ANTIOXIDANT ACTIVITY OF HALOPHILA OVALIS AND HALOPHILA BECCARII (HYDROCHARITACEAE): TWO IMPORTANT SEAGRASS SPECIES OF CHILIKA LAGOON, INDIA

Objective: The present study was undertaken to evaluate the total phenolic and flavonoid content and the antioxidant property of two important seagrass species namely, Halophila ovalis and Halophila beccarii occurring in Chilika lagoon, Odisha, India. Methods: Total Phenolic Content (TPC) of the extracts of Halophila species was determined by Folin-Ciocalteu method with little modifications and the total flavonoid content (TFC) was measured by aluminum chloride colorimetric assay. The antioxidant activity of different extracts was investigated by DPPH and ABTS radical scavenging activity. IC50 values were calculated for the DPPH and ABTS methods. Result: The study revealed that the methanol extract of H. ovalis has greater antioxidant activity than H. beccarii. Methanol extract of both the species (H. ovalis and H. beccarii) was found to possess high phenolic content at value of 70.25 mg GAE/g of extract and 48.53 mg GAE/g of extract respectively. Similarly flavonoid contents was found highest in methanol extract for both H. ovalis (76.82 mg quercetin equivalent/ g of extract) and H. beccarii (64.28 mg quercetin equivalent/ g of extract). The antioxidant activity of different extracts of these two species were evaluated using DPPH and ABTS radical assay. The methanol extract of both H. ovalis and H. beccarii showed high radical scavenging activity with IC50 values of 37.77 μg/ml and 52.25 μg/ml for DPPH and 25.62 μg/ml and 45.45 μg/ml for ABTS respectively. Conclusion: The study revealed the potential of the Halophila species as natural sources of antioxidants having considerable commercial importance.

Chilika, the largest tropical brackish water lagoon of Asia, is situated in the east coast of India in the state of Odisha. It is designated as a wetland of international importance under the Ramsar Convention and harbors rich flora and fauna characteristic of freshwater, brackish water, and marine habitats. As many as 5 seagrass species, namely Halophila ovalis, H. ovata, Halophila beccarii, Halodule pinifolia, and H. uninervis have been reported to occur in Chilika lagoon and the area under seagrass meadows are increasing after the opening of a new mouth connecting the lagoon to the Bay of Bengal [16]. As in other parts of the world, seagrasses of Chilika lagoon form dense meadows producing considerable biomass, provide excellent habitat for spawning fish and many juvenile marine invertebrates, perform multiple ecosystem services such as recycling of nutrients and stabilization of sediments. Although in a limited scale, the local fishermen communities use the whole plants of Halophila species as feed for buffaloes and goats after repeated washing, as packing materials for crabs and as green manure in their fields after decomposition. The leaf powder mixed with turmeric paste is also applied externally by fishermen for treatment of skin diseases.
In the face, increasing demand for antioxidants of plant origin, the importance of seagrasses as the natural source of antioxidants has increased many-fold. The antioxidant activity of plants might be due to the presence of phenolic compounds, flavonoids, α-tocopherol, and carotenoids [17]. In spite of the biological potential of seagrasses, the antioxidant activity of several seagrass species has not yet been extensively studies [18] for different coastal habits of India. In the present study, the antioxidant potential of two seagrass species occurring in Chilika lagoon such as H. ovalis and Halophila berccarii has been assessed along with measurement of total phenolic and total flavonoid contents (TFC) in different solvent extracts. H. beccarii is a globally threatened species classified under "Vulnerable" category by IUCN, and the antioxidant activity of this species has so far not been evaluated. Although some seagrasses of the west coast and southern coastal regions of India have been investigated in respect of their antioxidant, antimicrobial and anti-inflammatory properties [5,6,8,10,12,14], none of the species occurring in east coast of India which also includes Chilika lagoon, have been assessed for their antioxidant potential.
The present paper reports the antioxidant activities of two important seagrasses (H. ovalis and H. berccarii) of Chilika lagoon, Odisha, India, along with an estimation of total phenolic and TFC in different solvent extracts.

PLANT MATERIALS
Whole plants of H. ovalis (R.Br.) Hook.f. and H. beccarii Asch. belonging to the family Hydrocharitaceae were collected from sandy shores of Chilika lagoon close to Pathara village (19°37′10.1′′N Lat., 85°08′45.9′′E Long.; Alt. 11.6 m) of Ganjam district, Odisha by laying 1 m×1 m sample plots. The species were collected by our team and examined by Dr. P. C. panda and kept at RPRC herbarium having voucher no 10068 for H. ovalis and 10174 for H. beccarii. Plant samples were cleaned by repeated washing in lagoon water, removing clays, sands, and other epiphytic organisms growing on leaf blades. The plant samples were brought to the laboratory in zip lock polypack with the lagoon water to prevent dehydration. The samples then washed thoroughly with tap water to remove sands and debris, if any, leaves were removed manually and dried at room temperature.

