ASEAN Food Journal 14 (1): 61-68 (2007) Antioxidative Activities of Water Extracts of Some Malaysian Herbs 61 Antioxidative Activities of Water Extracts of Some Malaysian Herbs 1Huda-Faujan, N., *2Noriham, A., 3Norrakiah, A.S. and 3Babji, A.S. 1Food Biotechnology Programme, Faculty of Science and Technology, Kolej Universiti Islam Malaysia, Bandar Baru Nilai, 71800 Nilai, Negeri Sembilan, Malaysia 2Food Technology Programme, Faculty of Applied Science, Universiti Technology MARA, 40450 Shah Alam, Selangor, Malaysia 3Food Science Programme, School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia Abstract: This study was conducted on selected local herbs such as ulam raja (Cosmos caudatus), kesum (Polygonum minus), selom (Oenanthe javanica), pegaga (Centella asiatica) and curry leaves (Murraya koenigii) to investigate their antioxidative activities. The water extracts of the herbs were analysed for total phenolic content, reducing antioxidant power, ferric thiocyanate (FTC) and the thiobarbituric acid (TBA) test was also accried out. Polygonum minus showed the highest total phenolic content and reducing power among the herbs. Increasing the concentration of the extracts resulted in increased Fe3+ reducing antioxidant power for all the herbs. FTC and TBA tests on the extracts during seven days of storage showed that all the herbs extracts had the ability to reduce oxidation compared to the control (P < 0.05). From the FTC analysis, Murraya koenigii leaves was best in reducing the oxidation rate (67.67%) compared to the other herbs studied. Analysis of TBA showed that Centella asiatica extract had the highest antioxidant effect. However, both TBA and FTC analysis for these two herbs showed no significant difference (P > 0.05) from Polygonum minus and butylated hydroxyanisole (BHT) a synthetic antioxidant. Correlation analysis showed positive correlations between amount of total phenolic content and reducing power (r = 0.75) and antioxidative activities (r = 0.58) in linoleic acid emulsion system. This shows that antioxidative activities of these Malaysian herbal plants especially Polygonum minus may be a potential source of natural antioxidants with similar characteristics to the synthetic antioxidant, BHT. Keywords: Antioxidative activities, Malaysian herbs, total phenolic content, reducing power, Thiobarbituric Acid (TBA) INTRODUCTION activated oxygen. These molecules are exacerbating factors in cellular injury and Plants are potential sources of natural aging process (Gulçin et al., 2003). In addition, antioxidants. They produce various ROS induce some oxidative damage to bio- antioxidative compounds to counteract molecules like lipids, nucleic acids, proteins reactive oxygen species (ROS) in order to and carbohydrates. Their damage causes survive (Lu and Foo, 1995). ROS which aging, cancer and many other diseases include free radicals such as superoxide anion (Aruoma, 1994). In foods, ROS can cause lipid . - . radicals (O 2 ), hydroxyl radicals (OH ) and peroxidation, which leads to the deterioration non free-radical spesies such as H2O2 and of the food (Miller and Rice-Evans, 1997). The 1 single oxygen ( O2) are various forms of oxidative deterioration of the lipid-containing *Corresponding author. E-mail address: [email protected] (Noriham,ASEAN FoodA.) Journal Vol. 14, 61-68 62 Huda-Faujan, N., Noriham, A., Norrakiah, A.S. and Babji, A.S. food is responsible for the rancid odours and effects. Nowadays, there are numerous flavours during processing and storage, techniques that are available to evaluate consequently decreasing the nutritional antioxidant activities of compounds and of quality and safety of foods, due to the complex mixtures such as plant extracts formation of secondary and potentially toxic (Anatolovich et al., 2002). Despite the compounds. The addition of antioxidants is existence of these various methods and one way of increasing the shelf life of foods techniques, one procedure alone cannot (Cook and Samman, 1996). identify all possible mechanisms characterising Plant phenolics are probably had an antioxidant (Frankel and Meyer, 2000). multifunctional antioxidants. These Therefore this study was conducted to evaluate compounds were reported to quench oxygen- the antioxidative activity of several Malaysian derived free radicals by donating a hydrogen herbs using three different methods, and to atom or an electron to the free radical evaluate the relationship between the (Wanasundara and Shahidi, 1998). The antioxidative activity and total phenolic antioxidant effect of phenolic compounds has content of the herbs. been demonstrated in many systems through in vitro studies such as in human low density lipoprotein and liposomes (Leanderson et