ANTIMICROBIAL AND ANTIOXIDANT PROPERTIES OF SIRMO ( Allium vineale L . ) , MENDI ( Chaerophyllum macropodum Boiss . ) AND SIYABO ( Ferula rigidula DC . )

In the current study, antioxidative and antibacterial characteristics of the three various extracts such as methanol, ethanol and acetone of brine and fresh herbs containing Sirmo (Allium vineale L.), Mendi (Chaerophyllum macropodum Boiss.) and Siyabo (Ferula rigidula DC.) were investigated. When antioxidant activity was measured using the 2,2-difenil-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS) methods, the antimicrobial activity was measured by the agar well diffusion method. The DPPH values of all extracts ranged from 17.10±1.30 to 119.53±0.06 mg TEAC/kg lyophilized herb, while ABTS values ranged from 53.55±3.49 to 780.36±39.46 mg TEAC/kg lyophilized herb. It was determined that the exracts of Siyabo exhibited higher antibacterial activity in comparison to other plants. These results suggest that extracts of Sirmo, Mendi and Siyabo can be used as natural antimicrobials and antioxidants in food processing.


INTRODUCTION
It has been well known that plant spices have antimicrobial (Durmaz et al., 2006) and antioxidant activity since ancient times (Delaquis et al., 2002).In recent times, there is an increasing tendency in the antioxidant and antimicrobial role of vegetables, fruits, teas, wines, seeds and medicinal plants, that can be used as natural substances of antimicrobial and antioxidants (Dillard and German, 2000).The phenolic compounds in natural materials such as medicinal herbs, spices and plants has begun to gain more and more attention due to their antioxidant and antimicrobial activities (Cai et al., 2004;Dağdelen et al., 2014).
Especially in eastern Anatolia region of Turkey, Sirmo (Allium vineale L.), Mendi (Chaerophyllum macropodum Boiss.) and Siyabo (Ferula rigidula DC.) are traditionally used in production of herbed cheese for their flavor and aroma (Ocak et al., 2015;Köse, 2015).They are usually collected from mountains in May and June and are added into the cheeses with two ways.Firstly, the herbs are used freshly after washing and cutting into small pieces.The second way is making pickle from herbs.Approximately 30 days later, they are ready to produce herby cheese.They can also be stored for one year at ambient temperature and these herbs can be used whenever needed (Kavaz et al., 2013).
The chemical, microbiological, antimicrobial and antioxidant characteristics of some herbs used in the production of Herby cheese were investigated by several authors (Akyüz et al., 1996;Ağaoğlu et al., 2005;Sağun et al., 2005;Durmaz et al., 2006;Sağun et al., 2006;Çoruh et al., 2007;Çelik et al., 2008;Dağdelen, 2010;Dağdelen et al., 2014;Durmaz et al., 2015) However, in the reported studies, the effects of brine treatments on antioxidant and antimicrobial activity of herbs haven't been reported.Therefore, there is a need to examine the antimicrobial and antioxidant properties of herbs to compare treatments (fresh and brine), ensure product safety and produce high quality herby cheese.
The aim of this study was to evaluate in vitro antioxidant and antibacterial properties of methanol, ethanol and acetone extracts of fresh and brined Sirmo, Mendi and Siyabo.

Herbs and preparation of extracts
The investigation material consists of three local herbs such as Sirmo (Allium vineale L.), Mendi (Chaerophyllum macropodum Boiss.) and Siyabo (Ferula rigidula DC.) were used.These herbs are grown in Van province of Turkey.
The herbs were collected from mountain by villagers based on their own experiences and fresh herbs were sold in local markets.Samples were first washed with deionised water and then cut into small pieces.After that each herb was divided into two portions; the first portion (containing 20% whey in 10% brine) kept at 4 °C for one month and the second portion was placed in a freeze-drying device (Freeze-drier, Labconco, Czech Republic, Model:117) after being subjected to freezing at -18 °C (freezer) for 12 hours.One month later, brine herbs were subjected to freze drying like fresh herbs.
Methanol, ethanol and acetone solvents were used to obtain extracts from herbs for antimicrobial activity tests.About 1.25 g of grounded lyophilized brine and fresh herbs were extracted individually with solvents by using Soxhlet extractor for 6 h at the boiling point of the solvents (Lin et al.,1999).The solvents (methanol, ethanol and acetone) were concentrated using a vacuum rotary evaporator (IKA RV 10 rotary evaporator, Germany) and the final volume was dissolved in dimethyl sulfoxide (DMSO), then extracts were filtered using (Whatman no:1) filter paper and completed in 25 ml with DMSO.These stock solutions were kept at -18 °C until used.
For total phenolics and antioxidant activity extracts, 2.5 g of grounded lyophilized herb was put into a centrifuge tube, stirred with 22.5 mL of methanol and shaken in an incubator (Heidolph Unimax 1010, Germany) for 2 h in dark at 20 °C.After this, the homogenate was centrifuged at 9000 x g (Hettich Zentrifugen Universal 32 R, Germany) for 10 min at 20 °C and then supernatant was transferred into labeled sterile screw capped bottles.The above procedure was repeated twice using the pellet and then collected supernatants were combined.The supernatants were concentrated using a vacuum rotary evaporator (IKA RV 10, Germany) at 40 °C.The same procedure was repeated for ethanol and acetone extracts.Then residue was dissolved in 2 mL DMSO and the final volume was completed to 25 mL with methanol.

