Int J Pharm Pharm Sci, Vol 11, Issue 3, 54-61Original Article


SCREENING OF THE ANTIOXIDANT ACTIVITY OF CRUDE EXTRACTS IN 86 ALGAE SPECIES FROM EL JADIDA COAST (MOROCCO)

FATIMA CHIBI1, HALIMA RCHID1, WAFA ARSALANE1, RACHID NMILA1*

1Biotechnology and Valorization of Plant Resources: Algae and Plants, Department of Biology, Faculty of Sciences, Chouaib Doukkali University, El Jadida, Morocco
Email: rachid_nmila@yahoo.fr

Received: 21 Jul 2018 Revised and Accepted: 22 Jan 2019


ABSTRACT

Objective: This work aimed to screen the antioxidant activity of marine macroalgae from the Moroccan Atlantic coast (region of El Jadida).

Methods: Evaluation of the antioxidant activity of different collected species, lyophilized and extracted with a solvent mixture chloroform/methanol (2/1; v/v) was conducted according to two techniques, first by thin layer chromatography (tlc) then by spectrophotometry, using a free radical 2,2-diphenyl-1-picrylhydrazyl (dpph). The sampling on a distance of 110 km allowed to harvest 86 algal species (16 brown algae, 47 red algae, 14 green algae and 9 algae being identified).

Results: The analysis by thin layer chromatography reveals an antioxidant activity in nearly half of harvested algal species (52.32 %). This activity varies depending on the concentration of the extract and in function of incubation time in the presence of dpph. The monitoring of the kinetics of degradation of dpph by spectrophotometer in the presence of extracts which were active by tlc allowed to confirm the results and select the most active algal species based on the percentage of remaining dpph in the medium after 120 min of reaction: Fucus spiralis (17.02 %), Cyctoseira ericoides (12.16 %) (Phaeophyceae), and Gracilaria multipartita (36%), Halopitys incurvus (5%) (Rhodophyceae).

Conclusion: The results show that the methodology adopted in this work is reliable and can be used for rapid screening of antioxidant property in plants and the species: Fucus spiralis, Cyctoseira ericoides, Gracilaria multipartita, and Halopitys incurvus can be a promising source of natural compounds endowed with high antioxidant potential.

Keywords: Marine algae, Morocco, Extracts, Antioxidant activity, Dpph

© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2019v11i3.28666

INTRODUCTION

Since ancient times, marine algae have been used as food for livestock, fertilizers in agriculture, or as a source of medical agents. They are also widely used in traditional Chinese medicine to their pharmacological activities [1]. The algae are considered as a source of bioactive compounds. They are able to produce a large variety of secondary metabolites characterized by a large spectrum of biological activities and that can act at different levels [2, 3]. In addition to their use as a food source, especially in Asian countries [4], many algal compounds were studied for their variable activities as well as in health and environment fields [5]. The marine algae are considered an important source of bioactive substances such as antiviral [6], antioxidant [7], antitumoral [8, 9], antibacterial [10], anti-inflammatory, anticoagulant and antibiotic agents [11].

The intensive research has been conducted on the antioxidant activity due to the increasing demand for most food and pharmaceutical industries to develop bioactive natural compounds against aging and against cancer [7, 12-22]. These works led to the isolation of antioxidant molecules of very different natures: tocopherols [23], phenolic compounds [24], carotenoids and sterols [18] and sulfated polysaccharides [25, 26].

The Moroccan coast presents an undeniable richness in terms of diversity and quantity of macroalgae. Many of these algae are studied to assess different biological properties like antimalarial [27], antibacterial [28], antimicrobial [28], antiviral [30], anti-inflammatory [31], and antifungal activities [32]. However, the realized researches on the antioxidant activity of macroalgae from the Moroccan Atlantic coast remain to our knowledge generally modest [33, 34].

This study is done in the framework of the valorization of the Moroccan marine natural resources; it aims at the screening of antioxidant activity in the marine algae from the coast of El Jadida city. It is part of a project that aimed at the isolation and characterization of bioactive products from marine algae potentially usable in therapeutic and cosmetic fields.

MATERIALS AND METHODS

Algal materials and extraction

The algae inhabiting the Atlantic coast on a distance of 110 km, from Sidi Bounaim village (33 ° 22'37.2 "N 8 ° 14'56.7" W) to the city of Oualidia (32 ° 46'16.9 "N 9 ° 00'01.9" W) (fig. 1) were collected at low tide during several field outings (April 2013).

Fig. 1: Localization of collection sites

The algae were harvested manually, cleaned at first on site by sea water. In the laboratory, the algae were sorted and rinsed with running water and then with distilled water. After taxonomic identification, the specimens (table 1) were photographed and the voucher specimens were deposited in the laboratory of Biotechnology and Valorization of Plant Resources: Algae and Plants, Faculty of Sciences El Jadida, Morocco. The sorted samples were frozen at-80 °C and freeze-dried using a lyophilizer (Free Zone Plus 2.5 liters).

