2201
Anti Scavenging Property And Characterization
Using HPLC Of The Fucoidan extracted from
Turbinaria conoides.
Revathi Chitra Santhanam, Syed Ali Mohammed Yahoob, Anuradha Venkatraman.
Abstract : The role of Antioxidant is to inhibit the free radicals that are produced by a chemical reaction which damage the cells of the living tissues. Plant-source is found to be a rich in antioxidants. In this study, Brown seaweed, Turbinaria conoides was selected and fucoidan, a type of polysaccharide was extracted using a standard procedure and it was confirmed by HPLC using a standard fucose and the antioxidant assays like DPPH (2,2-diphenyl-1-picrylhydrazyl)assay, hydrogen peroxide scavenging assay, Nitric oxide scavenging assay and Deoxyribose radical scavenging assay was performed in the fucoidan extract .The above assays produced a significant results compared to the standards. From the results, it is proven that the selected seaweed Turbinaria conoides is a good source of antioxidant and it can be further analyzed for finding its anticancer properties.
Key words: Antioxidant, Brown Seaweed, DPPH, Fucose, fucoidan, polysaccharide, Turbinaria conoides.
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1.
Introduction
Over the past few years, macro algae plays a considerable role in many curative purposes. Like anticancer, antiviral, anti-inflammatory, hypercholesterolemia and hypolipidemic, antioxidant, antineoplastic and antibacterial activity in it [1]. In this present study fucoidan was extracted from Turbinaria conoides and it was analyzed using HPLC and subjected to antioxidant scavenging assays. The cell wall of the marine algae is found to be a rich source of mucopolysaccharide and polysaccharide [2, 3]. Polysaccharides biopolymers made up of simple monosaccharide units that are connected with each other by a glycosidic linkage. Polysaccharides are also found to have many usages like confectioners and solidifying agents etc [4, 5]. When we see the structural chemistry of Fucoidan it is made up of sulphated polysaccharide with a fucose backbone was observed mostly [ 6 ].The toxic effect of the free radical scavengers could be arrested by the antioxidant materials, which could solve the free radical and detoxicate the creatures.[ 7 ]. The antioxidant activities like DPPH, Hydrogen peroxide scavenging activity, nitric oxide scavenging activity and deoxyribose radical scavenging activity was performed and they gave a significant antioxidant activity .
2. EXPERIMENT SESSION
2.1 SEAWEED COLLECTION
Turbinaria conoides was collected from Mandapam coastal area, Rameshwaram. The collected sample was cleaned shade-dried and then it was further dried using a hot air oven. The dried seaweeds were subjected to grinding using a mixer machine. The powdered Turbinaria conoides sample was stored in an airtight container and used for carrying out the further analysis.
2.2 FUCOIDAN EXTRACTION
A new extraction method of fucoidan from the soaked water of brown seaweed (Xiaolin Chen et.al February 2012)[8] was used for the extraction of fucoidan from Turbinaria conoides with a slight modification. 100gms of powdered seaweed sample was mixed with 2.4l of distilled water and it was kept in the shaker for 24hours. Then it was filtered and the filtrate was separated. From the filtrate 150ml of the filtered solution was taken and mixed with 1% chitosan.1% chitosan was prepared in 1% acetic acid solution. The mixed solution was kept for 12 hours in a shaker for agitation. The obtained solution was centrifuged at 1500rpm for 5 minutes. The supernatant was removed and the pellet was dried in the hot air oven at 50°C. From the dried pellet, 4gms was obtained and it was dipped in 551ml of distilled water .pHwas adjusted and it was kept for 12 hours. After the period of 12 hours, the mixture was agitated in room temperature for 2 hours and it was filtered. The filtrate was washed with ethanol for 15minutes and it was centrifuged and the pellet was removed, air dried and it was used for further analysis.
3. HPLC FOR THE DETECTION OF FUCOIDAN
USING FUCOSE STANDARD
3.1 HYDROLYSIS OF POLYSACCHARIDE FOR THE ANALYSIS OF MONOSACCHARIDE COMPONENTS Polysaccharide was hydrolysed with 2 M Trifluorocetic acid at 100 °C for 8 hours in a sealed tube under nitrogen. The hydrolysate was evaporated to dryness and used for monosaccharide analysis.
