Antimicrobial activity of extracts of wormwood artemisia gmelinii

The article presents the data of microbiological study of the herb of Artemisia Gmelinii. The estimation of antimicrobial activity OfArtemisia gmelinii Weber ex Stechmextracts against test microorganisms is described. To study the antimicrobial activity of the biologically active components Artemisia gmelinii Weber ex Stechm, extracts obtained by different solvents were used: alcohol, chloroform and carbon dioxide.

Introduction.

Of special interest in search of antimicrobial substances are essential-oil plants, which are an inexhaustible source of biologically active substances (BAS). BAS of medicinal plants can be an effective adjunct in the complex treatment of infectious diseases [1]. Biological activity of different types of Artemisia Asteraceae family is described as producers metabolites with antimicrobial and antioxidative activity [2]. Essential oils Asteraceae spp. have been widely used for various medical purposes such as antimalarial, antibacterial, antiviral, nematocidal and fungicidal ones for many years [3]. The prospect of search of antimicrobials producers among Asteraceae spp., growing in Kazakhstan is undeniable, as there is a sufficient resource base. One species of wormwood is Artemisia gmelinii Weber ex Stechm. Determination of the potential antimicrobial and antifungal activity of extracts of Artemisia is a priority for the development of natural antimicrobial agents.

Goal Evaluation of the antimicrobial activity of extracts of Artemesiagmelinii against the test microorganisms.

Material and Methods.

To study the antimicrobial activity of bioactive components of Artemesiagmelinii extracts, obtained by processing a carbon dioxide, alcohol and chloroform, were used. The wormwood was collected in July and August 2015 at the foothills of the Trans-Ili Alatau of the Republic of Kazakhstan. Raw material drying was carried out by air-shadow method, in the well-ventilated accommodations, out of direct sunlight. The crushed leaves were extracted with chloroform, ethanol and CO2 using a Soxhlet apparatus. The resulting solutions were poured through a filter paper and stored at 4°C for further study of antimicrobial and anti-fungal activity using the micro-dilution method in accordance with the requirements of the European Committee for testing antimicrobial activity (European Committee on Antimicrobial Susceptibility Testing- EUCAST) 2003) and the guidelines of the Institute of clinical and laboratory standards [12].

To investigate the antibacterial and anti-fungal activity of Artemisia gmelinii reference strains obtained from the American Type Culture Collection have been used:

• gram-positive bacteria (Staphylococcus aureusATCC 25923, Staphylococcus aureusATCC 43300, Staphylococcus aureusATCC 6538, Staphylococcus epidermidisATCC 12228, Bacillus subtilisATCC 6633, Bacillus cereus ATCC 10876, Micrococcus luteus ATCC 10240, Streptococcus pyogenes ATCC19615, Streptococcus pneumoniaATCC49619, Streptococcus mutansATCC25175);
• gram-negative bacteria(Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiellapneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027);
• yeast-like fungi (Candida albicans ATCC 2091, Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019).

All test strains were subcultured on meat-peptone agar and Sabouraud medium at 35°C for 18 - 24 hours or 30°C for 24 - 48 hours for bacteria and fungi, respectively.

Samples containing test compounds of Artemisia were dissolved in dimethylsulfoxide (DMSO) to 50 mg/ml. Microbial suspensions were prepared in sterile saline (0,85% NaCl) with an optical density of 0,5 McFarland of the standard scale. The test bacterial suspensions were placed on Petri dishes with solid nutrient medium of Mueller-Hinton agar, containing 1 mg/ml of the test compounds, and then incubated at 37°C for 24 hours. Inhibition of microbial growth was assessed in comparison with a control culture sown on nutrient medium without the test substances.The minimum inhibitory concentration (MIC) of the test compounds was determined by two-fold micro dilutions in Mueller Hinton broth (for bacteria) and RPMI 1640 broth with MOPS (for fungi) in 96-well polystyrene plates. The final concentration of compounds ranged from 1000 to 7.81 pg/ml. The microbial suspensions were prepared by the above given scheme.

Then 1 mcl of bacterial or fungal suspension were added to each well, containing 100 mcl of broth and various concentrations of the test compounds. After incubation, MIC was evaluated by spectrophotometrical determining of the lowest compound concentrations, causing complete inhibition of growth of fungal or bacterial test strain. Medium without the test substances served as a control

The minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) was determined as the lowestconcentration of compounds which is required for death of certain types of bacteria or fungi. MBC/MFC was determined by inoculating of 5mcl of cultures from each well in the absence of growth while determining the MIC, followed by sowing on the appropriate agar medium. The result was evaluated after incubation in the thermostat at 35±2°C for 24 hours. The lowest concentration of the compounds without visible growth was evaluated as bactericidal/fungicidal concentration.

