REGLAMENTO DE LA LEY ORGÁNICA DE LA PROCURADURÍA GENERAL DE LA REPUBLICA

Cymodocea rotundata Ehrenb. & Hempr. ex Aschers. (Family: Cymodoceacea) is a flowering plant which inhabit Malaysian coastal waters. The seagrass is of ecological importance which forms dense meadow supporting high biodiversity and food to a variety of aquatic organisms. The species can adapt to certain level of anthropogenic disturbances, and thus is an important bio-indicator to examine the marine health condition. The aim of the present study is to determine the antioxidant activity of C. rotundata from several seagrass beds, and their relationship between morphometric and water quality characteristics. The seagrass was collected from 6 sites; (1) Teluk Pelanduk, Negeri Sembilan, (2) Pantai Punang-Sari-Lawas, Sarawak, (3) Pantai Sungai Bangat, Sarawak, (4) Teluk Sepanggar, Sabah, (5) Pantai Kampung Kelawat, Sabah and (6) Pulau Mabul, Sabah. Morphometric measurement of leaf blade length and blade width was taken from dry herbarium of the seagrass. Water samples were analyzed for ammonia, nitrate and nitrite. Seagrass methanol leaf extracts were evaluated for antioxidant activity including total phenolic content (TPC), total flavonoid content (TFC), DPPH and FRAP activities. This present study showed that C. rotundata leaf blade length was strongly correlated with nitrate (r=0.82) and inversely correlated with DPPH activity (r=-0.97). Long-leaved C. rotundata (13.6±2.3 cm) from Teluk Pelanduk (S1) possessed a very strong antioxidant DPPH EC50 (41.58±7.87 µg/mL). This suggests that the leaf morphometric of C. rotundata can be used as references for monitoring the water quality and antioxidant status of the seagrass.

Keywords: Antioxidant; Cymodocea rotundata; Seagrass; Morphometric Introduction

The search for natural antioxidant from marine plants (e.g., seaweed, seagrass and mangrove plants) has gained considerable attention in the past few decades, since the phytochemicals are cheaper, safer and displayed potent pharmacological effects against human diseases (Sithranga Boopahty & Kathiresan, 2010). Interest in employing natural antioxidant derived from marine source not only limited for commercial biomedicine industry, but also for their application in food production and cosmetic industry (Wells et al., 2016). Marine macroalgae, seaweed has been the center of focus in many research fields especially for the discovery of new drugs (Kavita, Singh, & Jha, 2014) due to its edibility and preference for human consumption, unlike seagrass and mangrove plants. However, this does not implies that other marine plants have no such beneficial values. In fact, seagrass has been long utilized as food (de la Torre-Castro & Rönnbäck, 2004; Bujang, Zakaria, & Arshad, 2006) and is an important traditional medicine for the coastal people (Newmaster et al., 2011). Seagrass extracts also exhibit antioxidant property on various antioxidant assays (e.g., total antioxidant activity, DPPH scavenging activity and reducing power) (Kannan, Arumugam, Thangaradjou, & Anantharaman, 2013). Few studies have been performed on antioxidant activity of seagrass from Tropical Indo-Pacififc bioregion (Santoso et al., 2012; Kannan, Arumugam, Thangaradjou, & Anantharaman, 2013; Jeyapragash, Subhashini, Raja, Abirami, & Thangaradjou, 2016); however, comparison between the antioxidant activities of seagrass from various habitats is still lacking. Previous study conducted by Grignon-Dubois, Rezzonico and Alcoverro (2012) on seagrass Zostera noltii from Atlantic and Mediterranean coasts showed that the extreme changes in physical parameter (e.g.,

52

temperature, light intensity and salinity) can greatly influences the accumulation of phenolic acids (rosmarinic acid, zosteric acid and caffeic acid) in the seagrass. Hence, the present study aims to investigate the morphometric and antioxidant properties of seagrass Cymodocea rotundata from various habitats.

