HPTLC Finger Print Analysis of Steroid, Flavonoid and Antiradical activity in Sargassum wightii from Gulf of Mannar

 

V. Madhan Chakkaravarthy* and Venkataraman Kumar

P.G. and Research Department of Plant Biology and Biotechnology, Botany Res., Laboratory, V. O. Chidambaram College, Tuticorin, 628 008, Tamil Nadu, India.

 

ABSTRACT:

The aim of this research was to estimate the steroid, flavonoid and antiradical activity of brown seaweed Sargassum wightii. The HPTLC analysis reveals the presence of polyphenolic signals at different ranges. The steroid and flavonoid content of ethanolic extract was observed in 0.35 Rf and presence of flavonoid in the given standard but not in the sample respectively. Scavenging activity was observed from 05 to 20mg/ml, 24 ± 1.3; 41.3 ± 2.4; 63.5 ± 2.3 and 73.1 ± 1.7, which showed significance of the active principles in S. wightii.

 

KEYWORDS: Sargassum wightii, Steroid, Flavonoid, Antiradical activity, HPTLC.

 

 

INTRODUCTION:

In marine ecosystems, macroalgae are ecologically and biologically important which provide medicinal constituents, nutrition, reproduction and an accommodating environment for other living organisms (McClanahan, 2002). Macroalgal polysaccharides are used in the food, cosmetics, paints, crop, textile, paper, rubber and building industries. In addition, they are used in medicine and in pharmacology for their antioxidant properties (Fleurence, 1999). Seaweeds have plenty of essential nutrients, especially trace elements and several other bioactive substances. That explains why today seaweeds are considered as the food supplement for 21st century as source for proteins, lipids, polysaccharides, mineral, vitamins and enzyme (Rimber, 2007). Interestingly, the best known component of the seaweed-derived industry is that of the phycocolloids, the gelling, thickening, emulsifying, binding, stabilizing, clarifying and protecting agents known as carrageenans, alginates and agars (Chopin, 2007). Steroid and flavonoid are one of bioactive elements in seaweeds exhibit several bio-properties such as anti-diabetic, anti-inflammatory, anti-hepatotoxic and anti-ulcer actions. Both Steroid and flavonoid help in protection of diseases by contributing, along with antioxidant vitamins and enzymes, to the defense system of the human body. They also potent, anti-allergic, anti-viral, anti-bacterial and have antiradical scavenging abilities. They also exhibited anti-coagulant, anti-hyperlipidase, anti-nephritic, vasodilative effects and human immune deficiency virus (HIV) type 1 integrase inhibition. Looking into the significance, the present work has been carried out to estimate antiradical scavenging of steroid and flavonoid in S. wightii, from Hare Island, Gulf of Mannar, India.

 

 

 

MATERIALS AND METHODS:

Seaweed collection and Ethanol extract preparation: Hare Island (Lat. 9o 12’21.6 N - Long. 79o 03’58.17 E), situated in the Gulf of Mannar Biosphere Reserve (Fig. 1), exactly 7 km away Mandabam Camp. S. wightii were collected at low tide in the monsoon season (May-December). Then the plants were washed thoroughly with sea water to remove extraneous materials and brought to the laboratory in plastic bags containing water to prevent evaporation. The collected specimen were dried and powdered with electric mixer stored at room temperature until used. The powder was defatted using petroleum ether, later extraction was done by 75% ethanol using soxhlet apparatus.

 

Phytochemical analysis: The presence of phytochemicals steroids (Liebermann’s Burchard test) were evaluated according to the methods described by Edeogal et al. (2005) and flavonoids by Xia Liu et al. (2005).

 

HPTLC analysis for steroid and flavonoid: Test solution 2.5µl and 3µl of standard solution was loaded as 5mm band length in the 3x10 Silica gel 60F254 HPTLC plate using Hamilton syringe and Camag Linomat 5 instrument. Mobile phase was Toluene-acetone (9:1) for Steroid and ethyl acetate-butanone-formic acid-water (5:3:1:1) for flavonoid were used. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at White light, UV 254nm and UV366nm (Srinivas Reddy et al., 2008).

 

DPPH radical scavenging activity: The free radical scavenging activity of the leaf extract and standard reference compound was analyzed by the 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH) assay as described by Sanchez-Moreno et al. (1998) with minor modification. In this assay, 1 ml of varying concentrations (5, 10, 15 and 20mg/ml) of ethanol extract of S. wightii, mixed with 1 ml of ethanol solution of DPPH (0.2 mM). The mixture was vortexed and incubated for 30min. The optical density of the solution was the measured at 517nm using Hitachi 2010. Spectrophotometer BHA (μg/ml) has been used as standard reference.

Statistical analysis: Calculation was performed by SPSS software (Version-11.5) and data were expressed as Mean±S.D.

