ANTIMICROBIAL ACTIVITY AND SOME PHYTOCHEMICAL ANALYSIS OF TWO EXTRACTS Vinca minor L .

This study investigated the antimicrobial activity as well as some phytochemical analysis of ethanol and diethyl ether ext acts from plant species Vinca minor L. In vitro antimicrobial activity of extracts was studied on 20 strains of microorganisms (16 bacteria and four yeasts). Testing was performed by microdilution method and minimum inhibitory concentration (MIC) and minimum microbic idal concentration (MMC) were determined. The strongest antimicrobial activity wa s detected on G+ bacteria of the genus Bacillus. Tested G bacteria and yeasts were not sensitive to the acti on of the extracts or the sensitivity was insignificant. Phytochemical analysis involved determining the am ount of total phenolics, flavonoids and tannins, as well as the determination of antioxidan t ctivity monitoring capability to neutralize free radicals (DPPH) and the reductive p otential. Phytochemical examination indicates that the total phenolic compounds were mo re in the ethanolic extract and the content of flavonoids and tannins marginally higher in the diethyl ether extract. The antioxidant activity (DPPH) of the ethanolic extrac t of V. minor was significantly stronger as compared to the diethyl ether extract, and the redu ction potential was approximately the same.


INTRODUCTION
The genus Vinca (Apocynaceae) comprises about seven species in the world.In Serbia, it is represented by Vinca herbacea, Vinca minor and Vinca major.V. minor or Lesser Periwinkle occurs throughout central Serbia (JOSIFOVIĆ eds., 1973).
The leaves are oblong-ovate to elliptical, approximately 2-5 cm long and 1-2.5 cm wide, with petioles 1-3 mm long.The flower is perfect and without odor.It is showy, purple to blue and occasionally white, borne singly in an axillary position on a 1-1.5 cm pedice.The fruit is a non-fleshy follicle, approximately 2-2.5 cm long.It releases three to five seeds (DARCY et al., 2002).V. minor flowers regularly from April to May and sporadically from May to September (FUNDERBUCK et al., 1976).It is adapted to the mild climate.It grows best in partial shade and moist, well-drained soil.It is often found in woods and bluffs, and along cemeteries, roads, and other disturbed areas (KURT and CAROL, 2000).In folk medicine, it is used internally for circulatory disorders, cerebral circulatory impairment and brain's metabolism support (FARAHA-NIKIAA et al, 2011).
Numerous literature data describe the compounds isolated from this plant.Phenols have been investigated for its antioxidant properties (NISHIBE et al., 1996).Also flavonoids were tested for the anti-inflammatory (BAHADORI et al., 2012) and antimicrobial effects (SZOSTAK and KOWALEWSKI, 1975).Extracts from V. minor species showed a pronounced antioxidant activity and the ability to reduce lipid peroxidation (NISHIBE et al., 1996) as well as the ability in treatment of cardiovascular and neurodegenerative diseases (KHANAVI et al., 2010;FERNANDES et al., 1996).
There is plenty of information about antibacterial (YILDIRIM et al., 2012;MEHRABIAN et al., 1995;TURKER et al., 2009) and antifungal (WILSON et al., 1997;DOMENICO et al., 2012) activities of V. minor ethanolic extract.In the available literature there are no data about the activity of V. minor diethyl ether extract.Considering that V. minor species is from the Balkans and has not been studied with these aspects, the aim of this study was to investigate the antibacterial and antifungal activities as well as phytochemical analysis with antioxidative activity of ethanolic and diethyl ether extracts of this plant.

Plant material
In April 2012 leaves of species V. minor L. were collected at the time of flowering.The leaves were harvested at noon on the west side of the mountain Goč.Identification and classification of the plant material was performed at the Faculty of Science, University of Kragujevac.The voucher sample is deposited at the Herbarium of the Department of Biology and Ecology, Faculty of Science, University of Kragujevac (No. MB01/12).The collected plant material was air-dried in darkness at ambient temperature.The dried plant material was cut up and stored in paper bags until needed.