Table 1: List of collected algae on the moroccan atlantic coast from sidi bounaim to oualidia city

Species of seaweed Family Order Collection localities
Brown algae (Phaeophyceae)
Bifurcaria bifurcata Sargassaceae Fucales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Cladostephus verticillatus Cladostephaceae Spacelariales Moulay Abdellah(33 °11'56.1"N 8 °35'32.4"W)
Colpomenia sinuosa Scytosiphonaceae Ectocarpales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Cystoseira myriophylloides Sargassaceae Fucales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Cystoseira ericoides Sargassaceae Fucales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Cystoseira sp Sargassaceae Fucales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Dictyopteris mambranacea Dictyotaceae Dictyotales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W)
Dictyopteris sp Dictyotaceae Dictyotales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W)
Dictyota dichotoma Dictyotaceae Dictyotales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Fucus spiralis Fucaceae Fucales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Halopteris scoparia Stypocaulaceae Spacelariales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Laminaria digitata Laminariaceae Laminariales Sid Daoui (33 °15'42.3"N 8 °30'10.9"W)
Padina pavonica Dictyotaceae Dictyotales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Saccorhiza polyschides Phyllariaceae Tilopteridales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Sargassum muticum Sargassaceae Fucales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Sargassum vulgare Sargassaceae Fucales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Red algae (Rhodophyceae)
Asparagopsis armata Bonnemaisoniaceae Bonnemaisoniales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Bornetia secundiflora Ceramiaceae Ceramiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Callithamnion tetricum Callithamniaceae Ceramiales Mazagan (33 °18'03.5"N 8 °21'55.9"W)
Callithamnion sp Callithamniaceae Ceramiales Oualidia (32 °46'16.9"N 9 °00'01.9"W)
Caulacanthus ustulatus Caulacanthaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Ceramium flabelligerum Ceramiaceae Ceramiales Harass (33 °14'39.6"N 8 °28'27.0"W)
Ceramium sp1 Ceramiaceae Ceramiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Ceramium sp2 Ceramiaceae Ceramiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Chondria coerulescens Rhodomelaceae Ceramiales Mazagan (33 °18'03.5"N 8 °21'55.9"W)
Chondria dasyphylla Rhodomelaceae Ceramiales Jorf (33 °08'32.0"N 8 °36'51.9"W)
Corallina sp Corallinaceae Corallinales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Falkenbergia rufolanosa Bonnemaisoniaceae Bonnemaisoniales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gelidium crinal Gelidiaceae Gelidiales Harass (33 °14'39.6"N 8 °28'27.0"W)
Gelidium latifolium Gelidiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Geliduim pulchelum Gelidiaceae Gelidiales Sid Daoui (33 °15'42.3"N 8 °30'10.9"W)
Gelidium sesquipedale Gelidiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gelidium spinulosum Gelidiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gelidium sp1 Gelidiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gelidium sp 2 Gelidiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gelidium sp 3 Gelidiaceae Gelidiales Sid Daoui (33 °15'42.3"N 8 °30'10.9"W)
Gigartina acicularis Gigartinaceae Gigartinales Sidi Abed (33 °03'11.6"N 8 °41'18.1"W) 
Gigartina teedii Gigartinaceae Gigartinales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gracilaria cervicornis Gracilariaceae Gracilariales Sidi Abed (33 °03'11.6"N 8 °41'18.1"W) 
Gracilaria conferta Gracilariaceae Gracilariales Harass (33 °14'39.6"N 8 °28'27.0"W)
Gracilaria confervoides Gracilariaceae Gracilariales Sidi Abed (33 °03'11.6"N 8 °41'18.1"W) 
Gracilaria multipartita Gracilariaceae Gracilariales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Gracilaria sp Gracilariaceae Gracilariales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Grateloupia filicina Halymeniaceae Halymeniales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W)
Gymnogongrus griffithsiae Phyllophoraceae Gigartinales Herchan (32 °52'12.8"N 8 °52'22.0"W)
Gymnogongrus norvegicus Phyllophoraceae Gigartinales Sid Daoui (33 °15'42.3"N 8 °30'10.9"W)
Gymnogongrus patens Phyllophoraceae Gigartinales Herchan (32 °52'12.8"N 8 °52'22.0"W)
Halopitys incurvus Rhodomelaceae Ceramiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Halurus equisetifolius Wrangeliaceae Ceramiales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Hypnea musciformis Cystocloniaceae Gigartinales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Jania longifurca Corallinaceae Corallinales Moulay Abdellah(33 °11'56.1"N 8 °35'32.4"W)
Laurencia pinnatifida Rhodomelaceae Ceramiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Laurencia sp Rhodomelaceae Ceramiales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Myriogramme costata Delesseriaceae Ceramiales Sidi Abed (33 °03'11.6"N 8 °41'18.1"W) 
Plocamium coccineum Plocamiaceae Plocamiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Plocamium sp1 Plocamiaceae Plocamiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Plocamium sp2 Plocamiaceae Plocamiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Pterocladia capillacea Pterocladiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Pterocladia sp Pterocladiaceae Gelidiales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Rhodymenia sp Rhodymeniaceae Rhodymeniales Sidi Abed (33 °03'11.6"N 8 °41'18.1"W) 
Scinaia furcellata Scinaiaceae Nemoniales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Sphaerococcus coronopifolius Sphaerococcaceae Sphaerococcales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Sphaerococcus sp Sphaerococcaceae Sphaerococcales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Green algae (Chlorophyceae)
Bryopsis balbisiana Bryopsidaceae Bryopsidales Jorf (33 °08'32.0"N 8 °36'51.9"W)
Cladophora ramossissima Cladophoraceae Cladophorales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Cladophora sp Cladophoraceae Cladophorales Harass (33 °14'39.6"N 8 °28'27.0"W)
Codium adhaerens Codiaceae Bryopsidales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Codium elongatum Codiaceae Bryopsidales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Enteromorpha intestinalis Ulvaceae Ulvales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Enteromorpha ramulosa Ulvaceae Ulvales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Enteromorpha sp Ulvaceae Ulvales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Valonia macrophysa Valoniaceae Cladophorales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Valonia utricularis Valoniaceae Cladophorales Oualidia (32 °46'16.9"N 9 °00'01.9"W)
Ulva fasciata Ulvaceae Ulvales Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Ulva lactuca Ulvaceae Ulvales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Ulva rigida Ulvaceae Ulvales Sidi Bouzid (33 °09’-33 °16’N, 8 °30’-8 °45’W)
Ulva sp Ulvaceae Ulvales Sid Daoui (33 °15'42.3"N 8 °30'10.9"W)
Algae being identified
Esp N. I.1 - - Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Esp N. I.2 - - Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Esp N. I.3 - - Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Esp N. I.5 - - Sidi Bounaim (33 °22'37.2"N 8 °14'56.7"W) 
Esp N. I.6 - - Sid Daoui (33 °15'42.3"N 8 °30'10.9"W)
Esp N. I.7 - - Moulay Abdellah(33 °11'56.1"N 8 °35'32.4"W)
Esp N. I.8. - - Oualidia (32 °46'16.9"N 9 °00'01.9"W)
Esp N. I.9 - - Oualidia (32 °46'16.9"N 9 °00'01.9"W)
Esp N. I.10 - - Oualidia (32 °46'16.9"N 9 °00'01.9"W)

