Antioxidant activity and total phenolic contents of selected seaweeds from Okha coast of Gujarat

Introduction

India has 7,500 km of coastline bounded to the south by the Indian Ocean to the west by the Arabian Sea to the east by the Bay of Bengal. The marine system with diverse habitats and developing some chronic diseases, various types of cancer, diabetes, obesity, improved endothelial function, and reduced blood pressure (Rahman et al., 2022) [10] . Seaweeds play the role of free radical scavengers; they prevent or repair damages caused by oxidative stress and have a high potential for the treatment of various diseases (Liu and Sun, 2020) [11] . The antioxidant characteristics of these seaweeds have been associated with their phenolic composition. Considering this, in the present study 69 seaweed species belonging to Chlorophyta, Phaeophyta, and Rhodophyta were collected from Okha Coast to assess their antioxidant activity and estimate phenolic content. The antioxidant potential and phenolic content of extracts from these seaweeds hold significant potential for applications in the food and pharmaceutical industries.

Methods and Materials Collection of Seaweed

Seaweeds were collected from Okha Coast (22º 28 ̍ 45N, 69º 04̍ 42E) Saurashtra from November to March (2023-2024).

A total of 69 algae samples were collected from Chlorophyta, Phaeophyta, and Rhodophyta (Plate 1-4). The algae were cleaned, washed in distilled water, air dried at room temperature, powdered, and stored.

Extraction of phenolic compound from the seaweeds

The 500 mg of dry seaweed powder was taken and crushed with 10 ml of 80% Methanol. The mixer was kept under dark for 48 h on shaking condition. The next day the extracts were centrifuged at 5000 g for 15 min. The supernatant was collected in a Petri plate and allowed for evaporation. The remaining dry residue was dissolved in sterile distilled water (5 ml) and used as a source of phenols. The methanol extract was used as an antioxidant measurement.

Total Phenolic content

The concentration of total phenols was determined according to Swain and Hills (1959) [12] . The reaction mixture contained 100 µl of extract, 10 µl of 1N Folin-Ciocalteau's reagent and 200 µl of saturated sodium carbonate. The reaction mixture was incubated at room temperature for 5 minutes. The absorbance was measured at 660 nm using Biotek microplate reader μQuant.

Measurements were carried out in triplicate and phenol content was calculated using a calibration curve of chlorogenic acid (100-1000 µg/ml).

DPPH free radical scavenging assay

Radical scavenging assay was determined according to Madhar et al., (2023) [13] . DPPH (1,1-diphenyl-2picrylhydrezyl) free radical scavenging assay is a stable free radical and is widely used to assess the radical scavenging activity of antioxidant compounds. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant due to the formation of the nonradical form DPPH-H by the reaction. This transformation results in a color change from purple to yellow, which is measured on a microplate reader (µ Quant, Bio Teak, USA). The disappearance of the purple color is monitored at 517 nm. The reaction mixture (200 µl) consists of 100 µl of DPPH (1 mM) and 100 µl methanol extract. It is incubated for 20 minutes at room temperature in the dark and then the absorbance is measured at 517 nm. Activity was expressed as mg equivalent to ascorbic acid/g dry powder, the experiment was performed in triplicate.

Results and Discussion

Free radicals play an important role in affecting human health by causing many diseases. Antioxidants scavenge free radicals from the cells and prevent or reduce the damage caused by oxidation. Seaweeds can serve as a good source of antioxidants. The protective effect of antioxidants continues to be studied around the world (Chakraborty et al., 2013; Liu and Sun, 2020) [11,14] . In the present work, radical scavenging activity was measured in mg/g dry powder and measured by DPPH free radical scavenging method. The stable free radical DPPH was reduced and decolorized. The antioxidant activity of 45 species of Rhodophyta seaweed is presented in Figure -1. The antioxidant activity was expressed in mg/gram dry powder. The radical scavenging activity of Rhodophyta species was observed in the range of 22 -52.9 mg/g dry powder.