Preparation of plant extracts
Dried ground plant material was extracted by maceration with ethanol and diethyl ether.30 g of plant material was soaked with 150 ml of solvent for 24 h at room temperature.The resulting extract was then filtered through filter paper (Whatman No.1).The residue from the filtration was extracted again twice using the same procedure.The filtrates obtained were combined and then evaporated to dryness using a rotary evaporator at 40 °C.The obtained amounts of crude extracts of V. minor were 4.52 g for ethanol extract and 1.28 g for ethyl ether extract.The extracts were kept in sterile sample tubes and stored at -20 °C.

Determination of total phenolic content
The total phenolic content was determined by using Folin-Ciocalteu's method (WOOTTON-BEARD et al., 2011).The reaction mixture was prepared by mixing 0.2 ml of methanolic solution of extract (1 mg/ml) and 1.5 ml of 1:10 Folin-Ciocalteu's reagent dissolved in water.The mixture was allowed to equilibrate for 5 min and then mixed with 1.5 ml 6 % Na 2 CO 3 solution.After incubation for 90 min at room temperature in darkness, the absorbance of the mixture was read at 725 nm against a blank using spectrophotometer.The blank was prepared with methanol instead of extract solution.The samples were prepared in triplicate and the mean value of absorbance was obtained.The same procedure was repeated for gallic acid which was used for calibration of standard curve.Total phenol content is reported as gallic acid equivalents by reference to linear equation of the standard curve (y = 0.008x + 0.0077, R2 = 0.998).Then the total phenolic content was expressed as a milligram of gallic acid equivalent per gram of extract (mg GAE/g of extract).

Determination of total flavonoid content
The concentrations of flavonoids were determined by using aluminium chloride method (QUETTIER-DELEU et al., 2000).The mixture contained 2 ml of methanolic solution of extract (1 mg/ml) and 2 ml of 2 % methanolic AlCl 3 ×6H2O solution.The mixture was vigorously shaken, and after 10 min of incubation at room temperature, the absorbance versus a prepared blank was read at 430 nm using spectrophotometer.The samples were prepared in triplicate and the mean value of absorbance was obtained.Rutin was used as a standard for calibration of standard curve.The concentrations of flavonoids were calculated from the linear equation of standard curve (y = 0.021x + 0.040, R2 = 0.999).Then the concentrations of flavonoids were expressed as milligram of rutin equivalent per gram of extract (mg RU/g of extract).

Determination of condensed tannins
Condensed tannins were determined by using the butanol-HCl method as described by PORTER et al., (1986).The mixture contained 0.5 ml of liquid extract was transferred to glass test tube, adding 3 ml of the butanol-HCl reagent (butanol-HCl 95:5 v/v) and 0.1 ml of the ferric reagent (2 % ferric ammonium sulfate in 2N HCl).Covered tubes were heated in a water bath at 97 to 100 °C for 60 min.After cooling, the absorbance was determined at 550 nm.A blank was measured as the absorbance of the unheated mixture.The samples were prepared in triplicate and the mean value of absorbance was obtained.Cyanidin chloride was used as a standard for calibration of standard curve.The concentrations of proanthocyanidins were calculated from the linear equation of standard curve (y ј 0.0094x ю 0.006, R2 ј 0.999).Then the concentrations of proanthocyanidins were expressed as milligram of cyanidin chloride equivalent per gram of extract (mg of CChE/g of extract).

