Research Journal of Biotechnology

Vol. 5 (1) Feb. (2010) Res. J. Biotech.

Antioxidant Potential and Phenolic Content of Ethanolic Extract of Selected Malaysian Plants
Arash Rafat1, Koshy Philip1* and Sekaran Muniandy2
1. Institute of Biological Sciencces, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, MALAYSIA 2. Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MALAYSIA * kphil@um.edu.my

Abstract
This study evaluated the antioxidant capacity and total content of phenolic compounds in an ethanolic extract of some commonly consumed raw Malaysian plants. The antioxidant activities of the samples were measured by two different methods while the Folin Ciocalteu reagent assay was used to estimate the phenolic contents of extracts. Cosmos caudatus extract showed the highest antioxidant potential followed by Centella asiatica, Oenanthe javanica, Euodia redlevi and Ocimum basilicum extracts respectively in both DPPH free radical scavenging activity and Superoxide Dismutase Activity Assays. Although the highest total phenolic content was found in Cosmos caudatus extract, there is no positive correlation between evaluated antioxidant activities and the phenolic contents of examined plants.
Keywords: Radical scavenging, superoxide dismutase, phenolic content.

antioxidant potential of several plant species. A positive linear correlation between total phenolic contents and antioxidant activities for aqueous and methanolic extracts of different Chinese medicinal plants5 and different Jordanian plant species27 was shown. There are different methods to estimate the antioxidant potential of a plant samples. 2,2diphenyl-1picrylhydrazil (DPPH) free radical scavenging assay is one of the most common applied methods. Evaluation of antioxidant capacity Cocoa, teas, red wine16, brown alga17, sorghum1, sweet potato8, mushrooms15 and fruits29 using 2,2diphenyl1-picrylhydrazil (DPPH) free radical scavenging assay show the capability of this method to be used for different types of samples. Superoxide Dismutase (SOD) Activity Assay is another commonly used technique which is applied to measure antioxidant activity of different samples. The method was first defined by McCord and Fridovich19 and later was modified by several of other researchers such as Oberley and Spitz21. Although antioxidant capacity of some ULAM methanolic10, 28 and aqueous11 extracts was examined, the antioxidant activities of ethanolic extracts of some selected ULAMs were evaluated in this study. Total phenolic content of the ethanolic extracts of specific plant parts was also measured to study the total content of phenolic compounds and the correlation of antioxidant potential in different parts of the AP plant body.

Introduction
Malaysians consume some raw plants as side dishes termed indigenously as ULAMs. The medical potential of some of these plants, which were also applied as traditional medicinal plants has been reported. Centella asiatica was shown to have the ability of reducing age-related decline in cognitive function and mood disorder in the healthy elderly18, improving venous wall alterations in chronic venous hypertension and protecting the venous endothelium12, 13. Antiviral and antigiardial capacities of Ocimum basilicum oil6, 7 and anti-hepatitis activity of Oenanthe javanica26 are some examples of reported medicinal uses for ULAMs. Different forms of free radicals such as nitric oxide and the alkoxyl radicals, were produced through aerobic respirations and increase the risk of chronic diseases23. Application of a healthy diet including edible antioxidants can help human body to neutralize these free radicals and reduce the oxidative stress damages. Plants are good source of edible antioxidants such as ascorbic acid, tocopherols, carotenoids and several phenolic compounds3, 9. Among all plant secondary metabolites which act as antioxidants phenolic compounds form a large and varied group. Phenolic compounds contribute significantly to the

Material and Methods

Chemicals: Hydrogen peroxide (H2O2) was obtained from UNI-CHEM (South Kearny, N.J.). 2, 2 diphenyl-1picrylhydrazil (DPPH*), Tert-butylated hydroxytoluene (BHT) and ascorbic acid (vitamin C) were purchased from Sigma-Aldrich (St. Louis, Mo) while 95% ethanol was Tween 80 obtained from Systerm ChemAR. (Polyoxyethylene sorbitan mono-oleate) was purchased from Hopkin & Williams (London). Sample Preparation and Extraction: Leaves of five local plants (Oenanthe javanica, Cosmos caudatus, Centella asiatica, Ocimum basilicum and Euodia redlevi) were collected. The explants were cleaned in tap water and airdried at room temperature in the dark separately. Dried leaves of each selected plant 3 grams by quantity were ground to

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produce fine homogenous powders. The fine plant powder was soaked in 40 ml of 95% ethanol at room temperature for 72 hours in the dark. The solution was then filtered through Whatman 5 filter paper (Whatman International, Maidstone) and evaporated to dryness using a rotary evaporator (Heidolph WB2000, Germany) at a temperature below 40 C. A stock solution (100 mg/ml) of the plant extracts was prepared in 5% Tween 80 dissolved in isotonic phosphate buffer (IPB), pH 7.4 and kept at 4 C until required for experiments. The working solution (10 mg/ml) for assays was made by diluting the stock solution with IPB.

