ASSESSMENT OF PHENOLIC COMPOUNDS, ANTIMICROBIAL ACTIVITY AND FREE RADICAL SCAVENGING POTENCY OF THREE SELECTED VEGETABLES

Phytochemicals are compounds derived from plants that are assumed to have defensive role against certain disease. They have antioxidants, anticancer, antimicrobial, antifungal, antiviral, antithrombic and anti-inflammatory properties. They have a high specificity to boost the immune system and play important role in the metabolism of hormones. The current study is based on qualitative and quantitative evaluation of total phenolics contents, phenolic compounds, antioxidant potential, free and bound phenolic acids in selected vegetables available at the local market of Hyderabad, Sindh, Pakistan. Two different extraction procedures ultrasonic-assisted base hydrolysis extraction and sonication extraction were used. Total 13 phenolic compounds were found and quantified by highperformance liquid chromatography (HPLC), in which Ferulic acid was quantified in a higher amount of 16.71 mg/g in bitter gourd. Total phenolic contents were determined by using Perklin-Elmer lambda UV/Visible spectrophotometer and higher concentration was found in Bitter gourd 92.56 mg 100/g as compared to Luffa and Brinjal with 79.03 and 66.56 mg 100/g, respectively. The antioxidant activity (DPPH assay) was measured at λmax of 517 nm, results revealed that Bitter gourd possessed the higher antioxidant activity with 182.61 μMol/g followed by Luffa and Brinjal with 112.94 and 82.96 μMol/g. The total Flavonoid contents were higher in Brinjal with 44.32 mg g -1 whereas Luffa and Bitter gourd possess the Flavonoid concentration in the range 38.02 and 34.64mg g -1 respectively, the total tannin contents also higher in Brinjal 31.40 mg/g follwed by in Luffa and Bitter gourd with 25.17 and 21.19 mg/g respectively. Antimicrobial activity showed that, all the extracts are the highly effective against S. aureus as compared to E. coli. Finally, it is concluded that all the selected vegetables are very good sources of Phenolic compounds as well as phytochemicals and should be included in the daily human diet for good health. On the basis of obtained results, it is also suggested that these samples will be further investigated for the determination of fatty acids by GC-MS and liquid chromatography-mass spectrum (LC-MS).


INTRODUCTION 1
The term vegetable typically includes consumable part of herbaceous plant, root, stem, leaves, flower, or fruits. Herbaceous are the non-woody plants that are cultivated in farms, home garden, kitchen gardens, gathered from backwoods tree and markets for the use of house (Eme-Okafor, 2016). Vegetables are the good source of fiber contain naturally low/no in fat/cholesterol and calories. They are rich in minerals and vitamins, phenolic acids, flavonoids, and glucosinolates, which act as antioxidants, blood sugar and blood pressure influencing substances. Vegetables contain various phytochemicals which have very * Corresponding author: jamal.mangi@usindh.edu.pk important effects including, anticarcinogenic, antiviral, cholesterol-lowering, antifungal, antibacterial, antiinflammatory or antithrombotic (Jaiswal et al., 2011). However ever most of these functional compounds have wide ranges of these properties. For example, antioxidative phytochemicals are phenolic compounds, saponins, protease, inhibitors, carotenoids, sulfides, phytoestrogens, monoterpenes, and phytosterols (Duyn and Pivonka, 2000;Tomas-Barberan and Espin, 2001). The European Prospective Study of Cancer (EPIC) and World Cancer Research Fund/American Institute for Cancer Research, the Health Education Authority (UK), the German Nutrition Society, and the German Cancer Society suggested that an inclusion of 50g and 375gm of vegetable in daily food intake lead to reduction in the 20% of the premature death and healthy life (Khaw et al., 2001). It is also recommended that, the daily food intake should contain functional compound that are rich in the phytochemicals in conjugation with non-alcoholic and fat consumption (Boeing et al., 2004). Recently, It was reported that, various crop production practices were used to enhance the phytochemical content and composition in many vegetables and fruits (Vaccinium spp) (Eichholz et al., 2012;Schreiner, 2005). Hu et al. (2004) reported that, the mechanisms of natural antioxidant are not efficient therefore, use of antioxidant in the diet is important. These antioxidant are also widely used in the food industry to increase the shelf life of food specially those foods which are rich in the polyunsaturated fat (Isabelle et al., 2010). In addition, these antioxidant are also used in the processed food to prevent the lipid oxidation (Ayaz et al., 2008;Andarwulan et al., 2010).
Formerly, it was revealed that, the chemicals which are synthesize in the laboratory are highly costly and toxic. Morover due to increase in the awareness of consumer about feed additives, the information about safety, and natural source of antioxidant must be mentioned on product (Zhou and Yu 2006;Dasgupta, 2007). It is reported that, growth conditions and genotypes are the evidence to alter the different properties includings phytochemicals, antioxidant and antibacterial properties (Stangeland et al., 2009). From last two decades, plants and vegetable are the only natural source of phytochemical which pays attention of scientist to explore their chemical composition by using different methods and techniques. The present work also based on the same phenomena i.e in this work we determine phytochemicals analysis of selected local vegetables, their antimicrobial activity, and radical scavenging activity.