Materials and Methods Seagrass collection

Cymodocea rotundata was collected from 6 seagrass beds; (S1) Teluk Pelanduk, Negeri Sembilan (2°25'7.2659"N, 101°53'2.928"E), (S2) Pantai Sungai Bangat, Sarawak (4°54'48.2880"N, 115°23'6.9540"E), (S3) Pantai Punang-Sari-Lawas, Sarawak (4°56'49.7459"N, 115°24'21.0360"E), (S4) Teluk Sepanggar, Sabah (6°3'17.4720"N, 116°6'43.2660"E), (S5) Pantai Kampung Kelawat, Sabah (6°18'49.2498"N, 116°19'39.9284"E) and (S6) Pulau Mabul, Sabah (4°14'53.6820"N, 118°37' 52.8659"E). In-situ water quality parameters (salinity, atmospheric pressure, temperature, dissolved oxygen, pH, conductivity and total dissolved solid) were measured using YSI multiparameter professional series (YSI, USA).

Morphometric measurement

Morphometric measurement of C. rotundata including leaf blade length (L, cm), leaf blade width (W, mm) and leaf sheath length (LS, mm) were measured from the dry herbarium deposited at Laboratory of Aquatic Botany, Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia (UPM) (Voucher specimen: TPCR01-03, PPSCR01-04, PSBCR01-04, TSCR01-03, PKKCR01-04 and PMCR01-03). Leaf blade length to width ratio (L/W) was calculated from the recorded values.

Water nutrient

Seawater ammonia (NH3), nitrate (NO3-) and nitrite (NO2-) concentration were measured by using a portable spectrophotometer Hach DR/2400 (Hach, USA) according to developer protocol (Hach Company, 2004).

Antioxidant activity

Fresh leaves of C. rotundata were carefully cleaned with distilled water from unwanted debris. The leaves were oven-dried at 40°C in an air recirculating oven (Memmert UF160, Germany), and then powderized using an electric blender. The powder was extracted with methanol for 24 hours on an orbital shaker at speed 150 rpm. The mixtures were filtered thrice through Whatman filter paper no. 1. The filtrates were pooled and dried under vacuum at 55ºC and 200-250 mbar in a rotary vacuum evaporator (IKA® RV 10, Germany). The extracts were assessed for antioxidant activity of total phenolic content (Zhang et al., 2006), total flavonoid content (He, He, He, & Wan, 2012), DPPH scavenging activity (Cheng, Moore, & Yu, 2006) and ferric reducing antioxidant power activity (Benzie & Strain, 1996). Statistical analysis

Morphometric measurement, water quality and antioxidant activity parameters were statistically analyzed with one-way analysis of variance (DMRT, p<0.05), principal component analysis (PCA, Pearson type) and agglomerative hierarchical clustering (AHC, similarities of Pearson correlation coefficient) using XLSTAT 2014 (Addinsoft, 2015).

53 Results and Discussion

Antioxidant activity

Seagrass C. rotundata collected from S2 and S1 possessed the highest total phenolic content (TPC) with the values of 28.20±0.38 and 27.82±1.14 mg gallic acid equivalent (GAE) per g extract, respectively > S3 (26.17±1.23 mg GAE/g extract) and S4 (25.37±1.87 mg GAE/g extract) > S5 (17.63±0.0.65 mg GAE/g extract) > S6 (13.87±0.57 mg GAE/g extract) (Figure1a). This present study showed higher TPC values (S1-S4) compared to studied sample from Palk Bay, India (15.38 mg GAE/g extract, Jeyapragash, Subhashini, Raja, Abirami, & Thangaradjou, 2016), Chinapallam, India (12.64 mg GAE/g extract, Kannan, Arumugam, Thangaradjou, & Anantharaman, 2013) and Pramuka Island, Indonesia (0.02-3.35 mg GAE/g extract, Santoso et al., 2012). Polyphenols in seagrass tends to dissolve in polar and semi- polar solvent, as reported by Santoso et al. (2012) which demonstrated high phenolic content in methanol as compared to a more non-polar solvent ethyl acetate and n-hexane. This present study also showed that extraction using absolute methanol yielded more polyphenol content compared to using aqueous ethanol (15.38 mg, Jeyapragash, Subhashini, Raja, Abirami, & Thangaradjou, 2016) and aqueous methanol (12.64 mg GAE/g extract, Kannan, Arumugam, Thangaradjou, & Anantharaman, 2013). Production of phenolic content may also highly associated with the seagrass environmental conditions, e.g., tidal nature, temperature, light intensity and salinity, by which inter-tidal seagrass contained high phenolic acids compared to sub-tidal (Grignon-Dubois, Rezzonico, & Alcoverro, 2012). Total flavonoid content (TFC) of sample collected from S2 (53.93±4.42 mg quercetin equivalent (QE) per g extract), S4 (50.16±2.67 mg QE/g extract) and S5 (51.08±2.63 mg QE/g extract) were significantly higher (p<0.05) than > S3 (41.56±3.63) and S6 (41.01±1.08) > S1 (33.78±2.53 mg QE/g extract). The TFC values were higher than aqueous methanol extract as reported by Kannan, Arumugam, Thangaradjou and Anantharaman (2013) for C. roundata (4.56 mg QE/g extract) and C. serrulata (5.12 mg QE/g extract).

