International Journal of Drug Development and Research

Keywords

Codium adhaerens, antioxidant, scavenging activity.

INTRODUCTION

Seaweeds are commonly called as marine macroalgae are classified into three divisions based on their colors such as Chlorophyta, Rhodophyta and Phaeophyta. Algae are renewable natural sources potentially rich in sulfated polysaccharides, with a predominance of one or another polysaccharide depending on the group. In red algae, there are mainly sulfated galactans, green algae have more heterogeneous sulfated polysaccharides that are rich in galactose, mannose, xylose, arabinose, glucose and brown seaweed are rich in fucans, laminarin and alginic acid [1]. Seaweeds are growing in extreme environment, this factor implies that seaweed have some protective mechanisms and compounds (Matasukawa et al., 1997). They are considered as a major source of secondary metabolites with broad spectrum of biological activities. Seaweeds are rich in nutritional value mainly due to the presence of proteins, polysaccharides, minerals and vitamin content [2] A free radical is a molecule with more unpaired electrons in the outer orbital. Most of these free radicals are in the form of high reactive oxygen and nitrogen species produce due to oxidative stress leads to serious damages in the tissue. In search of novel source of antioxidants in the last few decades, marine algae have been widely studied for their antioxidant activity. A recent report reveals that marine algae are rich sources of antioxidant compounds [3]. Polyphenols are the major compounds for the antioxidant properties of seaweeds. Polyphenols antioxidant property is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers [4]. The objective of the current study was to evaluate the antioxidant potential of ethyl acetate extract of Codium adhaerens.

MATERIALS AND METHODS:

Sample collection

Green alga Codium adhaerens collected from the Pudumadam region, Mandapam, Southeast coast of India during the period of January 2014 by hand picking. After collection, they were washed thoroughly with seawater followed by distilled water thrice to remove salt and debris present. They were shade dried, grounded using mechanical grinder, packed in a sterile container and stored in the refrigerator until use.

Marine algal extract preparation

Soxhlet extraction of C. adhaerens was carried out with ethyl acetate solvent. Ten grams of algal powder was added with 200 ml of ethyl acetate was used for extraction and it was carried out in soxhlet apparatus for a period of 18 hours at room temperature and condensed using rotary vaccum evaporator and stored in the refrigerator at 4º C for further analysis [5].

Preliminary phytochemical screening

Phytochemicals present in the crude extract of C. adhaerens such as alkaloids, flavonoids, phenolics, proteins, saponins, steroids, tannins, terpenoids was determined by following the standard protocol described by Harborne, Trease and Evans [6-7]

Total phenolic content

The total phenolic content present in the crude extract of C. adhaerens were evaluated according to the method of Malik and Singh [8]. 1 ml of extracts were taken and made up to 3 ml with distilled water. Then, 0.5 ml of 1:1 dilution of Folin’s ciocalteau reagent and 2 ml of 20% Sodium carbonate were added to it. The mixture was boiled for a 1 min, cooled, incubated at 30 min at dark and absorbance was measured at 650 nm against the blank. A standard graph was plotted using known concentration of gallic acid. The concentration of phenols present in the graph were calculated from the calibration curve and expressed as mg/g of Gallic acid equivalents

Total flavonoid content

The total flavonoid content present in the extract of C. adhaerens was determined by following the method of Zishen et al. with little modifications [9]. 1 ml of extract was mixed with 5 ml of distilled water and 300μl of 5% Sodium nitrite solution and incubated for 6 min. Then, 500 μl of 10% aluminium chloride was added, after 5 min, 2 ml of 1M sodium hydroxide was added to the mixture and then the volume was made up to 10 ml with distilled water and absorbance was measured at 510nm against the blank. Quercetin was used to calibrate the standard curve and the results were expressed as mg/g Quercetin equivalents.

Total antioxidant activity

The total antioxidant activity of the crude extract of C. adhaerens was determined by the phosphomolybdneum method [10]. Various range of the extracts (200-1000) μg/ml was prepared. 1ml of each extract was mixed with 2 ml reagent (0.6M sulphuric acid, 28mM sodium phosphate, 4mM ammonium molybdate), incubated at 95ºC for 90 min and the absorbance was measured at 635nm using UV spectrophotometer. The percentage of antioxidant activity was calculated using the following formula.

