International Journal of Nutrition
ISSN: 2379-7835
Current Issue
Volume No: 4 Issue No: 4
share this page

Research Article | Open Access
  • Available online freely | Peer Reviewed
  • In Vitro Assessment of Antioxidant Enzymes, Phenolic Contents and Antioxidant Capacity of the Verdolaga (Portulacaceae)

    Yaaser Q. Almulaiky 1 2       Musab Aldhahri 3 4     Fahad A. Al-abbasi 3     Sami A. Al-Harbi 5     Mohamed H. Shiboob 6    

    1Chemistry Department, Faculty of Sciences and Arts, University of Jeddah, Khulais, P.O. Box 355, Khulais, 21921, Saudi Arabia

    2Chemistry Department, Faculty of Applied Science, Taiz University, Taiz, Yemen

    3Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia

    4Center of Nanotechnology, King Abdulaziz University, Jeddah, Saudi Arabia

    5Department of Chemistry, University College in Al-Jamoum, Umm Al-Qura University, Makkah, Saudi Arabia

    6Environmental Department, Faculty of Meteorology, Environment and Arid Land Agriculture King Abdulaziz University, Jeddah, Saudi Arabia

    Abstract

    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.

    Received 24 Dec 2019; Accepted 02 Jan 2020; Published 06 Jan 2020;

    Academic Editor:Ishan Wadi, National Institute of Malaria Research, India.

    Checked for plagiarism: Yes

    Review by:Single-blind

    Copyright©  2020 Yaaser Q. Almulaiky, et al.

    License
    Creative Commons License    This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

    Competing interests

    The authors have declared that no competing interest exists.

    Citation:

    Yaaser Q. Almulaiky, Musab Aldhahri, Fahad A. Al-abbasi, Sami A. Al-Harbi, Mohamed H. Shiboob (2020) In Vitro Assessment of Antioxidant Enzymes, Phenolic Contents and Antioxidant Capacity of the Verdolaga (Portulacaceae). International Journal of Nutrition - 4(4):36-47.
    Download as RIS, BibTeX, Text (Include abstract )
    DOI10.14302/issn.2379-7835.ijn-19-3144

    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, 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.

    Materials and Methods

    Chemicals

    2,2’-Diphenyl-1-picrylhydrazyl (DPPH), gallic acid, Folin-Ciocalteu reagent (FCR), and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 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 H2O2, 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 103 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 H2O2 (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.

    Measurements of Other Contents

    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 NaNO2 solution (5%) and permitted to stand for 6 min. Then 150 μL of AlCl3 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:







    Antioxidant Assays

    DPPH Radical Scavenging Activity

    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 IC50 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 (EC50) was interpolated from the diagram of absorbance at 700 nm.

    Hydrogen Peroxide Scavenging Activity

    Ruch’s method was used to evaluate the H2O2 scavenging activities of the methanol extracts 24. A solution of H2O2 (2 mM) was mixed with 50 mM sodium phosphate buffer (pH 7.4). The concentration of H2O2 was calculated using the molar extinction coefficient of H2O2 (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 H2O2 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 H2O2. 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.

    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 (Table 1). In this study, the peroxidase enzyme activity was high in verdolaga root strips, stem strips and leaves (225 ± 0.124, 49 ± 0.86 and 71.5 ± 0.101 units per gram of tissue, respectively). The action of polyphenol oxidase, one of the terminal oxidases in the plant cell, was improved under unfavorable conditions. This enzyme, alongside peroxidase, engaged in the oxidation of phenolic components, which are cancer prevention agents that favor cell resistance 26. Therefore, a measurement of the concentration of oxidizing phenols could be representative of cell’s vulnerability. In the present investigation, polyphenol oxidase was highly active in the verdolaga’s root strips, stem strips, and leaves (132 ± 0.79, 83.2 ± 0.43 and 61.7 ± 0.56 units per g tissue, respectively). Catalase activity was present in the verdolaga’s leaves, root strips, and stem strips as well (60.25 ± 0.25, 23±0.22 and 15.13 ± 0.64 units per g tissue, respectively). These enzymes have been examined in other plants, including the peroxidase from the latex of Euphorbia cotinifolia27. The activity of catalase and antioxidant capacity were screened in nine medicinal plants that are customarily provided in Chinese prescriptions 28. The partial characterization of the activity of polyphenol oxidase in the herb Thymus longicaulis subsp. chaubardii var has been described 29.

