PHYTOCHEMICAL AND BIOACTIVITY DIVERSITY IN THE EXTRACTS FROM BULBS AND LEAVES OF DIFFERENT POPULATIONS OF Allium jesdianum , A VALUABLE UNDERUTILIZED VEGETABLE

Allium jesdianum Boiss. & Buhse (Yazdi onion) belonging the family Alliaceae, is an endemic species of Iran that grows wild in the Zagros Mountains range, southwestern Iran. The indigenous people of Iran use the leaves and bulbs of A. jesdianum for the treatment of colds and kidney problems. The bulbs and leaves of various populations of the plant were collected from the alpine regions in Chaharmahal va Bakhtiari province, southwestern Iran. The total phenolic content of ethanol extract was determined by FolinCiocalteu method, the antioxidant activity was evaluated measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the antibacterial activity of the extracts against four bacteria was determined by serial dilution assay. Results indicated that the total phenolic content in ethanol extracts from leaves and bulbs of A. jesdianum ranged between 27.83 to 98.23 mg GAE/g extract. A comparison of all plant extracts in the DPPH assay indicated that ethanol extracts from the populations of A. jesdianum leaves were the most effective free radical scavenging agents. The extracts indicated moderate-to-good inhibitory activities against four bacteria, especially against B. cereus. This finding suggests that the bulbs and leaves of A. jesdianum may be considered a natural source of antioxidants and antimicrobial agents.


INTRODUCTION
Oxidation, deterioration, and microbial contamination that occur in food products can lead to sickness of consumers and economic loss to processors [Yanishlieva et al. 2006].Herbs can contain a wide variety of free radical scavenging molecules, especially phenolic compounds such as phenolic acids, flavonoid, quinones, coumarins, lignans, stilbenes, and tannins that have antioxidant activity [Shahidi and Naczk 1995, Conforti et al. 2009, Ghasemi Pirbalouti et al. 2013, Bajalan et al. 2017, Vosoughi et al. 2018].Primary sources of naturally occurring antioxidants are whole grains, fruits, spices, and vegetables [Rahman et al. 2012].For this reason, the herbs and their constituents have been used in the food industry for their flavoring and biological activities since ancient times [Katalinic et al. 2006].Phenolic compounds present in spice plants, as dietary sources, possess bioactive properties protecting cellular systems against oxidative stress [Bajalan et al. 2017, Vosoughi et al. 2018].Recently, interest in finding naturally occurring antioxidants to replace synthetic antioxidants in foods has increased considerably, primarily due to the possible carcinogenicity of synthetic antioxidants [Velioglu et al. 1998].An antioxidant is a substance present at low concentrations compared to those of an oxide substrate, significantly delays or prevents oxidation of that substrate [Li et al. 2007].
The genus of Allium L. is the largest and important representative genus of the Alliaceae family comprising 700 species; each with different tastes, forms and colors; nonetheless, they are close in biochemical, phytochemical, and nutraceutical properties [Tepe et al. 2005].Allium species are revered to possess antibacterial, antifungal, antiviral, antiprotozoal, and anthelmintic activities and they contain the powerful antioxidants, sulfur and other numerous phenolic compounds, that have aroused great interests for food industries [Griffiths et al. 2002, Benkeblia 2005, Ariga and Seki 2006, Taran et al. 2006].The Allium species have been used for a long time as a medicinal for the prevention and treatment of certain diseases such as diabetes, arthritis, colds and flu, stress, fever, coughs, headache, hemorrhoids, asthma, arteriosclerosis, cancer, respiratory, gastrointestinal, rheumatic, and inflammatory disorders [Amin 1991, Hirsch et al. 2000, Kojuri et al. 2007, Bagheri et al. 2011].Biological and medical functions of Allium species are due to their sulfur compounds, such as S-alk(en)yl-Lcysteine sulfoxides [Fritsch and Keusgen 2006], however, presence of phenolic compounds are also beneficial for human health [Corzo--Martinez et al. 2007].
Allium jesdianum is an endemic species of Iran that grows wild in the high altitude (1800-2600 m) in the Zagros Mountains chain.Known as "Yazdi onion" or "Bonsorkh" in Persian, the leaves and bulbs of A. jesdianum are used for the treatment of abdominal pain, rheumatic pain, colds, and kidney problems by the indigenous people of Chaharmahal va Bakhtiari, southwestern Iran [Ghasemi Pirbalouti 2009].Results of studies have shown analgesic effects of the extract of A. jesdianum [Yoshihiro and Minpei 1999].Furthermore, there are some steroids in its bulb, which have shown cytotoxic and cytostatic effects against malignant tumor cells [Grases et al. 2009].
To our knowledge, there are no published reports on total phenolic content, antibacterial and antioxidant activities of various populations of A. jesdianum.The main objective of this study was to evaluate the content of phenolic compounds, antioxidants and antibacterial activities of ethanol extracts from the bulbs and leaves of A. jesdianum.