3.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
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Corresponding Author: S.Revathi Chitra, Assistant professor, Department of Biotechnology, Kumararani Meena Muthiah College of Arts and Science, Affiliated to the University of Madras, Adyar, Chennai, Tamilnadu. Mail Id: prof.revathichitra@gmail.com Contact Number: 9786351213
2202 A 250 mm long column with an outer diameter of 4 mm
(Hypersil; Thermo-Scientific) using acetonitrile:water (80:20) containing 1 % (v/v) acetic acid as an eluents at a flow rate of 0.8 mL. Sugar identification was done by comparison with reference sugar Fucose. Identification of the sugar components were performed on HPLC equipped with Refractive Index detector (HPLC RID-20A Shimadzu, Tokyo, Japan).
4. DPPH RADICAL SCAVENGING ASSAY
DPPH radical scavenging assay was performed using the following procedure of Yen, G. H [9] Extract was prepared and it was mixed with 2.0ml of the sample were mixed with 2.0 ml of 0.16 mM DPPH methanolic solution. The mixture was vortexed for 1 min and then left to stand at room temperature for 30 min in the dark. The absorbance of all the sample solutions was measured at 517 nm. Which was given by Manimala M. R [10] the sample was taken in different concentration from 100 to 500µg/mlScavenging effect (%) = (Absorbance of control – Absorbance of the test solution /Absorbance of control X 100)
4.1 HYDROGEN PEROXIDE SCAVENGING ASSAY This assay was done based on the standard method [11]. Hydrogen peroxide was prepared using the phosphate buffer. The prepared solution was mixed with the test extract and it was kept for 10 of incubation at room temperature. Ascorbic acid was used as standard [12] the percentage of H2O2 scavenging was calculated using the following formula
H2O2 scavenging effect (%) = (Absorbance of control – Absorbance of test solution)/Absorbance of control) × 100
4.2 NITRIC OXIDE SCAVENGING ACTIVITY
Nitric oxide scavenging activity was accomplished using the following method [13]. Sodium NitroPrusside was prepared using phosphate buffer saline mixed with test solution & incubated at 25o c. Test solution was mixed with equal volume of freshly prepared Griess reagent, and freshly prepared phosphoric acid and the colour change in the solution was measured using UV spectrometry at 546nm. Nitric oxide activity (%) = (Absorbance of control-Absorbance of test) / control-Absorbance of control × 100.
4.3 DEOXYRIBOSE RADICAL SCAVENGING ACTIVITY
2.0 ml of test samples were added to the test tube containing reaction mixture of 2.0 ml FeSO4.7H2O 0.2 ml EDTA(10mM) and 0.2 ml deoxyribose. The volume was made up to 1.8 ml with phosphate buffer (0.1M, pH-7.4) and to that 0.2 ml H2O2 was added. The mixture was incubated at 37°C under dark for 4 hours. After incubation, 1ml of TCA (2.8%) and TBA (1%) were added to the mixture, and kept in boiling water bath for 10 min. After that, the mixture was absorbance at 532nm using UV spec. In case of turbidity, the absorbance was measured after filtration. Ascorbic acid was used as standard according to Indu H [14].
Scavenging activity (%) was calculated using the equation: Deoxyribose radical scavenging activity (%) = (Absorbance of control - Absorbance of test) / Absorbance of control × 100
5. RESULTS
100gms of powdered Turbinaria conoides sample gave a yield of around 2gms of Fucoidan and the antioscavenging activities were performed using the extracted Turbinaria conoides. HPLC results showed a single peak with a retention time of 6.307 for standard Fucose. Fucoidan extracted from Turbinaria conoides showed multiple peaks and the presence of fucose was confirmed at 6.293 retention time(Fig 1) DPPH i radical scavenging activity was observed from 21.39% to 80.05%for the test sample and 13.72% to 64.71% for the standard ascorbic acid with varying concentration from 100 to µg/ml which resulted in the remarkable IC50 value of 314.225 for the test extract, while the Std sample gave a IC 50 403.815(Fig 2).Hydrogen peroxide scavenging activity showed 25.71% to 85.40% for the test sample and 23.68% to 84.26% for the standard ascorbic acid which leads to the significant IC50 value of 267.8 for the standard and 253.87 for the test extract (Fig 3). Nitric oxide scavenging activity showed a commendable percentage of 29.93% to 86.40% for the test sample and 23.0% to 82.45% for the standard which resulted in the IC50 value of 279.1 for the standard and 244.353 for the test extract(Fig 4).Deoxyribose radical showed a scavenging activity from 29.96% to 78.66% for the standard and 16.96% to 80.44% for the test sample at varying concentration from 100 to 500µg/ml with a significant IC50 value of 260.203 for standard and 303.33 for the test extract.(Fig 5)same concentration was used for all the
2203 Fig: 1 The HPLC graphical representation for the test sample Fucoidan extracted from the Turbinaria conoides.
Fig:2 Graphical representation of the DPPH radical scavenging activity showing IC 50 for standard and test Extract.