Preparation of extracts

A part of dried samples was milled into powder manually in the mortar and the obtained powder (1 g) was extracted with a solvent mixture chloroform/methanol (2/1; v/v) under continuous agitation during 1h at room temperature.

After filtration, the samples were extracted a second time with the same new solvent under ultrasound (Transonic-420) for 15 min and filtered. The two extracts were assembled and concentrated under reduced pressure (temperature ≤45 °C) using a rotary evaporator (Büchi Rotavapor R-3000). The concentrated samples were reduced to powder by lyophilization and the obtained powders were stored in a desiccator.

Evaluation of antioxidant activity by tlc plate

The chemical assay used to evaluate the antioxidant activity was developed in the laboratory; it is inspired by the method of Takao et al. [35]. The evaluation of antioxidant activity was realized by the technique of thin layer chromatography using the free radical dpph (Sigma-Aldrich).

The powder of each extract was dissolved in methanol (Fluka) using ultrasound for 5 min. Aliquots of each sample (2, 4 and 8 µl) were deposited on a silica gel plate (tlc Sil G25 UV 254 mm-Marcherey-Nagel, 10 x 20 cm, ep. 0.25 mm)using a micropipettes RINGCAPS (Hirschmann DIN/ISO7750). A methanol solution of dpph 6.10-4 mol/l was sprayed uniformly on the plate in shelter from the light. After development, the plates were read at the well determined time intervals. The images were made using a scanner (HP Deskjet 2050A) at the end of each period. The screening of antioxidant activity was realized by comparing two controls: ascorbic acid and (+) δ-Tocopherol (Sigma-Aldrich).

The antioxidant activity of the extracts was estimated by the fading of spots compared to controls spots.

Evaluation of the antioxidant activity by spectrophotometry

The antioxidant activity of the algae extracts which were reacted positively on tlc plate is evaluated by spectrophotometry based on the method described by Brand Williams et al. [36]. The antioxidant activity of different algae was evaluated by measuring the dpph degradation kinetics in the presence of obtained extracts at 5 mg/ml. The results were compared to the profile of dpph alone or in the presence of δ-tocopherol. A methanolic solution of dpph at 6.10-5 mol/l was prepared in advance and stored at 4 °C in the dark. The powders of crude extracts were dissolved in methanol. 0.1 ml of the extract solution was mixed with 3.9 ml of methanolic dpph. The reaction mixture was stirred vigorously with a vortex. The absorbances at 517 nm were measured at regular time intervals: 0 min, 1 min, 5 min, and every 15 min until reached a plateau. The negative control was prepared of 0.1 ml of methanol and 3.9 ml of dpph.

RESULTS

The harvested algae of the different sites on the coast of El Jadida show an important algal richness. This richness has characterized by the abundance of red algae compared to the brown and green algae. This distribution varies from site to site depending on coastal nature and in the function of human activities.

Screening of antioxidant activity on TLC plate

The preliminary screening of antioxidant activity in different algal extracts by tlc was based on the discoloration of dpph in the spots of deposits. The more deposit is discolored more antioxidant activity is important. The observed discoloration in the ascorbic acid spot and (+) δ-Tocophérol represents the reference.

Fig. 2: Chromatogram of the algae extracts and controls after 120 min of reaction with dpph 1: E. intestinalis sp1, 2: E. intestinalis sp2, 3: F. rufolanosa, 4: C. ramosissima, 5: F. spiralis sp1, 6: F. spiralis sp2, 7: G. acicularis, T1: Tocopherol, T2: Ascorbic Acid, 8: C. myriophylloides, 9: P. capillacea, 10: U. lactuca, 11: E. ramulosa, 12: G. spinulosum, 13: B. bifurcata, 14: G. multipartita sp1, 15: G. multipartita sp2, 16: G. teedii, 17: C. ericoides, 18: H. musciformis, 19: D. dichotoma, 20: C. adhaerens, 21: G. sesquipedale, 22: P. coccineum, 23: S. coronopifolius, 24: Corallina sp, 25: V. utricularis, 26: C. sinuosa, 27: C. elongatum and 28: P. pavonica, A: 2 µl, B: 4 µl et C: 8 µl

The analysis of the plate (fig. 2) reveals different reactions with dpph:

-Extracts that do not show any reaction with dpph (complete absence of discoloration): Falkenbergia rufolanosa, Sphaerococcus coronopifolius, Enteromorpha ramulosa.

-Extracts whose reaction was rapid, discoloration was visible from the first minutes: Gelidium sesquipedale, Cystoseira ericoides, Fucus spiralis, Dictyota dichotoma.

-Extracts in which the reaction was slow, fading appears only belatedly: Cystoseira myriophylloides, Plocamium coccineum, Pterocladia capillacea, and Esp N. I.1.

The chromatograms also show that the reaction with dpph, when it takes place, varies depending on the concentration of the deposited extract and the discoloration was more intense from bottom to upward of the tlc plate.