al., MATERIALS AND METHODS 1997). Several studies had been conducted to Chemicals evaluate the correlation between phenolic Ammonium thiocyanate and ferrous chloride compounds and antioxidant activity. The were purchased from Merck (Merck KGaA, antioxidant activity of Du-Zhong (Eucomnia Darmstadt, Germany). Ferric chloride, linoleic ulmoides) (Yen and Hsieh, 1998), Mung Bean acid (99.5%), potassium ferricyanide, Hulls (Duh et al., 1997), ear mushrooms butylated hydroxytoluene (BHT), butylated (Chao, 2001) and anise seed (Pimpenella hydroxyanisole (BHA), thiobarbituric acid anisum L.) (Gulçin et al., 2003) were found to (TBA) and trichloroacetic acid (TCA) were correlate with the phenolic compounds. obtained from Sigma Chemical Co. (St. Louis, Studies on local Malaysian plants such as MO, USA). Commercial rosemary (Rosemary turmeric (Curcuma domestica), betel leaf (Piper Herbalox ®) was obtained from Kalsec UK Ltd. betel), pandan leaf (Panadanus odorus), asam (Suffolk, UK). gelugur (Garnicia atroviridis), mengkudu (Morinda citrifolia), pegaga (Centella asiatica), Materials and Extraction Method ginger (Zingiber officinale), cassava shoot All herbs were obtained from a local wet (Manihot asculenta) (Jayamalar and Suhaila, market in Bandar Baru Bangi, Selangor, 1998; Mohd. Zin et al., 2002, Zainol et al. 2003; Malaysia. The herbs were ulam raja (Cosmos Noriham et al., 2004) have shown that they caudatus), kesum (Polygonum minus), selom also exhibit good antioxidant activity. Wang et (Oenanthe javanica), pegaga (Centella asiatica) al., (1999) reported that the antioxidative and curry leaves (Murraya koenigii). The edible properties of some vegetables and fruits are portions of the fresh samples were cleaned and partly due to the low molecular weight washed using running tap water. Then phenolic compounds, which are known to be excessive water was drained out and the potent as an antioxidant. samples were air-dried using a fan. Samples In Malaysia, herbs are commonly eaten were blended with distilled water in a ratio of fresh as a vegetable (salad and ulam), especially 3:1 (water: herb) and filtered using Whatman among the Malay communities. Most of these No. 1 paper. The filtrates were then evaporated herbs are believed to be associated with using a vacuum evaporator at 50oC to give antioxidant activities and have many beneficial viscous mass. The crude extracts were weighed ASEAN Food Journal Vol. 14, 61-68 Antioxidative Activities of Water Extracts of Some Malaysian Herbs 63 and stored at 0-4oC for further experiments method (Osawa and Namiki, 1981) with slight and analysis. modification. Four milligrammes of each herb extract samples was dissolved in 4.0 ml ethanol Determination of Total Phenolic Content (99.5%) and kept in a dark bottle (d = 40.0 Total phenolic content was determined using mm, t = 75.0 mm). Each sample was then Folin–ciocalteu reagent following the method mixed with 4.1 ml linoleic acid (2.5% in of Singleton and Rossi (1965) with slight ethanol 99.5%), 8 ml phosphate buffer (0.02 modification using tannic acid as a standard. M, pH 7.0) and 3.9 ml distilled water to make Briefly, 1 ml of extract solution (5 mg ml-1) up to 20 ml. BHT was used as a positive control was added in a 100 ml volumetric flask that and an empty bottle (no sample) was used as contained about 60 ml distilled water. Then, 5 a negative control. The mixture/solution was ml of Folin–ciocalteu reagent was added and incubated at 40 – 45oC. After incubation, 9.7 the content of the flask thoroughly mixed. ml ethanol (75%) and 0.1 NH4SCN (30%, as After 1-8 minutes, 15 ml Na2CO3 (20%) was a colour reagent) was added to 0.1 ml of the added and the volume was made up to 100 ml solution. Precisely 3 min after the addition of using distilled water. The mixture was allowed 0.1 ml of FeCl2 (0.002 M) in HCl 3.5% to the to stand for 2 hours with intermittent shaking. reaction mixture, the absorbance of the The absorbance was measured at 760 nm using resulting red colour was measured at 500 nm a UV-Vis spectrophotometer (Jenway 6100, using spectrophotometer (Jenway 6100, Dunmow, Essex, U.K). Total phenolic content Dunmow, Essex, U.K) every 24 h until a day was determined as mg of tannic acid equivalent after the absorbance of the control reached (TAE) using an equation obtained from the maximum value (day seven). The inhibition standard tannic acid calibration graph. of lipid peroxidation was calculated as follows: Reducing Antioxidant Power % Inhibition = 100 – [(A1/Ao) X 100] The reducing antioxidant power of water extracts of the herbs was determined using the Where Ao is the absorbance of the control method of Oyaizu (1986). Different reaction and A1 is the absorbance in the concentrations of herb