MATERIAL AND METHODS
Plant material.Samples of bulb and leaves of A. jesdianum, collected from wild populations of plants growing in various alpine regions of southwestern Iran were used in this study (Fig 1. and Tab. 1).In total, three replicate samples of 30 plants were gathered from three natural habitats at the early flowering until April 30 th to May 20 th 2012.Plant identity was confirmed by Prof. V. Mozaffarian, and a representative voucher specimen (No. 100×) was been placed in the Herbarium of RCANR of Chaharmahal va Bakhtiari province, Iran.Extract preparation.Immediately following the collection, the separated leaves and bulbs of each A. jesdianum plant sample were bagged independently.The bulbs were rinsed with tap water and cut into small slices using a kitchen mixer.The tissue samples were subsequently air-dried in a shaded room at 30°C.A 100 g sample was extracted with 250 ethanol (99%, Merck, Darmstadt, Germany) at 45°C for 8 h followed by a Soxhlet apparatus.The ethanol was subsequently removed under reduced pressure on a rotary evaporator (Model Zirbus 302 W, Italy) at 40°C.The extracts were filtered using a Whatman No. 2.
Determination of total phenolic content.The total amount of phenolic compounds in each extract was determined using the Folin-Ciocalteu method following procedure of Singleton and Rossi [1965] with some modifications.Briefly, 0.5 ml of the sample was mixed with 2.5 ml of Folin-Ciocalteu's (Sigma-Aldrich Co., Steineheim, Germany) phenol reagent for 5 min at 37°C, 2 ml of saturated Na 2 CO 3 (7.5%)(Merck Co., Darmstadt, Germany) was added and the mixture was brought to 10 ml with the addition of deionized, distilled water.The mixture was maintained at room temperature in the dark for 120 min and then the absorbance was measured at 765 nm against a reagent blank using a Perkin-Elmer Lambda UV/Vis spectrophotometer.Gallic acid (Merck Co., Darmstadt, Germany) was used as the reference standard and the total phenolic content was expressed as mg of gallic acid equivalents per gram of each extract on dry basis (mg GAE/g extract).
Antioxidant test.The DPPH radical scavenging activity of the ethanol extract was determined using the method proposed by Huang et al. [2005].The extracts (100 µl) at concentrations of 8 to 500 µg/ml were mixed with 3.9 ml an equal volume of 0.2 mM ethanol solution of DPPH (Sigma-Aldrich Co., Steineheim, Germany).The disappearance of the DPPH after 30 min of incubation at room tempera-ture was determined using a Perkin-Elmer Lambda UV/Vis spectrophotometer at 515 nm against a blank, i.e. without DPPH.Ethanol was used to zero the spectrophotometer and the absorbance of the DPPH radical without antioxidant and measure daily served as the control.The amount of sample necessary to decrease the absorbance of DPPH by 50% (IC 50 ) was calculated graphically and the percentage inhibition was determined according to the equation: where: AC 0 is the absorbance of the control at t = 0 min and AA t is the absorbance of the antioxidant at t = 30 min.The inhibitory concentration (IC 50 ) of the extract needed to inhibit 50% of the DPPH radicals obtained from the standard curve was compared to that of standard/commercial antioxidants.The food preservative butylated hydroxytoluene (BHT) was used as positive control.All measurements were replicated three times.
Antibacterial test.Antibacterial activities of the extracts were tested using clinical isolates of four bacteria strains, the Gram-positive bacteria (Bacillus cereus and Listeria monocytogenes) and the Gramnegative bacteria (Proteus vulgaris and Salmonella typhimurium).The bacteria, originally obtained from chicken meat samples, were provided by the Food Microbiology Laboratory, Veterinary Medicine Faculty, I.A.U., Iran.The population of each bacterial strain was increased by culturing in an overnight Mueller-Hinton broth (MHB) at 37°C.To quantify the antibacterial activity of extracts, bacteria populations were prepared for testing by adjusting each population to 1.0 McFarland standards (1.0 × 10 7 CFU/ml), using a spectrophotometer.Minimum inhibitory concentrations (MIC) were determined using the broth-serial dilution method following standardized methods [CLSI 2012].The extracts and the antimicrobial agents (ciprofloxacin, and flume-quine) were each dissolved in 5% DMSO and then diluted to the highest test concentration.Subsequent test concentrations were made in a series of two-fold dilutions to develop concentration levels of 8 to 500 µg/ml in sterile, 10 ml test tubes containing MHB.A population of bacteria was subsequently added to each tube containing extract and/or antimicrobial agent and then incubated at 37°C for 48 h.After the incubation period, the absorbance of each incubated solution was measured at 630 nm using a spectrophotometer as a measure of bacterial growth to indicate MIC values [Zampini et al. 2005].The minimum bactericidal concentration (MBC) of each essential oil was determined according to the MIC values by transferring 5 µl from MIC tubes to agar plates and incubating at 37°C for 48 h.The MBC was recorded as the minimum concentration of extract, in which no viable bacterial growth was observed.All experimental tests were replicated three different times.
Statistical analysis.Data were analyzed by oneway analysis of variance with three replications using the SPSS 19.0 statistical software.Means of the total phenolic content and IC 50 of various extracts for antioxidant assays were compared with Duncan test at p ≤ 0.05 level.

