In Vitro Assessment of Antioxidant Enzymes, Phenolic Contents and Antioxidant Capacity of the Verdolaga (Portulacaceae)

In this study, the antioxidants and photosynthetic compounds of Verdolaga were examined. Compounds were extracted from distinctive segments of the verdolaga using various solvents such as methanol (40, 60, 80%), ethanol (40, 60, 80%), acetone (40, 60, 80%), and deionized water. The use of 80% methanol led to the highest extracted concentration of phenolic substances and flavonoids. The extracted products (Leaves, Stem strips, and Root strips) were evaluated for their radical scavenging capabilities with DPPH (IC50= 22.26, 20.56, and 32.10), and ABTS (IC50= 2.86, 3.70, and 5.24), reducing power (EC50= 15.70, 16.39, and 21.69), and peroxide scavenging activity (1C50= 1.717, 2.937, and 3.255), respectively. The extracted products were analyzed by a gas chromatography-mass spectrometer. Peroxidase, catalase, and polyphenol oxidase assays were completed for the crude extract of verdolaga’s leave, stem strips, and root strips. As indicated by these tests, extracts of the verdolaga’s roots, stems and leaves using 80% methanol yielded high antioxidant activity. The most elevated concentrations of extracted chlorophyll, lycopene, and carotenoids were from the leaves and the highest concentration of extracted tannin was noted from strips of stems. The highest measures of peroxidase and polyphenol oxidase were identified in root strips and the highest units of catalase was identified in leaves. DOI: 10.14302/issn.2379-7835.ijn-19-3144 Corresponding author: Yaaser Q. Almulaiky, Chemistry Department, Faculty of Sciences and Arts, University of Jeddah, Khulais, P.O. Box 355, Khulais, 21921, Saudi Arabia, Tel. +966566880264. E-mail: yaseralmoliki@hotmail.com


Introduction
Reactive oxygen species (ROS) are produced in higher volumes during tissue injury. An excessive amount of ROS can denature deoxyribonucleic acid (DNA) and proteins, disrupt cell layers, and negatively affect lipids through chain reactions [1]. The production of reactive oxygen species (ROS) mediated lipid peroxidation plays a key role in cell death, including autophagy, ferroptosis, and apoptosis. Cell reinforcements, such as atoms that have the capability to neutralize radical, protect against these damages. These compounds can assist in forestalling diseases, including malignancy, hepatitis, asthma, atherosclerosis, joint inflammation, coronary illness, and diabetes [2]. Recently, plants and herbs have been placed in dietary supplements as cancer prevention agents, as natural alternatives to manufactured cell reinforcements [3].
The reports of side effects from synthetic ingredients of supplements have prompted buyers to explore options that are associated with less egregious results [4]. It has been reported that extracts from herbs are able to support cell defense mechanisms and stimulate antimicrobial activity [3,5]. The extraction of polyphenols from a plant through diverse solvents and the percent yield primarily depends on the strategy for extraction [6,7]. The extraction strategy must result in the maximum yield of the target compounds [8]. There are a few studies that have utilized blends of ethanol, methanol, acetone and water, to extract polyphenols from plants [3][4][5][6][7][8][9][10]. Portulaca oleracea, (common verdolaga) is an imperative restorative plant with range of pharmacological benefits, including the ability to increase the rate of tissue repair and antimicrobial activity. It also contains vitamins A and C, omega-3 unsaturated fats, β-carotene, and α-linolenic acid [11].
The airborne segments of the plant are utilized in different cultures as a diuretic, antibiotic, antispasmodic, and antihelminthic [12]. It also eaten with other greens in the Middle East and Mediterranean, as the stems and leaves are succulent with a salty and acidic taste that is similar to spinach. Verdolaga is an almost certain competitor as a valuable cosmetic ingredient. Thus, further exploration of its unexamined uses can benefit humanity [11]. It is widespread, quickly developing and self-compatible and creates vast quantities of seeds that have long reasonability [13]. The target of this study was to explore the use of different solvents (methanol, ethanol, acetone and water) to extract phenolic substances and flavonoids, and to appraise whether extracts from the leaves, root strips, and stem strips of Verdolaga had the best cancer preventing characteristics.
Catechin, ferric chloride, catechol, hydrogen peroxide, and guaiacol were purchased from Extrasynthese (Lyon, France). The rest of the standard solvents were purchased from Sigma-Aldrich (USA).

