• 1 •Chin J Integr Med

Marine organisms are rich sources of structurally new and biologically active metabolites.(1) In recent years, there have been many reports of macroalgae derived compounds that have a broad range of biological activities, such as antibiotic, antiviral, antioxidant, antifouling, anti-inflammatory, cytotoxic and antimitotic activities.(2) Many metabolites isolated from marine algae have been shown to possess bioactive effects.(3) In fact, the discovery of metabolites with biological activities from macroalgae increased signifi cantly in the past three decades.(4) On the other hand seaweeds have recently received significant attention for their potential as natural antioxidants.(5)

Marine algae extract have been demonstrated to have strong antioxidant properties,(6) protective effects against liver injury caused by carbon tetrachloride,(7) antimicrobial activity(8) and antiviral properties.(9) Antioxidants are used to preserve food quality mainly by prevention of oxidative deterioration of constituent of lipids. There is an increasing interest in natural antioxidants because of the safety and toxicity problems of synthetic antioxidants, such as butylated hydroxylanisol and butylated hydroxytoluene, which are commonly used in lipid-containing food.(10) Natural antioxidants can protect the human body from reactive oxygen species and free radicals, and retard the

progress of many chronic diseases as well as lipid oxidative rancidity in food.(11) Therefore, the aim of present study is to investigate the in vitro antimicrobial and antioxidant activities of different seaweed extracts from Hare Island to evaluate their potential antioxidative resources for food and medicinal industry.

METHODSCollection of the Algal Material

T h e m a r i n e m a c r o a l g a e v i z . , P a d i n a tetrastromatica, Stocheopremum marginatum and Phorphyra sp. were collected from the Hare Island, Tuticorin coast of Tamil Nadu, India (Lat 8°40'–8°55'N and Long 78°0'–78°15'E) during September–October 2010. The collected samples were thoroughly washed with fresh water to remove salt, sand, epiphytes, debris and other marine organism, then transported to laboratory for identifi cation.

ORIGINAL ARTICLEIn Vitro Antimicrobial and Antioxidant Activities of Seaweeds

from Hare Island, Tuticorin Coast of India

Sangappillai Thangaraj, Subramanian Bragadeeswaran, and Nadarajah Srikumaran

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2013Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai (608 502), Tamil Nadu, IndiaCorrespondence to: Dr. S. Thangaraj, Tel: 91-9952550844, E-mail: thangamjayam@gmail.com DOI: 10.1007/s11655-013-1548-x

ABSTRACT Objective: To assess the antimicrobial and antioxidant activities from methanolic extracts of brown and red seaweeds. Methods: Padina tetrastromatica, Stocheopremum marginatum and Phorphyra sp. were evaluated by in vitro standard methods. In antimicrobial assay, P. tetrastromatica extract showed highest zone inhibition against E. coli and S. typhi. The S. marginatum extract exhibited the maximum zone inhibition against S. typhi, minimum against K. pneumonia. Phorphyra sp. extract showed higher sensitivity against T. mirabilis. The antioxidant activities of seaweeds were determined at different concentration of 100, 300 and 500 μg/mL. Results: In 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total phenolic content assays, the S. marginatum extract was found the highest reducing power and the lowest showed by Phorphyra sp. and S. marginatum extracts. In the Linoleic acid peroxidation assay, the P. tetrastromatica extract showed the highest reducing power and minimum showed in S. marginatum extract. The Fourier-transform infrared spectra of the three seaweed extracts exhibited a strong broad, medium, weak and broadly-stretched peak at around 3,400 cm-1. Conclusion: Our study indicates that the seaweeds are potential source for antimicrobial and antioxidant agents and further study will fulfi ll for the purifi cation and structural characterization of the compounds.KEYWORDS Padina tetrastromatica, Phorphyra sp., 1,1-diphenyl-2-picrylhydrazy and total phenolic content

• 2 • Chin J Integr Med

Preparation of Algal ExtractsThe algae samples were shade dried for 15 days

and then pulverized into fi ne powder using pestle and mortar. The each 20 g powder of algae samples were soaked in MeOH separately and maintained for 5 days at room temperature. The extracts were filtered and evaporated to dryness under pressure using a rotary evaporator (Lark Innovative, India) to get a semi-solid residue.