Vol. 5 (1) Feb. (2010) Res. J. Biotech.

l using distilled water. Lastly 300 l of sodium carbonate solution (0.2 mg/ml) was added and incubated at 37 C for 45 minutes. Absorbances of the solutions were measured at 760 nm. Total phenolic contents were determined as a gallic acid equivalent (GAE) based on Folin-Ciocalteau calibration curve using gallic acid (ranging from 50 to 1000 mg/ml) as the standard and expressed as mg gallic acid equivalent per gram of dry sample. Statistical Analysis: All determinations were carried out in triplicates and data were subjected to one-way analysis of variance (ANOVA) using SPPS version 15 programme. The Duncans Multiple Range Test (DMRT) was used to separate the means.

Antioxidant Activity Evaluation Methods

Free Radical Scavenging Activity: This assay was based on the method described by Bozin et al4 with some modifications. Briefly, 950 l of 90 M 2,2 diphenyl-1picrylhydrazil (DPPH*) solution was added to 50 l of the working samples and made up to a final volume of 4 ml with 95% ethanol. After the mixtures were vigorously shaken, they were incubated at room temperature in the dark for 2 hours. The reduction of solution colour caused by scavenging of the free radicals (DPPH*) was measured at 515 nm using a spectrophotometer. The capability of samples to scavenge DPPH* was obtained by comparison of sample colour reduction effect with the control (mixture without working solution) using the following equation and expressed as percentage values. DPPH radical scavenging activity (%) = [(A control A sample) / A control] 100 The positive control was prepared using Tertbutylated hydroxytoluene (BHT) at a concentration of 10 mg/ml. Superoxide Dismutase (SOD) Activity Assay: SOD Assay Kit-WST (Dojindo Molecular Technologies, Gaithersburg) was applied to determine the Superoxide Dismutase (SOD) activity. Reaction mixtures of the kit were mixed with 20 l of working solution samples and after a gentle shaking they were inoculated at 37 C for 20 minutes. The inhibition activity of SOD on the reaction of xanthine oxide generated superoxide with a tetrazolim salt was then determined by measuring the absorbance of the mixtures at 450 nm using a microplate reader. The positive control in this study was made by adding 20 l of ascorbic acid at a concentration of 10 mg/ml instead of working solution sample. All inhibition rates were expressed as a percentage of the uninhibited reaction. Total Phenolic Content Determination: A Folin-Ciocalteau method based on Slinkard and Singleton25 report was applied to determine the total amount of phenolic compounds in different parts of AP. Working solution samples (20 l) of each plant part were added to 100 l of 2N Folin-Ciocalteu reagents. The mixture was made up to a final volume of 1600

Results and Discussion

DPPH Free Radical Scavenging Activity: Decolouration of the stable free DPPH radical by antioxidants in samples was measured spectrophotometerically. According to the results (Table 1), the highest free radical scavenging potential was obtained from C. caudatus extract (86.85%) followed by C. asiatica (84.00%), O. javanica (56.87%), E. redlevi (53.15%) and O. basilicum (24.09%) respectively. The DPPH radical scavenging capacities of all examined extracts were significantly lower than the applied positive control (BHT). SOD Activity: The result of Superoxide Dismutase (SOD) activity assay (Figure 2) showed C. caudatus extract (98.56%) obtained the highest activity followed by C. asiatica (81.86%), E. redlevi (78.97%), O. javanica (73.51%) and O. basilicum (63.61%) respectively. SOD activities of all extracts are significantly different and only C. caudatus extract capacity is not significantly different with positive control (ascorbic acid). Total Phenolic Content: The calculation of total phenolic content of plant extracts was carried out using the standard curve of gallic acid and presented as gallic acid equivalents (GAE) per gram (Table 3). E. redlevi extract contained the highest amount of phenolic compounds and the lowest amount is present in C. asiatica extract. All examined plant extracts had significantly different contents of phenolics except O. basilicum which was not significantly different with C. asiatica and O. javanica. In the study, the antioxidant activities of five local Malaysian plants usually consumed as salad vegetables were evaluated. Due to the significant contribution to antioxidant activity by phenolic compounds in plants, the total phenolic content of the ethanolic extracts of these five plants were measured. The results of both DPPH free radical scavenging and SOD assays showed that C. caudatus extract has the highest antioxidant activity followed by C. asiatica extract. O. basilicum showed the lowest antioxidant potential in both assays. Although DPPH radical scavenging activity of O. javanica extract was higher than E. redlevi, it showed the