Verdolaga Samples
The sample used in this experiment were collected from Al-Baha city, Saudi Arabia, situated at 20.0119N 41.2607E, Oct, 2019. Leaves, as well as strips of root and stems, were obtained by manual separation, washed, and air-dried at ambient temperature. The air-dried samples were ground and placed in a dry environment until further use except samples used for determination of antioxidant enzymes, fresh sample was used.

Determination of the Antioxidant Enzymes
Crude Enzyme Extracts Two grams each of fresh leaves, root strips and stem strips were ground in a mortar separately and then combined with 20 mM Tris-HCl buffer, which had a pH of 7.2. The homogenates were centrifuged at 13,000 rpm for 10 min at 4℃. The supernatants were stored as crude extract at -18℃ for further analysis.
Class III Plant Peroxidase (EC 1.11.1.7) Assay Miranda's method was used to estimate the level of peroxidase activity [14]. The crude extract (0.1 mL) was mixed with 40 mM guaiacol (extinction coefficient, ξ= 26.6 mM -1 cm -1 ), 8 mM H 2 O 2 , 50 mM acetate buffer (pH 5.5) for a total 1 mL. The level of absorbance of the mixture was recorded at 470 nm every 1 min using a spectrophotometer.

Polyphenol Oxidase Assay
The polyphenol oxidase activity was measured with catechol (extinction coefficient, ξ=2.2 x 10 3 mM -1 cm -1 ), as the substrate, as stated by the method used by Siddiq and Cash [15]. The reaction contained 0.9 mL of 20 mM catechol reagent prepared in 10 mM phosphate buffer (pH 6.8) and 0.1 mL crude extract. The absorbance of the mixture, at 420 nm, is recorded for 3 min.

Catalase Assay
The activity of catalase enzymes was detected using the method in Ref [16]. 2 ml of substrate were made by mixing 25 mM H 2 O 2 (extinction coefficient, ξ= 43.6 mM-1 cm -1 ), in a 75 mM phosphate buffer (pH 7.0) with 0.5 mL crude extract. The absorbance at 240 nm, was registered for 1 min.

Preparation of Plant Extracts
10 gr each of dried verdolaga leaves, root strips, and stem strips were extracted by combining with different concentrations of organic solvent (80%, 60%, 40% methanol, 80%, 60%, 40% ethanol, and, 80%, 60%, 40% acetone) and 30 mL of deionized water, then shaken at 120 rpm for 24 h. The mixtures were poured through filter paper. The extracts were placed in a cooler at 4°C until they were used in further biochemical assays. The extracts were analyzed in triplicate.

Determination of Total Phenolic Content
The phenolic contents were measured using FCR by the method explained by Velioglu [17]. Distilled water (800 μL) and 100 μL FCR were mixed with 100 μL of the plant extract for 5 min at ambient temperature. Then 500 μL of 20% sodium carbonate was added to the reaction mixture. After 30 min the absorbance was recorded at 750 nm. Gallic acid was used as the standard phenolic compound. The results were expressed as an equivalent mg gallic acid/g dry matter (mg GAE/g DM).

Total Flavonoid Content
The flavonoid contents of plant extracts were detected using the method described by Zhishen [18] with a slight modification: a solution of catechin was used as the standard. The reaction mixture was produced by mixing 250 μL plant extract, 1.25 mL distilled water and 75 μL NaNO 2 solution (5%) and permitted to stand for 6 min. Then 150 μL of AlCl 3 solution (10%), 0.5 mL of 1M NaOH and 275 μL of distilled water was added to the reaction mixture, and permitted to stand for 5 min. After that, the absorbance of the solutions was recorded at 510 nm. The results were calculated as mg catechin equivalent/g dry matter (mg CE /g DM).

Total Condensed Tannin Contents
The tannin contents of each portion of the verdolaga were measured using the methods in [19] with a slight modification, in which catechin was used as standard. The reaction mixture was prepared by mixing 400 µL of plant extract, 3 mL vanillin solution (4% in methanol) and 1.5 mL concentrated hydrochloric acid. The absorbance was recorded at 500 nm after 15 min of incubation at ambient temperature. The tannin contents recorded with the units of µg CE /g DM.