Determination of antioxidant activity DPPH radicals scavenging capacity assay
The ability of the plant extract to scavenge DPPH free radicals was assessed by using the method described by TAKAO et al., (1994).The stock solution of the plant extract was prepared in methanol to achieve the concentration of 2000 µg/ml.Further, two-fold dilutions were made to obtain concentrations of 1000, 500, 250, 125, 62.5 µg/ml.Diluted solutions of extract (1 ml each) were mixed with 1 ml of DPPH methanolic solution (80 µg/ml).After 30 min in darkness at room temperature, the absorbance was read in a spectrophotometer at 517 nm.The control samples consisted of 1 ml of methanol added to 1 ml of DPPH solution.Аscorbic acid was used as a positive control.The experiment was performed in triplicate.Scavenging activity is expressed as the inhibition percentage calculated using the following equation:

Scavenging activity (%) = 100 × [(A control -A sample ) / A control )]
where A control is the absorbance of the control and A sample is the absorbance of the extract.
The IC 50 value is the effective concentration at which 50 % of DPPH radicals were scavenged.It was obtained from the graph of scavenging activity (%) versus concentration of samples.Low IC 50 value indicates strong ability of the extract to act as DPPH scavenger.

Reductive power of plant extract
As well as in previous assay the tested concentrations of the extracts ranged from 62.5 µg/ml to 1000 µg/ml.The mixture contained 0, 5 ml of methanolic solution of extract (1 mg/ml), 1.25 ml of 1 % К 3 Fe(CN) 6 and 1.25 ml of phosphate buffer (рH =6.6; 200 mM).
After 20 min of incubation at temperature 50 °C, 1.25 ml of a 10% solution of trichloroacetic acid was added.In 1.25 ml of prepared mixture 1.25 ml of distilled water and 0.25 ml of 0.1% solution of iron chloride.After incubation (30 min), the absorbance versus a prepared blank was read at 700 nm using spectrophotometer.Increase in absorbance of the reaction mixture indicates greater reducing capability.Ascorbic acid was used as a standard.The samples were prepared in triplicate and the mean value of absorbance was obtained, through which the activity was expressed (LIM et al., 2009).

Suspension preparation
Bacterial and yeast suspensions were prepared by the direct colony method.The turbidity of initial suspension was adjusted by comparing with 0.5 McFarland's standard (ANDREWS, 2005).Initial bacterial suspensions contain about 10 8 colony forming units (CFU)/ml and yeast suspensions contain 10 6 CFU/ml.1:100 dilutions of initial suspension were additionally prepared into sterile 0.85 % saline.

Microdilution method
Antimicrobial activity was tested by determining the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) by using microdilution method with resazurin (SARKER et al., 2007).The 96-well plates were prepared by dispensing 100 µl of nutrient broth, Mueller-Hinton broth for bacteria and Sabouraud dextrose broth for yeast, into each well.A 100 µl from the stock solution of tested extracts (concentration of 5000 µg/ml) was added into the first row of the plate.Then, twofold, serial dilutions were performed by using a multichannel pipette.The obtained concentration range was from 2500 to 19.53 µg/ml.A 10 µl of diluted bacterial and yeast suspension was added to each well to give a final concentration of 5 ×10 5 CFU/ml for bacteria and 5 × 10 3 CFU/ml for yeast.Finally, 10 µl resazurin solution (6.75 mg/ml) was added to each well inoculated with bacteria and yeast.Resazurin is an oxidationreduction indicator used for the evaluation of microbial growth.It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells.The inoculated plates were incubated at 37 °C for 24 h for bacteria, 28 °C for 48 h for the yeast.MIC was defined as the lowest concentration of tested substance that prevented resazurin color change from blue to pink.
Minimum microbicidal concentration (MMC) was determined by plating 10 µl of samples from wells, where no indicator color change was recorded, on nutrient agar medium.At the end of the incubation period the lowest concentration with no growth (no colony) was defined as minimum microbicidal concentration.
Doxycycline and fluconazole, dissolved in nutrient liquid medium, were used as positive controls.Stock solutions of crude extracts were obtained by dissolving in DMSO and then diluted into broth to achieve a concentration of 10 % DMSO.Solvent control test was performed to study the effects of 10 % DMSO on the growth of microorganism.It was observed that 10 % DMSO did not inhibit the growth of microorganism.Also, in the experiment, the concentration of DMSO was additionally decreased because of the two-fold serial dilution assay (the working con-centration was 5 % and lower).Each test included growth control and sterility control.All tests were performed in duplicate and MICs were constant.