The antioxidant activity of the Halymenia genus group H. esperi (44 mg) was given the highest activity compared to H. macrocarpa (40.33 mg), H. floresii (37.69 mg), H. porphyraeformis (32.49 mg), H. durvillei (25 mg) and H. maculata (24 mg). The Hypnea muscuformis (52.92 mg) was given the maximum amount of antioxidant activity followed by Hypnea valentiae (38 mg), H. spinella (35.69 mg), H. esperi (31.26 mg) and H. cervicornis (30.14 mg). From the group of Laurencia genera, a moderate amount of activity was observed in L. obtuse (42.53 mg), L. platyclade (38.7 mg), L. perforata (33.86 mg), L. glandulifera (32.09 mg), L. papillosa (30.59 mg) and L. cruciate (30 mg). The Gracillaria genera were given antioxidant activity in the range (30.55-36 mg). The G. foliferi (36 mg) was given higher activity compared to other species of the genus. The Scinaia fascicularis (51 mg) was given the maximum amount of antioxidant activity compared to others S. confusa (41.77 mg) and S. complanate (22 mg). The higher to moderate amount of free radical scavenging was observed in all seaweeds such as Acanthophora spicifera (35.6 mg), Amphiroa anceps (28.5 mg), Amphiroa fragilissima (31.2 mg), Botryocladia leptopoda (36.6 mg), Centroceras clavulatum (30.0 mg), Champia indica (44.0 mg), Coelarthrum opuntia (31.3 mg), Coralina elongate (39.0 mg), Dermonema virens (32.0 mg), Gelidiella acerosa (38.1 mg), Gelidiopsis variabilis (27.0 mg), Gelidium coulteri (40.0 mg), Gelidium micropterum (47.0 mg), Halopegma duperreyi (30.71 mg). Liagora ceranoides (31.90 mg), Rhodymenia dichotoma (23 mg) and Sarconema filiforme (42.53 mg). These red algae have high amounts of pigments such as chlorophyll (a and d), carotenoids (β-carotene, fucoxanthin, astaxanthin, xanthophyll), and other pigments such as phycoerythrin, phycocyanin, and allophycocyanin (Cian et al., 2015) [15] . Rhodophyta usually presents a higher content of proteins, carbohydrates, and minerals than green and brown algae (Belghit et al., 2017, Torres, 2019; Øverland et al., 2019) [16,17,18] . Numerous studies have confirmed that Rhodophyta has high contents of bioactive compounds (~1600 described) (Leal et al., 2013) [19] . Regarding their properties, antioxidant, anti-inflammatory, anti-tumor, antiviral, anti-hyperlipidemic, anticancer, and immunomodulatory activities have been described both on in vitro and in vivo experiments (Silva et al., 2012; Rudtanatip et al., 2018) [20,21] . The antioxidant compounds in seaweeds, like carotenoids, polysaccharides, and polyphenols, can neutralize ROS through their oxidation process since their affinity to those oxidative compounds is very high (Moubayed et al., 2017) [22] . The antioxidant activity of 14 species of Chlorophyta seaweed is presented in Figure 2. The green algae Chaetomorpha antennina was given the higher antioxidant activity (54 mg) compared to Acrosiphonia orientalis, Bryopsis plumosa, Caulerpa racemose, Caulerpa verticillate, Chaetomorpha aerea, Chaetomorpha crassa, Cladophoropsis javanica, Enteromorpha flexuosa, Enteromorpha linza, Ulva lactuca, Valonia aegagropila, Halimeda macroloba and Acrosiphonia arcta. The antioxidant activity of 10 species of Phaeophyta seaweeds is presented in Figure -3. The brown algae Padina boryena (47 mg) produced the maximum amount of antioxidant activity compared to Dictyopteris delicutula (38 mg), Dictyota dichotoma (35 mg), Padina tetrastomatica (33 mg), Spatoglossum asperum (42 mg) and Cystoseira indica (30 mg). The Sargassum sp. was given antioxidant activity in range (31-41 mg/ g dry powder). Algae exhibit higher antioxidant activity due to a higher content of nonenzymatic antioxidant components, such as ascorbic acid, reduced glutathione, phenols, and flavonoids (Wu et al., 2010) [23] . Previous research has documented the antioxidant potential of numerous seaweeds. The study of antioxidant metabolites in algal physiology suggested the involvement of a large number of secondary metabolites (Souza et al., 2007) [24] . In recent studies, laminarin extracted from brown seaweed has demonstrated significant antioxidant properties (Rajauria et al., 2021) [25] . The genus Halimeda contains phytopharmaceuticals and possess pharmacological properties, such as antimicrobial activity, stimulation of apoptosis, and anti-trichomonal and antiinflammatory activity (Silva et al., 2017) [26] . Dietary supplementation of seaweeds (Sargassum spp.) and their derivatives enhanced the immune responses and antioxidant capabilities (Telles et al., 2018) [27] . As a result, many marine bio-sources in the last decades have attracted attention for their potential to discover natural bioactive compounds for the development of novel drugs and nutritious foods. Phenolic compounds represent a broad and varied category of molecules encompassing various families of aromatic secondary metabolites found in plants (Waterman and Mole, 2005) [28] . These compounds have been recognized for their diverse biological activities, including antioxidative properties. Previous studies have indicated that extracts from marine seaweeds, particularly their polyphenols, exhibit antioxidant activity (Yan et al., 1999; Kuda et al., 2005) [29,30] . Phlorotannins and fucoxanthin are reported as the primary active compounds in different seaweed extracts (Yan et al., 1999) [31] . In the present work, phenolic content was measured in mg/g dry powder. The phenolic content of 45 species of Rhodophyta seaweed is presented in Figure -4. The nine red algae were produced the higher amount of phenolic content such as Gelidium coulteri (75.67 mg), Grateloupia turuturu (58.55 mg), Grateoloupia elliptica (47.38 mg), Acanthophora spicifera (40.88 mg), Laurencia perforate (40.17 mg), Laurenia platyclade (38.90 mg), Hypnea musciformis (35.60 mg) and Gracilaria corticate (33.08 mg).