Chemicals and ReagentsSod ium n i t r op russ ide , 1 ,1 -d ipheny l -2 -

picrylhydrazyl (DPPH), potassium chloride were purchased from Himedia Laboratory, Mumbai, India. While carbon tetra chloride, trichloro acetic acid, thiobarbituric acid, potassium hydroxide, potassium sulphate buffer were purchased from Subra Scientifi c Company, Pondicherry, India.

Evaluation of Antimicrobial ActivityAntibacterial activity was measured using a

disc diffusion method according to the Bauer, et al.(12) Chloramphenicol was used as a positive control. The plates were incubated at 37 ℃ for 24 h. The antibacterial test was assessed by measuring the diameter of the area in which bacterial growth was inhibited around the disc.

DPPH Radical ScavengingThe DPPH free-radical scavenging assay was

carried out in triplicate, based on the method used by Leong and Shui.(13) Two milliliters of 0.15 mmol/L DPPH were added to the different dilutions of the extract (amounting to 1 mL) and the reaction mixture was incubated for 30 min after which its absorbance was measured at 517 nm.

Determination of Total Content of Phenolic Compounds

The total content of phenolic compounds of three seaweeds were determined by a modification of the Folin-Ciocalteu method.(14) Briefly, 0.4 mL of 10% Folin-Ciocalteu solution was added to 0.2 mL of a sample. After an interval of 3 min, 0.8 mL of a 10% sodium carbonate was added. The mixture was allowed to stand for 1 h at ambient temperature and the absorbance was then measured at 750 nm.

Linoleic Acid PeroxidationThe antioxidant activity of the algal extracts were

determined by a spectrophotometric procedure using the hemoglobin catalyzed peroxidation of linoleic acid assay.(15,16) The algal extract (0.1 mL) was mixed with 0.025 mL linoleic acid-ethanol solution (0.1 mol/L) and 0.075 mL phosphate buffer (0.2 mol/L, pH 7.2) and 0.05 mL hemoglobin solution (0.08%) was added, the linoleic acid peroxidation was started. The reaction mixtures were incubated at 37 ℃ for 1 h, thereafter the reaction was stopped using 5 mL HCl-ethanol solution (0.6%). The thiocyanate method was then used to quantify the peroxidation: 0.02 mL of FeCl2 solution (0.02 mol/L) and 0.01 mL of ammonium thiocyanate (Aldrich) solution (30%) were added to 1.6 mL of the sample solution. Absorbance measurement was obtained at 490 nm. Catechin was used as positive control.

Fourier-Transform Infrared SpectroscopyFourier-transform infrared (FTIR) measurements

were performed in the transmission mode with a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector of the FTIR microscope (Bruker IRScope Ⅱ) coupled to an FTIR spectrometer (Bruker Equinox model 55/S). The spectra were obtained in the wave number range of 400–4,000 cm-1 at room temperature on a Nicolet-Avater-360. The solid samples of (10 μg) seaweed extracts were mixed with 100 μg of dried Potassium Bromide (KBr) and compressed to prepare a salt disc (10 mm diameter) for reading the spectrum further.

RESULTSSamples Collection

The seaweeds P. tetrastromatica (248.14 g), S. marginatum (34.85 g) and Phorphyra sp. (63.31 g) were collected. The solvents of seaweeds were concentrated under reduced pressure to give a dark greenish gummy mass of 3.27–20.73 g in dry weight.

Evaluation of Antimicrobial Activity of the Seaweeds

The methanolic extract of P . tetrastromatica extract showed highest zone inhibition (18 mm and 12 mm) against E. coli and S. typhi. Whereas the S. marginatum extract showed the maximum zone inhibition (12 mm) against S. typhi and minimum of 5 mm was observed against E. coli and T. mirabilis. In the case of Phorphyra sp. extract showed high sensitive against T. mirabilis with 9 mm of zone inhibition and lower zone of inhibition were observed

• 3 •Chin J Integr Med

against S. typhi and K. pneumoniae (Figure 1).