DPPH EC50 is the effective concentration quenching 50% of generated DPPH radical. EC50 value can be categorized according to its quenching ability; very strong (<50 µg/mL), strong (50–100 µg/mL), intermediate (100-150 µg/mL), strong (150–200 µg/mL) and (>200 µg/mL) (Blois, 1958). Sample collected from S1 had the lowest EC50 value of 41.58±7.87 µg/mL and was categorized as a very strong antioxidant agent. Sample from S2, S3 and S4 demonstrated strong antioxidant activity, and required twice the concentration to achieve S1 DPPH activity, with values of 90.51±3.70, 99.46±2.21 and 95.05±3.30 µg/mL, respectively. Sample from S5 (109.17±0.45 µg/mL) and S6 (141.59±17.37 µg/mL) demonstrated intermediate antioxidant activity. Antioxidant activities of seagrass were comparable to tropical edible seaweed Caulerpa racemosa with TPC, TFC and DPPH EC50 values of 19.8±2.01 mg GAE/g, 16.0 mg cathecin equivalents/ g and 90.00 µg/mL, respectively (Chia, et al., 2015).

54

Besides, Pearson’s correlation analysis showed that the leaf blade length of C. rotundata was inversely strong correlated with DPPH activity (r=-0.97, p<0.05) (Figure 1b). This proved that extract from long-leaved C. rotundata exhibited a very strong antioxidant activity. Moreover, TPC was also inversely correlated with DPPH (r=-0.80, p<0.05). The high TPC contribute to high DPPH antioxidant activity similar to Santoso et al. (2012) and Jeyapragash, Subhashini, Raja, Abirami and Thangaradjou (2016). Reducing power of the seagrass extracts were evaluated based FRAP assay using Fe2+ _{as equivalent standard. Cymodocea rotundata collected} from S4 possessed high FRAP activity with value 3.71±0.69 mM Fe2+_{E/g extract,} followed by S1 (2.86±0.18 mM Fe2+_{E/g extract) > S2 (1.90±0.06) and S3 (1.90±0.16} mM Fe2+_{E/g extract) > S5 (1.15±0.05) and S6 (0.71±0.06 mM Fe}2+_{E/g extract).} Direct comparison of FRAP activity with available literature was difficult due to different standard and unit used. However, previous study showed that effective concentration (EC50) of C. rotundata leaves (2.58 µg/mL) exhibited more potent FRAP activity than rhizomes (1.39 µg/mL) (Jeyapragash, Subhashini, Raja, Abirami, & Thangaradjou, 2016). 0.00 50.00 100.00 150.00 200.00 S1 S2 S3 S4 S5 S6 TPC (mg GAE/g extract) TFC (mg QE/g extract) DPPH (µg/mL) FRAP (mM Fe2+E/g extract) 0.71 (d) 141.59 (a) 41.58 (d) 109.17 (bc) 99.46 (bc) 95.05 (c) 90.51 (c) 41.01 (b) 33.78 (c) 51.08 (a) 41.56 (b) 50.16 (a) 53.93 (a) 13.87 (d) 27.82 (a) 17.63 (c) 26.17 (b) 25.37 (b) 28.20 (a) 1.90 (c) 1.90 (c) 3.71 (a) 1.15 (d) 2.86 (b)