The thirty species of red algae were produced the moderate amount of phenolic content i.e. Rhodymenia dichotoma (31.45 [32] . Additionally, seaweeds contain distinct polyphenols such as phenolic terpenes like rosmanol, carnosol, and carnosic acid (Zhong et al., 2020) [33] , as well as terpenoids like chromene, chromanol, and plastoquinone (Cotas et al., 2020) [34] . Bromophenols such as 2,4-bromophenol, 2,6-bromophenol, and 2,4,6-tribromophenol are also present in seaweeds (Rodríguez-Bernaldo et al., 2010; Cotas et al., 2020) [32,34] . Seaweeds are rich in hydrophilic polyphenolic compounds including epigallocatechin gallate, epicatechin, and phlorotannins (Boonchum et al., 2011) [35] . Several studies suggest that polyphenols play a role in preventing cardiovascular diseases, cancers, inflammation, and osteoporosis (Das., 2012) [36] . Furthermore, polyphenols appear to be involved in hindering neurodegenerative diseases and diabetes mellitus (Scalbert et al., 2005) [37] . Phenolic compounds produced from marine algae have been investigated for their anticancer, antibacterial, anti-inflammatory, and antioxidant properties (Wang et al., 2017) [38] .

Conclusion

The results indicated that all the tested seaweeds in this investigation possess antioxidant activity and total phenolic content. The Hypnea musciformis (Rhodophyta), Chaetomorpha antennina (Chlorophyta), and Padina boryena (Phaeophyta) possess higher antioxidant activity. The Gelidium coulteri (Rhodophyta), Acrosiphonia orientalis (Chlorophyta), and Sargassum sp. (Phaeophyta) possess a higher amount of phenolic content compared to other seaweeds. Most seaweeds are given the maximum amount of antioxidant and phenolic contents. These seaweeds can be used as a good source of natural antioxidants. Further study is required to find their biologically active compounds.

Acknowledgement

AcknowledgmentsAuthors are thankful to the Department of Bioscience, Saurashtra University, Rajkot, and Vimal Research Society Agro-Biotech and Cosmic Power Rajkot, Gujarat India for providing the lab facility.