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reverse in SOD assay result. These different results indicate that one method alone is not reliable to evaluate the antioxidant activities. These results are in agreement with both HudaFaujan et al studies on ferric thiocyanate (FTC) method applied for aqueous extracts11 and methanolic extracts10 of some Malaysian herbs. They showed that C. caudatus extract inhibited linoleic acid oxidation higher than C. asiatica while C. asiatica extract obtained higher linoleic acid oxidation inhibition compared to O. javanica extract. Although, there are several reports which showed good correlation between total phenolic content and antioxidant capacity in different plants 2, 5, 22, 27, some studies contradicted this statement. For example, antioxidant potential of Chenopodium quinoa and Amaranthus spp. seeds obtained using three different methods (FRAP, DPPH and carotene bleaching) poorly correlated with total phenolic content20. No significant relationship was found between antioxidant activity and total phenolic content of fifteen genotypes of selected Turkey Zizyphus jujube Mill. Fruits14. Based on Folin-Ciocalteau experimental results obtained in this study, no positive correlation is detected between antioxidant activity of ethanolic extracts of selected plants and their total phenolic content. Ethanolic extract of E. redlevi contains the highest amount of phenolic compounds compared to other examined extracts. Total phenolic content of Cosmos caudatus is followed by E. redlevi although there is no significant difference between them. The lowest total phenolic content is shown in C. asiatica extract even though C. asiatica showed the second highest antioxidant capacity in both DPPH scavenging and SOD assays. These results confirmed the previous studies by Huda-Faujan et al10, 11 which showed the amount of total phenolics in methanolic and water extracts of C. caudatus to be higher than both C. asiatica and O. javanica. While O. basilicum was reported to have the lowest antioxidant potential among all tested plants in both DPPH scavenging and SOD assays, it did not show the lowest total phenolic content. E. redlevi which showed the highest amount of phenolic compounds between all analyzed plants, was not the best antioxidant among all plants studied. These results can show that even if phenolic compounds play an important role as antioxidants, there are other compounds in plants which have oxidation inhibition activity which must be considered. Another point is the influence of non-phenolic compounds on Folin-Ciocalteu reagent. Some other compounds such as ascorbic acid might also react with the Folin-Ciocalteu reagent and affect colour measurement.24

Vol. 5 (1) Feb. (2010) Res. J. Biotech.

(86.85%) and the highest SOD activity (98.56%). Total phenolic content of C. caudatus (60.56 mg of GAE/g) was also not significantly different with the highest amount obtained by E. redlevi (65.33 mg of GAE/g). The present study indicates that the tested Malaysian vegetables (ULAMs) are potent sources of natural antioxidants. In vivo antioxidant assays for selected plants remains to be carried out as all employed methods in this study were done in vitro. Study on antioxidant activities of non-phenolic components of these vegetables must be considered for future investigation.

Acknowledgement
The authors would like to thank Universiti Malaya for providing a research grant (FS257/2008C) and the use of laboratory facilities to carry out this study.

Table I
Neutralization of DPPH radical of plant extracts in the free radical scavenging activity assay Plant Sample BHT (Positive Control) Cosmos caudatus Centella asiatica Oenanthe javanica Euodia redlevi Ocimum basilicum DPPH Radical Scavenging Activity (%) 94.86 0.15 a 86.85 1.02 b 84.00 1.21 c 56.87 1.43 d 53.15 1.02 e 24.09 0.85 f

The sample means were compared by one-way ANOVA followed by Duncans Multiple Comparison Test. Samples represented with different letters are significantly different (p<0.05).