(1)

Ac = Absorbance of control; As = Absorbance of sample.

Reducing power assay

Different concentration of the extracts (200-1000) μg/ml was prepared using distilled water. 1 ml of each extract were mixed with 2.0 ml of 0.2 M phosphate buffer and 2.5 ml of 1% potassium ferric cyanide and incubated for 20 min at 50ºC. 2.5 ml of 10% Trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. 2.5ml distilled water and 0.5 ml of 1% Ferric chloride was mixed with 2.5 ml of supernatant and the absorbance was measured at 700nm.[11]

DPPH free radical scavenging assay

The scavenging activity of C. adhaerens extract against DPPH free radical was determined according to the procedure of Blios [12]. Various range of concentration (200-1000) μg/ml was prepared using distilled water. 1 ml of each extract was mixed with 2ml of 0.16mM DPPH in methanol and incubated at dark for 20 min at room temperature. After incubation, the absorbance of the sample was measured at 520nm using UV spectrophotometer against the blank. The percentage of scavenging activity of C. adhaerens against the DPPH was calculated using the equation 1.

Superoxide free radical scavenging assay

The superoxide anion radical scavenging activity of C. adhaerens was evaluated by the method of Liu [13]. Various range of concentration (200-1000) μg/ml was prepared using distilled water. 1ml of each extract was mixed with 0.5 ml of 2.52mM NBT, 0.5ml of 64μM NADH and 60μM phenanzine methosulphate and incubated at room temperature for 15 min. Absorbance of the sample was read at 560nm using UV spectrophotometer. The percentage of scavenging activity of C. adhaerens against superoxide anion was calculated using the equation1.

Hydrogen peroxide radical scavenging assay

The capability of C. adhaerens extract was determined by the method of Ruch [14]. Various range of concentration (200-1000) μg/ml was prepared using distilled water. 40mM hydrogen peroxide was prepared using phosphate buffer (pH 7.4). 1ml of each extract was added with 600μl of 40mM hydrogen peroxide and incubated for 20 min at room temperature against the blank. The percentage of scavenging activity of C. adhaerens against hydrogen peroxide radical was calculated using the equation1.

Nitric oxide scavenging assay

Nitric oxide scavenging activity of C. adhaerens extract was measured by the Griess reaction [15]. Various range of concentration (200-1000) μg/ml was prepared using distilled water. 500μl of 5mM sodium nitroprusside in phosphate buffer was added to 1 ml of each extract and incubated at 25ºC for 30 min.2 ml of Griess reagent (1% sulphanilamide, 0.1% napthylethylene diamine dichloride and 3% phosphoric acid) was added to the reaction mixture and absorbance was measured at 560nm using UV spectrophotometer against blank. The percentage of scavenging activity of C. adhaerens against nitric oxide free radical was calculated using the equation 1.

RESULTS

In the present study, Green alga Codium adhaerens was collected and screened for the presence of major phytoconstituents. The ethyl acetate extract of Codium adhaerensreveals the presence of phytochemicals such as alkaloids, flavonoids, phenolics, glycosides, proteins, tannins, terpenoids and steroids as shown in the table 1 which could be the responsible for major bioactivity.

of biological activities mainly antioxidant activity [16].The total phenolics present in the C. adhaerensethyl acetate extract were determined by Folin’s – ciocalteau reagent and the results were expressed as mg/g Gallic acid equivalents. The phenolic contents were found to be 53.11 ± 0.29 mg/g GAE equivalents. Flavonoids are the most important natural polyphenolic due to their wide range of chemical and biological activities including antioxidant and free radical scavenging activities [17]. The total flavonoids present in the ethyl acetate extract of C. adhaerens were evaluated by aluminium chloride method and the results were expressed as52.45 ± 0.37 mg/g Quercetin equivalents. The total antioxidant activity of green alga C. adhaerens was evaluated by the phosphomolybdate method. C. adhaerens ethyl acetate extract exhibited the maximum antioxidant activity at 1000μg/ml as (74.61 ± 1.55) %.