The analysis of several obtained chromatograms showed in some seaweed extracts that the reaction with dpph is increasingly important depending on incubation time (table 2).

All brown algae tested react positively with dpph and seem to possess antioxidant activity. In this class, the first batch of algae (64.28 %): Bifurcaria bifurcata, Colpomenia sinuosa, Cyctoseira ericoides, Cystoseira sp, Dictyota dichotoma, Fucus spiralis, Saccorhiza polyschides, Sargassum muticum, and Sargassum vulgare induce degradation of the dpph in the first 15 min.

Table 2: TLC screening of antioxidant a

Seaweed species Extraction yield (%) Reaction with dpph according to the incubation time
15 min 120 min
Brown algae (Phaeophyceae)
Bifurcaria bifurcata 0.62 +
Cladostephus verticillatus 0.39 NT
Colpomenia sinuosa 0.75 +
Cystoseira myriophylloides 4.22 -
Cystoseira ericoides 7.00 +
Cystoseira sp 1.10 +
Dictyopteris membranacea 3.87 -
Dictyopteris sp ND NT
Dictyota dichotoma 6.72 +
Fucus spiralis 5.47 +
Halopteris scoparia 0.13 -
Laminaria digitata ND -
Padina pavonica 9.07 -
Saccorhiza polyschides 3.64 +
Sargassum muticum 3.11 +
Sargassum vulgare 7.09 +
Red algae (Rhodophyceae)  
Asparagopsis armata 4.49 -
Bornetia secundiflora 6.36 -
Callithamnion tetricum 1.43 -
Callithamnion sp 1.63 -
Caulacanthus ustulatus 4.61 +
Ceramium flabelligerum 1.06 -
Ceramium sp1 ND -
Ceramium sp2 1.50 -
Chondria coerulescens 1.22 -
Chondria dasyphylla 1.79 -
Corallina sp 1.38 +
Falkenbergia rufolanosa ND -
Gelidium crinal ND NT
Gelidium latifolium 1.43 +
Geliduim pulchelum ND NT
Gelidium sesquipedale 2.92 +
Gelidium spinulosum 5.16 -
Gelidium sp1 0.67 +
Gelidium sp 2 1.86 +
Gelidium sp 3 ND +
Gigartina acicularis 0.74 +
Gigartina teedii 3.17 -
Gracilaria cervicornis ND -
Gracilaria sp ND -
Gracilaria multipartita 0.78 +
Gracilaria conferta 0.87 -
Gracilaria confervoides 2.21 -
Grateloupia filicina 1.60 -
Gymnogongrus griffithsiae 1.12 -
Gymnogongrus norvegicus 1.76 -
Gymnogongrus patens 6.27 -
Halopitys incurvus 4.61 +
Halurus equisetifoluis ND NT
Hypnea musciformis 5.31 -
Jania longifurca 1.35 -
Laurencia pinnatifida 6.16 +
Laurencia sp 2.27 NT
Myriogramme costata 0.56 NT
Plocamium coccineum 3.26 -
Plocamium sp1 4.58 -
Plocamium sp2 4.43 -
Pterocladia capillacea 1.16 -
Pterocladia sp 0.65 -
Rhodymenia sp 0.21 -
Scinaia furcellata ND +
Sphaerococcus coronopifolius ND -
Sphaerococcus sp 1.52 -
Green algae (Chlorophyceae)
Bryopsis balbisiana 1.83 -
Cladophora ramossissima 7.79 +
Cladophora Sp ND -
Codium adhaerens ND -
Codium elongatum 1.18 -
Enteromorpha intestinalis 0.76 +
Enteromorpha ramulosa ND -
Enteromorpha sp ND -
Ulva fasciata 3.51 -
Ulva lactuca ND +
Ulva rigida 1.11 -
Ulva Sp 2.10 -
Valonia utricularis 4.93 -
Valonia macrophysa ND NT
Algae being identified
Esp N. I.1 0.43 -
Esp N. I.2 2.15 -
Esp N. I.3 ND -
Esp N. I.5 ND NT
Esp N. I.6 2.82 NT
Esp N. I.7 4.34 NT
Esp N. I.8. ND NT
Esp N. I.9 ND NT
Esp N. I.10 1.49 NT

ctivity in different seaweed extracts at 15 min and 120 min of reaction with dpph

ND: not determined, NT: not tested, (-): No degradation of dpph in the spot of deposited extract, (+): Degradation of dpph in the spot of deposited extract

The second batch of algae (35.72 %) composed of Dictyopteris membranacea, Halopteris scoparia, Laminaria digitata, and Padina pavonica shows a slower reaction with the radical dpph, the spots are visible after an hour of incubation.

For the Rhodophyceae, of the 42 tested extracts, only 25 showed a positive reaction with dpph. 12 extracts (28.57 %) were reacted during the first 15 min, which is the case of Caulacanthus ustulatus, Corallina sp, Gelidium latifolium, Gelidium sesquipedale, Gelidium sp1, Gelidium sp2, Gelidium sp3, Gigartina acicularis, Gracilaria multipartita, Halopitys incurvus, Laurencia pinnatifida, and Scinia furcellata.

The algae Asparagopsis armata, Bornetia secundiflora, Ceramium flabelligerum, Ceramium sp1, Gelidium spinulosum, Gracilaria cervicornis, Gracilaria conferta, Gracilaria sp, Pterocladia capillacea, Plocamium coccineum, Plocamium sp1, Plocamium sp2, and Rhodymenia sp, were induced belatedly the discoloration of the dpph radical. For other algae tested, no reaction with dpph was detected after 120 min of incubation.

Moreover, a strong dpph discoloration was observed in the first 15 min in the presence of Halopitys incurvus extract, it was strongly accentuated after 120 min.