Evaluation of Antioxidant Activity of the Seaweeds

The values of percent DPPH scavenging activities of P. tetrastromatica, S. marginatum and Phorphyra sp. extracts were summarized in Figures 2–4. The highest percent (2.98%) DPPH radical scavenging activity was observed in S. marginatum at 500 μg/mL followed by standard (gallic acid), and lowest scavenging activity (0.14%) showed by Phorphyra sp. extract (Figure 2).

The linoleic acid peroxidation radical-scavenging

activities of proteins are shown in Figure 3. The crude extract of P. tetrastromatica at a concentration of 100–500 μg/mL showed a scavenging effect of 4.362%, 4.214% and 4.102%. The methanolic crude of P . tetrastomatica showed significantly higher phenol content of (2.98%) and S. marginatum exhibit lower content of (1.11%) as compared with other two seaweeds.

The IR spectra of the three seaweed extracts are shown in Figure 5 and Table 1. All samples exhibited strong and broadly-stretched peak at around 3,400 cm-1 that represent the characteristic of alcohols, phenols groups and a medium C-H band at around 2,950 cm-1 represent of alkanes. The peak at 1,638 cm-1 express medium N-H bend 1°amines, 1,508 cm-1 strong represent the N-O asymmetric stretch nitro compounds, the medium peak at 1,458 cm-1 shows the presence of C-H bend alkanes, and 1,403 cm-1 represents the presence of C-C stretch (in ring) aromatics. The strong 1,321 cm-1 represents of C-N stretch aromatic

Figure 1. Antibactrial Actvity of Three Seaweeds Extracts against Human Pathogens

Zone

of i

nhib

ition

(mm

)

T. mirabilis

18

16

14

12

10

8

6

4

2

0

P. tetrastromatica Positive Negative

S. typhi K. pneumoniae E. coli

AZo

ne o

f inh

ibiti

on (m

m)

T. mirabilis

16

14

12

10

8

6

4

2

0

S. marginatum Positive Negative

S. typhi K. pneumoniae E. coli

B

Zone

of i

nhib

ition

(mm

)

T. mirabilis

16

14

12

10

8

6

4

2

0

Phorphyra sp. Positive Negative

S. typhi K. pneumoniae E. coli

C

Figure 2. Scavenging of DPPH Radical

Per

cent

age

of D

PP

H

scav

engi

ng (%

)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

P. tetra

strom

atica

S. mar

ginatum

Phorp

hyra

sp.

Gallic

acid

100 μg/mL

300 μg/mL

500 μg/mL

Sample tesed

Figure 4. Total Phenolic Content

P. tetra

strom

atica

S. mar

ginatum

Phorp

hyra

sp.

Gallic

acid

100 μg/mL

300 μg/mL

500 μg/mL

5.04.54.03.53.02.52.01.51.00.5

0Per

cent

age

of S

cave

ngin

g (%

)

P. tetra

strom

atica

S. mar

ginatum

Phorp

hyra

sp.

Gallic

acid

100 μg/mL

300 μg/mL

500 μg/mL

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

Phe

nolic

con

tent

(%)

Figure 3. Linoleic Acid Peroxidation

• 4 • Chin J Integr Med

amines, the medium of bond at 1,243 cm-1 express C-H wag -CH2 X bonds that show the presence of alkyl halides, medium bond 1,079 cm-1 around express the C-N stretch bonds shows aliphatic amines, the 966 cm-1 represents =C-H bend that express the alkenes compound. The peak around 879 cm-1 represents strong C-H "oop" aromatics group, the broadly peak around 668 cm-1 shows the characteristic of strong N-H wag that represents of 1°, 2°amines group, finally the medium bond of 931 cm-1 shows the O-H bend characteristic of carboxylic acids.