References

1. Rotter A, Barbier M, Bertoni F, Bones AM, Cancela ML, Carlsson J, et al. The essentials of marine biotechnology. Frontiers in Marine Science. 2021;16(8):629629.
2. Vijay K, Balasundari S, Jeyashakila R, Velayathum P, Masilan K, Reshma R. Proximate and mineral composition of brown seaweed from Gulf of Mannar. International Journal of Fisheries and Aquatic Studies. 2017;5(5):106-112.
3. Jha B, Reddy CRK, Thakur MC, Rao MU. Seaweeds of India. The diversity and distribution of seaweeds of Gujarat Coast. Developments in Applied Phycology. Springer, Dordrecht. 2009;3:216.
4. Xu J, Liao W, Liu Y, Guo Y, Jiang S, Zhao C. An overview on the nutritional and bioactive components of green seaweeds. Food Production, Processing and Nutrition. 2023;5(1):18.
5. Hughes LJ, Black LJ, Sherriff JL, Dunlop E, Strobel N, Lucas RM, et al. Vitamin D content of Australian native food plants and Australian-grown edible seaweed. Nutrients. 2018;10(7):876.
6. Lozano Muñoz I, Díaz NF. Minerals in edible seaweed: Health benefits and food safety issues. Critical Reviews in Food Science and Nutrition. 2020;62(6):1592-1607.
7. Kumar H, Choudhary N, Varsha KN, Suman SR. Phenolic compounds and their health benefits: A review. Journal of Food Research and Technology. 2014;2(2):46-59.
8. Lomartire S, Gonçalves AM. An overview of potential seaweed-derived bioactive compounds for pharmaceutical applications. Marine Drugs. 2022;20(2):141.
9. Rahman MM, Rahaman MS, Islam MR, Rahman F, Mithi FM, Alqahtani T, et al. Role of phenolic compounds in human disease: current knowledge and future prospects. Molecules. 2021;27(1):233.
10. Liu Z, Sun X. A critical review of the abilities, determinants, and possible molecular mechanisms of seaweed polysaccharides antioxidants. International Journal of Molecular Sciences. 2020;21(20):7774.
11. Swain T, Hillis WE. The phenolic constituents of Prunus domestica. The quantitative analysis of phenolic constituents. Journal of the Science of Food and Agriculture. 1959;10(1):63-68.
12. Madhar BH, Monpara JK, Chudasama KS, Vekaria ML, Patel VJ, Thaker VS. Phytochemical Screening, Total Phenolics and Antioxidant Activities of Selected 100 Medicinal Plants of Semi-arid Region of Gujarat, India. Acta Botanica Brasilica. 2023;V02i02:58-64.
13. Chakraborty K, Praveen NK, Vijayan KK, Rao GS. Evaluation of phenolic contents and antioxidant activities of brown seaweeds belonging to Turbinaria spp. (Phaeophyta, Sargassaceae) collected from Gulf of Mannar. Asian Pacific Journal of Tropical Biomedicine. 2013;3(1):8-16.
14. Cian RE, Drago SR, Sanchez de Medina F, MartínezAugustin O. Proteins and carbohydrates from red seaweeds: evidence for beneficial effects on gut function and microbiota. Marine Drugs. 2015;13(8):5358-5383.
15. Belghit I, Rasinger JD, Heesch S, Biancarosa I, Liland N, Torstensen B, et al. In-depth metabolic profiling of marine macroalgae confirms strong biochemical differences between brown, red and green algae. Algal Research. 2017;26:240-249.
16. Torres MD, Flórez-Fernández N, Domínguez H. Integral utilization of red seaweed for bioactive production. Marine Drugs. 2019;17(6):314.
17. Øverland M, Mydland LT, Skrede A. Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals. Journal of the Science of Food and Agriculture. 2019;99(1):13-24.
18. Leal MC, Munro MH, Blunt JW, Puga J, Jesus B, Calado R, et al. Biogeography and biodiscovery hotspots of macroalgal marine natural products. Natural Product Reports. 2013;30(11):1380-1390.