Antibacterial assay
An agar well diffusion method was used to determine the antibacterial activities of herb species (NCCLS, 1999).50 mg/mL concentrations of all the extracts in DMSO solvents were used and sterilized by filtration (pore size 0.47 mm).All the microorganisms mentioned above were incubated at 37±0.1 °C for 24 h after inoculation into Mueller Hinton Broth (Oxoid).After the incubation, bacterial inoculum was diluted with sterile physiological solution with 0.5 Mc Farland turbidity standard tubes.Mueller-Hinton Agar was inoculated by 100 µL of prepared culture over the entire agar surface.One hundred microliters of extract solution was filled into the wells of agar plates directly.The plates were kept at 20-25°C for 2 h to enable diffusion of extracts and were incubated for 24 h at 37°C for all bacterial species.Standard antibiotic discs (6 mm in diameter) of Tetracycline (30 μg) and Ampicillin (10 μg) were used as positive controls for comparison.Methanol, ethanol, acetone and DMSO solvents were also tested as negative controls.
Antibacterial activity of the extracts were evaluated by measuring the inhibition zone against tested organisms.Each assay was performed in triplicate.

Determination of total phenolic compounds
The total phenolic content of extracts were analysed by the Folin-Ciocalteu method (Yemiş et al., 2008).A total of 150 µL sample extract and 3.0 mL (of 2%) sodium carbonate (w/v in water) were transferred into a test tube.After about two minutes 150 µL Folin-Ciocalteu's reagent (1:1, v/v in water) was added and mixed throughly.The reaction was kept in the dark for 45 min at room temperature.The absorbance values of the mixture was recorded at 765 nm in a spectrophotometer (UV Mini-1240, Shimadzu, Japan).Each assay was performed in triplicate.Gallic acid was used to calculate calibration curve and the results were expressed as mg of gallic acid equivalents per kg of lyophilized herb (mg GAE/kg of lyophilized herb ).

Antioxidant activity DPPH assay
The free radical scavenging activity of the extracts of various herb species were measured using DPPH• free radical scavenging method (Brand-Williams et al., 1995) with slight modifications.100 µl of the herb extract were added to 2.4 mL of DPPH solution (25 mg DPPH/L methanol).The mixture was shaken vigorously and maintained in dark for 30 min.After the incubation, the absorbance of resulting solution was recorded at 520 nm in a spectrophotometer.Results were expressed as mg Trolox (Sigma-Aldrich, Canada) equivalent antioxidant capacity (TEAC) per kg of lyophilized herb.Methanol was used instead of the sample for the control measurements.

ABTS assay
ABTS (Sigma-Aldrich, Canada) radical cation solution was prepared by reacting 7.0 mM ABTS stock solution with 2.45 mM (final concentration) potassium persulfate for 16 h in the dark at room temperature until reaching a stable oxidative state (Re et al. 1999).Then the ABTS •+ solution was diluted with methanol to get an absorbance of 0.700 ± 0.020 at 734 nm.For the spectrophotometric assay, a diluted solution of ABTS + (2.9 mL) and 100 mL of herb extract in methanol was mixed thoroughly and the absorbance was determined at 734 nm at 6 min after mixing.Results were expressed in terms of mg TEAC per kg lyophilized herb.

Statistical analysis
Data were recorded as means ± standard deviation of triplicate measurements.SAS version 9.3 (SAS, 2012) was used to analyse statistical calculations.Duncan multiple comparison test was performed on herb species and the effect of main treatments was determined.Statistical differences were considered significant at P <0.05.

Antimicrobial activity
Table 1 shows the results of the antimicrobial activity of plant extracts.The inhibition zones varied depending on the nature of solvent, plant species, treatments of plant species used for extraction and bacterial species.In general, methanol extracts of the three species were found to be more efficient than other extracts on Gram positive and negative bacterias.These results are in accordance with those from previous studies of plants for antimicrobial activity, where it was reported that methanol is a good solvent for extraction of antibacterial substances from plants in comparison with other solvents (Ahmad et al., 1998;Eloff et al., 1998;Ojala et al., 2000).
Methanol extracts of brine Siyabo showed the largest inhibition zone against S. aureus (23.50 mm).The fact that methanol extract of Siyabo has higher antibacterial activity may be due to the high content of phenolics in this plant (Table 2).
Sirmo and Siyabo extracts had weak antibacterial activity and Mendi extracts didn't have any antibacterial activity on E. faecalis.Also, Siyabo extracts had weak antibacterial activity, Sirmo and Mendi didn't have any antibacterial activity on S.typhimurium.
All of fresh and brine herb extracts showed higher antibacterial activity than reference antibiotics against P. aeruginosa.Therefore, extracts from these plants could be used as natural food preservatives, which could inhibit growth of P. aeruginosa.Solvents (negative control) used for extraction showed no activity against any bacteria tested.
Brine Siyabo extracts had higher antibacterial activity than Sirmo and Mendi extracts against both Gram positive and negative bacterias.The higher antibacterial activity of this extract may be due to the higher terpenes of this plant.The most abundant terpenes in Siyabo are a-terpinene, apinene, L-limonene (Dağdelen, 2014) and these compounds have been reported to antibacterial ability (Griffin et al., 1999).