    Table 1. The antioxidant enzyme activities of Verdolagasamples.
    portulaca oleracea Units of Peroxidase/g Tissue Units of Polyphenol Oxidase/g Tissue Units of Catalase/g Tissue
    Leaves 71.5 ± 0.101 61.7 ± 0.56 60.25 ± 0.25
    Stem strips 49 ± 0.86 83.2 ± 0.43 15.13 ± 0.64
    Root strips 225 ± 0.124 132 ± 0.79 23 ± 0.22

    Values are presented as means± SE (n=3)

    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 Caesalpiniadecapetala30 and Lantana camara31. 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. In most cases CTs are available in the leaves 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 1O2 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 Figure 1a, b, and c in the Supplementary Material). The IC50 of methanol extraction products from leaves, stem strips, and root strips were 25.26, 20.56 and 32.10 μg GAE/mL, respectively. The correlation coefficient (R2) between the phenolic contents and DPPH scavenging activities for leaves, and stem strips and root strips were 0.952, 0.965, and 0.966, respectively, which indicated a strong correlation. The IC50 values measured for Yemeni guava segments ranged from 19 to 22 μg /mL 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 (Figure 2a, b, and c in the Supplementary Material). The IC50 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 (Figure 3a, b, and c in the Supplementary Material). The presence of reducers nearby causes the transformation of the Fe3+/ferricyanide complex to become the ferrous structure. The development of Perl's Prussian blue was recognized at 700 nm and shows a higher reducing power. The reducing power of root strips was observed to be higher than that of stem strips and leaves (EC50 21.69, 16.39, and 15.70 μg GAE, respectively) (Table 4). The R2 between the phenolic group for the leaves, strips of stems and roots, and the formation of the ferrous complexes were observed to be 0.969, 0.969, and 0963, respectively, demonstrating a strong relationship. It was noted that the reducing properties were because of the proximity of reductones. Reductones are able to act as a cancer prevention agent by breaking the free radical chain, by providing a hydrogen molecule 44. The EC50 values for the medicinal plant, Coleus forskohlii, was found to be 96.15 and 14.6 μg phenolic concentrations/mL for its stem and leaves, respectively 45. The scavenging capacities of methanol extraction products from verdolaga on H2O2 are in Figure 1 and Table 5. They are compared to gallic acid. The verdolaga extracts were intense in scavenging H2O2 in a sum subordinate way. To exhibit 50% scavenging activity on H2O2, 1.717, 2.937, and 3.255 μg gallic acid equivalent was required to equal the strength of leaves, stem strips, and root strips, respectively. On the other hand, the elimination of H2O2 was 50% with 1.296 μg gallic acid. Because H2O2 has no unpaired electrons, it does not qualify as a radical. However, it tends to be lethal to a cell because it might deliver hydroxyl radicals to the cells 46. Therefore, the disposal of H2O2 is beneficial for the antioxidant defense system in the cell. The GC-MS chromatograms for strips of stem, root, and leaves are shown in Figure 2. The analytes from stem strips, root strips, and leaves, their maintenance time, and peak area (%) are shown in Table 6. The greater part of component derivatives contained hydroxyl groups, which could possess antioxidant potential.