DISCUSSIONSeaweeds are a renewable natural source

with diverse biological activities. Hence, in this present study P. tetrastromatica, S. marginatum and Phorphyra sp. were investigated for their antimicrobial and antioxidant activities. The methanolic extract of P . tetrastromatica extract showed the highest zone inhibition activity against E. coli and S. typhi. Whereas the S . marginatum extract showed the maximum inhibition against S . typhi and minimum activity was observed against K. pneumonia. In the case of Phorphyra sp. extract showed high sensitive against T. mirabilis. Del Val, et al(17) also reported the antibacterial activities of the methanol extract of Enteromorpha sp. against several pathogens. Similarly, the present study in methanolic extract of P. tetrastromatica showed a broad spectrum of inhibition as compared with other two seaweed extracts. In this study the seaweed extracts such as P. tetrastromatica and Phorphyra sp. showed a sensitivity against

Figure 5. FTIR Spectra of the Polysaccharide of Three Seaweeds Extract

Notes: (a) P. Tetrastromatica, (b) S. Marginatum and (c) Phorphyra sp.

0.60

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0

A

4000 3600 3200

34093400

29362360

22742079

1639

15351459

14511404

13241249

10811022

966931

889874

719668

505

1054

1509

2342

2800 2400cm-1

2000 1800 1600 1400 1200 1000 800 600 400

b

415

0.900.850.750.700.650.600.550.500.450.400.350.300.250.200.150.100.05

0

A

4000 3600 3200

34033388

3755

29342854

23602342

22692076

1720

16561641

152914611511

14511440

1403

1303

1248

1082

1025

967931

889875

719668

6245061324

13511552

2800 2400cm-1

2000 1800 1600 1400 1200 1000 800 600 400

c

1.51.41.31.21.11.00.90.80.70.60.50.40.30.20.1

0

A

4000 3600 3200

3459

34363425

34193406

3394

3372

2958

2922 28512360

1638

15081499

14581403

13211243 1079

1039

879

6682342

207122662872

3466

2800 2400cm-1

2000 1800 1600 1400 1200 1000 800 600 400

a

Table 1. Tentative Identifi cation of FTIR Peaks for Seaweed Extracts P. Tetrastromatica, S. Marginatum and Phorphyra sp. (wave number, cm-1)

No P. tetrastromatica S. marginatum Phorphyra sp. Peak assignments Functional group

1 3436 (s, b) 3400 3400 O-H stretch, H-bond Alcohols, phenols

2 2958 (m) 2936 2934 C-H stretch Alkanes

3 1638 (m) 1639 — N-H Bend, Amide I band 1°amines, secondary structure

4 — — 1656 -C=C- stretch Alkenes

5 1508 (s) 1535 1529 N-O asymmetric stretch Nitro compounds

6 1458 (m) 1459 1461 C-H bend Alkanes

7 1403 (m) 1404 1403 C-C stretch (in ring) Aromatics

8 1321 (s) 1324 1324 C-N stretch Aromatic amines

9 1243 (m) 1249 1248 C-H wag –CH2 X Alkyl halides

10 1079 (m) 1081 1082 C-N stretch Aliphatic amines

11 — 966 (s) 967 =C-H bend Alkenes

12 879 (s) 889 875 C-H "oop" Aromatics

13 668 (s, b) 668 668 N-H wag 1°, 2°amines

14 — 931 (m) — O-H bend Carboxylic acids

Notes: s, strong; b, broad; m, medium; w, weak

• 5 •Chin J Integr Med

K . pneumonia and T . mirabi l is strains. Such unusual response could be attributed to masking of antibacterial activity by the presence of some inhibitory compounds in the extract by Sastry and Rao.(18) Some studies concerning the effectiveness of extraction methods highlight that methanol extraction yields higher antimicrobial activity than n-hexane and ethyl acetate.(18,19) The present investigation revealed that the methanolic extract of P. tetrastromatica extract showed high inhibition zones than S. marginatum and Phorphyra sp. extracts as compared with standard antibiotic. The variation of antibacterial activity of the organic solvent extracts might be due to the presence of different antibacterial substances among these species as suggested by Lustigman and Brown.(20)

