DETERMINATION OF ANTIMICROBIAL ACTIVITY OF ExTRACTS OF CALENDULA OFFICINALIS FLOWERS

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Flavonoids are well known to have a significance in the forming the most important pharmacological effects of medicinal plants: antimicrobial, diuretic, anti-inflammatory, antiviral, choleretic, spasmolitic activities [1, 2]. Due to the contents of flavonoids in pot marigold flowers (Calendula officinalis L.) as a leading group of biologically active compounds (BAC), the herbal materials of this plant officially used as an antiseptic, anti-inflammatory medicines [2, 3]. For well-known phytopreparatons on the basis of pot marigold («Phytohepatol», «Pectoral tea no. 4», «Cale- phlonum», «Phytonephrol», «Rotocanum», ointment «Calendula», suppositories «Calendula», «Calendula tincture») the therapeutic activity and relative safety are marked. For that very reason, in our opinion, at present there is a new stage of the research on the investigation of pot marigold antimicrobial activity [4-9]. A lot of modern publications describe that foreign scientists were studied antimicrobial properties of plant samples of herbal materials growing in Brazil, Pakistan, India and Iraq [4-9]. It has been found that water pot marigold extract exhibits expressed antibacterial activity against enteropathogenic bacteria such as Salmonella, Shigella dysenteriae, Shigella fl, Shigella sonnei and Escherichia coli, particu- larly at a concentration of 100 μg/ml [7]. These pathogens were studied at the concentrations of 25 μg/ml, 50 μg/ml and 100 μg/ml. The object of comparison (standard) was an antibiotic “Tsefotan” at a concentration of 10 μg/ml. It is important to point out that Shigella sonnei was the most sensitive in all investigated concentrations, marked the highest growth inhibition zone (23 mm) at the concentration of 100 μg/ml in the comparison with “Tsefotan” (growth inhibition zone 16 mm), taken at value of 10 μg/ ml. The concentration of 25 μg/ml revealed less represented activity against all the bacterial iso- lates, except for Shigella sonnei. It is interesting that Shigella sonnei even in this value showed a growth inhibition zone of 20 mm. It is interesting, that the antimicrobial activity for all agents in a concentration of 50 μg/ml was also comparable to that of the effect of the study drug. Values of growth inhibition zone in a concentration of 50 μg/ml for Salmonella, Shigella dysenteriae, Shigella flexneri Shigella sonnei and Esche- richia coli were 10 mm, 13 mm, 15 mm, 21 mm, 15 mm, in comparison to the antibiotic in a dose of 10 μg/ml (9 mm, 17 mm, 22 mm, 16 mm and 18 mm, respectively) [7]. In connection with it is possible to recommend phytopharmaceuticals on basis of pot marigold in the treatment of intestinal infections this etiology. The investigation of water, acetone and meth- anol extracts of fresh Calendula flowers against five bacteria: Staphylococcus aureus, Bacillus cereus, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa showed rather high antibacterial activity in the result of growth zone inhibition in the plate with agar inoculum [4]. Some researchers proved the importance of multipurpose using of Calendula officinalis. Thus, the antimicrobial activity of methanol and water extracts of leaves, roots, flowers and stems of Calendula was investigated. The best results were found out against Escherichia coli and Salmonella typhi for the methanol extract of pot marigold roots. In terms of multiresistant microorganisms the most effective substance was the composition of extracts obtained from different plant organs [9]. Some scientists indicate the high antimicrobial activity of Calendula stems [8]. Besides, antifungal activity in methanol and water extracts of leaves, roots, stems and flowers of pot marigold againt Aspargillus niger, Candida albicans was found [9]. Other researchers have found the advantage of methanolic extract of Calendula flowers in comparison with ethanol extract in terms of antimicrobial activity on a group of microorganisms isolated from patients [5]. However, the equivalent activity of both extracts against fungi of the genus Candida and genus Aspergillus was found, comparable to the «Fluconazole» [9]. Brazilian scientists described advance researches of homeopathic preparations based on pot marigold in terms of revealing of anti- microbial activity [6]. Calendula tincture of industrial production has been analyzed (Brazil, pharmacy dosage forms). The dilution of tincture was conducted from the value 1:1 (original form) through a concentration of 1:2, 1:4, 1:8, 1:16, 1:32 to 1:64 to the value of 70% ethanol, which was created on the base of this medicine. This approach was used in order to evaluate a possible influence of ethanol on the antibacterial activity of tincture. It is known, that 70% ethanol is routinely used in the preparation of phytotherapeutic tinctures. Several bacterial strains were used for the test. Chlorhexidine was used as a positive control, and the purified water as a negative control. Antibacterial activity of Calendula tincture is detected at the maximum dilution against Streptococcus oralis 1:16 (6.5 mm growth inhibition zone) against Streptococcus mutans and Streptococcu salivarius 1:32 (growth inhibition zone of 7.0 and 7.5 mm, respectively) against Enterococcus faecalis and Eikenella corrodens 1:64 (growth inhibition zone of 6.0 mm and 6.5 respectively). In this case 70% ethanol did not show any antibacterial activity against studied bacterial strains [6]. Consequently, literature data indicate the importance of studying of