Preparation of extract
The collected leaves were thoroughly washed, cut into pieces and dried for 15-20 days under shade. Then, the dried materials were grounded to a coarse powder. For extraction, different solvents such as methanol, n-hexane, and chloroform were added to 100 g of powdered leaves separately and placed in Soxhlet apparatus for 24 h. The extracts were filtered with Whatman 40 filter paper and then concentrated using a rotary evaporator to give rise to a semisolid mass. Each solvent extraction method was repeated thrice for the purpose of accuracy. The residues obtained were stored in refrigerator for further analysis.

Determination of total phenolic content (TPC)
TPC of the extracts of two Halophila species was determined by the Folin-Ciocalteu method [19] using gallic acid as a standard phenolic compound. To record the readings within the standard curve range of 0.0-600 μg of gallic acid/ml, the extracts were diluted with distilled water to a known concentration. In a test tube, 250 μl of gallic acid solution or diluted extract was added to 1 ml of distilled water followed by the addition of 250 μl of Folin-Ciocalteu reagent. To allow complete reaction with the Folin-Ciocalteu reagent, the samples were shaken well for mixing and then allowed to stand at room temperature for 5 min. Then, 2.5 ml of aqueous solution of 7% Na 2 CO 3 was added to the test tube, and the final volume was made up to 6 ml with distilled water. After incubating the samples for 90 min, the absorbance of the subsequent blue colored solution was measured using spectrophotometer at 760 nm. The result was expressed as mg of GAE/g of the extract using an equation that was obtained from the standard gallic acid graph. All the experiments were carried out in triplicates.

Determination of TFC
TFC of different extracts of two Halophila species was assessed by the protocol suggested by Ordon et al. [20] with little modification. This process is based on the establishment of a complex flavonoidaluminum. 1 ml of ethanol solution of 2% AlCl 3 was mixed with 1 ml of extract solution and kept in the dark for 1 h at room temperature. After incubation, the absorbance of the solution was measured at 420 nm using ultraviolet-visible (UV-VIS) spectrophotometer. From the calibration curve of Quercetin, TFC was calculated and expressed in terms of mg quercetin equivalent/g of the extract. All the determinations were performed in triplicates.

1, 1diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity
Different extracts of Halophila species were subjected to DPPH radical scavenging activity, using the method of Blois with little modification [21]. According to this method, 1 ml of extracts with different concentrations was added to 2 ml of 0.1mM of freshly prepared DPPH methanol solution. The vigorously shaken mixture was left to stand for 30 min in the dark at room temperature. Then, the colorimetric changes of the resulting solution, i.e. from deep violet color to light yellow color were measured by a UV/Visible spectrophotometer at 517 nm. In this experiment, ascorbic acid was used as positive control.
The absorbance of the DPPH radical without any samples (the control) was measured. All the observations were carried out in triplicate.
Radical scavenging activity was expressed as percentage inhibition of DPPH radical and was calculated using the following equation-

%Inhibition = [(A control -A test )/A control ]×100
Where, A control is the absorbance of the control and A test is the absorbance of samples.
The antioxidant activity of each sample was expressed in terms of IC 50 value calculated from the graph after plotting inhibition percentage against sample concentration.
ABTS radical scavenging assay ABTS radical scavenging activity of different extracts of Halophila species was determined by following the method of Re et al. [22]. The working solution was prepared by mixing the two stock solutions, i.e. 7 mM ABTS solution and 2.4 mM potassium persulfate solution in equal quantities. Then, it was allowed to react for 12 h at room temperature in the dark. To obtain an absorbance of 0.706±0.001 units at 734 nm using the spectrophotometer, the subsequent solution was then diluted by adding 1 ml of freshly prepared ABTS solution. ABTS solution was freshly prepared for each experiment. 1 ml of different extracts were mixed with 2.5 ml of the ABTS solution and it was allowed to react. Then after 7 min, the absorbance was measured at 734 nm. BHT was used as positive control. The ABTS scavenging capacity of the extract was compared with the positive control and percentage inhibition was calculated as:

"ABTS radical scavenging activity (%) = [(A control -A test )/A control ]×100
Where A control is the absorbance of ABTS radical+methanol; A test is the absorbance of ABTS radical+sample."