Of the 13 tested Chlorophyceae, the result shows the presence of the antioxidant activity in Bryopsis balbisiana, Cladophora ramossissima, Enteromorpha intestinalis, and Ulva lactuca.

For Bryopsis balbisiana, the degradation of dpph was observed beyond the first 15 min. The species of Cladophora sp, Codium adhaerens, Codium elongatum, Enteromorpha ramulosa, Enteromorpha sp, Ulva fasciata, Ulva rigida, Ulva sp, and Valonia utricularis show no activity with dpph.

Evaluation of the antioxidant activity by spectrophotometry

The antioxidant activity was also evaluated by spectrophotometer in the extracts of Rhodophyceae, Chlorophyceae, and Phaeophyceae. This second work step is performed on the only extracts which have reacted positively with dpph by tlc technique.

The dpph degradation kinetics in the presence of extracts from different species of Phaeophyceae is represented in fig. 3. At 120 min of incubation, all the tested algal extracts cause degradation of the free radical dpph. The percentage of remaining dpph in the medium varies depending on the tested extract. It is particularly greatly reduced in the presence of the Fucus spiralis extract (17.02 %) and Cystoseira ericoides extract (12.16 %).

Fig. 3: Kinetic degradation of dpph (%) alone, in the presence of Brown algae extracts or of tocopherol according to the time (min) I.11: Sargassum muticum, I.12: Cystoseira sp, I.13: Dictyopteris membranacea, I.8: Padina pavonica, I.6: Dictyota dichotoma, I.5: Cyctoseira ericoides, I.2: Fucus spiralis sp1 and I.4: Bifurcaria bifurcata

In addition, in the presence of Cystoseira ericoides extract, more than 50% of dpph was degraded in the first 5 min.

For the other tested algae: Bifurcaria bifurcata, Cystoseira sp, Dictyopteris membranacea, Dictyota dichotoma Padina pavonica and Sargassum muticum the dpph degradation is not negligible, the percentage of remaining dpph in the reaction medium at 120 min varies from 82.43 % (Dictyota dichotoma) to 54.87% (Cystoseira sp).

The tested Rhodophyceae also cause degradation of the dpph (fig. 4). The results show a modest degradation in the presence of the Corallina sp, Gigartina (Gigartina acicularis, Gigartina teedii), Gelidium (Gelidium latifolium, Gelidium sp1, Gelidium sp 2), and Pterocladia capillacea extracts. In fact, at 120 min of incubation, the percentage of remaining dpph in the medium varies from 69.53 % to 95.66 %. However, in the presence of Gracilaria multipartita or Halopitys incurvus extracts, dpph is highly degraded. For Gracilaria multipartita, nearly 62.78 % dpph is degraded at 120 min.

In the presence of Halopitys incurvus extract, the degradation of dpph is very important and very rapid; nearly 80% of dpph was degraded in the first 5 min of incubation. At 30 min, the remaining dpph in the reaction medium does not exceed 6.65 %.

Fig. 4: Kinetic degradation of dpph (%) alone, in the presence of Red algae extracts or of tocopherol according to the time (min) II.2: Pterocladia capillacea, II.12: Gelidium sp1, II.14: Gelidium latifolium, II.16: Gelidium sp 2, II.5: Gracilaria multipartita sp2, II.1: Gigartina acicularis, II.11: Corallina sp, II.6: Gigartina teedii and II.19: Halopitys incurvus

For Chlorophyceae, the crude tested extracts revealed a moderate antioxidant activity. The results in fig. 5 show a low degradation of dpph (5 to 20 %) in the presence of an algal extract of Codium adherens (9.94 %), Valonia utricularis (14.01 %), Ulva lactuca (17.39 %), and Codium elongatum (22.38 %).

Fig. 5: Kinetic degradation of dpph (%) alone, in the presence of green algae extracts or of tocopherol according to the time (min) III.6: Codium adherens, III.4: Ulva lactuca, III.7: Valonia utricularis et III.8: Codium elongatum

DISCUSSION

The Moroccan coast is rich in marine flora, nowadays, 612 species have been recorded: 102 Chlorophyceae, 131 Phaeophyceae, and 379 Rodophyceae [37].

The Moroccan Atlantic coast is particularly rich in algal biodiversity with a considerable species resource of economic importance. For this, Morocco is among the world's largest producers of agar or carrageenan [38] and the major part of algae used in this processing industry comes from the region of El Jadida [39]. This demand goes globally on increasing due to the development of new biotechnological applications.

The results of our sampling show that the coast of the region of El Jadida presents a significant algal biodiversity. This algal richness is dominated by red algae. These results are consistent with previous work realized by Hanif et al. [40] in this region.

Along the coast, the spatial distribution varies considerably between sites, sometimes because of pollution from inland waters of various types (industrial or domestic wastewaters, leachate of agricultural land).

Several methods were used to test the antioxidant activity in the macroalgae [4, 12, 41-43]. In this work, we have developed a method based on the works of Takao et al. [35] and Brand Williams et al. [36] who used the dpph assay.

dpph is a stable free radical widely used as a means to estimate the radical scavenging activity of natural antioxidant substances in algae [17, 44] or in the algal products [11, 25-26] by reason of its stability, the simplicity of the method and of the reproducibility of results.

The results obtained during the screening of antioxidant activity by tlc plate in the tested algae show that all tested Phaeophyceae possess antioxidant activity. These results are consistent with those reported for various brown algae harvested on different sites and using the free radical dpph: three species of Sargassum [12], Colpomenia sinuosa [7], Cystoseira sp, Dictyota dichotoma, Saccorhiza polyschides [17], Colpomenia sinuosa, Dictyota sp [45], Bifurcaria bifurcata, Dictyopteris membranacea, Dictyota dichotoma, Fucus spiralis, Laminaria digitata, Sargassum muticum, Sargassum vulgare [44] Sargassum vulgare [11], Fucus spiralis, Laminaria digitata and Sargassum muticum [22].