Total phenolic compounds and radicalscavenging activities
Total phenolic compounds of various solvent extracts were shown in Table 2. Total phenolics of herb species ranged from 14.99±0.86 to 164.20±3.44 mg GAE/kg lyophilized herb.While methanol extract of fresh Siyabo had the highest total phenolic, acetone extract of brine Mendi had the lowest total phenolic.Data values are expressed as means±standard deviation, Values in the same column followed by adifferent letters (a-c) are significantly different (P <0.05),Values in the same row followed by adifferent letters (A-C) are significantly different (P <0.05),General averages were also evaluated between themselves (P <0.05).
The amounts of total phenolics found in the methanol extracts were at very high concentrations in comparison to other solvents.These results are in accordance with those from previous literature (Arabshahi-Delouee and Urooj, 2007;Dağdelen et al., 2014).Total phenolic compounds in the methanol extracts ranged from 38.77 (in Mendi) to 164.20 (in Siyabo) mg of GAE/kg lyophilized herb.
The average total phenolic content of fresh herbs were found to be higher than brined samples of herbs.Similarly, Durmaz et al. (2015) reported that dried Sirmo and Mendi herbs contain higher levels of total phenolics than that in brined samples of the herbs.
Both DPPH and ABTS assays are widely used for evaluating antioxidant activities of natural extracts (Dağdelen et al., 2014).Antioxidant activities of different extracts expressed as mg TEAC/kg lyophilized herb (Table 3 and 4).
Antioxidant activity ranged from 17.10 to 120.21 mg TEAC equivalents/kg lyophilized herb weight and ethanol extract of fresh Siyabo exhibited the highest antioxidant activity (120.21mg TEAC/kg lyophilized herb), followed by methanol extract of fresh Siyabo (118.36 mg TEAC/kg lyophilized herb) with the DPPH method.Similarly, it was determined that the ethanol extract of Siyabo gave the highest results among the solvents used by Dağdelen et al., (2010).
DPPH relatively stable organic radical, has been widely used in the determination of antioxidant activity of single compounds, as well as of various herb extracts (Katalinic et al., 2006).Antioxidant activity determined by ABTS showed the same relationships as did DPPH method, but TEAC values were higher.Differences in antioxidant activity between the investigated herbs may be due to the variation in antioxidant species as well as polarity and solubility behaviors.Phenolic compounds are the major components responsible for the antioxidant activity of herbs (Rice-Evans et al., 1996).
According to the findings obtained from the herb extracts, DPPH and ABTS values were found to be lower than leaf and flower extracts of A. roseum (Najja et al. 2011).In another study, Çoruh et al. (2007)

CONCLUSION
As a result, utilization of methanol and ethanol play a vital role in the extraction of plant constituents.Therefore, the total phenolic compounds, antibacterial and antioxidant activity found in the methanol and ethanol extracts of herbs were determined as very high in comparison to acetone extracts.
Studied extracts of herbs had antibacterial activity against Gram positive and negative bacterias.Antibacterial ability of the extracts might depend on their some phenolics and other bioactive components.This investigation is first study on the antibacterial and antioxidant activity of fresh and brine herbs studied.The compounds responsible for the antimicrobial and antioxidant ability are still unclear.Therefore, further phytochemical investigations are needed to determine, isolate and characterize the bioactive components.

Table 2 .
Total phenolic compounds (mg of GAE/kg lyophilized herb) of herb extracts obtained using different herb species and solvents

Table 3 .
Antioxidant activities of herb extracts obtained using different herb species and solvents determined by the DPPH methods (mg TEAC/kg lyophilized herb)

Table 4 .
Antioxidant activities of herb extracts obtained using different herb species and solvents determined by the ABTS methods (mg TEAC/kg lyophilized herb) Ebrahimabadi et al. (2010)ty of C. macropodum from Van province in Turkey and found that 50% inhibitory concentration (IC50) values were 0.623 and 0.852 mg/mL for DPPH radical scavenging and lipid peroxidation inhibition, respectively.Antioxidant activities of the leaves and flowers extracts of C. macropodum from Isfahan province in Iran were found 196.8 and 167.1 μg/mL as IC50 values for DPPH test fromEbrahimabadi et al. (2010), respectively.The antioxidant activities of C. macropodum obtained from these studies cannot be compared with those obtained from our study because their values given in different units.