10 7 CFU/ml.
In a Petri dish set on a horizontal surface, 10 ml of melted "hungry" agar was added.After solidification of this lower agar layer, 3-6 sterile thin-walled steel cylinders were placed on its surface at equal distance from each other and from the edge of the cup (inner diameter -6.0±0.1 mm, height -10.0±0.1 mm).Around the cylinders, the top layer was filled, which consisted of 14 ml of molten and cooled up to 45-48 °С agar, which was previously mixed with the seed dose of the test microorganism.When working with bacterial cultures for the second layer, meat-peptone agar (MPA) was used; when working with yeast fungi agar Saburo was used.After cooling of the upper layer the cylinders were removed with sterile tweezers and the test samples of the medicine were added to the obtained wells until they were completely filled.The Petri dishes were held for 30-40 minutes at room temperature and placed in a thermostatbacterial cultures at the temperature 32.5±2.5 °С for 18-24 h, culture of yeast fungi -at the temperature 22.5±2.5 °С for 48 h.
The interpretation of the results was performed by the examination of microbial growth inhibition zone including the diameter of the wells.The measurement was carried out with an accuracy of 1 mm, while focusing on the complete absence of visible growth.
The diameter of microorganisms' growth inhibition zones characterizes the antimicrobial activity of the samples: -the absence of microorganisms' growth inhibition zones around the wall, as well as the growth inhibition area up to 10 mm in diameter, was assessed as insensitivity of microorganisms to the introduced sample; -growth inhibition areas 11-15 mm in diameter were evaluated as a weak sensitivity of the culture to the concentration of active substances in the sample; -zones of growth inhibition with a diameter of 16-25 mm were assessed as an indicator of moderate sensitivity of microorganisms to the test sample; -zones of growth inhibition, the diameter of which exceeded 25 mm, indicates a high sensitivity of microorganisms to the test sample.

Results
The obtained results of the carried out research of the antimicrobial properties of the investigated mixture samples are represented in Table 2.

Discussion
Research of antimicrobial activity of herbal mixture with the composition described above is original for such combination of active substances.However, it is known that an increased content of coagulase-positive staphylococci and enterococci is observed in patients with helminthiases, namely, ascariasis [12].
Data obtained experimentally and presented in It should be noted that the samples revealed weak antimicrobial activity in relation to the test culture of Staphylococcus aureus: the diameters of growth inhibition are less than 15 mm, namely 13.6±0.5 mm.
The studied mixture showed a moderate antimicrobial activity (diameter of growth inhibition zones of test culture is 16-25 mm) in relation to bacterial cultures of Bacillus subtilis (19.8±0,4) and to gram-negative cultures of Escherichia coli (17.8±0.4) and Pseudomonas aeruginosa (17.6±0.5).
Thus, the obtained findings showed that samples of herbal mixture for oral administration have an antimicrobial spectrum in relation to gram-positive (Staphylococcus aureus ATCC 25293, Bacillus subtilis ATCC 6633), gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) bacterial cultures of microorganisms and can be characterized by moderate activity towards test cultures.It should be noted that fungicidal activity towards the yeast fungi Candida albicans ATCC 885-653 was not found.
The investigated samples of the herbal mixture and the offered phytocomposition are promising for further work on the development of the new dosage form with plant extracts for oral administration and require continuation of the research.

Table 1
Classification of common nematodes that cause disease in humans

Table 2
Results of antimicrobial activity research (n=5)

Table 2
indicate that the investigated samples of prepared herbal mixture show an antimicrobial action spectrum in relation to the used bacteria test cultures, namely, bacterial gram-positive (Staphylococcus aureus ATCC 25293 and spore culture of Bacillus subtilis ATCC 6633), gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) cultures.The antifungal activity in relation to the yeast fungi of the genus Candida (Candida albicans ATCC 885-653) was not found.