Fe (III) reduction is often used as an indicator of electron donating activity, which is an important mechanism of phenolic antioxidant action [18]. The reductive potential of the crude ethyl acetate extract of C. adhaerens is the reduction of ferric to ferrous ion transformation. Increasing absorbance indicates the increasing reducing potential of extract. In this study, the reducing power increases with the increasing concentration of the sample as shown in the fig 2.

DPPH is a quick and extensively used method for evaluating the scavenging ability of the extracts. In this assay, the extracts have a capability to reduce the stable DPPH radical to the yellow color product, diphenyl picryl hydrazine. This assay is based on the reduction of DPPH alcoholic solution in the presence of a hydrogen-donating antioxidant due to the formation of the nonradical form DPPH-H by the reaction [19].It was found that the scavenging activity of the ethyl acetate extracts of C. adhaerens as increases with increasing concentration of sample as depicted in the fig 2. At 1000μg/ml, the maximum activity was found to be (72.92 ± 0.23)%,

The ethyl acetate extract of C. adhaerens was evaluated for superoxide scavenging assay by the nitroblue tetrazolium reduction method and the results are depicted in the figure 4. At 1000 μg / mL, maximum scavenging activity of (59.41 ± 1.58) %,was observed. This result indicates that the scavenging activity is directly proportional to the concentration of the compound. Nitric oxide scavenging activity of the C. adhaerens extract was measured using Griess reaction. This assay is based on the principle at physiological pH, sodium nitroprusside in aqueous solution generates nitric oxide which interacts with oxygen to produce nitrite ions. Scavengers present in the extract compete with oxygen leads to the decreased production of nitrite ions [20]. The extracts have shown the scavenging activity in a dose dependent manner as shown in the fig 5. At 1000μg/ml, the C. adhaerens has shown the scavenging activity as(52.41 ± 1.40)%

The capability of the C. adhaerens extract to effectively scavenge hydrogen peroxide was determined. Scavenging of H2O2 by extracts may be attributed to their phenolics, which can donate electrons to H2O2, thus neutralizing it to water [21]. The extracts were shown the scavenging activity in a concentration dependent manner. The scavenging activity increased with the increasing concentration of the sample as shown in the fig 6. At 1000μg/ml, it

DISCUSSIONS

Seaweeds are considered as a major food ingredient in Asian countries includes Japan, Korea, Malaysia and china. In Asia, it has been used for centuries in the preparation of salads and soups [22]. Naturally seaweeds are contains novel antioxidant compounds which control the free radical formation from metabolic reaction. Recent years, it has drawn attention of many researchers worldwide due to the rich source of antioxidant compounds [23]. In preliminary phytochemical screening, major phytoconstituents were found in the ethyl acetate extract of C. adhaerens. Many types of seaweed were reported to contain active compounds that can cure diseases. Now a day, most of the people started to use remedies of natural origin for curing illness [24]. Phytoconstituents such as phenolics, flavonoids, tannins have been found in the ethyl acetate extract of C. adhaerens. The presence of polyphenolic compounds is a good indicator of antioxidant activities in terms of their ability to scavenge free radicals [25]. The uniqueness of their molecular skeleton and structures has contributed to the strong antioxidant activity. Polyphenols for instant uses its phenol rings as electron traps for free radicals [26]. Many methods are available to detect the antioxidant activity of the seaweeds. In this present study, the antioxidant activity was determined by the phosphomolybdate method, scavenging activity against free radicals DPPH, Superoxide, nitric oxide and hydrogen peroxide was done. In this present study, the green alga C. adhaerens has shown high phenolic content, antioxidant activity and scavenging activity. Likewise, previous reports have been demonstrated that green algae have high antioxidant potential and scavenging activity. Several studies have been demonstrated that the correlation between the phenolic content and antioxidant activity of certain seaweeds [27]. Antioxidant potential of the ethyl acetate extract of C. adhaerens was assayed by the phosphomolybdate method. It has shown the maximum antioxidant activity at the concentration of 1000 μg/ml. Reducing potential of the extract was measured by the potassium ferric cyanide method and it has shown high reducing capability. The reducing ability of the compound mainly depends on the presence of reductones, which exhibits the antioxidant potential by breaking the free radical chain by donating the hydrogen atom [16]. It is a significant indicator of antioxidant potential. Similar studies have also been reported by several researchers in marine macro algae [28-29].

