Qualitative and Quantitative Determination of Secondary metabolites and Antioxidant Potential of Eruca sativa

Eruca sativa known as Taramira (family Brassicaceae), is a minor oil crop and medicinal plant in various parts of Middle East and IndoPak subcontinent. Since long it has been used in traditional medicines as remedies for different diseases [1]. Therefore phytochemical composition and respective biological activities are important to understand the therapeutic potential of medicinal herbs. Among other, phenolic compounds are the most widely explored phytochemicals for therapeutic potential in different medicinal plants. Most of these studies conclude that pharmacological activities of any medicinal plant are due to the presence of secondary metabolites. Secondary metabolites usually consist of the phenolic compounds, alkaloids, tannins, saponins, carbohydrates, glycosides, flavonoids, steroids, etc. Most phenolic compounds such as flavonoids, glycosides, triterpenoids, flavonons, carbohydrates and anthraquinones are commonly present in most of the medicinal plants. All of these secondary metabolites and particularly phenolic compounds have been reported as scavengers of free radicals and also have been considered as good therapeutic candidates for free radical related pathologies [2]. Nowadays, there is an increasing focus for the search of anti-oxidants (non-synthetic) from medicinal plant such as carotenoids, ascorbic acid (vitamins), phenolic and flavonoids. There are also several studies related to the analysis of phenolic composition in other members of the Brassicaceae family [3-6]. So far, whole plant extracts and purified Glucosinolates (GSLs) Brassica oleracea L. has been studied for their antioxidant potential by evaluating its effects on lipid peroxidation (deoxyribose assay), the Bleomycin assay and a Radical cation 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) [7]. It has been reported that E. sativa seed extracts are potent antioxidants, exhibit diuretic effects and provide renal protection. Previous phytochemical studies of E. sativa showed that leaves and seeds contain glucosinolates. Three new quercetins have been isolated and identified from E. sativa leaves [8]. However, there is not sufficient information in the form of scientific analysis about detailed phytochemical composition of E. sativa and their respective bioactivities [1]. Anti-oxidative activity is a measure of capability of compound to scavenge free Hydroxyl groups and Oxygen species. It is very important property of medicinal plants because there are number of reports which mention that in biological systems, free radicals are causative agents for different disease such as cancer. Therefore, antioxidant properties are an index of antioxidant potential against reactive oxygen species (free radicals) [2].


Introduction
Eruca sativa known as Taramira (family Brassicaceae), is a minor oil crop and medicinal plant in various parts of Middle East and Indo-Pak subcontinent. Since long it has been used in traditional medicines as remedies for different diseases [1]. Therefore phytochemical composition and respective biological activities are important to understand the therapeutic potential of medicinal herbs. Among other, phenolic compounds are the most widely explored phytochemicals for therapeutic potential in different medicinal plants. Most of these studies conclude that pharmacological activities of any medicinal plant are due to the presence of secondary metabolites. Secondary metabolites usually consist of the phenolic compounds, alkaloids, tannins, saponins, carbohydrates, glycosides, flavonoids, steroids, etc. Most phenolic compounds such as flavonoids, glycosides, triterpenoids, flavonons, carbohydrates and anthraquinones are commonly present in most of the medicinal plants. All of these secondary metabolites and particularly phenolic compounds have been reported as scavengers of free radicals and also have been considered as good therapeutic candidates for free radical related pathologies [2]. Nowadays, there is an increasing focus for the search of anti-oxidants (non-synthetic) from medicinal plant such as carotenoids, ascorbic acid (vitamins), phenolic and flavonoids. There are also several studies related to the analysis of phenolic composition in other members of the Brassicaceae family [3][4][5][6]. So far, whole plant extracts and purified Glucosinolates (GSLs) Brassica oleracea L. has been studied for their antioxidant potential by evaluating its effects on lipid peroxidation (deoxyribose assay), the Bleomycin assay and a Radical cation 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) [7]. It has been reported that E. sativa seed extracts are potent antioxidants, exhibit diuretic effects and provide renal protection. Previous phytochemical studies of E. sativa showed that leaves and seeds contain glucosinolates. Three new quercetins have been isolated and identified from E. sativa leaves [8]. However, there is not sufficient information in the form of scientific analysis about detailed phytochemical composition of E. sativa and their respective bioactivities [1]. Anti-oxidative activity is a measure of capability of compound to scavenge free Hydroxyl groups and Oxygen species. It is very important property of medicinal plants because there are number of reports which mention that in biological systems, free radicals are causative agents for different disease such as cancer. Therefore, antioxidant properties are an index of antioxidant potential against reactive oxygen species (free radicals) [2].
Our early studies showed that E. sativa seeds are rich source of phytochemicals including flavonoids, phenolics, alkaloids and ascorbic acid [9]. Traditionally, in folk medicine people also use leaves, stem and inflorescence of E. sativa in their food [2]. So this prompted us to investigate all part of plant to identify its nutritional value. Therefore this study was conducted with the aim to investigate phytoconstituents present in all aerial parts of E. sativa (stem, leaves, flowers and seeds) and assessment of their antioxidant characteristics. Further, considering the reported presence of phenolics in of E. sativa, we decided to carry out the isolation and identification of different phenolic compounds through Reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC exhibits a great potential in separating complex mixtures of phenolic compounds [10,11] by using combination of mobile phases with different elution gradients and specific run times.