2204 Fig: 4 Graphical representation of the Nitric oxide scavenging activity showing the IC50 value for the standard and test extract
Fig: 5 Graphical representation of the Deoxyribose Radical scavenging activity showing the IC50 Valu .
6. DISCUSSION
2, 2 diphenyl1- picrylhydrazyl is a dark purple coloured crystalline powder composed of stable free radical molecules with a nitrogen atom. Thus the DPPH scavenging is related to the DPPH reduction and gives rise to stable free radicals. Number of free radicals in the DPPH with an unpaired electron will give maximum absorption at 517nm [15].Paring of the hydrogen donor decrease the absorption strength which result in the decolourization to yellow from purple colour [16]. In this assay our test sample showed a significant result compared to the standard. The same result was observed in the three seaweeds, P. tetrastromatica, C. racemosa and T. ornata demonstrated higher DPPH radical scavenging activity [17] Hydrogen Peroxide Radical Scavenging Activity of our test sample showed a significant result compared to the standard and
the similar result was obtained in the T. zeylanicum
2205 property in them [23]. Deoxyribose radical scavenging
assay is used to find the chemical reaction rate of the hydroxyl radical with the antioxidant compounds [24] our extract showed a significant hydroxyl scavenging assay .The same concept was prove [25]Free radical scavenging activity was performed on Taiwanese native plants and it was found to show a good IC50 value for the radical scavenging assays.
7. CONCLUSION
Endo and exogenous metabolic process in the human body results in the highly reactive oxygen species. macromolecules are oxidized by the highly reactive oxygen species they can result in more number of degenerative diseases like cancer, emphysema, cirrhosis, atherosclerosis, arthritis and neurological disorders[26,27]The main role of antioxidant compounds is to stop attack of free radical and will decrease the chances of these disorders.[28]Thus it is very clear from our results that, the fucoidan extracted from Turbinaria conoides contains the antioxidant potential in them which gives us an indication that it may contains the anticancer potential in them. On further analysis, the anticancer potential in them can be confirmed.
Acknowledgment
The Author thanks, Dr. Syed Ali Mohammed Yahoob , Head Department of Biotechnology, Mohammed Sathak college of Arts and Science for his constant support and encouragement.
REFERENCES
[1] El-Baroty GS, Moussa MY, Shallan MA, Ali MA, Sabh AZ, Shalaby EA.Contribution to the Aroma, Biological Activities, Minerals, Protein, Pigments and Lipid Contents of the Red Alga: Asparagopsis anticancer (Delile) Trevisan. J App Sci Res. 2007, 3, 1825–1834.
[2] Kumar CS, Ganesan P, Suresh PV, Bhaskar N (2008b) Seaweeds as a source of nutritionally beneficial compounds—a review. J Food Sci Technol 45:1–13
[3] Murata M, Nakazoe J (2001) Production and use of marine algae in Japan. Jpn Agr Res Q 35:281–290 [4] McHugh DJ (1987) Production and utilization of
products from commercial seaweeds. FAO Fisheries Technical Paper No. 288, pp 1–189 [5] Tseng CK (2001) Algal biotechnology industries
and research activities in China. J Appl Phycol 13:375–380
[6] Berteau O, McCort I, Goasdoue N, Tissot B, Daniel R. 2002. Characterization of a new alpha-L-fucosidase isolated from the marine mollusk Pecten maximus that catalyzes the hydrolysis of alpha-L-fucose from algal fucoidan (Ascophyllum nodosum). Glycobiology 12: 273–282.
[7] Balakumar K, Ramanathan S Kumaresan and Suresh R (2010) DNA damage by sodium arsenite in experimental rats: ameliorative effects of antioxidant vitamins C and E.
