1216-1221The seagrass Cymodocea serrulata was collected from Ramanathapuram coastal region and its antioxidant potential was determined. The ethanol extract showed the highest phenolic content of 284.94 mg/ml gallic acid equivalence and the ethyl acetate extract showed the highest flavonoids content of 40.18 mg/ml quercetin equivalence. The tannin content was higher at 264.71 mg/ml tannic acid equivalence in aqueous extract. The ethanol extract exhibited the highest 2,2 diphenyl-1-picrylhydrazyl (DPPH) and 2,2¢-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity with IC50 values of 44.47 µg and 2.5 µg, respectively. The ferric reducing ability and nitric oxide scavenging activity were efficient in both ethanol and aqueous extracts. The superoxide scavenging activity was high in hexane extract. The comparative antioxidant study of the subsequent extract of C. serrulata showed that the ethanol extract possesses the highest free radical scavenging property compared to other extracts. This may be due to the presence of high phenolic compounds. The study brings out the medicinal value of C. serrulata which can be used as a nutraceutical compound in various food and pharmaceutical industries

Amudha, P.

Bharathi, N. Pushpa

Jayalakshmi, M.

Vanitha, V.

English

NOPR

Indian Journal of Geo Marine Sciences Vol. 48 (08), August 2019, pp. 1216-1221       Phytochemical screening and in vitro antioxidant activity of the seagrass Cymodocea serrulata N. Pushpa Bharathi, M. Jayalakshmi, P. Amudha, & V. Vanitha* Department of Biochemistry, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai, Tamil Nadu, India *[E-mail: pushpa3_10@rediffmail.com] The seagrass Cymodocea serrulata was collected from Ramanathapuram coastal region and its antioxidant potential was determined. The ethanol extract showed the highest phenolic content of 284.94 mg/ml gallic acid equivalence and the ethyl acetate extract showed the highest flavonoids content of 40.18 mg/ml quercetin equivalence. The tannin content was higher at 264.71 mg/ml tannic acid equivalence in aqueous extract. The ethanol extract exhibited the highest 2,2 diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity with IC50 values of 44.47 µg and 2.5 µg, respectively. The ferric reducing ability and nitric oxide scavenging activity were efficient in both ethanol and aqueous extracts. The superoxide scavenging activity was high in hexane extract. The comparative antioxidant study of the subsequent extract of C. serrulata showed that the ethanol extract possesses the highest free radical scavenging property compared to other extracts. This may be due to the presence of high phenolic compounds. The study brings out the medicinal value of C. serrulata which can be used as a nutraceutical compound in various food and pharmaceutical industries. [Keywords: Cymodocea serrulata; Antioxidant activity; DPPH assay; Phytochemical analysis; Seagrass; ABTS assay; Phenols; Flavonoids; Tannin] Introduction With an increase in the lack of disease resistance and adverse effects of synthetic drugs, it has become essential to explore our traditional use of medicine and search for a natural compound that can act as a drug or supportive agent to treat various diseases. Thus, the search for a novel, potent natural compound from marine angiosperm is best suited for this purpose. The marine angiosperms are hydrophytes that grow and complete their lifecycle in a completely submerged saline environment1. Since their habitat is in the stressed environment, they can produce structurally diverse secondary metabolites, such as alkaloids, phenols, flavonoids, terpenoids, tannins, steroids, and so on. These secondary metabolites were proved to serve as medicine and supplement against various human diseases. Compared to algae, seagrasses remain less exploited despite the fact that they offer tremendous opportunities to find new commercially valuable phytochemicals2. In this study, the seagrass Cymodocea serrulata has been chosen and justified for traditional use as medicine as it possesses antioxidant potential.  Materials and Methods Fresh seagrass C. serrulata was collected in June from the coastal waters of Thiruppalaikudi, Ramanathapuram district. C. serrulata was identified and authenticated by CMFRI (ICAR, Govt. of India). The whole seagrass C. serrulata was washed thoroughly and shade-dried at room temperature.  The dried seagrass was then powdered using a mechanical grinder. About 150 g of the powder was soaked in 1:2 ratio of five different solvents successively in increasing order of polarity, namely, hexane, chloroform, ethyl acetate, ethanol, and water, for 15 days (three days for each solvent) at room temperature and shaken mildly. The extracts were collected and the solvent was evaporated by using a vacuum distillation unit. Both dried and powdered forms of the extracts were weighed and subjected to further analysis. The net weight of the subsequent extract was measured. The proximate analysis of the subsequent extracts of C. serrulata was carried out by the standard procedure3,4. The method and inference of phytochemical analysis are given in Table 1.  