19. Silva TH, Alves A, Popa EG, Reys LL, Gomes ME, Sousa RA, et al. Marine algae sulfated polysaccharides for tissue engineering and drug delivery approaches. Biomatter. 2012;2(4):278-289.
20. Rudtanatip T, Lynch SA, Wongprasert K, Culloty SC. Assessment of the effects of sulfated polysaccharides extracted from the red seaweed Irish moss Chondrus crispus on the immune-stimulant activity in mussels Mytilus spp. Fish & Shellfish Immunology. 2018;75:284-290.
21. Moubayed NM, Al Houri HJ, Al Khulaifi MM, Al Farraj DA. Antimicrobial, antioxidant properties and chemical composition of seaweeds collected from Saudi Arabia (Red Sea and Arabian Gulf). Saudi Journal of Biological Sciences. 2017;24(1):162-169.
22. Wu SC, Wang FJ, Pan CL. The comparison of antioxidative properties of seaweed oligosaccharides fermented by two lactic acid bacteria. Journal of Marine Science and Technology. 2010;18(4):8.
23. Sousa JM, De Souza EL, Marques G, Meireles B, de Magalhães Cordeiro ÂT, Gullón B, et al. Polyphenolic profile and antioxidant and antibacterial activities of monofloral honeys produced by Meliponini in the Brazilian semiarid region. Food Research International. 2016;84:61-68.
24. Rajauria G, Foley B, Abu-Ghannam N. Characterization of dietary fucoxanthin from Himanthalia elongata brown seaweed. Food Research International. 2017;99:995-1001.
25. Silva AMO, Novoa AV, Gutierrez DD, Mancini-Filho J. Seaweeds from Halimeda genus as sources of natural antioxidants. Journal of Analytical and Pharmaceutical Research. 2017;5(6):1-5.
26. Telles CB, Mendes-Aguiar C, Fidelis GP, Frasson AP, Pereira WO, Scortecci KC, et al. Immunomodulatory effects and antimicrobial activity of heterofucans from Sargassum filipendula. Journal of Applied Phycology. 2018;30:569-578.
27. Yan X, Chuda Y, Suzuki M, Nagata T. Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Bioscience, Biotechnology, and Biochemistry. 1999;63(3):605-607.
28. Kuda T, Tsunekawa M, Goto H, Araki Y. Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. Journal of Food Composition and Analysis. 2005;18(7):625-633.
29. Yan X, Nagata T, Fan X. Antioxidative activities in some common seaweeds. Plant Foods for Human Nutrition. 1998;52:253-262.
30. Rodríguez-Bernaldo de Quirós A, Frecha-Ferreiro S, Vidal-Pérez AM, López-Hernández J. Antioxidant compounds in edible brown seaweeds. European Food Research and Technology. 2010;231:495-498.
31. Zhong B, Robinson NA, Warner RD, Barrow CJ, Dunshea FR, Suleria HA. LC-ESI-QTOF-MS/MS characterization of seaweed phenolics and their antioxidant potential. Marine Drugs. 2020;18(6):331.
32. Cotas J, Leandro A, Monteiro P, Pacheco D, Figueirinha A, Gonçalves AM, et al. Seaweed phenolics: From extraction to applications. Marine Drugs. 2020;18(8):384.
33. Boonchum W, Peerapornpisal Y, Kanjanapothi D, Pekkoh J, Pumas C, Jamjai U, et al. Antioxidant activity of some seaweeds from the Gulf of Thailand. International Journal of Agriculture and Biology. 2011;13:95-99.
34. Das S. Antimicrobial and antioxidant activities of green and ripe fruits of Averrhoa carambola Linn. and Zizyphus mauritiana Lam. Asian Journal of Pharmaceutical and Clinical Research. 2012;5(3):102105.
35. Scalbert A, Johnson IT, Saltmarsh M. Polyphenols: antioxidants and beyond. American Journal of Clinical Nutrition. 2005;81(1):215S-217S.
36. Wang ZJ, Xu W, Liang JW, Wang CS, Kang Y. Effect of fucoidan on B16 murine melanoma cell melanin formation and apoptosis. African Journal of Traditional, Complementary and Alternative Medicines. 2017;14(4):149-155.