55

Figure 1. (a) Antioxidant activity of seagrass Cymodocea rotundata from various seagrass beds. Bars sharing the same alphabets (a>b>c>d) are not significantly different (p<0.05, DMRT). (b) Pearson’s correlation matrix (n) of the variables with values in bold are different from 0 with a significance level alpha=0.05. Abbreviation: L= leaf length, W= leaf width, LS= leaf sheath length, L/W: leaf length to width ratio, T= temperature, P= atmospheric pressure, DO= dissolved oxygen, C= conductivity, TDS= total dissolved solid, S= salinity, NH3= ammonia, NO3-= nitrate, NO2-= nitrite, TPC= total phenolic content

Habitat influence on seagrass morphometric and antioxidant activity

Seagrasses under studied are clustered into 3 groups according to the PCA and AHC variables (Figure 2a-c). PCA factor of F1 (37.52%) and F2 (31.40%) were accounted for 68.93% of the total variance (Figure 2a & b). LS, pH, DPPH, DO and TFC, were positively correlated with F1, while L/W, T, TDS, S, C, DPPH and DO were positively correlated with F2. Based on the PCA score biplot and AHC dendogram, the seagrasses are clustered into 3 groups. Group A comprises of sample from S1 and S2 which were characterized with long leaf blade (13.6±2.3 cm and 9.4±1.6 cm, respectively), short leaf sheath (29.29±3.63 and 33.71±4.37 mm, respectively) and inhabit high water salinity (26.07-32.78 PSU), nitrate (1.6-2.0 mg/mL) and nitrite (0.023-0.036 mg/mL) environments. Seagrasses collected from these areas exhibited a potent DPPH EC50 activity of very strong (S1) to strong (S2) according to antioxidant power classification (Blois, 1958). Nitrate was strongly correlated with leaf blade length (r=0.82, p<0.05) which proved that C. rotundata grows in high water nitrate environment possessed long leaf blade (13.6±2.3 cm). However, nitrate enrichment combined with low light treatment may reduce seagrass leaf growth rate, since the light, energy and carbon skeletons were insufficient to support nitrate reduction (Jiang, Huang, & Zhang, 2013). Group B includes samples from S4, S5 and S6 from Sabah, which inhabit clear water with high DO (5.97-7.59 mg/L), low nitrate (1.0-1.1 mg/mL) and low nitrite (0.002-0.013 mg/mL) level. Samples collected from Sabah possessed intermediate (S5 and S6) to strong (S4) DPPH EC50 activity. The absent of the nitrate stressor reduces the antioxidant activity of the seagrass. Group C consists only sample from S3 with longest leaf sheath (42.48±6.68 mm) inhabit in low water conductivity (21621±4209 S/cm), total dissolved solid (16380±4896 mg/L)

Var L W LS L/W T P DO C TDS S pH NH3NO3-NO2-TPC TFC DPPH FRAP L 1 W 1 LS 1 L/W 1 T -0.85 1 P 1 DO 1 C -0.86 0.86 1 TDS -0.85 0.88 0.99 1 S -0.82 0.95 0.90 1 pH 0.94 1 NH3 1 NO3- _{0.82 0.98 1} NO2- _{0.94 0.92 1} TPC 1 TFC 1 DPPH-0.97 -0.80 -0.80 1 FRAP 1 0 1

56

and salinity (10.69±3.15 PSU). Pearson’s correlation analysis showed that (Figure 1b) the water conductivity, total dissolved solid and salinity were inversely strong correlated with seagrass leaf sheath length (r= -0.86, -0.85 and -0.82, respectively), which suggest that seagrass lives under these parameter values could produce longer leaf sheath.

Figure 2. (a) Biplot of principal component analysis (PCA) variables, (b) Biplot of PCA score and (c) Dendogram of agglomerative hierarchical clustering (AHC) of variables.

Conclusion

The findings from this present study showed that water nitrate influences the morphometric changes of C. rotundata’s leaf blade length (r=0.82, p<0.05). High seawater nitrate produced long-leaved C. rotundata, e.g., sample from Teluk Pelanduk (S1, 13.6±2.3 cm). Long-leaved C. rotundata also possessed a very strong DPPH antioxidant activity (r=-0.97, p<0.05) with EC50 value of 41.58±7.87 µg/mL. This suggests that the leaf morphometric of C. rotundata can be used as references for monitoring the water quality and antioxidant status of the seagrass.