Data analysis
All data were presented as means ± standard deviations (mean ± SD) where appropriate.All calculations were performed using Microsoft Excel software.

Total phenol content, flavonoid and tannins concentrations
Since there are various possibilities of the extraction of biological active plant compounds, we used different solvents (ethanol and diethyl ether) in this study.Concentration of total phenols, flavonoids and tannins in extracts of V. minor are shown in Table 1.
Using the method with Folin-Ciocalteu reagent, the concentrations of total phenols were examined.The highest concentration was found in ethanol extract of V. minor (36.142 mg GA/g).
Using the method with aluminium chloride we obtained the concentrations of flavonoids.The highest concentration of flavonoids had diethyl ether extract (39.079 mg RU/g).In determining the amount of tannins, a method with butanol-HCl reagent was used.The obtained results show that diethyl ether extract has higher concentration of tannin than the ethanolic extract (Table 1).

Antioxidant activity
The antioxidant activity of different plant extracts of V. minor is determined by the use of two methods: DPPH radicals scavenging capacity assay and reductive power of plant extract.
The capability of ethanol and diethyl ether extracts of V. minor to neutralize free radicals (DPPH) is expressed in the form of IC 50 values (µg/ml).The results are shown in Table 2.In addition, activity was determined for the control substance as a standard parameter in the experiment, also expressed in the form of IC 50 values.Antioxidative efficiency of ethanolic extract was significantly higher compared to diethyl ether extract.Reduction potential of tested extracts was lower than control (ascorbic acid) and extracts did not differ for its reduction power (Table 3).The main phenolics in V. minor were found.Kaempferol-3-O-(6-O-rhamnosylglucoside)-7-O-glucoside, 2,3-dihydroxybenzoic acid, vincoside and chlorogenic acid were isolated from the leaves of V. minor.2,3-Dihydroxybenzoic acid showed a potent radicalscavenging activity (NISHIBE et al., 1996).
Other authors consider that antioxidant activity is derived from alkaloids present in large quantities in this plant (SCHEINDLIN and RUBIN, 2006).It has been found that V. minor aquatic alkaloid extract exhibited significant DPPH free radical scavenging activity, while their aquatic non-alkaloid extract showed only weak activity (BAHADORI et al., 2012).