Table II
SOD activities of the examined samples are presented as inhibition rate Plant Sample Ascorbic Acid (Positive Control) Cosmos caudatus Centella asiatica Oenanthe javanica Euodia redlevi Ocimum basilicum DPPH Radical Scavenging Activity (%) 99.20 0.27 a 98.56 0.69 a 81.86 0.58 b 73.51 0.54 c 78.97 0.66 d 63.61 .061 e

Conclusion
C. caudatus extract among all examined plants showed the highest free radical scavenging potential

The sample means were compared by one-way ANOVA followed by Duncans Multiple Comparison Test. Samples represented with different letters are significantly different (p<0.05).

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Research Journal of Biotechnology Table III

Total phenolic contents of local plant leaves extracts Plant Euodia redlevi Cosmos caudatus Oenanthe javanica Ocimum basilicum Total Phenolic Contents (mg of GAE/g) 65.33 1.91 a 60.56 3.57
a

Vol. 5 (1) Feb. (2010) Res. J. Biotech.

Angiology, 52 (2), S61-S67 (2001) 13. Incandela L. et al, Total triterpenic fraction of Centella asiatica in chronic venous insufficiency and in high-perfusion microangiopathy, Angiology, 52 (2), S9-S13 (2001) 14. Kamilolu1 . et al, Total phenolics and antioxidant activity of jujube (Zizyphus jujube Mill.) genotypes selected from Turkey, Afr. J. Biotech., 8, 303 (2009) 15. Lakshmi B. et al, Evaluation of Antioxidant Activity of Selected Indian Mushrooms, Pharm. Biol., 42 (3), 179-185 (2004) 16. Lee K.W., Kim Y.J., Lee H.J. and Lee C.Y., Cocoa Has More Phenolic Phytochemicals and a Higher Antioxidant Capacity than Teas and Red Wine, J. Agric. Food Chem., 51, 7292-7295 (2003) 17. Lekameera R., Vijayabaskar P. and Somasundaram S.T., Evaluating antioxidant property of brown ALGA Colpomenia sinuosa (DERB. ET SOL), Afr. J. Food Sci., 2, 126-130 (2008) 18. Mator J. et al, Positive modulation of cognition and mood in the healthy elderly volunteer following the administration of Centella asiatica, J. Ethnopharmacol., 116 (2), 325-32 (2008) 19. McCord J.M. et al, Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein), J. Biol. Chem., 244, 6049 (1969) 20. Nsimba R., Kikuzaki H. and Konishi Y., Antioxidant activity of various extracts and fractions of Chenopodium quinoa and Amaranthus spp. Seeds, Food Chem., 106, 760-766 (2008) 21. Oberly L.W. and Spitz D.R., Nitroblue Tetrazolium, In: Greenwald R.A. (Ed.), CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, 217-220 (1985) 22. Othman A. et al, Antioxidant capacity and phenolic content of cocoa beans, Food Chem., 100, 1523-1530 (2007) 23. Rice-Evans C.A. and Gopinathan V., Oxygen toxicity, free radicals and antioxidants in human disease: biochemical implications in atherosclerosis and the problems of premature neonates, Essays Biochem., 29, 3963 (1995) 24. Shahidi F. and Naczk M., Food Phenolic: Sources, Chemistry, Effect and Applications, CRC Press, US, 489-490 (1995) 25. Slinkard K. et al, Total phenol analysis: automation and comparison with manual methods, Am. J. Enol. Vitic., 28, 49 (1977) 26. Wang W.N., Yang X.B., Liu H.Z., Huang Z.M. and Wu G.X., Effect of Oenanthe javanica flavone on human and duck hepatitis B virus infection, Acta Pharmacologica. Sinica., 26 (5), 587 (2005) 27. Tawaha K. et al, Antioxidant activity and total phenolic content of selected Jordanian plant species, Food Chem., 104, 1372 (2007) 28. Zainol M.K. et al, Antioxidative activity and total phenolic compounds of leaf, root and petiole of four accessions of Centella asiatica (L.) Urban, Food Chem., 81, 575581 (2003) 29. Zhang L.H. et al, In Vitro Antioxidant Activities of Fruits and Leaves of Pomegranate, Acta Hort., 765, 31-34 (2008).

38.75 4.44 b 32.23 4.45 bc

Centella asiatica 26.13 4.82 c The sample means were compared by one-way ANOVA followed by Duncans Multiple Comparison Test. Samples represented with different letters are significantly different (p<0.05).

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(Received 10th September 2009, accepted 20th December 2009)

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