57 Acknowledgement

This project was funded by Forest City, Country Garden Pacific View, Johor entitled “Monitoring of Seagrass Species, Coverage and Adaptability”. We would like to acknowledge Universiti Putra Malaysia (UPM) for technical support and assistance in this research project.

References

Addinsoft. (2015). XLSTAT V. 2015.1. 01: Data Analysis and Statistics Software for Microsoft Excel. Addinsoft, USA.

Benzie, I. F. & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239(1), 70-76.

Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181(4617), 1199-1200.

Bujang, J. S., Zakaria, M. H. & Arshad, A. (2006). Distribution and significance of seagrass ecosystems in Malaysia. Aquatic Ecosystem Health & Management, 9(2), 203-214.

Cheng, Z., Moore, J. & Yu, L. (2006). High-throughput relative DPPH radical scavenging capacity assay. Journal of Agricultural & Food Chemistry, 54(20), 7429- 7436.

Chia, Y. Y., Kanthimathi, M. S., Khoo, K. S., Rajarajeswaran, J., Cheng, H. M. & Yap, W. S. (2015). Antioxidant and cytotoxic activities of three species of tropical seaweeds. BMC Complementary & Alternative Medicine, 15(339), 1-14.

de la Torre-Castro, M. & Rönnbäck, P. (2004). Links between humans and seagrasses—an example from tropical East Africa. Ocean & Coastal Management, 47(7), 361-387.

Grignon-Dubois, M., Rezzonico, B. & Alcoverro, T. (2012). Regional scale patterns in seagrass defences: Phenolic acid content in Zostera noltii. Estuarine, Coastal & Shelf Science, 114, 18-22.

Hach Company (2004). DR/2400 Spectrophotometer Procedure Manual. Colorado, USA.

He, Y., He, Z., He, F. & Wan, H. (2012). Determination of quercetin, plumbagin and total flavonoids in Drosera peltata Smith var. glabrata YZ Ruan. Pharmacognosy Magazine, 8(32), 263-267.

Jeyapragash, D., Subhashini, P., Raja, S., Abirami, K. & Thangaradjou, T. (2016). Evaluation of in-vitro antioxidant activity of seagrasses: signals for potential alternate source. Free Radicals & Antioxidants, 6(1), 77-89.

Jiang, Z., Huang, X. & Zhang, J. (2013). Effect of nitrate enrichment and salinity reduction on the seagrass Thalassia hemprichii previously grown in low light. Journal of Experimental Marine Biology & Ecology, 443, 114-122.

58

Phytochemical constituents, antioxidant properties and p-coumaric acid analysis in some seagrasses. Food Research International, 54, 1229-1236.

Kavita, K., Singh, V. K. & Jha, B. (2014). 24-Branched Δ5 sterols from Laurencia papillosa red seaweed with antibacterial activity against human pathogenic bacteria. Microbiological Research, 169, 301-306.

Newmaster, A. F., Berg, K. J., Ragupathy, S., Palanisamy, M., Sambandan, K. & Newmaster, S. G. (2011). Local knowledge and conservation of seagrasses in the Tamil Nadu State of India. Journal Of Ethnobiology & Ethnomedicine, 7(37), 1-17.

Santoso, J., Anwariyah, S., Rumiantin, R. O., Putri, A. P., Ukhty, N. & Yoshie-Stark, Y. (2012). Phenol content, antioxidant activity and fibers profile of four tropical seagrasses from Indonesia. Journal of Coastal Development, 15(2), 189-196.

Sithranga Boopathy, N. & Kathiresan, K. (2015). Anticancer drugs from marine flora: an overview. Journal of Oncology, 2010, 1-18.

Wells, M. L., Potin, P., Craigie, J. S., Raven, J. A., Merchant, S. S., Helliwell, K. E., Smith, A. G., Camire, M. E. & Brawley, S. H. (2016). Algae as nutritional and functional food sources: revisiting our understanding. Journal of Applied Phycology, 29(2), 949-982. Zhang, Q., Zhang, J., Shen, J., Silva, A., Dennis, D. A. & Barrow, C. J. (2006). A simple 96-well microplate method for estimation of total polyphenol content in seaweeds. Journal of Applied Phycology, 18(3), 445-450.

59

2.9 Nutrient content of wild pepper, Piper umbellatum from Sg. Asap Koyan, Belaga,