 

RESULTS AND DISCUSSION

The phytochemical analysis of ethanol extract had showed the presence of steroids. It is well known that antioxidant activity of plants might be due to their phenolic compounds. Polyphenolic compounds with known properties which include free radical scavenging ability, inhibition of hydrolytic and oxidative enzymes (Frankel, 1995). The presence of polyphenolic compound in S. wightii prompted us to study the free radical scavenging activity. HPTLC profile of ethanol extract, the peak table of chromatogram quantification, leaves of S.wightii were 8682.7 area; 315.5 height and 0.35 Rf in the peak 1 was high in the standard when compare with peak 2 of the S. wightii sample 1450.5 area; 46.7 height and 0.35 Rf were observed respectively, which confirmed the presence of steroid in the given sample (Fig. 1). With reference to the flavonoid in the given sample blue, yellow coloured fluorescent zone at UV 366nm mode was present in the standard track, it was observed from the chromatogram after derivatization, which confirmed the presence of flavonoid in the given standard but not in the sample. In Rutin standard 10447.2 area; 339.4 height and 0.24 Rf in the peak 1 were observed (Fig. 2). The present study investigated the scavenging activity of ethanol extract of S. wightii, which showed significant antiradical activity generated by DPPH. Scavenging activity was observed from 05 to 20mg/ml, 24 ± 1.3 (12%); 41.3 ± 2.4 (21%); 63.5 ± 2.3 (31%) and 73.1 ± 1.7 (36%). Since more than 50% of DPPH radical inhibition is considered to be significant, the inhibition was observed from 10mg/ml. BHA showed strong free radical scavenging activity at all concentrations (Table 1). Currently available drugs regimens for management of diseases have certain drawbacks and therefore, there is a need for safer and more effective drugs (Shu, 1998). This finding helps in further research in the investigation of seaweeds with different solvent fraction for their antiradical activity and also useful to utilize the marine plants as a source food and medicine.

 

Table 1. Antiradical activity of BHA & S. wightii

Concentration (mg/ml)

% Inhibition of DPPH free radical

BHA (μg/ml)

Sw (mg/ml)

5

42.1 ± 2.9

24 ± 1.3

10

61.3 ± 1.2

41.3 ± 2.4

15

79.1 ± 2.3

63.5 ± 2.3

20

83.5 ± 1.8

73.1 ± 1.7

 

ACKNOWLEDGEMENT:

We thankful to the Principal, V. O. Chidambaram College and M. S. University, Tamil Nadu, for providing all facilities during the study.

 

REFERENCES:

1.       Chopin, T., 2007. Closing remarks of the new president of the international seaweed Association. The 19th International Seaweed Symposium Kobe-Japan.

2.       Fleurence, J., CoeurCle, S., Mabeau, M., Maurice, Landrein, A. and LeCoeur, C. 1995. Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata. J. Appl. Physio., 7: 577-582.

3.       Rimber Ir. Indy, 2007. Why is seaweed so important? M.Sc thesis Faculty of Fisheries and Marine Sciences, Sam Ratulangi University, Jln. Kampus Bahu, Manado 95115, Indonesia.

4.       McClanahan, T.R., Cokos, B.A. and Sala, E. 2002. Algal growth and species composition under experimental control of herbivory, phosphorus and coral abundance in Glovers Reef, Belize. Mar. Poll. Bull. 44: 441-451.

5.       Fleurence, J. 1999. Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends in Food Sci. and Technol. 10: 25-28.

6.       Srinivas Reddy, B., Kiran Kumar Reddy, R., Naidu, V.G.M., Madhusudhana Sachin, K., Agwane, B., Ramakrishna. S., Prakash and Diwan, V. 2008 Evaluation of antimicrobial, antioxidant and wound healing potentials of Holoptelea integrifolia. J. Ethnopharmacol. 115: 249-256.

7.       Sanchez-Moreno, Larrauri, J.A. and Saura-Calixto, F. 1998. A procedure to measure the antiradical efficiency of polyphenols. J. Sci. Food Agric. 76: 270-276.

8.       Edeogal, H.O., Okwu, D.E. and Mbaebie, B.O. 2005. Phytochemical constituents of some Nigerian medicinal plants. African J. Biotechnol. 4: 685-688.

9.       Xia Liu, R., Wang, Q., Zhuguo, H., Li, L., ShuBi, K. and An Guo, D. 2005. Simultaneous determination of 10 major flavonoids in Dalbergi Oderifera by high performance liquid chromatography. J. Pharmaceutical and Biomedical Analysis, 39: 469-476.

10.     Shu, Y. Z. 1998. Recent natural products based drug development: a pharmaceutical industry perspective. J. Nat. Prod. 61: 1053-71.

 

 

Received on 21.12.2010

Accepted on 26.12.2010     

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Research Journal of Pharmacognosy and Phytochemistry. 3(2): March- April 2011, 72-74