DPPH assay is a reliable, accurate method for assessing the scavenging activity of the compound. DPPH is a purple color compound generally fades into yellow color when an antioxidant is present in the medium. Thus antioxidant molecules can quench the free radical DPPH by donating hydrogen atom or accepting electrons in hydrogen atom. Rapid decrease of absorbance indicates the more potent antioxidant activity of the extract [30]. C. adhaerens has shown the maximum scavenging of (72.92 ± 0.23) %,Superoxide anion radicals are produced by various flavoenzymes like xanthine oxidase, which converts hypoxanthine to xanthine and subsequently touric acid in ischemiareperfusion. In the PMS-NADH-NBT system, superoxide anion derived from dissolved oxygen by PMS-NADH coupling reaction reduces NBT [31]. The absorbance decrease at 560nm indicates the scavenging ability of extract in the reaction. C. adhaerens ethyl acetate extract has shown the scavenging activity against superoxide free radical as (59.41 ± 1.58) %,

Different species of seaweeds possess scavenging ability of hydrogen peroxide. It could cross membrane and might oxidize a compound. H202 alone is not a reactive but rarely it cause damage to the cells due to the production of hydroxyl radicals in the cells [32]. Hydrogen peroxide scavenging activity of C, adhaerens ethyl acetate extract as found to be (49.17 ± 0.83) % Antioxidant properties of phenolics might be due to their ability to act as reductones, hydrogendonors, free radical quenchers which prevents the formation of free radicals [33]. C. adhaerens antioxidant activity might be due to the presence of higher polyphenolic content in it.

CONCLUSION

Based on the results observed, it can be concluded C. adhaerens has high antioxidant and free radical scavenging activity. The present study reveals C. adhaerens extract might be utilized as a natural source of antioxidant, supplement in food and pharmaceutical industry. Further study is must to isolate and characterize the specific bioactive compound present in it responsible for antioxidant activity.

AKNOWLEDGEMENT

We would like to thank the management of VIT University for providing the facilities and constant encouragement for the research.