The total phenol content was determined by the Folin–Ciocalteu’s method and the values obtained were expressed as gallic acid equivalence (GAE) (mg/ml)5. The total flavonoids were measured following Singh et al.6 and the values obtained were expressed as quercetin equivalence (QE) (mg/ml). The tannin content was determined by the Folin–BHARATHI et al.: ANTIOXIDANT ACTIVITY OF SEAGRASSCYMODOCEA SERRULATA   1217Denis method7 and the values obtained were expressed as tannic acid equivalence (TAE) (mg/ml). The protocol of Mensor et al. was followed for the DPPH (2,2 diphenyl-1-picrylhydrazyl) radical scavenging assay8. A solution of 0.135 mM DPPH was prepared in methanol. The 0.5 ml of radical solution was mixed with different concentrations of extracts and also with the standard (5–200 g/ml) and left in the dark at room temperature for 30 min. The absorbance was measured at 518 nm. The 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity was determined by the method of Re et al.9 About 1.9 ml of ABTS solution (7 mM) was added both to the extracts and to the standard of different concentrations (5–200 µg) and mixed well. Then the mixture was allowed to stand at room temperature for 20 min. The absorbance was measured at 734 nm by using a spectrophotometer. Nitric oxide (NO) scavenging activity was determined according to the method reported by Tsai et al.10 The scavenging activity of C. serrulata extracts toward superoxide (SO) anion radicals was measured by the method of Liu et al.11 The absorbance was measured by a spectrophotometer at 560 nm. The free radical scavenging activity of the assays was calculated according to the following equation:   where A0 is the absorbance of the control (blank, without extract) and A1 is the absorbance in the presence of the extracts. The ascorbic acid was kept as standard.  The ferric-reducing ability of the extract was determined by the procedure followed by Benzie  and Strain12. The absorbance measured at 593 nm spectrophotometrically was proportional to the combined ferric-reducing ability and antioxidant property of the extract. The relative activity of the sample was compared with the standard ascorbic acid.  Results and Discussion The preliminary phytochemical analysis of  C. serrulata extracts showed the presence of phytocompounds, such as alkaloids, flavonoids, phenol, quinone, terpenoids, steroids, and glycosides. The results obtained were similar to the report published by Zapata et al.13 Table 2 presents the qualitative analysis of the phytocompounds present in the extracts of C. serrulata. These phytochemicals, which are the integral part of the defense system, may be responsible for the survival of C. serrulata  in the sea. Phenolic compounds are very important  Table 1 — Methods and inference of phytochemical analysis Phytochemicals Method Inference Flavonoids 10% lead acetate Change of white color on adding acid Steroids Con. H2SO4 Ring formation Terpenoids Salkowski method Reddish brown color formation Tannin 0.1% ferric chloride Brownish green or blue–blackcolor formation Saponin Shaking Foam persist for 10 min Alkaloids Meyers test and Wagner test White-colored precipitate Reddish brown color Phenols Ferric chloride test Bluish green color formation Quinine Con. H2So4 Color formation Carbohydrate Fehling’s test Formation of red precipitate Glycosides Keller Killani’s test Lower reddish brown, upper bluish green layer  Table 2 — Phytochemicals present in C. serrulata extracts Compounds S1 Hexane S2  Chloroform S3 Ethyl acetate S4 Ethanol S5 Aqueous Alkaloids + + + Nil Nil Flavonoids + + + + + Phenols + + + + Nil Quinone + + + + + Tannin Nil + + + Nil Carbohydrates Nil Nil Nil + + Steroids Nil + + + Nil Glycosides Nil + + Nil + Terpenoids + Nil Nil + + Saponin Nil Nil Nil Nil +  INDIAN J. MAR. SCI., VOL. 48, NO. 08, AUGUST 2019 1218to plants and have multiple functions. They play a major role in the plant defense system by acting against pathogens and also as an antioxidant thereby preventing cells from damage14.  The total phenol content was higher in the  ethanol extracts (284.94 mg/ml) followed by aqueous (1878.36 mg/ml) and ethyl acetate extracts  (83.89 mg/ml). The lowest phenol content was reported in chloroform (47.58 mg/ml) and hexane extracts (35.21 mg/ml) (Fig. 1).  Flavonoids are naturally occurring biological compounds that can act as potent antioxidants and can prevent cardiovascular disease by preventing the oxidation of LDL. They also reduce the risk of cancer by acting as a natural scavenger of free radicals15. The aqueous extract shows the highest (45.89 mg/ml) flavonoids content followed by the ethyl acetate extract consisting of 40.18 mg/ml of flavonoids content (Fig. 2). Ethanol extract consists of considerable flavonoids content (34.56 mg/ml). The high content of phenols and flavonoids estimated in ethanol extracts of C. serrulata implies the application of this extract not only as an anticancer and anti-inflammatory agent but also as an agent to treat various cardiac ailments16. The amount of phenolic compounds extracted depends on the solvent used. C. serrulata is a plant living in salt water,  and it produces unique phenolic compounds as a phytoprotectant to act against stress. These phenols have the antioxidant property that can scavenge free radicals which are responsible for various dreadful diseases. Kannan et al. observed that the phenols and flavonoids contributed significantly to the antioxidant property of the seagrass17. Nanthakumar et al. reported that C. serrulata methanolic extract exhibits antioxidant property and cytotoxicity activity against the HeLa cells with growth inhibition of 40.47% at 100 µg/ml18.  