Determination of Carotenoids, Chlorophyll, and Lycopene
The presence of chlorophyll, carotenoids, and lycopene were confirmed by the method that was described by Wang [20]. One gram of each part of the fresh plant was cut into small pieces, mixed well and ground with 10 mL acetone and hexane (40:60). The organic supernatant was transferred into a capped tube that was placed on ice. The remaining aqueous layer was re-extracted with 10 mL of the same solvent and the organic layer was transferred to the same tube, and this process was repeated until the aqueous layer became colorless. One milliliter from the total volume of the organic extract was utilized to determine the absorbance at 450, 502, 645, and 663 nm. The following formulae were used to calculate the concentrations of carotenoids, lycopene, and chlorophylls: The free radical scavenging activities (FRSAs) of crude methanol extracts were detected using the DPPH radical scavenging assay [21]. Each methanol extract (100 µL) was added to 900 µL of 0.1 mM freshly prepared DPPH reagent and placed in the dark at 30℃ for 30 min. The absorbance was recorded at 517 nm. The scavenging activity (%) was determined by the following equation: The results were plotted as the % of scavenging activity versus the sample concentration. The IC 50 value, which was the concentration that was required to produce 50% FRSA, was interpolated from the plots.

ABTS Radical Cation Decolorization Assay
Re's method was used to estimate the capacity of the extracts to scavenge ABTS radicals (ABTS•+) [22]. The ABTS•+ solution was diluted in 0.1 M sodium phosphate buffer (pH 7.4) to provide an absorbance of 0.750 at 734 nm, then 1 mL of the diluted ABTS •+ solution was added to 0.5 mL crude methanol extract. The absorbance was recorded at 1 min after mixing, and the percentage of radical scavenging was relative to a blank that containing no scavengers. The scavenging activity of test compounds was determined using the following equation:

Reducing Power Assay
To evaluate the reducing power of the extracts, we used Oyaizu's method [23] with a slight modification: One mL of reaction mixture was made from combining the various concentration of 80% methanol extract, 250 µL of 0.2 M phosphate buffer (pH 6.6) and 250 µL of 1% potassium ferricyanide. The mixture was incubated at 50ºC for 20 min. After, 250 µL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 3000 rpm for 10 min. The supernatant (0.5 mL) was mixed with 0.5 mL distilled water and 0.1 mL of 0.1% ferric chloride, and the absorbance was immediately recorded at 700 nm. The concentration of extract that would produce 0.5 of absorbance (EC 50 ) was interpolated from the diagram of absorbance at 700 nm.

Hydrogen Peroxide Scavenging Activity
Ruch's method was used to evaluate the H 2 O 2 scavenging activities of the methanol extracts [24]. A solution of H 2 O 2 (2 mM) was mixed with 50 mM sodium phosphate buffer (pH 7.4). The concentration of H 2 O 2 was calculated using the molar extinction coefficient of H 2 O 2 (81 mol -1 cm -1 ). The 1 mL reaction mixture contained (0.5-2.5 µg/mL methanol extracts, and the volume was made up of 0.4 mL of 50 mM phosphate buffer (pH 7.4), and then 0.6 mL H 2 O 2 was added). The reaction mixture was vortexed and its absorbance was recorded at 230 nm over 10 min against a blank solution containing 50 mM phosphate buffer without H 2 O 2 . Gallic acid (0.5-2.5 µg/mL), was used as a positive control. The scavenging activity was calculated using the following equation:

Gas Chromatography-Mass Spectrometer (GC-MS) Analysis
The methanol extracts of the verdolaga were analyzed using a TRACE GC ultra-system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a 30 m X 0.25 mm X 0.25 μm Elite-5-MS capillary column (Thermo Fisher Scientific). The column temperature was increased from 40ºC to 220ºC at a rate of 4ºC/min. The injector temperature was 250ºC; injection volume, 1 μL; helium carrier gas flow rate was 20 mL/min; transfer temperature was 280ºC. MS parameters were as follows: EI mode, with an ionization voltage of 70 eV, an ion source temperature of 180ºC, and a scan range of 50-600 Da. The peaks were tentatively identified based on a library search using NIST and Wiley Registry 8 Edition.

Statistical Analysis
Data were analyzed by a one-way ANOVA and the Student's t-test. The results were expressed as mean ± SD. The results were significant when P < 0.05.  IJN CC-license DOI: 10.14302/issn.2379-7835.ijn-19-3144 Vol-4 Issue 4 Pg. no.-40