antimicrobial characteristics of the pot marigold in terms of further new opportunities of Calendula pharmacological application. The aim of the investigation was a screening study of the antibacterial activity of the Calendula flowers extracts. The objects and methods. The objects of the study were calendula extracts: water extract (infusion) of Calendula flowers (1:20); tincture of Calendula flowers (1:5) (70% ethanol) (elaboration of pharmacognosy department SamSMU), tincture of Calendula flowers (1:10) (70% ethanol) OOO “Tula pharmaceutical factory” drug «Calendula tincture», series 91214, useful time 1217; liquid extract of Сalendula flowers (1:2) 40% ethanol; liquid extract of Сalendula flowers (1:2) 70% ethanol. In all cases herbal materials was air-dried sample of Сalendula flowers highly productive variety «Kalta», industrial cultivated in the Samara region (organization «Samaralektravy»). Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Candida albicans were used as test cultures for the determining of the antimicrobial activity of the water and Calendula ethanol extracts. Determination of the minimum inhibitory concentration was determined by double serial dilutions in broth according to MUK 4.2.1890- 04 [10]. Culture media was prepared from the dry medium industrially manufactured according to the specification of manufacturer. Several isolated colonies were selected to prepare inoculum from the broth culture. Then the minor amount of material was transferred by loop to a tube with 4.0-5.0 ml of non-selective liquid broth. After that it was adjusted to optical density 0.5 McFarland units by adding sterile broth or isotonic sodium chloride solution. Micromethod was used for the study. Testing was conducted on the fi volume 0.2 ml. 96-cavity sterile plate for immunological studies (flat bottomed) with the lid was filled with a double serial dilution of the investigated substances by the help of multichannel pipettes. After preparing the dilutions it was inoculated with the prepared suspension of the tested microorganism. Incubation was carried out in ordinary atmosphere at the temperature 35 ºС. During the incubation, the plate was covered to prevent drying of the content of the cavity. Calculation of the results was perfomed visually. To determine the presence of microorganism growth cavity with bacterial inoculation were viewed in transmitted light. Minimum growth inhibitory dilution is deter- mined by the cavity, which was suppressed by the visible growth of microorganisms. It was considered the influence on the results of the study of antimicrobial action of alcohol. For this aim this effect was determined on test cultures of microorganisms by double serial dilutions (positive control). Results and discussion. During microbiological analysis the following results were obtained. For infusion of calendula flowers there was a revealed high activity against all the studied strains. In particular, the infusion has antimicrobial activity against Escherichia coli to 4 times dilutions, against Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus to 8 times dilution, and in relation to Candida albicans to 32 times dilution (table 1). Industrial sample of the Tula pharmaceutical factory, tincture (1:10) at 70% ethanol showed antibacterial activity against all these strains, except for E.coli. In particular, the tincture has antimicrobial activity against Staphylococcus aureus to 8 times dilutions, against Pseudomonas aeruginosa to 16 times dilution, as well as against Bacillus cereus and Candida albicans to 64 times dilution (table 2). The alalysis for tincture of Сalendula (1:5) at 70% ethanol revealed antimicrobial activity against such pathogens as Pseudomonas aeruginosa and Staphylococcus aureus to 8 times dilution, as well as against Bacillus cereus to 256 times dilution (table. 3). Liquid extract of Сalendula flowers (1:2) on 40% ethanol in comparison to the liquid extract (1:2) 70% ethanol has the widest range of effects on microorganisms, namely: Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and Candida albicans. Against Staphylococcus aureus liquid extract on 70% ethanol has no antimicrobial effect. However, the liquid extract on 70% ethanol has the most intense on the antimicrobial activity to Bacillus cereus to 256 times dilution, while the liquid extract at 40% alcohol showed more intensive antimicrobial activity on Candida albicans to 16 times dilution. Turn out that against Pseudomonas aeruginosa both extracts has equal antimicrobial activity to 8 times dilution (table 4 and table 5). Conclusions The screening study of antibacterial activity of extracts of Calendula flowers was carried out. It was revealed that the widest range of antibacterial activity has an infusion of pot marigold flowers. Against Pseudomonas aeruginosa the most active object was tincture (1:10) with 70% ethanol, against Escherichia coli the only object that have demonstrated antimicrobial activity was infusion of Calendula flowers (1:20), against Bacillus cereus the highest activity was shown by Calendula flowers tincture (1:5) at 70% ethanol, and the liquid extract (1:2) on 70% ethanol, against Candida albicans tincture showed the highest activity (1:10) on 70% ethanol.

About the authors

P. V Afanasyeva

Samara State Medical University

Samara

A. V Kurkina

Samara State Medical University

Samara

V. A Kurkin

Samara State Medical University

Samara

A. V Lyamin

Samara State Medical University

Samara

A. V Zhestkov

Samara State Medical University

Samara

References

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