    Table 2. Solvent effect on phenolic contents and flavonoids of Verdolaga
    Solvent phenolic contents GAE/g DM Flavonoids CE/ g DM
      Leaves Stem strips Root strips Leaves Stem strips Root strips
    Methanol 80% 15.7±1.05 12.8±0.88 9.1±0.53 5.5±0.22 4.1±0.75 2.6±0.64
    Methanol 60% 13.5±1.11 10.6±0.54 8.4±0.64 3.5±0.16 3.3±0.43 2.3±0.34
    Methanol 40% 10.2 ± 1.02 8.7 ± 0.37 7.3 ± 0.78 2.9 ± 0.26 2.8 ± 0.23 2.5 ± 0.72
    Ethanol 80% 12.3 ± 1.04 10.5 ± 0.21 8.2 ± 0.45 3.2 ± 0.24 3.2 ± 0.11 2.9 ± 0.24
    Ethanol 60% 10.1 ± 1.05 8.9 ± 0.85 7.4 ± 0.32 2.5 ± 0.15 2.3 ± 0.16 1.9 ± 0.26
    Ethanol 40% 7.8 ± 0.75 6.9 ± 0.71 6.6 ± 0.12 1.3 ± 0.19 1.8 ± 0.10 1.6 ± 0.28
    Acetone 80% 13 ± 0.82 11.1 ± 0.92 8.9 ± 0.12 4.4 ± 0.49 3.1 ± 0.14 3.4 ± 0.23
    Acetone 60% 12.2 ± 0.53 9.0 ± 0.11 8.1 ± 0.015 3.8 ± 0.73 2.6 ± 0.19 2.8 ± 0.63
    Acetone 40% 10.6 ± 0.77 7.4 ± 0.78 7.3 ± 0.013 2.6 ± 0.34 2.3 ± 0.16 2.1 ± 0.38
    water 9.5 ± 0.014 7.8 ± 0.026 5.4 ± 0.01 1.8 ± 0.52 1.5 ± 0.13 2.5 ± 0.43

    GAE, gallic acid equivalent, CE, catechin equivalent. Values are presented as means± SE (n=3)
    Table 3. The total concentration of antioxidant in Verdolagasamples
      Leaves Stem strips Root strips
    Chlorophyll (μg/g DM) 531.78 ± 0.69 83.75 ± 0.35 2.75 ± 0.10
    Lycopene (μg/ g DM) 28.63 ± 0.03 4.84 ± 0.05 3.32 ± 0.13
    Total carotenoids (μg/ g DM) 271.9 ± 0.41 48.8 ± 0.26 9.41 ± 0.17
    Tannin contents (μg CE/g DM) 497.8 ± 0.34 533.9 ± 0.39 368 ± 0.12

    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.
    portulaca oleracea DPPH ABTS reducing power
      IC50 (μg GAE) R2 IC50 (μg GAE) R2 EC50 (μg GAE) R2
    Leaves 25.26 0.952 2.86 0.938 15.70 0.969
    Stem strips 20.56 0.965 3.70 0.994 16.39 0.969
    Root strips 32.10 0.966 5.24 0.974 21.69 0.963

    IC50 is the inhibition concentration, which is the concentration that is required to produce 50% free radical scavenging activity. EC50 is the efficient concentration, which is the concentration of extract that would produce 0.5 units of absorbance. R2 is the correlation coefficient between the phenolic contents and the DPPH scavenging activities, ABTS scavenging activities, and reducing power.
    Table 5. The antioxidant effect of the gallic acid equivalent of Verdolagaon Hydrogen peroxide hydrolysis scavenging.
    portulaca oleracea H2O2 hydrolysis scavenging %
    IC50 (μg GAE) R2
    Leaves 1.717 0.993
    Stem strips 2.937 0.973
    Root strips 3.255 0.969
    Gallic acid 1.296 0.992