Collection and identification of plant sample
Crude oil, Seeds and Fresh plant sample of E. sativa consist of all aerial parts were purchased from the local herbal store (district Islamabad, Punjab) on January, 2013 and specimen was identified by Dr. Muhammad Qasim Hayat in the Department of Plant Biotechnology, Atta-ul-Rehman School of Applied Biosciences, National University of Science and Technology Islamabad, Pakistan. The identified voucher specimen (No. 23) was deposited in the Medicinal Plant Research Laboratory for future record. All plant samples were dried under shade and then ground into fine powder form (80 mesh sieve size) by electrical grinder. Powdered sample of all parts stored in clean paper bags [12] and preserved at 4ºC for further analysis.

Preparation of plant extracts
Organic extracts of seeds and aerial parts of plant (leaves, stem and flowers separately), were prepared by using two different solvents (85% Methanol and water with increasing polarity) [13]. Dried plant powder weighed carefully and used for extract preparation through soxhlet apparatus at respective temperature. The extract obtained was filtered and concentrated in rotary evaporator [14,15]. The concentrated plant extracts (semi-solid mass) were lyophilized and than store the dried organic extracts in air tight brown bottles.

Estimation of Total Phenolics content in different parts of E. sativa
Folin-ciocalteu method has been used for analysis of total phenol from all plant samples (leaves, stem, flowers and seeds) after extraction. Organic extracts of plant powder (1 gm) were prepared in combination of solvents (20 mL) acetone-methanol-water with the ratio of 7:7:6 volume/volume. Afterward these extracts were subjected to centrifugation (6000 rpm, 10 mins). Than analyzed for quantification of total phenolic contents following the methodology [22,23]. 1 mL of organic extract, 10 mL of water (deionized) and 2 mL of Folin-Denis reagent has been taken in test tube. Saturated sodium carbonate solution (2 mL) added to reaction mixture and kept in dark at room temperature for 1 hour of incubation. Absorbance was measured at spectrophotometerically at 640 nm. The total phenolics concentration was calculated from a calibrated curve of standard phenolic compound Gallic acid and phenolic contents of plant extracts were expressed as mg GAE/g, Gallic acid equivalent.

Antioxidant activity
DPPH radical scavenging activity: DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity of all extracts (leaf, stem, flowers and seeds of E. sativa) were measured by the spectrophotometric method for the presence of DPPH as a free radical. Plant extract (25 μl) is added into 975 μL DPPH solution, which is prepared by dissolving 2.5 mg DPPH in 100 mL of Methanol. Whole mixture was shaken vigorously and then subjected for incubation (30 min) in the dark place at the room temperature. Finally measure the absorbance at 517 nm through spectrophotometer. Following equation has been used to calculate the percentage of DPPH scavenging activity: Whereas A control=Absorbance of the control sample A sample=Absorbance of the sample containing plant extract/ standard.
Antioxidant activity of all different extracts of E. sativa have been expressed in term of IC50 values, which is specific concentration of the extract that caused 50% neutralization of DPPH radicals [24]. Calibration curve for Ascorbic acid (vitamin C) used as a standard [25].
Hydrogen peroxide scavenging activity: H 2 O 2 Scavenging potential of plant extracts was analyzed by reported method [26,27] with slight modifications. H 2 O 2 solution has been prepared in PBS (Phosphate Buffer Saline). 0.6 mL of 4 mM H 2 O 2 has been added to 4mL of extract and incubated for 10 min. The absorbance of whole mixture was measured at 230 nm through spectrophotometer. Following formula has been used to estimate % age of H 2 O 2 free radical scavenging activity. Ascorbic acid has been taken as standard.