Antimicrobial activity
The results of in vitro antibacterial and antifungal activities of ethanol and diethyl ether leaves extracts of V. minor are shown in Table 4 and 5.For comparison, the tables also give the results of the activities of doxycycline and fluconazole.The solvent (10 % DMSO) had no effect on the growth of tested microorganisms.Antimicrobial activities of tested extracts were assessed by determining the MIC and MMC values for 20 species of microorganisms.
The values of minimum inhibitory concentrations and minimum microbicidal concentrations obtained in this experiment ranged from 19.53 to >2500 µg/ml.Efficacy of antimicrobial activity depended on the species of microorganism.
The strongest antimicrobial activity was showed on G+ bacteria, especially from genus Bacillus, while the activity on other bacteria was moderate.Ethanol and diethyl ether extracts were active in concentration from 19.53 µg/ml to >2500 µg/ml.The most sensitive bacteria were B. pumilus NCTC 8241 (MIC 19.53 µg/ml) and B. subtilis (MIC 78.13 µg/ml) for the ethanol extract and B. pumilus NCTC 8241 (MIC 19.53 µg/ml) and B. subtilis (MIC 156.25 µg/ml) for diethyl ether extract.G-bacteria did not show sensitivity within the tested extract concentrations.The exception is P. aeruginosa ATCC 27853 where MIC was 2500 µg/ml.
When analyzing the effectiveness of extracts in preventing the growth of yeasts, that activity was generally weak.All species of yeasts have shown high resistance.MMC values are often coincided with MICs.Ethanol extract stood out with a stronger effect on all the tested yeasts compared to other extract with the noticeable difference.Although there are numerous phytochemical and biochemical studies of this plant, the diethyl ether extract from V. minor leaves has not been investigated enough.
The effect of ethanolic extract of V. minor on pathogenic bacteria isolated from aquatic organisms was studied by TURKER et al. (2009).They came to conclusion that the ethanolic extract of V. minor exhibited a broad-spectrum activity against both G+ (Streptococcus agalactiae, Lactococcus garvieae and E. faecalis) and G-bacteria (Aeromonas hydrophila and Yersinia ruckeri).As well as in our study, the effect of the ethanolic extract was significantly lower than positive controls and effect was better in G+ bacteria with the exception of E. faecalis.In vitro antibacterial activities of V. minor plant extracts, growing in Turkey, showed the best antibacterial activity, just as in our study, with ethanolic extract.Cosidering results obtained against S. typhimurium there was no match between this study and our study.Ethanolic extract of V. minor leaves exhibited moderate inhibitory activity against Staphylococcus epidermidis, Streptococcus pyogenes and S. aureus (YILDIRIM et al., 2012).MEHRABIAN et al., (1995) reported the antimicrobial effect of V. minor on some pathogen bacteria (S. aureus and P. aeruginosa) where similar effect of extracts was observed on both mentioned bacteria.In our study the results varied between S. aureus and P. aeruginosa.Ethanolic extract hadn't expressed activity or it was a soft spot for G-bacteria.
With these results, tested plant has some scientific justification as a medicinal plant.In the future, identification of active components can be studied for plant extracts having strong bioactivity.This study indicates that extracts of V. minor showed a certain level of antimicrobial activity and may be an additional biologic potential in pharmaceutical use, but that additional research is needed, especially in order to identify the active components.

CONCLUSION
The results of antimicrobial activity indicate that tested extracts showed different degree of antimicrobial activity in relation to the tested species.Extracts of V. minor demonstrated more potent inhibitory effects on the growth of G+ bacteria than to the other tested microorganisms.Therefore, the leaves of this plant can be a potential source of antibacterial substances.
The results of our study suggest the great value of the V. minor species, suggesting it for use in pharmaceutical, phytotherapy and food industry.
Antimicrobial activity of diethyl ether and ethanol extracts were tested against 20 microorganisms including sixteen strains of bacteria (standard strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633, B. pumilus NCTC 8241, Proteus mirabilis ATCC 12453; and clinical isolates: Escherichia coli, Staphylococcus aureus, S. aureus PMFKG-B12, Enterococcus faecalis, Pseudomonas aeruginosa, Proteus mirabilis, Salmonella enterica, S. typhimurium and Bacillus subtilis) and four species of yeast: Candida albicans ATCC 10231, C. albicans (clinical isolate), Rhodotorula sp.PMFKG-F27 and Saccharomyces boulardii PMFKG-P34.All clinical isolates were a generous gift from the Institute of Public Health, Kragujevac.The other microorganisms were provided from a collection held by the Microbiology Laboratory, Faculty of Science, University of Kragujevac.

Table 1 .
Concentration of total phenols, flavonoids and tannins in extracts of V. minor.
*Each value shown is the mean value ± standard deviation
*Each value shown is the mean value ± standard deviation.

Table 3 .
The reductive potential of investigated plant extracts and standard substance.
*Each value shown is the mean value of absorbance ± standard deviation

Table 4 .
Antibacterial activities of ethanol and diethyl ether extracts of V. minor leaves against tested bacteria.

Table 5 .
Antifungal activities of ethanol and diethyl ether extracts of V. minor leaves against tested yeast