    IC50 is the inhibition concentration as μg gallic acid equivalent of the test sample that eliminate 50% of hydrogen peroxide.
    Table 6. GC-MS analysis of compounds in the methanol extract from strips of verdolaga
      Compound Name Retention Time (min.) Peak area (%)
    Stem 9,12,15-Octadecatrienoic acid, 2-((trimethylsilyl)oxy)-1-(((trimethylsilyl)oxy) methyl)ethyl ester, (Z,Z,Z)- 4.26 0.01
    2,5-Octadecadiynoic acid, methyl ester 4.40 0.0
    Gibberellic acid 5.28 0.0
    Hexanoic acid, 3,5,5-trimethyl, 1,2,3-propanetriyl ester 14.71 0.37
    Cyclopropanedodecanoic acid, 2-octyl, methyl ester 8.70 0.00
    Cholestan-3-ol, 2-methylene, (3á,5à) 30.25 0.09
    Bisdi(trimethylsiloxy)phenylsiloxytrimethylsil oxyphenylsiloxane 39.38 0.13
    D-Homo-24-nor-17-oxachola 1,20,22-triene 3,7,16-dione, 14,15:21, 23-diepoxy4,4,8trimethyl, (5à,13à,14á,15á,17aà) 47.17 0.57
    Root 2,5-Octadecadiynoic acid, methyl ester 4.22 0.01
    Cyclotetrasiloxane, octamethy- 5.26 0.01
    9,10-Secocholesta 5,7,10 (19) triene1,3diol, 25 (( trimethylsilyl)oxy), (3á,5Z,7E) 7.17 0.01
    2,5-Octadecadiynoic acid, methyl ester 5.73 0.00
    Eucalyptol 6.01 0.03
    Hexadecanoic acid, 2-hydroxy1(hydroxymethyl) ethyl ester 42.92 0.64
    Leaves Pterin 6-carboxylic acid 5.01 0.00
    1-Monolinoleoylglycerol trimethylsilyl ether 5.33 0.02
    9,10-Secocholesta 5,7,10 (19) triene1,3diol, 25 (( trimethylsilyl)oxy), (3á,5Z,7E) 7.25 0.00
    Cyclopropanedodecanoic acid, 2-octyl, Methyl ester 7.39 0.01
    3,6,9,12-Tetraoxatetradecan 1ol, 14 (4 (1,1,3,3-tetramethylbutyl)phenoxy) 9.18 0.01
    Cholest-22-ene-21-ol, 3,5-dehydro6methoxy, pivalate 47.95 0.02

    Figure 1. Hydrogen peroxide scavenging activity of leaves, strips of roots and stems. All experiments were carried out in triplicates and values are presented as mean ± SE.
    Figure 1.

    Figure 2. GC-MS chromatograms of leaves (a), stem strips (b), and root strips (c) crude methanol extract.
    Figure 2.

    Conclusion

    This study revealed that Verdolaga contained a high concentration of phenolic, flavonoids, CTs, and compounds with powerful antioxidant activities. This plant contained a high concentration of free radical scavengers, which are useful in postponing the aging process, the development of malignancy, and the progression of other pathophysiological illnesses. This paper reveals the enzymes in Verdolaga responsible for combating and avoiding diseases. The GC-MS investigation of the methanol extracts of verdolaga enabled the quantification of cell-protecting agents. The applications of this investigation support the development of nutritional supplements and nutraceutical products using verdolaga extracts.

    Acknowledgements

    We would like to acknowledge the Department of Biochemistry at the University of Jeddah for its support.