Results and Discussion
The capacity for antioxidant activity in plant tissues is related to the level of cell-reinforcing substances that are present, including phenolic compounds, carotenoids, tocopherol, ascorbic acid, and compounds that are able to catalyze the scavenging of free radicals (e.g., catalase, polyphenol oxidase, and peroxidase) [25]. Furthermore, phenolics and anthocyanins, as cancer prevention agents, are associated with oxidative reactions. Catalase, peroxidase, and polyphenol oxidase have been identified in verdolaga extracts ( Chinese prescriptions [28]. The partial characterization of the activity of polyphenol oxidase in the herb Thymus longicaulis subsp. chaubardii var has been described [29]. In order to extract phenolic and flavonoid compounds, the most ideal solvent must be selected. Table 2 shows the ability of different solvents in extracting phenolic and flavonoid substances from various parts of verdolaga. After the extraction process, methanol (40, 60, 80%), ethanol (40, 60, 80%), acetone (40, 60, 80%) and water were examined to assess for the most ideal solvent to be utilized in this investigation. Among four solvents, the methanol (80%) yielded the highest concentration of elevated absolute phenolic compounds (15.7, 12.8 and 9.1mg GAE/g DM for leaves, stem strips, and root strips, respectively).
Flavonoid contents were also of higher concentration in 80% methanol (5.5, 4.1 and 2.6 CE/g DM for Leaves, stem strips, and root strips, respectively). Methanol was the most appropriate solvent to extract polyphenolic compounds from plant tissues, because of its capacity to repress the activity of polyphenol oxidase, which is an enzyme that causes the oxidation of polyphenols and its ease of dissipation, in contrast to water [9]. Methanol extracts have been utilized in the investigation of antioxidant activities and flavonoids compounds in the wood pulp and pericarp of Caesalpinia decapetala [30] and Lantana camara [31]. Tannins are phenolic plant secondary compounds and are present throughout the plant kingdom. They exist in structures called condensed tannins (CTs) [32]. Jones and Mangan wrote that CTs can attach to proteins at close proximities (pH 3.5-7.5) to form CT-protein structures, which separate at a pH of 3.5 [33].  Values are presented as means ± SD (n=3)  Table 4. The antioxidant effects of gallic acid equivalents in Verdolagaon the reduction of DPPH, ABTS radicals, and reducing power.
IC 50 is the inhibition concentration, which is the concentration that is required to produce 50% free radical scavenging activity. EC 50 is the efficient concentration, which is the concentration of extract that would produce 0.5 units of absorbance. R 2 is the correlation coefficient between the phenolic contents and the DPPH scavenging activities, ABTS scavenging activities, and reducing power. and stems of plants while in a few studies, CTs have been found only in the petals of flowers, including white and red clover [34]. The concentration of CTs in P. oleracea are shown in Table 3. The highest concentration of CTs identified in stem strips (533.9 μg CE/g DM) compared to leaves and root strips (497.8 and 368 μg CE/g DM, respectively). Methanol has been shown to be excellent in extracting CT, demonstrated by a process involving Limonium delicatulum (48.38 mg/g DM) [35]. Chlorophyll enables the conversion of light energy into plant substance. Chlorophyll, lycopene, and other carotenoids were found in fresh leaves, stem strips, and root strips (Table 3). Chlorophyll (531.78 μg/g DM), total carotenoids (271.9 μg/g DM), and lycopene (28.63 μg/g DM) were higher in leaves (P < 0.05) than in stem strips and root strips. Singlet oxygen quenching via carotenoids occurs through physical or chemical quenching which has been discussed in a few studies [36,37]. The viability of physical quenching surpasses that of chemical quenching and includes the exchange of electrons from 1 O 2 to the carotenoid, leading to the formation of a ground-state oxygen and an energized, triplet-state carotenoid. Carotenoids are situated in chromoplasts, as they color vegetables and other organic products. Along with chlorophyll, they are engaged in two photosystems.
Vechetel and Ruppel [38] detailed that carotenes provided the most critical photosynthetic colors, and they protected chlorophyll and thylakoid films from peroxidation. The scavenging of a stable DPPH radical is a widely used method to estimate antioxidant activity [39]. The phenolic substance in three segments of verdolaga demonstrated a low level of scavenging of the DPPH radical (Table 4 and Fig. 1a [40]. ABTS is a basic and frequently utilized technique to assess the activity of cancer prevention agents [41,42]. The phenolic substances in verdolaga demonstrated their reliance on the convergence of the ABTS radical, which might contribute to its reducing capacity (Table 4). Each of these concentrates presented a linear variety of inhibition power with the additional concentration of extract (Fig. 2a, b, and c in the Supplementary Material). The IC 50 value for leaves, stem strips, and root strips were found to be 2.86, 3.70 and 5.24 μg GAE/mL, respectively. Compounds with reducing power demonstrate that they are electron providers and can reduce the oxidized intermediates of lipid peroxidation products. Thus, they are essential and are auxiliary antioxidants [43]. It was evident that methanol became more powerful in extracting compounds from verdolaga ( Fig.3a, Figure 1 and