RESULTS AND DISCUSSION
Extraction yield.The colors of the ethanol extracts from the leaves and bulbs of A. jesdianum were light red and dark red, respectively.Statistical analysis indicated that there was significant difference (p ≤ 0.01) among various populations for extract yield.The highest extract yield was obtained from the leaves of Dastena population of A. jesdianum with 61.7% w/w on dry weight basis (Tab.2).No information on extracts yield of the leaves and bulbs of A. jesdianum is available.
Total phenolic content.Phenolic compounds are believed to account for a major portion of antioxidant capacity and antimicrobial activity in many plants [Yogesh et al. 2012].A significant difference (p ≤ 0.01) for total phenolic content was measured among the extracts (Tab.2).The maximum total phenolic content was obtained from the extract of A. jesdianum leaves collected from the Shirmard population with 98.23 ±15.5 mg GAE/g extract (Tab.2).The lowest total phenolic content was achieved for the extract from leaves of A. jesdianum collected from Dastena with 27.83 ±2.5 mg GAE/g extract (Tab.2).In a study, the amount of total phe-nolic in the extracts from three Allium species ranged from 75 to 80000 mg/kg freeze-dried plant material [Nuutila et al. 2003].Results of a study [Prakash et al. 2007] indicated that the total phenolic contents in extracts from four varieties of A. cepa varied from 4.6 to 74.1 mg GAE/g.Within the vegetable family, the composition and quantity of the phenolics vary significantly according to different intrinsic and extrinsic factors, such as plant genetics and cultivar, soil and growing conditions, maturity state and harvest conditions [Jeffery et al. 2003].
Antioxidant test.The potential antioxidant activity of extracts was determined by the scavenging activity of stable free radical DPPH.This is a quick, reliable and reproducible method to assess the in vitro antioxidant activity of pure compounds as well as plant extracts [Ghasemi Pirbalouti et al. 2014].The effect of antioxidants on DPPH is based on their ability to donate a hydrogen atom to DPPH, thus converting the radical into a stable molecule [Diouf et al. 2009].Lower IC 50 value indicates a stronger ability of the extract to act as a DPPH scavenger, while higher IC 50 value indicates a lower scavenging activity of the scavengers as more scavengers were required to achieve 50% scavenging reaction.In our study, the antioxidant activity of the extract from various populations of A. jesdianum was expressed as IC 50 with values from 0.33 to 2.15 mg/ml indicating that extracts act as moderate to good DPPH scavenger (Tab.2).Significant differences (p < 0.01) in IC 50 values were found for different parts and populations of A. jesdianum.A comparison of all plant extracts in the DPPH assay indicated that ethanol extracts from the leaves of three populations and the bulbs of a population of A. jesdianum (IC 50 values ranged from 0.32 to 0.48 mg/ml) were the most effective free radical scavenging agents (Tab.2).The extracts from three populations of A. jesdianum leaves have the highest total phenolic content (80.31 to 89.23 mg GAE/g extract).The total phenolic in these plant extracts provided substantial antioxidant activity.The antioxidant activity of Alliums species have been reported by numerous investigators [Velioglu et al. 1998, Ghasemi Pirbalouti et al. 2015].
Chaithradhyuthi Gayathri et al. [2009] reported that the antioxidant properties of methanol extracts from some Allium species, with A. ascalonicum, had the maximum radical scavenging activity and total phenolic content.In present study, the extracts from leaves had higher antioxidant activity than bulbs.This study confirms the results obtained in previous researches [Nencini et al. 2007, Ghasemi Pirbalouti et al. 2015]; in fact, these studies showed that the wildtype species of Allium and in particular leaves are more active and efficient in respect to bulbs.Antibacterial test.Extracts from various populations of A. jesdianum demonstrated relative inhibitory activities against pathogenic bacteria tested, the MICs and MBCs of the tested samples are presented in Table 3. Results indicated that different bacteria species demonstrated different levels of sensitivity to the extracts.The MICs of extracts were within concentration ranges from 0.062 to 0.25 mg/ml, and respective MBCs were from 0.125 to >0.50 mg/ml.Generally, the ethanol extracts from leaves and bulbs of A. jesdianum indicated moderate to good inhibitory activities against four bacteria.The highest antibacterial activity was obtained from the extracts of leaves of the Chalroghani and Shirmard populations against L. monocytogenes.Probably in present study, the phenolic compounds are responsible of the antibacterial activity of the extracts from A. jesdianum, especially in the leaves of A. jesdianum.Amin and Kapadnis [2005] reported the antimicrobial activity of the water extract from A. ascalonicum (shallot).The mechanisms, due to which plant extracts can inhibit microorganisms vary.Phenolic compounds can act at two different levels: the cell membrane and cell wall of the microorganisms [Taguri et al. 2006].They can interact with the membrane proteins of bacteria by means of hydrogen bonding through their hydroxyl groups, which can result in changes in membrane permeability and cause cell destruction.Phenolic compounds can also penetrate the bacterial cells and coagulate the cell content [Tian et al. 2009].Several investigators have observed antibacterial activity of Allium species extracts and have attributed this activity to thiosulfinates compounds, which arise from Allium species [Benkeblia et al. 2007, Ghasemi Pirbalouti et al. 2015].These compounds are unstable and give rise to transformation products; however, polar compounds as polyphenols are more stable in cooking and storage [Ahmad and Beg 2001, Lanzotti 2006, Ghasemi Pirbalouti et al. 2014, Ghasemi Pirbalouti et al. 2015].