Tannins are high molecular weight compounds. They usually bind to proteins, lipids, or carbohydrates. In Ayurveda, tannin-rich plant is used to treat various diseases because of its excellent healing property19.19 The determination of tannin shows that the total tannin content was higher in the aqueous extract (264.71 mg/ ml), followed by ethyl acetate extract (115.24 mg/ml), whereas the ethanol extract consists of low amount of tannin (32.55 mg/ml) (Fig. 3). The presence of high amount of tannin shows that C. serrulata can be used both as an antiseptic and as a healing agent. DPPH is a stable free radical that can donate a hydrogen ion to the molecule with its delocalization property and results in a deep violet color20. It is stable at ambient temperature owing to its dimerization capacity. The ethanol extract showed the highest DPPH scavenging activity compared to other extracts. Its activity is almost near to the scavenging activity of standard ascorbic acid (Fig. 4). An IC50 value of ethanol extract (44.47 µg/ml) was near to the Fig. 1 — Total phenolic content of C. serrulata extracts Fig. 2 — Total flavonoids content of C. serrulata extracts Fig. 3 —Total tannin content of C. serrulata extracts BHARATHI et al.: ANTIOXIDANT ACTIVITY OF SEAGRASSCYMODOCEA SERRULATA 1219Fig. 4 — DPPH scavenging activity of C. serrulata extracts Fig. 5 — ABTS scavenging activity of C. serrulata extracts IC50 value of standard ascorbic acid (13.57 µg/ml). This study reports that C. serrulata has a compound that has the ability to donate electrons or hydrogen ions to scavenge DPPH free radicals, thus they can act as a potent antioxidant. When compared to the phytochemicals present in the ethanol extract, they showed a high content of phenol compounds, showing that DPPH scavenging activity is due to the presence of phenol in the ethanol extract of C. serrulata.  ABTS is a radical cation (toxic) reactive oxygen species that can disrupt various cell signaling pathways and lead to cell death21. Antioxidant acts on these radicals and prevents the formation of cation. The maximum ABTS scavenging activity was reported in the ethanol extract which was equivalent to standard ascorbic acid (Fig. 5). This reveals the efficiency of C. serrulata to treat radical cation-related diseases. NO is a potent pleiotropic mediator of various physiological processes, such as smooth muscle Fig. 6 — Nitric oxide scavenging activity of C. serrulata extracts Fig. 7 — Super oxide scavenging activity of C. serrulata extracts relaxant, neuron signaling, inhibition of platelet aggregation, and regulation of cell-mediated toxicity22. The NO radical scavenging activity of  the extracts showed that the aqueous extract of  C. serrulata has higher percentage of inhibition (near to the standard ascorbic acid) (Fig. 6). The ethanol extract has less inhibition activity than the aqueous extract, but higher than hexane, chloroform, and ethyl acetate extracts. C. serrulata extract can act against the inflammation caused by NO species during the action of phagocytosis of the pathogen by leukocytes.SO anion radical is one of the strongest reactive oxygen species among the free radicals that are generated after oxygen is taken into living cells. There was a high SO scavenging activity of hexane extract when compared to the standard (Fig. 7). Other extracts do not show considerable SO scavenging activity and when compared to the phytochemical analysis of the extracts, hexane shows the higher content of tannins, which shows that the tannins present in the hexane extract may be responsible for SO scavenging activity23.INDIAN J. MAR. SCI., VOL. 48, NO. 08, AUGUST 2019 1220Fig. 8 — Percentage curve of FRAP assay by C. serrulata extracts Ferrous ion can initiate lipid peroxidation by the Fenton reaction as well as accelerating peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals. FRAP (Ferric-Reducing Ability of Plasm) assay is based on reducing the colorless ferric–tripyridyl triazine complex to blue color ferrous (II). The color formation is due to the reducing power of the electron-donating antioxidants present in the test solution which can be measured24. In this study, the aqueous extract and ethanol extract showed a better reducing ability (Fig. 8) followed by ethyl acetate and chloroform extracts. The hexane extract showed the lowest reducing ability. The ferrous reducing ability of the subsequent extracts was concentration dependant. Conclusion This study not only demonstrates the phytochemicals present in C. serrulata but also their antioxidant activity by extracting successively, using solvents based on polaritys. When compared with other extracts, the ethanol extract of C. serrulata shows the maximum scavenging activity of free radicals and it has a high content of total phenols and flavonoids. The efficient free radicals scavenging property and reducing property of the ethanol extract of C. serrulata proves that it possesses excellent antioxidant properties, and hence it can be used to treat various free radical-mediated diseases. The antioxidant properties exhibited by C. serrulata justify its traditional medicinal use. 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Phytochemical screening and in vitro antioxidant activity of the seagrass Cymodocea serrulata

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Phytochemical screening and in vitro antioxidant activity of the seagrass Cymodocea serrulata

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