Of the 42 tested species of Rhodophyceae, nearly two thirds (58.5%) reacted positively against dpph during this first screening by tlc. Several studies describe the presence of antioxidant activity in red algae. For Chondria Coerulescens, Chondria dasyphylla, Gracilaria confervoides, Grateloupia filicina, Hypnea musciformis, Plocamium coccineum, Plocamium sp1, Pterocladia capillacea and Sphaerococcus coronopifolius, our findings are entirely consistent with those of Martins et al. [45], Meenakshi et al. [46] and Bouhlal et al. [34].

For the Green algae tested (13 species), only Bryopsis balbisiana, Cladophora ramossissima, Enteromorpha intestinalis and Ulva lactuca have shown a presence of antioxidant activity, in the three last species, the reaction is early with dpph and is more accentuated at 120 min. For Enteromorpha ramulosa and Ulva fasciata no activity was show. A similar result was mentioned in other works [45, 46, 53]. However, for Ulva lactuca, an important activity is observed in the first 15 min as opposed to the results of Martins et al. [46] who reported a low activity against the dpph.

The antioxidant activity of the harvested algae is also evaluated by spectrophotometry using the dpph radical, only the algal extracts have shown activity by tlc technique are evaluated in order to confirm or refute the results of the previous screening.

Three extracts show a strong antioxidant activity: Cystoseira myriophylloides, Fucus spiralis and Cyctoseira ericoides. Therefore, the percentage of remaining dpph in the medium after 120 min of incubation is respectively 35.07, 17.02 and 12.16%. A high activity was also highlighted in the species of Fucus spiralis by Farvin and Jacobsen [22].

Ruberto et al. [47] showed strong activity in 8 species of Mediterranean Cystoseira other than Cyctoseira ericoides and Cystoseira myriophylloides. In our study Cystoseira ericoides has proven the most active brown seaweed; the results show degradation of more than 60% of dpph in the medium as soon as the first 10 min.

In the species of Rhodophyceae, the results show a modest antioxidant activity in most of the tested extracts except that of Gracilaria multipartita and Halopitys incurvus. In Gracilaria nearly two-thirds of dpph present in the reaction medium are degraded after 120 min of incubation. Heo et al. [41] and Martins et al. [45], showed the presence of antioxidant activity in extracts of Gracilaria collected from the Brazilian coasts and the Icelandic coasts.

The Halopitys incurvus extract shows a strong antioxidant activity. A strong degradation of the dpph was observed in the first minutes. The dpph degradation kinetic in the presence of the extract is very comparable to that in the presence of tocopherol. This result is in opposition to those of Bouhlal et al. [34], who emphasized no activity in the methanol extract of this species.

The analyzed extracts of Chlorophyceae species showed a modest antioxidant activity. Martins et al. [45] also report a low activity of the Ulva lactuca extract and Codium extract against the dpph. This low activity in the extract of Ulva lactuca was also obtained by Meenakshi et al. [46] and Farvin and Jacobsen [22].

By these methodological approaches, we have been able to highlight the presence of the antioxidant activity in 45 algae extracts from a screening of 86 species of algae harvested from El Jadida Coast. Some of these algae have been studied and their antioxidant activity has been proven; Fucus spiralis [21-22, 48-51], Cystoseira ericoides [17, 21, 50], Colpomenia sinuosa [7, 16, 19], Padina pavonia [21], Dictyota dichotoma [16-18, 22], Bifurcaria bifurcata [12, 16], Halopitys incurvus [52], Gelidium sesquipedale [51], Sargassum vulgare [11, 14-15, 21, 52], Asparagopsis armata and Saccorhiza polyschides [21]. These studies confirm the results of our work on these algal species.

In this work, the obtained results for the various tested extracts were compared to tocopherol, a reference antioxidant molecule. These results remain interesting because it must be emphasized that the tocopherol is a known pure antioxidant molecule, while our samples are crude extracts (a mixture of several molecules). Moreover, it can be suggested that the fractionation operations and purification of these extracts could give results where the antioxidant activity would be more promising.

CONCLUSION

To our knowledge, this study is the largest screening of the antioxidant activity of macroalgae in the Moroccan Atlantic coast (region of El Jadida) at the present time.

In addition to the algal richness biodiversity represented by the three groups Phaeophyceae, Rhodophyceae, and Chlorophyceae, our study highlights a strong activity in Cyctoseira ericoides, Cystoseira myriophylloides, Fucus spiralis (Phaeophyceae) and in Gracilaria multipartita and Halopitys incurvus (Rhodophyceae). Moreover, the strong activity found in the extract of Cystoseira ericoides, and that of Halopitys incurvus is obtained for the first time on species living in the Moroccan coasts. Additionally, the methodological approach used in this study is reliable and can be used for screening the antioxidant activity on a wide range of plant extracts from the marine or terrestrial natural source.

Our results show the importance of marine algae as a potential source of active compounds, principally, antioxidant substances. These can have a major importance in the development of new drugs.

ACKNOWLEDGMENT

We are immensely grateful to Mr Mifdal Mohamed for his linguistic support.

AUTHORS CONTRIBUTIONS

The authors declare that they contributed to this article according to the information mentioned below:

CONFLICT OF INTERESTS

The authors declare that they have no conflicts of interest

REFERENCES

  1. Liu L, Heinrich M, Myers S, Dworjanyn SA. Towards a better understanding of medicinal uses of the brown seaweed Sargassum in Traditional Chinese Medicine: a phytochemical and pharmacological review. J Ethnopharm 2012;14:591-619.