CONCLUSIONS
The present study is apparently the first report of quantitative total phenol profile, antioxidant and antibacterial activities of the ethanol extracts from leaves and bulbs of A. jesdianum, as an Iranian endemic herb.This medicinal plant is ordinarily used for food and pharmaceutical purposes and as health foods in Iran.Results of this study indicated that the extracts from leaves of A. jesdianum had the highest antibacterial properties.The results of current study demonstrated that the ethanol extracts from A. jesdianum with the maximum total phenolic content espe-cially in the leaves had the highest antioxidant activity.Phenolic compounds present in the plant are responsible for its effective free radical scavenging, antioxidant and antimicrobial activities.In conclusion, A. jesdianum could be an important dietary source of phenolic compounds with antioxidant capacity and antibacterial properties.Nonetheless, in order to gain better views on the antioxidant levels and activities in Allium species, further studies on purification, identification and quantification of each phenolic compound and other non-phenolic compounds are necessary in future.

Table 1 .
Geographical and climate of natural habitats of Allium jesdianum P -annual precipitation (mm), T -average temperature (°C), E.C. -electrical conductivity (dS/m), O.C. -organic carbon (%) Meteorological information was obtained from weather stations located within the study area and the surrounding zone; each value in the mean of

Table 2 .
Extract yield, antioxidant activity, and total phenolic content of the ethanol extracts from Allium jesdianum

Table 3 .
Antibacterial activity (MICs and MBCs) of the ethanol extracts from Allium jesdianum against four bacteria