Chemicals, standards and vegetables
Fresh three selected vegetables Bitter gourd (Momordica charantia), Luffa (Luffa acutangular), and Brinjal (Solanum melongena) were collected from the local market of district Hyderabad Sindh, Pakistan during April and May 2020. All chemicals and reagents used in this research work were pure analytical grade.

Processing of vegetable material
The collected vegetables were washed with tape and then distilled water, to remove dust particles and dried in shade for 2 weeks, after drying each vegetable ground separately with an electric grinder and kept in different glass storage bottles that have clamp lids before analysis.

Phenolic compounds evaluation
Ultrasonic-assisted base hydrolysis extraction method was used in order to extract the phenolic compounds, 0.5g of powdered vegetables was hydrolyzed with 10ml of base solution (0.372g EDTA, 2N sodium hydroxide and 1g of ascorbic acid) in 100ml polypropylene tubes by purging nitrogen (N 2 ) preceding covering. At that point, covered tubes were vortexed and sonicated for 30 minutes at 56°C. After hydrolysis, the samples were cooled and acclimated to hydrochloric acid (pH 2.5), and the phenolic mixture were taken out with 5x 2ml of acidic ether (ethyl acetate C 4 H 8 O 2 ) and then vertexed for 25-30 followed by centrifuged at 5,000 rpm for 10 minutes to separate the whole, aqueous layers and higher organic layer of phenolic compounds. After the solidification of phenolics compounds, they were dried by removing nitrogen followed by 5ml of 80% methanol was added. Finally, the solution was filtered via 0.45 μm syringe filters before HPLC investigation. The comparative concentrates were likewise utilized in favor of assessment of total phenolic contents (TPC) and antioxidant activity (Memon et al., 2013).

Assessment of total phenolic contents (TPC)
The quantification of total phenolic contents (TPC) assay was carried out by Folin-Ciocalteu (FC) reagent method with slight modification (Memon et al., 2012;Boakye et al., 2015). The reaction mixtures consist 0.2ml of diluted extracts, 0.8ml of Folin-Ciocalteu (FC) reagent and 2ml of sodium carbonate (Na 2 CO 3 ) Saturated solution (7.5%). Finally, mixtures were filled with demonize water and adjusted the volume up to 7ml then for the completion of the reaction placed the mixtures in darkness at ambient temperature for 2 hours. After incubation time absorbance was measured on Perkin Elmer lambda 35, UV/Vis spectrophotometer at 765nm. The total phenolic content was calculated from a standard, the obtained results of various extracts were expressed as Gallic acid equivalents (mg/g).

Assessment of total Flavonoid contents
Total flavonoid contents (TFC) were analyzed by reported method (Laghari et al., 2011). The reaction mixture was sonicated extract contains 2ml of sodium nitrite (5% w/v), 0.5ml AlCl 3 (10%w/v) and 3ml NaOH (4.3% w/v) and distilled water up to 10ml. After that, the solution was placed in the dark for 15 mins for completion of the reaction (Laghari et al., 2011), and the absorbance was measured on UV/VIS spectrophotometer, Perkin Elmer lambda 35, at 500 nm, 80% aqueous methanol was taken as reference, Rutin was used as a standard with the concentration range from 1-100 µg/ml The results were expressed as rutin equivalents (Rutin eq. µg/ml) and predicted from a standard curve.