Percentage H 2 O 2 scavenging activity=(A control-A Sample) x100
A control Whereas A control=Absorbance of the control sample A sample=Absorbance of the sample containing plant extract/ standard.

Reversed-phase high-performance liquid chromatographic identification of Phenolics from E. sativa
Sample preparation for RP-HPLC analysis: Powdered plant samples (200 mg) was subjected for extraction with 6 mL of hydrochloric acid (25%) and 20 mL methanol for 1 h. Plant extracts were filtered into volumetric flask. Then combine the whole extract, filter and dilute with HPLC grade methanol. Take 5 mL of extract and filter through a Chromafil, transfer extract into a volumetric flask and further diluted with HPLC grade methanol (up to 10 mL) [28].

Specification of the RP-HPLC instrument:
Analysis of all samples was performed using RP-HPLC, Perkin Elmer Series 200 pump and equipped with 200 UV/VIS detectors, Total chrom V2.6.0.01 with LC instrument control software having reverse phase water guard Column: Symmetry C18 (5μm, 250mm) and Hamilton microliter syringe using an injection volume of 20 μl. RP-HPLC methods is consist of gradient elution of two solvents -Solvent A (Methanol) and Solvent B (Acetic acid in water (1:25)). Detector wavelengths range set at 280 nm and 360 nm. Flow rate adjusted at 1.0 ml/min.The gradient program start with 100% B and then held at this concentration for the first 4 minutes. This was followed by 50% eluent A for the next 6 minutes after which concentration of A was increased to 80% for the next 10 minutes and then reduced to 50% again for the following 2 minutes. Therefore total run time was of 22 minutes. Standards: The standard phenolic compounds including Quercetin Dihydrate, Salicylic Acid, Aspirin, L-Ascorbic Acid, Tannic Acid, Benzoic Acid, Gallic Acid, Catechol, Vanillin, Ellagic Acid, Phloroglucinol GR, Rutin, Kaempferol and Resorcinol were purchased from reputed manufacturers. Preparation of standard samples: Standard compound (6 mg) was dissolved in HPLC grade Methanol (10 mL). This mixture was subjected to sonication for mixing. Before injecting into the column this mixture was filtered by using Whattman Nylon Membrane Filter (0.45 μm). The HPLC analysis of 14 standard phenolic compounds was performed at the same wavelength, flow rate and sample concentration [2,28,29].
Statistical analysis: Statistical evaluation of results was carried out by applying student´s t-test, and results were analyzed as mean ± SD (Standard Deviation) and percentage values of different Phytochemicals. Statistical Significance of results was considered at P-values, 0.05 and 0.01. Concentrations yielding 50% inhibition (IC50) was calculated by interpolation from linear regression analysis. All statistical analyses have been performed by using Graph Pad Prism 5 software.