[8] XiaolinChena,b, Ronge Xing, Huahua Yu, Pengcheng li .A new extraction method of fucoidan from the soaked water of Brown seaweed
(Laminaria japonica) Desalination and Water Treatment, 40 (2012) 204–208
[9] Yen, G. H. and Chen, H. Y. 1995. Antioxidant Activity of Various Tea Extracts in Relation to Their Antimutagenicity, Journal of Agricultural and Food Chemistry,Vol. 43, pp. 27–32
[10]Manimala, M. R. A. and Murugesan, R. 2014. In vitro antioxidant and antimicrobial activity of carotenoid pigment extracted from Sporobolomyces sp. isolated from natural source, Journal of Applied and Natural Science, Vol. 6, No. 2, pp. 649 – 653.
[11]Ruch, R. J., Cheng, S. J. and Klaunig, J. E. 1989. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea,Carcinogenesis, Vol. 10, pp. 1003.
[12]Gulcin, D. 2006. Antioxidant and antiradical activities of l-carnitin, Life Science, Vol. 78, pp. 803-811
[13]Fadzai Boora, Elaine Chirisa and Stanley Mukanganyama (2014), Evaluation of Nitrite Radical Scavenging Properties of Selected Zimbabwean Plant Extracts andTheirPhytoconstituents, Journal of Food Processing, pp.1-7.
[14]Indu, H. and Seenivasan, R. 2013. In Vitro Antioxidant Activity of Selected Seaweeds from Southeast Coast of India. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 5, No. 2, pp. 474-484
[15]B. Thendral Hepsibha, S. Sathiya , C. Saravana Babu , V. Premalakshmi and T. Sekar In vitro studies on antioxidant and free radical scavenging activities of Azima tetracantha. Leam leaf extracts Indian Journal of Science and Technology Vol. 3 No. 5 (May 2010) ISSN: 0974- 6846[10]
[16]Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature. 181, 1199– 1200.
[17]Yin Yin Chia , M S Kanthimathi, Kong Soo Khoo , Jayakumar Rajarajeswaran , Hwee Ming Cheng and Wai Sum Yap Antioxidant and cytotoxic activities of three species of tropical seaweeds Chia et al. BMC Complementary and Alternative Medicine (2015) 15:339 DOI 10
[18]Frank Ngonda In- vitro Anti-oxidant Activity and Free Radical Scavenging Potential of roots of Malawian Trichodesma zeylanicumm (burm. f.) Asia journal of biomedical and pharmaceutical Sciences e-ISSN: 2249-622X Received: 7 th May 2013 Received in revised form: 20 th May 2013 Accepted: 31 st May 2013 Available online: 10 th June 2013pg no 21 to 25
[19]H. Lata, G.K. Ahuja. Role of free radicals inhealth and disease. Ind. J. Physiol. Allied Sci.57: 124‐28 (2003).
[20]Ialenti, S. Moncada, M. Di Rosa. Modulation of adjuvant arthritis by endogenous nitric oxide.Br. J. Pharmacol. 110: 701‐705 (1993).
2206 [22]Rozina Parul1*, Sukalayan Kumar Kundu 2 and
Pijush Saha2 In Vitro Nitric Oxide Scavenging Activity Of Methanol Extracts Of Three Bangladeshi Medicinal Plants THE PHARMA INNOVATION – JOURNAL ISSN: 2277- 7695CODEN Code: PIHNBQZDB-Number: 2663038-2IC Journal No: 7725Vol. 1 No. 12 2013Online Available at www.thepharmajournal.com
[23]Revathi Chitra S1, *Syed Ali M*1, Anuradha V2, Shantha M3, Yogananth . Antioxidant activity of polysaccharide from Sargassum sp IOSR Journal Of Pharmacy www.iosrphr.org (e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219 Volume 8, Issue 8 Version. II (August 2018), PP. 24-31
[24]Halliwell B, Gutteridge JM, Aruoma OI (1987) The deoxyribose method: a simple ―test-tube‖ assay for
determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 165: 215–219 [25]W.-C. Hou1 , R.-D. Lin2 , K.-T. Cheng3 , Y.-T.
Hung1 , C.-H. Cho1 , C.-H. Chen4 , S.-Y. Hwang4 , and M.-H. Lee1 Free radical-scavenging activity of Taiwanese native plants Phytomedicine 10: 170– 175, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed [26]. Halliwell B, Gutteridge JM. Oxygen toxicity,
oxygen radicals, transition metals and diseases. Biochem J. 1984;219:1–4. [PMC free article] [PubMed] [Google Scholar]
[27]Maxwell SR. Prospect for the use of antioxidant therapies. Drugs. 1995;49:45–361. [Google
Scholar]