Table 1

Results.

Action of chloroform-extract.

As seen from Table 1, the chloroform extract exerts an inhibitory activity against all the tested gram-positive bacteria: MIC ranged from 1,25 mg/ml to 10 mg/ml. The extract showed the greatest activity to Staphylococcus aureus ATCC 43300 - 1,25 mg/ml and Streptococcus pneumonia ATCC49619- 2,5 mg/ml. MIC 5 mg/ml was revealed against pathogenic strains of Staphylococcus (Staphylococcus aureus ATCC 25923; Staphylococcus aureus ATCC 6538) as well as Staphylococcus epidermidis (Staphylococcus epidermidis ATCC 12228) and Micrococcus luteus ATCC 10240, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876. Most MIC value was observed relative to Streptococcus mutans (MIC of 10 mg/ml).

Satisfactory results have been achieved with Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027. They were sensitive at higher extract concentrations (MIC ≥20 mg/ml). The test compound showed quite high inhibitory activity to a yeast-like fungi genus Candida (Candida albicans ATCC 2091, Candida albicans ATCC 10231, Candidaparapsilosis ATCC 22019), MIC was 2.5 - 5 mg/ml.

MBC ranged widely within 0,62-20 mg/ml. MIC 20 mg/ml was needed for destruction of 99,9% Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Bacillus cereus ATCC 10876 and all tested gram-negative bacteria. The extract showed enough high fungicidal activity against the yeasts - MIC 5 mg/ml. Chloroform extract in low concentration showed bactericidal activity against Staphylococcus aureus ATCC 43300, Streptococcus pyogenes ATCC19615, fungicidal- to yeasts. In determining the MIC/MBC ratio this indicator for all microorganisms tested was within ≤4, which allows making a conclusion about a sufficiently high antimicrobial and fungicidal activity of chloroform extract. Action of ethanol-extract.

Sufficiently high inhibitory activity ethanol extract exhibited to 15 test microorganisms; MIC ranged from 1,25 mg/ml to 10 mg/ml, with exception of Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027 (MIC 20 mg/ml). MBC ranged from 2,5 mg/ml to 20 mg/ml. The highest concentration was needed to exhibit bactericidal activity for microorganisms of the genus Bacillus, Escherichia, and Pseudomonas (MBC 20 mg/ml). The ratio of the MIC / MBC for all microorganisms tested ranged within ≤4, which indicates antibacterial and antifungal activity of the extract.

When compared with the results of the chlorophyll-extract one can see that the ethanol extract possesses pronounced antibacterial and antifungal activity (MIC/MBC was mostly in range 1).

Action of CO2-extract.

CO2 extract antibacterial activity was expressed with regard to all the tested gram-positive bacteria (MIC of 1.25 mg/ml to 5 mg/ml). For all the tested gram-negative bacteria MIC was ≥ 20 mg/ml. The best results compared to otherswere those of CO2- extract: it showed high fungistatic activity, for yeasts MIC was 2.5 - 5 mg/ml.

MBC of CO2-extract had a fairly wide range from 0.62 mg/ml to 20 mg/ml. The minimum concentration required for bactericidal action was against Streptococcus pneumonia ATCC49619 (MBC 62 mg/ml), Streptococcus pyogenes ATCC19615 (MBC 1.25 mg/ml), Streptococcus mutans ATCC2517 (2,5 mg/ml). MIC/MBC ranged from 0.248 to 8, which indicatesvarious effects of CO2 extract. Fungistatic and fungicidal activities were high enough (2.5-5 mg/ml), MIC/MBC ≤ 2.

Analyzing the results one can state as follows:

• in spite of their different chemical profiles all kinds of extracts of Artemisia gmelinii exhibit antimicrobial and antifungal activity;
• S.aureus, S.epidermidis showedthe highest sensitivityto the extract components; their sensitivity was exhibited to fairly low concentrations of the extracts (from 1.25 to 5 mg/ml);
• extracts showed expressed activity against the yeast fungi of the genus Candida; on the contrary inhibition of bacteria of the genus Pseudomonas and Proteus is observed at high concentrations of the extracts;
•  extractshave high activity, more pronounced in the CO2-extract against Streptococcus mutans, which contributes significantly to the development of caries.

Thus, extracts of wormwood Artemisia gmeliniihave shown a wide spectrum of antibacterial and antifungal activity. Determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.

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