  2. El Gamal AA. Biological importance of marine algae. Saudi Pharm J 2010;18:1-25.

  3. Stengel DB, Connan S, Popper ZA. Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application. Biotechnol Ad 2011;29:483–501.

  4. Kuda T, Tsunekawa M, Goto H, Araki Y. Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. J Food Compos Anal 2005;18:625–33.

  5. Shalaby EA. Algae as promising organisms for environment and health. Plant Signaling Behav 2011;6:1338-50.

  6. Zandi K, Fouladvand M, Pakdel P, Sartavi K. Evaluation of in vitro antiviral activity of a brown alga (Cystoseira myrica) from the persian gulf against herpes simplex virus type 1. Afr J Biotechnol 2007;6:2511-4.

  7. Lekameera R, Vijayabaskar P, Somasundaram ST. Evaluating antioxidant property of brown alga Colpomenia sinuosa (DERB. ET SOL). Afr J Food Sci 2008;2:126-30.

  8. Taskin E, Caki Z, Ozturk M, Taskin E. Assessment of in vitro antitumoral and antimicrobial activities of marine algae harvested from the eastern mediterranean sea. Afr J Biotechnol 2010;9:4272-7.

  9. Ashwini S, Suresh babut V, Sarith A, Manjula S. Seaweed extracts exhibit anticancer activity against HeLa cell lines. Int J Curr Pharm Res 2017;9:114-7.

  10. Jayasree P, Thiruchelvi R, Balashanmugam P. Evaluation of antibacterial, antioxidant, and anticancer potentials from marine red algae Gracilaria corticata. Asian J Pharm Clin Res 2018;1:347-50.

  11. Dore CMPG, Faustino Alvesa MGC, Pofírio Will LSE, Costa TG, Sabry DA, de Souza Rego LAR, et al. A sulfated polysaccharide, fucans, isolated from brown algae Sargassum vulgare with anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects. Carbohydr Polym 2013;91:467-75.

  12. Matsukawa R, Dubinsky Z, Kishimoto E, Masaki K, Masuda Y, Takeuchi T, et al. A comparison of screening methods for antioxidant activity in seaweeds. J Appl Phycol 1997;9:29-35.

  13. Connan S, Deslandes E, Ar Gall E. Influence of day–night and tidal cycles on phenol content and antioxidant capacity in three temperate intertidal brown seaweeds. J Experimental Mar Biol Ecol 2007;349:359-69.

  14. Nahas R, Abatis D, Anagnostopoulou MA, Kefalas P, Vagias C, Roussis V. Radical-scavenging activity of aegean sea marine algae. Food Chem 2007;102:577-81.

  15. Abd El Mageid MM, Salama NA, Saleh MAM, Abo Taleb HM. Antioxidant and antimicrobial characteristics of red and brown algae extracts. 4th Conference on Recent Technologies in Agriculture; 2009. p. 818-28.

  16. Demirel Z, Yilmaz Koz FF, Karabay Yavasoglu UN, Ozdemir G, Sukatar A. Antimicrobial and antioxidant activity of brown algae from the Aegean Sea. J Serb Chem Soc 2009;74:619-28.

  17. Zubia M, Fabre MS, Kerjean V, Le Lann K, Stiger Pouvreau V, Fauchon M, et al. Antioxidant and antitumoural activities of some phaeophyta from brittany coasts. Food Chem 2009;116:693-701.

  18. Ayyad SEN, Makki MS, Al-kayal NS, Basaif SA, El-Foty KO, Asiri AM, et al. Cytotoxic and protective DNA damage of three new diterpenoids from the brown alga Dictoyota dichotoma. Eur J Med Chem 2011;46:175-82.

  19. Kelman D, Kromkowski Posner E, McDermid KJ, Tabandera NK, Wright PR, Wright AD. Antioxidant activity of hawaiian marine algae. Mar Drugs 2012;10:403-16.

  20. Vijayabaskar P, Shiyamala V. Antioxidant properties of seaweed polyphenol from Turbinaria ornate (Turner) J. Agardh, 1848. Asian Pacific J Tropical Biomed 2012;S:90-8.

  21. Andrade PB, Barbosa M, Matos RP, Lopes G, Vinholes J, Mouga T, et al. Valuable compounds in macroalgae extracts. Food Chem 2013;138:1819-28.

  22. Farvin KHS, Jacobsen C. Phenolic compounds and antioxidant activities of selected species of seaweeds from danish coast. Food Chem 2013;138:1670-81.

  23. Yuan YV, Bone DE, Carrington ME. Antioxidant activity of dulse, Palmaria palmata, extract evaluated in vitro. Food Chem 2005;91:485-94.

  24. Plaza M, Cifuentes A, Ibanez E. In the search of new functional food ingredients from algae. Trends Food Sci Tech 2008;19:31-9.

  25. Ananthi S, Raghavendran HRB, Sunil AG, Gayathri V, Ramakrishnan G, Vasanthi HR. In vitro antioxidant and in vivo anti-inflammatory potential of crude polysaccharide from Turbinaria ornata (brown alga). Food Chem Toxicol 2010;48:187-92.

  26. Vijayabaskar P, Vaseela N, Thirumaran G. Potential antibacterial and antioxidant properties of a sulfated polysaccharide from the brown marine algae Sargassum swartzii. Chin J Nat Med 2012;10:0421−8.

  27. Etahiri S, Bultel Ponce V, Caux C, Guyot M. New bromoditerpenes from the red alga Sphaerococcus coronopifolius. J Nat Prod 2001;64:1024-7.