Evaluation of total tannin contents
In brief, 0.2g powder samples of each vegetable were extracted in 1% hydrochloric acid in methanol for 20 mins at 30°C then 1ml of extract and 5ml of vanillin reagent (50:50 mixture of 1% vanillin 8% HCl in methanol) was treated for 20 mins at 30°C (Siddiqui et al., 2017), and the absorbance was measured on UV/VIS spectrophotometer, at 500nm. HCl (4%) as a substituent of vanillin reagent was added to the extract and used as a blank. While catechin was used for standard. The results were estimated by the standard curve of catechin which obtained in the concentration range 6.25 µg/ml to 200 µg/ml (Siddiqui et al., 2017).

Radical scavenging activity (RSA)
Two ml of 0.1 mmol of DPPH solution was mixed to 2mL sonicated extract of every vegetable followed by shaking and placed in the dark for up to 25 minutes. The quercetin standards were prepared, and the range of concentration was used 1 to 10 μmol. Subsequently, the absorbance was determined through a UV/Vis spectrophotometer at 517nm. The antioxidant activity equivalent to quercetin was adapted from the standard curve and expressed as µmol/100g of the sample (Memon et al., 2013;Amron and Konsue, 2018).

Identification and Quantification of phenolic compounds by (HPLC-DAD)
HPLC Thermo Finnigan, SCM 1000 (California, USA) connected with a DAD (diode array detector) system was used for the analysis of phenolic compounds. Reverse-phase Hypersil Gold C-18 (Thermo Corporation, USA) was used to separate different phenolic compounds, (Memon et al., 2013). The gradient mobile phase solvent 0.1% formic acid in water (A) and in methanol (B), were used. The rate of flow was kept as 1 ml/min and the gradient linear was 5% to 30% for 25 min, followed by 30% for 45 min, gradient linear elution was altered as of 30% up to 100% for 52 minutes. After 52 min, elution was carried back to 5% for 3 min in order to equilibrate the column. Furthermore, with the help of DAD, the UV analysis was done, set at 270, 310, and 325nm. The software Chromquest Version 4.2 was applied for data interpretation. All phenolic compound structures were separately confirmed by comparison their UV spectra and retention time (tR) with related standards. For the construction of each standard curve, the standard solutions in the different concentration series from 1-50 μg/ml were introduced into the HPLC-DAD system. The concentrations of the different phenolic constituents were calculated through peak area. In accordance with their individual standard curves.

Determination of antimicrobial activities
The antibacterial action of vegetables was determined by the strategy depicted by (Rahman et al., 2017). Four concentrations of 125, 250, 500 and 1000 μgmL -1 were set up in 100% DMSO (dimethyl sulfoxide), and DMSO was utilized as the negative control. Whatman No. 1 paper with 20μL of different obsessions (100% diluted) was arranged outwardly of the petri dishes place in an incubation center for 24 hours at 37°C. After that, the antibacterial movement estimating by the territory of hindrance width in mm around the circles and determined MIC (minimum inhibition concentration) values (Bouchekrit et al., 2016).

DISCUSSION
These vegetables are found to be rich sources of phenolic compounds, flavonoid and tannin contents, in quantitative analysis. The classification of phenolic content (mg/g) lies as given bitter gourd> luffa >brinjal ranging from 92.56 to 66.56 (mg/g). Phenolic compounds are known as regular antimicrobial (Raju and Rao, 2012). Tannins have additionally been perceived because of their remedial potential. The most elevated measure of tannins was accounted for in brinjal (31.40 mg/g). Leafy foods contain various kinds of phenolic compounds in various degrees; go about as potential antioxidant and a vital aspect for saving health and keeping away from various diseases (Krinsky and Johnson, 2005). Beforehand, in comparative investigations (Aqil et al. 2006;Ramya et al. 2011) similar values for the Total Phenolic Contents of methanol (74.33±5.13 mg/g) and ethanolic extracts (4.9 mg/g) were reported. Contrasts between the findings might be because of different conditions like temperature, area, environment, diseases and bug openness inside the species, selection of parts tried a season of taking examples, and techniques for declaration (Singh et al., 2014).

CONCLUSION
It concluded that ultra-sonic assisted base hydrolysis extraction is the most efficient method for the extraction, one can get maximum yield. The selected vegetables are very good sources of phenolic compounds, they are highly effective against microorganisms and oxidants because they contain a wide and broad range of significant organic compounds which highly beneficial for good health. It is also concluded that selected vegetables need much more attention from researchers to assess their leftover wellbeing impacts by deciding unsaturated fats, minerals, and heavy metal contamination by using different analytical techniques.