In the present work, the highest percent of DPPH radical scavenging activity was observed in P. tetrastromatica than other seaweed extracts. These results illustrate that compounds with the strongest radical scavenging activity in medium polarity. The results corroborates well with earlier reports in other higher plants including brown/red seaweeds(21) and their enzymatic extracts.(22) The crude extract of P. tetrastromatica at a concentration from 100–500 μg/mL showed a high scavenging effect from 4.362%, 4.214% and 4.102% than other seaweed extracts. The result corroborates well with those studies by Duan, et al(23) and Wang, et al(24) who observed a higher yield of aqueous fraction in the red alga, Polysiphonia urceolata (36.36%) and Rhodomela confervoides (72.30%), due to many polysaccharide was extracted from the algae. However, in contrast with results of Chandini, et al,(5) higher yield was observed in petroleum ether (28.40%), ethyl acetate (27.54%) and aqueous fraction (30.27%) of T. conoides, P. tetrastomatica and S. marginatum, respectively. It is thought that phenolic compounds are one of the most effective antioxidants in brown algae.(25,26) As shown in Figure 4, the content was high in methanolic extract of P. tetrastromatica and lower in S. marginatum extract. Jimenez-Escrig, et al(25) reported that the decrease of phenolic compounds due to drying and storage was different according to the variety of algae. Many researches stated that phenolic compounds are one of the most effective antioxidants in brown algae.(5,26) In our study, the methanolic crude of P. tetrastomatica showed significantly higher phenol content of 2.98% and S. marginatum showed lower phenol content of 1.11%. However, as compared with the results of

Duan, et al(23) and Wang, et al(24) phenolic content of methanolic extracts in the fi ve brown alga studied was obviously lower than the red algae R. confervoides and P . urceolata. Phenolic compounds are widely distributed in the plant kingdom and have been reported to have several biological activities including antioxidant properties. Earlier reports revealed that marine seaweed extracts especially their polyphenols have antioxidant activity.(23,27,28) The major active compounds in different seaweed extracts have been reported to be phlorotannins and fucoxanthin.(27)

The present results of the IR spectra coincided with Parasakthi(29) observed the FTIR peaks at 534.61, 1,024.16, 1,321.88, 1,380.81 and 1,640.40 cm-1 in chitin from the shell of S. aculeata which resembles the peaks of crab carapace, legs and the claw. Saraswathy, et al(30) observed the major absorption band between 1,220 and 1,020 cm-1 which represents the free amino group (-NH2) at C2 posit ion of glucosamine, a major group present in chitosan. Thus the current studies revealed the presence of potent antimicrobial and antioxidant compounds in marine seaweeds which could be used in pharmacological research.

AcknoledgmentsAuthors wish to thank the authority of Annamalai

University, Annamalai nagar for providing necessary facilities.

REFERENCESEly R, Supriya T, Naik CG. Antimicrobial activity of marine 1.

organisms collected off the coast of South East India. J Exp

Mar Biol Ecol 2004;09:121-127.

Salvador N, Garreta AG, Lavelli L, Ribera M. Antimicrobial 2.

activity of Iberian macroalgae. Sci Mar 2007;1:101-113.

Faulkner DJ. Marine natural products. Nat Prod Rep 3.

2002;19:1-49.

Smit AJ. Medicinal and pharmaceutical uses of seaweed 4.

natural products: a review. J Appl Phycol 2004;16:245-262.

Chandini SK, Ganesana P, Bhaskar N. 5. In vitro antioxidant

activities of three selected brown seaweeds of India. Food

Chem 2007;107:707-713.

Yuan YV, Walsh NA. Antioxidant and antiproliferative 6.

activities of extracts from a variety of edible seaweeds.

Food Chemical Toxicol 2006;44:1144-1150.

Wong CK, Ooi VEC, Ang PO. Protective effects of seaweeds 7.

against liver injury caused by carbon tetrachloride in rats.

Chemosphere 2000;41:173-179.

• 6 • Chin J Integr Med

Valdebenito H, Bittner M, Sammes PG, Silva M, Watson 8.

WH. Phytochemistry 1982;21:1456-1457.

Chatterji A, Dhargalkar VK, Sreekumar PK, Parameswaran 9.

PS, Rodrigues R, Kotnala S. National Inst i tute of

Oceanography, Goa, India, 2004;24.

Amarowicz R, Naczk M, Shahidi F. Antioxidant activity of 10.

various fractions of non tannin phenolics of canola hulls. J

Agric Food Chem 2000;48:2755-2759.

Kinsella JE, Frankel E, German B, Kanner J. Possible 11.

mechanisms for the protective role of antioxidants in wine

and plant foods. Food Tech 1993;47:85-89.

Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibitic 12.

susceptibility testing by a standardized single disc method.

Am J Clin Patol 1996;45:493-496.

Leong LP, Shui G. An investigation of antioxidant capacity 13.

of fruits in Singapore markets. Food Chem 2002;76:69-75.

Oki T, Masuda M, Furuta S, Nishiba Y, Terahara N, 14.

Suda I. Involvement of anthocyanins and other phenolic

compounds in radical scavenging activity of purple-fl eshed

sweet potato cultivars. J Food Sci 2002;67:1752-1756.

Kuda T, Tsunekawa M, Hishi T, Araki Y. Antioxidant 15.

properties of dried kayamo-nori, a brown alga Scytosiphon

lomentaria (Scytosiphonales, Phaeophyceae). Food Chem

2005;89:617-622.

Fant J, Grandon B, Rathinasabapathi B, Olson ST, Lyrene 16.

P. Antioxidant potential of fruits: evaluation of two blueberry

cultivars using an in vitro assay. Proc Fla State Hort Soc

2005;118:250-252.

Del Val AG, Platas G, Basilio A, Cabello A, Gorrochateui 17.

J, Suay I, et al. Screening of antimicrobial activities in red,

green and brown macroalgae from Gran Canaria (Canary

Islands, Spain). Int Microbiol 2001;4:35-40.

Sastry VMVS, Rao GRK. Antibacterial substances from 18.

marine algae: successive extraction using benzene,

chloroform and methanol. Bot Marina 1994;37:357-360

Moreau J, Pesando D, Bernad P, Caram B, Pionnat 19.

JC. Seasonal variations in the production of antifungal

substances by some Dictyotales (brown algae) from French

Mediterrean coast. Hydrobiology 1988;162:157-162.

Lustigman B, Brown C. Antibiotic production by marine 20.

algae isolated from the New York/New Jersey Coast. Bull

Environm Contamin Toxicol 1991;46:329-335.

Kuda T, Tsunekawa M, Goto H, Araki Y. Antioxidant 21.

properties of four edible algae harvested in the Noto

Peninsula, Japan. J Food Compos Anal 2005;18:625-633.

Park PJ, Shahidi F, Jeon YJ. Antioxidant activity of 22.

enzymatic extracts from a brown seaweed Ecklonia cava

by electron spin resonance spectrometry and comet assay.

J Food Lipids 2004;11:15-28.

Duan XJ, Zhang WW, Li XM, Wang BG. Evaluation of 23.

antioxidant property of extract and fractions obtained from a

red alga Polysiphonia urceolata. Food Chem 2006;95:37-43.

Wang BG, Zhang WW, Duan XJ, Li XM. 24. In vitro antioxidative

activities of extract and semi-purifi ed fractions of the marine

red alga Rhodomela confervoides (Rhodomelaceae). Food

Chem 2009;113:1101-1105.

Jimenez-Escrig A, Jimenez-Jimenez I, Pulido R, Saura-25.

Calixto F. Antioxidant activity of fresh and processed edible

seaweeds. J Sci Food Agricult 2001;81:530-534.

Nagai T, Yukimoto T. Preparat ion and funct ional 26.

properties of beverages made from sea algae. Food Chem

2003;81:327-332.

Yan X, Chuda Y, Suzuki M, Nagata T, Yan XJ. Fucoxanthin 27.

as the major antioxidant in Hijikia fusiformis a common edible

seaweed. Biosci Biotechnol Biochem 1999;63:6050-607.

Ganesan P, Chandini SK, Bhaskar N. Antioxidant 28.

properties of methanol extract and its solvent fractions

obtained from selected Indian red seaweeds. Biores Tech

2008;99:2717-2723.

Parasakthi MS, ed. Extraction of chitin from two cephalopods. 29.

India. M.Sc., Dissertation, Annamalai University; 2004:18.

Saraswathy G, Pal S, Rose C, Sastry TP. A novel bio-30.

inorganic bone implant containing deglued bone, chitosan

and gelatin. Bull Mater Sci 2001;24:415-420.

(Received December 3, 2011)Edited by YUAN Lin