  28. Chiheb I, Riadi H, Martinez Lopez J, Dominguez Seglar JF, Gomez Vidal JA, Bouziane H, et al. Screening of antibacterial activity in marine green and brown macroalgae from the coast of morocco. Afr J Biotechnol 2009;8:1258-62.

  29. Younes F, Etahiri S, Assobhei O. Activité antimicrobienne des algues marines de la lagune d’Oualidia (Maroc): criblage et optimisation de la periode de la récolte. J Appl Biosci 2009;24:1543-52.

  30. Bouhlal R, Riadi H, Bourgougnon N. Antiviral activity of the extracts of rhodophyceae from. Morocco Afr J Biotech 2010;9:7968-75.

  31. Farid Y, Chennaoui M, Assobhei O, Etahiri S. Evaluation de l’effet du lieu de recolte des algues marines des cotes atlantiques marocaines sur L’activite antibacterienne et anti–inflammatoire. Microbiol Ind San Environn 2012;6:54-66.

  32. Oumaskour K, Boujaber N, Etahiri S, Assobhei O. Screening of antibacterial and antifungal activities in green and brown algae from the coast of Sidi Bouzid (El Jadida, Morocco). Afr J Biotech 2012;11:16831-7.

  33. Valls R, Piovetti L, Praud A. The use of diterpenoids as chemotaxonomic markers in the genus Cystoseira. Hydrobiologia 1993;26:549-56.

  34. Bouhlal R, Riadi H, Bourgougnon N. Antioxidant activity of rhodophyceae extracts from atlantic and mediterranean coasts of morocco. Afr J Plant Sci 2013;7:110-7.

  35. Takao T, Kitatami F, Watanabe N, Yagi A, Sakata K. A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shell fish. Biosci Biotechnol Biochem 1994;58:1780-3.

  36. Brand Williams W, Cuvelier E, Berset C. Use of free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 1995;28:25-30.

  37. Benhissoune S. Contribution à letude de la flore des macroalgues marines du Maroc (mediterranee et atlantique). PhD Thesis, Mohammed V University; 2002. 

  38. Givernand T, Sqali N, Barbaroux O, Orbi A, Semmaoui Y, Rezzoum NE, et al. Mapping and biomass estimation for a harvested population of Gelidium sesquipedale (Rhodophyta, Gelidiales) along the Atlantic coast of Morocco. Phycologia 2005;44:66-71.

  39. Hanif N, Chair M, Idrissi MC, Naoki T. Contribution to the algal biodiversity study in the moroccan atlantic coast. Int J Innov Res Sci Eng Tech 2014;3:12507-24.

  40. Hanif N, Chair M, Idrissi MC, Naoki T. L'exploitation des algues rouges Gelidium dans la région d’El Jadida: aspects socio-économiques et perspectives. Afr Sci 2014;10:103-26.

  41. Heo SJ, Cha SH, Lee KW, Jeon YJ. Antioxidant activities of red algae from jeju island. Algae 2006;21:149-56.

  42. Ragubeer N, Beukes DR, Limson JL. Critical assessment of voltammetry for rapid screening of antioxidants in marine algae. Food Chem 2010;121:227–32.

  43. Tierney MS, Smyth TJ, Rai DK, Soler Vil A, Croft AK, Brunton N. Enrichment of polyphenol contents and antioxidant activities of Irish brown macroalgae using food-friendly techniques based on polarity and molecular size. Food Chem 2013;139:753-61.

  44. Balboa EM, Conde E, Moure A, Falque E, Dominguez H. In vitro antioxidant properties of crude extracts and compounds from brown algae. Food Chem 2013;138:1764–85.

  45. Martins CDL, Ramlov F, Carneiro NPN, Gestinari LM, Santos BFD, Bento LM, et al. Antioxidant properties and total phenolic contents of some tropical seaweeds of the Brazilian coast. J Appl Phycol 2012;25:1179–87.

  46. Meenakshi S, Umayaparvathi S, Arumugam M, Balasubramanian T. In vitro antioxidant properties and FTIR analysis of two seaweeds of gulf of mannar. Asian Pacific J Tropical Biomed 2011;1:S66–S70.

  47. Ruberto G, Baratta MT, Biondi DM, Amico V. Antioxidant activity of extracts of the marine algal genus Cystoseira in a micellar model system. J Appl Phycol 2001;13:403–7.

  48. Cerantola S, Florian B, Erwan AG, Deslandes E. Co-occurrence and antioxidant activities of fucol and fucophlorethol classes of polymeric phenols in Fucus spiralis. Bot Mar 2006;49:347–51.

  49. Pinteus S, Azevedo S, Alves C, Mouga T, Cruz A, Afonso C, et al. High antioxidant potential of Fucus spiralis extracts collected from peniche coast (Portugal). New Biotechnol 2009;25:S296.

  50. Ferreres F, Lopes G, Gil-Izquierdo A, Andrade PB, Sousa C, Mouga T, et al. Phlorotannin extracts from fucales characterized by HPLC-DAD-ESI-MS: approaches to hyaluronidase inhibitory capacity and antioxidant properties. Mar Drugs 2012;10:2766-81.

  51. Grozdanic N, Stanojkovic TP, Kljajic Z, Etahiri S, Assobhei O, Konic Ristic A, et al. In vitro evaluation of antioxidant and antitumoral activities of marine algae Gelidium Sesquipedale and Fucus Spiralis. Eur J Cancer 2012;48:S26.

  52. Plaza M, Amigo Benavent M, del Castillo MD, Ibanez E, Herrero M. Facts about the formation of new antioxidants in natural samples after subcritical water extraction. Food Res Int 2010;43:2341–8.

  53. Satyalakshmi S. Determination of biological activities of three marine algae collected from visakhapatnam coast. Asian J Pharm Clin Res 2017;10:274-9.