    Supplementary Figure

    References

    1.Yoon A, Hughley T, R H Williams. (2019) . , The FASEB 33, 440.
    2.Anelise S, Carla R, Matheus S, Claudia A, Maria C et al. (2014) . , Antioxidants 4, 745.
    3.Guerrero B, M G Granda-Albuja, Guevara M, G A Iturralde, Jaramillo-Vivanco T et al.. Alvarez-Suarez J.M. (2019).Nat. prod. Res 1.
    4.A E Hayouni, Abedrabba M, Bouix M, M.. Hamdi,Food Chem.2007 3-1126.
    5.Dorman H, S G Deans, J.. , App. Micro.2000,2,308
    6.J M Vieira, R A Mantovani, M F Raposo, M A Coimbra, A et al. (2019) . , Cunha, Carbo. ploy,213: 1217.
    7.A H Goli, Barzegar M, M A Sahari. (2005) . , Food Chem 3, 521.
    8.Zuo Y, Chen H, Deng Y. (2002) . , Talanta 2, 307.
    9.Yao L, Jiang Y, Datta N, Singanusong R, Liu X et al. (2004) . , Food Chem 2, 253.
    10.S C Opie, Robertson A, M N Clifford, Agric J.. Food Chem.1990,50,561
    11.M H, Ahmad B, S R Mir, B A Zargar, N et al. (2011) . , Res 9, 3044.
    12.Xiang L, Xing D, Wang W, Wang R, Ding Y et al. (2005) . , Phytochemistry 21, 2595.
    13.Liu L, Howe P, Y F Zhou, Xu Z-O, Hocart C et al. (2000) . , Chromatogr 1, 207.
    14.M V, Lahor H M Fernandez, Cascone O. (1995) . , App. Biochem. Biotechnol 2, 147.
    15.Siddiq M, J N Cash, J. (2000) . Food Proc. Preserv 24, 353.
    16.Aebi H. (1974) . In Methods of Enzymatic Analysis (Second Edition) 2-673.
    17.Y S Velioglu, Mazza G, Gao L, B D Oomah, Agric J. (1998) Food Chem. 10-4113.
    18.Zhishen T Mengcheng J.. W.Jianming(1999).Food Chem,4 555.
    19.R B Broadhurst, W T jones, Sci J. (1978) . , Food Agric 3, 788.
    20.Z F Wang, T J Ying, B L, X D Huang, J. (2005) . , Zhejiang Univ. Sc 6, 502.
    21.Ao C, Li A, Elzaawely A A, Xuan T D, Tawata S. (2008) . , Food control 10, 940.
    22.Re R, Pellegrini N, Proteggente A, Pannala A, Yang M et al. (1999) . , Free Radical Bio. Med 10, 1231.
    23.Oyaizu M. (1986) . , Jap. J. Nutr. Diet 6, 307.
    24.R J, S J Cheng, E J. (1989) . , Klaunig, Carcinogenesis 6, 1003.
    25.Bartosz G. (1997) . , Acta Physiologiae Plantarum 1, 47.
    26.M G Polovnikova, O L Voskresenskaya. (2008) . , Russ. J. Plant Phys 5, 699.
    27.Kumar R, K A Singh, V K Singh, M V Jagannadham. (2011) . , Process Biochem 6, 1357.
    28.S E Lee, H J, J S Ha, H S Jeong, J H Kim. (2003) . , Life Science 2, 167.
    29.Dogan S, Dogan M. (2004) Food chem. , Mar 1, 69.
    30.C R Pawar, S J, J.. Young pharm.2010,1, 45
    31.Mahdi-Pour B, S L Jothy, L Y, Chen Y, Sasidharan S. (2012) . , Asian Pac J Trop Biomed 12, 960.
    32.Haslam E. (1989) Plant Polyphenols. Vegetable Tannins Revisited. , Cambridge, UK 154.
    33.W T Jones, J L Mangan. (1977) . , J. Sci. Food Agric 2, 126.
    34.T N Barry. (1985) . , British J Nutr 1, 211.
    35.Medini F, Fellah H, Ksouri R, Abdelly C, J. (2014) . , Taibah Univ. sci 3, 216.
    36.G W Burton, U K. (1984) . , Ingold, Science 224, 569.
    37.Sies H, Stahl W. (1995) . , Am. J. Clinic Nutr 6, 1315.
    38.B W Vechetel, H G Ruppel. (1992) . , Plant Cell Phys 1, 41.
    39.A Von Gadow, Joubert E, C F Hansmann. (1997) . , Food chem 1, 73.
    40.Almulaiky Y, Zeyadi M, Saleh R, Baothman O, Al-shawafi W et al. (2018) . , Agric. Biotechnol 16, 90.
    41.Y Z Cai, Luo Q, Sun M, Corke H. (2004) . , Life Science 17, 2157.
    42.R Y Gan, Kuang L, X R, Zhang Y, E Q Xia et al. (2010) . , Molecules 9, 5988.
    43.Chanda S, Dave R. (2009) . , African J. Micro. Res 13, 981.
    44.Shimada K, Fujikawa K, Yahara K, Nakamura T, Agric J. (1992) . , Food Chem 6, 945.
    45.Y Q Almulaiky, Kuerban A, Aqlan F, S A Alzahrani, M N Baeshen et al. (2017) . , J. Pharm. Res. International 3, 1.
    46.Halliwell B. (1991) . , Am. J. Med 3, 14.