Tables at a glance

Table 1

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

References

1. 1) Rocha HAO, Farias EHC, Bezerra LCLM, Albuquerque IRL, Medeiros VP, Queiroz KCS, Leite EL. Polissacarídeossulfatados de algasmarin has com atividadeanticoagulante. Infarma 2004; 16: 1-2.
2. 2) Ruperez P, Calixto F S, Dietary and physicochemical properties of edible Spanish seaweeds. European Food Research and Technology2001; 212:349-354.
3. 3) ShyamalaViswanathan, Ebciba C, Santhiya R ThangarajuNallamuthu. Phytochemical Screening and In Vitro Antibacterial, Antioxidant and Anticancer Activity of AmphiroaFragilissima (Linneaus) J V Lamoroux. International Journal of Innovative Research in Science, Engineering and Technology 2014; 3(5): 12933-47.
4. 4) Rice-Evans CA. Measurement of total antioxidant activity as a marker of antioxidant status in vivo. Procedures and limitations, Free Radical Research 2000; 33:59-66.
5. 5) Parekh J, Nair R, Chanda S. Preliminary screening of some folkloric plants from Western India for potential antimicrobial activity. Indian Journal of Pharmacology 2005; 37: 408-409.
6. 6) Harborne JB, Williams CA. Advances in flavonoid research.Phytochemistry 2000; 55:481-485.
7. 7) Trease GE, Evans WC. Pharmacognosy, 11th edn.,BailliereTindall, London 1989; 45-50.
8. 8) Malik EP, Singh MB. Plant Enzymology and Hittoenzymology (1st Edn).1980; 292.
9. 9) Zishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry 1999; 64: 555-59.
10. 10) Prieto P, Pineda M, Aguilar MM. Spectrophotometric quantification of antioxidant capacity through the formation of a Phosphomolybdenum complex; specific application to the determination of vitamin E. Analytical Biochemistry 1999; 269: 337-341.
11. 11) Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Japanese Journal of Nutrition 1986; 44: 307-15.
12. 12) Blios MS. Antioxidant determinantions by the use of a stable free radical. Nature 1958; 26: 1199 - 1200.
13. 13) Liu F, Ng TB. Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sciences 2000; 66: 725-735.
14. 14) Ruch RJ, Cheng SJ, Klaunig JE. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 1989; 10: 1003-1008.
15. 15) Govindarajan, R.,Rastogi, S., Vijayakumar, M. Studies on antioxidant activities of Desmodiumgangeticum. Biological and Pharmacological Bulletin 2003; 26: 1424.
16. 16) Tao Wang, Rosa Jonsdottir, GuorunOlafsdottir. Total phenolic compounds, radical scavenging and metal cheatation of extracts form Icelandic seaweeds. Food Chemistry 2009; 116: 240-248.
17. 17) Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry 1999; 47: 3954-3962.
18. 18) Nabavi SM, Ebrahimzadeh MA, Nabavi SF, FazelianM ,Eslami B. In vitroantioxidant and free radical scavenging activity of Diospyros lotus and Pyrusboissieriana growing in Iran. PharmacognosyMagazine2009;4(18): 123-127.
19. 19) Shon MY, Kim TH, Sung NJ. Antioxidants and free radical scavenging activity of Phellinusbaumii (Phellinus of Hymenochaetaceae) extracts. Food Chemistry 2003; 82: 593-597.
20. 20) Ebrahimzadeh MA, Ehsanifar S and Eslami B .Sambucusebuluselburensis fruits: A good source for antioxidants. Pharmacognosy Magazine 2009; 4(19): 213-218.
21. 21) Nabavi SM, Ebrahimzadeh MA, Nabavi SF and Jafari M. Free radical scavenging activity andantioxidant capacity of EryngiumcaucasicumTrautv and Froripiasubpinnata. Pharmacology online 2008; 3: 19-25.
22. 22) Jiménez-Escrig A, Sánchez-Muniz FJ. Dietary fibre from edible seaweeds: chemical structure, physicochemical properties and effects on cholesterol metabolism. Nutrition Research 2000; 20: 585-598.
23. 23) Alekseyenko TV, Zhanayeva SY, Vendiktova AA, Zvyagintseva TN, Kuznetsova TA, Besednova N. Antitumor and antimetastatic activity of fucoidan, a sulfated polysaccharide isolated from the Okhotsk sea Fucusevanescens brown alga. Bulletin Experimental Biology and Medicine 2007; 147: 730-732.
24. 24) Tyagi, N. and Bohra, A. Screening of Phytochemicals of fruit plant and antibacterial potential against Pseudomonas aeruginosa. Biochem. Cell Architecture 2002; 2: 21-24.
25. 25) 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): 8-14
26. 26) Meenakshi, ManickaGnanambigai D, Tamil Mozhi S, Arumugam M, Balasubramanian T. Total Flavonoid and in vitro Antioxidant Activity of Two Seaweeds of Rameshwaram Coast, Global Journal of Pharmacology 2009; 3(2): 59-62.
27. 27) Siriwardhana N, Lee KW, Kim SH, Ha JW, Jeon YJ. Antioxidant activity of Hizikiafusiformis on reactive oxygen species scavenging and lipid peroxidation inhibition. Food science and technology international 2003; 9 (5): 339–346.
28. 28) Kumaran A, Karunakaran RJ. In vitro antioxidant properties of methanol extracts of five Phillanthus species from India.LebensWissen and Technologie 2007; 40: 344-352.
29. 29) Kumar Chandini S, Ganesan P, Bhaskar N. In vitro antioxidant activities of three selected brown seaweeds of India. Food Chemistry 2008; 107: 707-713.
30. 30) Indu.H, Seenivasan.R. In Vitro Antioxidant Activity of Selected seaweeds From Southeast Coast of India.International Journal of Pharmacy and Pharmaceutical Sciences 2013; 5(2). 474 - 84.
31. 31) IhamiGulcin, HaciAhmetAlici, Mehmet Cesur. Determination of in vitro antioxidant and radical scavenging activities of propofol.Chemical and Pharmaceutical Bulletin2005; 53: 281 – 285.
32. 32) Siriwardhana N, Lee KW, Kim SH, Ha JW, Jeon YJ. Antioxidant activity of Hizikiafusiformis on reactive oxygen species scavenging and lipid peroxidation inhibition. Food Science and Technology International 2003; 9 (5): 339–346.