Total phenolic and TFC
In the present study, methanolic extract of H. ovalis was found to possess high phenolic (70.25 mg GAE/g of extract) and high flavonoid contents (76.82 mg quercetin equivalent/g of extract) as compared to H. beccarii. The phenolic content of H. ovalis of Chilika lagoon was significantly higher (70.25 mg GAE/g) than that of the same species collected from Mandapam coast, Tamil Nadu, India (0.424 mg GAE/g) as reported by Ragupathi et al. [10]. The TPC of methanol extract of H. beccarii was 48.53 mg GAE/g of extract, and TFC was 64.27 mg quercetin equivalent/g of extract whereas the extract of n-hexane showed the lowest TPC and TFC contents for both the species (Table 1).
TPC was calculated using the standard curve of gallic acid ( Fig. 1) (standard curve equation: Y=0.004x+0.063, R 2 =0.998) and TFC was calculated using the standard curve of Quercetin (Fig. 2) (standard curve equation: Y= 0.013x+0.487, R 2 =0.886). It was reported that plant secondary metabolites such as phenolics and flavonoids are the potent free radical scavengers [23]. Plant antioxidants which include phenolic compounds showed significant free radicals scavenging activity. Thus, the antioxidant potential of a sample can be accredited mostly to its phenolic compounds [24].
The antioxidant activity of different extracts obtained from H. ovalis and H. beccarii was evaluated using DPPH radical assay. Here, the extracts showed substantial DPPH radical inhibiting property at 200 μg/ml concentration ( Table 2). The DPPH radical scavenging activity of both the Halophila species compared with that of standard ascorbic acid was calculated. It was evidenced from the result that In both the species of Halophila, DPPH free radical scavenging capacity of extracts and positive controls was from strongest to weakest:ascorbic acid > methanol extract > chloroform extract > n hexane extract.
In the present study, the extracts of Halophila species were effectively scavenge the ABTS radical. The methanol, chloroform, and n-hexane extracts of both the species exhibited potent scavenging effects against ABTS (Table 3).
At 100 μg/ml concentration, the extracts exhibited higher inhibitory property against ABTS radicals. This assay is centered on the inhibition of the absorbance of radical cation ABTS + , which has a characteristic wavelength at 734 nm, by antioxidants. The principle involves the relationship between ABTS and potassium persulfate to produce the ABTS radical cation (ABTS + ) which is a blue-green chromogen. The colored radical is changed back to colorless ABTS when it reacted with antioxidant reductant [25]. The IC 50 value of the methanol extract of H. ovalis and H. beccarii is 25.62 μg/ml and 45.45 μg/ml, respectively ( Table 4).
The extracts were able to reduce stable free radicals and hence can be correlated with the higher phenolics and flavonoids contents that are known to be potential antioxidants [26,27]. It is well known that the antioxidant activity of plant extracts containing polyphenol components is due to the capacity to be donors of hydrogen atoms or electrons and to capture the free radicals. Hence, it can be concluded that the higher antioxidant activity of methanol extract of both the Halophila species observed in both DPPH and ABTS method might be due to higher phenolics and flavonoids content. The study showed that methanol extract of H. ovalis and H. beccarii collected from Chilika lagoon exhibited 50% of scavenging activity on DPPH radicals at 37.77µg/ml and 25.62 µg/ml and ABTS radicals at 52.25µg/ml and 45.45 µg/ml, respectively. Relatively less scavenging activity has been reported in H. ovalis from Mandapam coast, Tamil Nadu by Rengasamy et al. [10] and from Chunnambar estuary, Pondicherry by Yuvaraj et al. [14]. Hence, we consider that the higher antioxidant activity of the extracts might be due to its high content of phenolics and flavonoids in H. ovalis occurring in Chilika lagoon in eastern India. However, till date, there is no published report on the antioxidant activity of H. beccarii in India or elsewhere.

CONCLUSION
The antioxidant activity of extracts of H. ovalis and H. beccarii two seagrass species of Chilika lagoon was evaluated on the basis of DPPH and ABTS radical scavenging activities. The result of the present study revealed that the methanolic extract of H. ovalis of Chilika lagoon exhibited the highest antioxidant activity in comparison to the antioxidant potential of this species in other habitats reported so far. The methanolic extract of H. ovalis contained a high level of phenolics and flavonoids, which can be attributed to strong antioxidant activity observed against ABTS and DPPH radicals. This strong antioxidant property identifies the particular seagrass species as a potential source

AUTHORS' CONTRIBUTIONS
PCP conceived the project and taxonomically identified the species. PCP and SKN supervised and guided the research work and preparation of the manuscript. SKK collected and processed the samples and performed analysis of extracts with different solvents. SS and BK studied the scavenging activity and analyzed the data. BK, SKK, and SS interpreted the data and prepared the manuscript. All authors read and approved the final manuscript.