Results and Discussion
Vegetables belonging to family Brassica are rich in phytochemical constituents particularly polyphenols and they are known to play an important role in human nutrition. We analyzed three different extracts (methanol, water and methanol-water) from leaves, stem, flowers and seeds of E. sativa for evaluation of secondary metabolites. We have measured the antioxidant activity of these extracts and correlated the data with total phenolic compounds. Further, we have done identification for different phenolics compounds present in extracts of E. sativa (stem, leaves, seeds and flowers) through RP-HPLC. Qualitative analysis of secondary phytoconstituents has been carried out from aqueous and methanolic extracts of stem, leaves, flower, seeds and crude oil. These results showed that all parts of E. sativa are rich source of Steroids, Terpenoids, Tannins, Diterpenes, and Cardiac glycosides, Polyphenols, Alkaloids, Flavonoids and Phytosterols (Table 1). Saponins are only present in extracts of seed and leaf. While Leucoanthocyanins and Anthocyanin are absent from all aerial parts. Comparative phytochemical analysis of aerial parts of plants with crude oil which is available at local herb stores, indicate that crude oil contain less number of phytoconstituents, might be due to the method of extraction. Crude oil contains only steroids, terpenoids, coumarins and flavonoids. Previous study related to phytochemical analysis of E. sativa seeds report similar findings [30]. Therefore presence of significant amount of phytoconstituents confers medicinal properties, including antioxidant activities on these extracts of E. sativa.
Qualitative analysis showed that all aerial parts of E. sativa are rich in Phenolics, which are medicinally important phytoconstituent [2]. Therefore Folin-ciocalteu method has been used to estimate total Phenolic from all different parts of E. sativa. Results showed that seeds are rich in phenolics as they contain highest amount of phenolics 27.1 ± 0.2 mg GAE/g extract as compared to all other aerial parts. Eruca leaf and flower also contain significant amount of phenolics 23.07 ± 0.11, 19.9 ± 0.3 mg GAE/g respectively. Results indicate that whole plant is a good source of phenolics which support its use in most of the regions where people consume this herb as a whole plant (leaf, flowers, stem and seed) or various combinations in the form of fresh salad8. Antioxidant potential of different extracts of E. sativa (leaves, seeds, stem and flowers) has been evaluated by DPPH radical scavenging assay and compared with Gallic acid as a standard phenolic compound. Seeds extract has highest antioxidant potential at 50 mg/mL concentration (66.93 ± 0.2). Antioxidant activity of the tested extracts and the positive control (vitamin C), expressed as the percentage of deactivation of the DPPH free radicals ( Table 2). Comparatively radical scavenging potential in all extracts follow this trend seeds>leaves>flowers> stem (Table 3). Antioxidants potential of phenolics in the extracts was determined by the IC50 values (the concentration of the sample required to scavenge 50% of the DPPH free radicals). High antioxidant activity and phenolic content observed in seeds extracts of E. sativa. Methanolic seeds extracts also exhibit lowest IC50 (100. 6   other extracts. Recent studies [31] suggested that the ethanolic extracts of E. sativa seeds possessed a potent antioxidant activity and exert a protective effect on mercuric chloride induced renal toxicity. Studies report that these health-promoting activities of E. sativa are partially related to their strong antioxidant properties [32][33][34]. Free radical H 2 O 2 scavenging potential of all plant extracts including stem, leaves, seeds and flowers has been evaluated spectrophotometrically (Table 3). Results indicate that seeds, and leaf extracts have highest scavenging potential at the concentration of 200 μg/mL (64.12 ± 4.6, 56.2 ± 1.1 respectively). Although Hydrogen peroxide (H 2 O 2 ) is a weak reactive agent, but in biological systems it is also a source of free hydroxyl radicals, so production of more HO-(Hydroxyl groups) free radicals disturb the normal balanced HO-level in biological systems, hence it cause toxicity in the cells. Therefore, the eliminating excessive amount of H 2 O 2 is very important for antioxidant defense in cell [26]. Recent scientific reports revealed the role of free radicals in the emergence of such lifestyle diseases such as atherosclerosis, heart attack, stroke, cancer, diabetes, senile cataracts and accelerated aging. Structurally polyphenols contain number of Hydroxyl groups (HO-), which are responsible for antioxidant characteristics and potential of polyphenols. Because of these OHgroups, polyphenols are able to chelate transition metal ions, such as iron, and copper. Generally, these metal ions are involves to initiate various free radical chain reactions. Additionally they are also capable to inhibit enzymes such as xanthine oxidase and NADPH oxidase. These enzymes catalyze the production of large amounts of reactive oxygen [23]. In present study we are reporting the phenolic composition in methanolic extracts of different parts of E. sativa (stem, leaves, flowers and seeds) for the first time. Four different extracts of E. sativa has been analyzed and eleven different phenolics have been identified through RP-HLC. There is limited data on the phenolic and flavonoid content of Eruca species. Qualitative analyses by liquid chromatography/mass spectrometry (LC/MS) and NMR of identified quercetin triglucosides in E. sativa. Initial qualitative thin-layer chromatography analysis identified various isorhamnetin glycosides, kaempferol and quercetin in leaves of flowering Diplotaxis species [33,35]. Limited literature is present related to the chemical constituents of E. sativa leaves and flowers [35], which do not provide the sufficient information. In present study RP-HPLC conditions, provide good separation of the peaks which could be identified in the chromatogram and revealed that different types of phenolic compounds are present in different parts of E. sativa. These phenolic compounds were identified in comparison         with reference compounds under same conditions and UV spectrum. Initially separated phenolics peaks were identified by direct comparison of their retention times with those of respective standards compounds.

Conclusion
Quantitative analysis of phenolics from E. sativa was achieved first time in this work. The observed level of phytoconstituents revealed that E. sativa is a rich source of antioxidant compounds. The RP-HPLC analysis of phenolics enables reproducible and accurate determination of eleven common phenolic compounds from E. sativa.

Conflicts of Interest
All authors have no conflict of interest.