Phytochemical screening and in vitro antioxidant activity of Merremia emarginata (Burm. f.)

Transcription

1 Chapter I Phytochemical screening and in vitro antioxidant activity of Merremia emarginata (Burm. f.) 1. INTRODUCTION The plant Merremia emarginata belongs to Merremia genus and Convolvulaceae family. This is also called as Morning-Glory family. Merremia emarginata (Burm.f.) is claimed to be bitter, acrid, refrigerant, deobstruant, diuretics, alterant, anthelmintic, carminative and digestive. It is also useful in nephropathy, uropathy, pneumonisis, cardiac diseases, gastropathy, metropathy, fever, anaemia, leucoderma, otalgia, cephalgia and rat bite etc (Khare, 2007). Merremia emarginata (Burm. f.) synonym Ipomoea reniformis is prevalent throughout India, Malaysia and tropical Africa. The plant is well known as Elikadhu keerai or Paratai keerai in Tamil and Kidney leaf morning-glory in English. M. emarginata is uncultivated food crop consumed by poor people in India as green leaf vegetable (Figure 2.1). It is a creeping perennial herb rooting at the nodes. Leaves are simple, long stalked, reniform or ovate-cordate. Flowers are yellow, auxiliary and 1-3 flowered with very short peduncles. Fruits looks like sub globose capsules with 2-4 glabrous light brown seeds. The plant is therapeutically used as deobstruent, diuretic, for rheumatism, neuralgia, cough and headache (Singh et al., 2002). Babu et al. (2009) have reported that M. emarginata (ME) holds antioxidant activity, alpha amylase inhibitory activity and cytotoxic activity. M. emarginata leaves have antibacterial activity against both gram positive and gram negative bacterial species (Elumalai et al., 2011). 23

2 Figure 2.1. Merremia emarginata (Burm. f.) Scientific classification: Kingdom : Plantae Division : Magnoliophyta Class : Magnoliosida Order : Convolvulales Family : Convolvulaceae Genus : Merremia Species : Emarginata Botanical name: Merremia emarginata Tamil : Elikathu Keerai or Paratai Keerai English : Kidney leaf morning glory Bengali : Bhuikamri; Indurkani Hindi : Musaakaani Sanskrit : Akhukarni, Mooshikakarnee Marati : Undirkaani Telugu : Elika-Jemudu 24

3 Habitat and distribution Merremia emarginata (Ipomoea reniformis) is a procumbent herb spreading upto 1m and possess yellow coloured flowers. Mainly grows in rainy season and winter season. Its properties appear to be fanciful. Weedy in fields, roadsides, grasslands, on clay to sandy soils, forest floors; spreads upto 200 m. throughout India in humid areas. Phenology Flowering; November, Fruiting: March, November. Parts used: Leaves. 2. MATERIALS AND METHODS 2.1. Plant material Leaves of M. emarginata were collected in November from Dharmapuri, Tamil Nadu in India. The identity of the plant was authenticated by the Department of Plant Science, Bharathidasan University, Tiruchirappalli. The leaves were picked and washed with water to remove all unwanted debris, shade dried at a room temperature of 28º C for 10 days, ground into powder using an electronic grinder and stored in an airtight container until further use Preparation of extract Powdered plant material (10 g) was extracted with distilled water (250 ml; C) on a shaker (Orbitec-scigenics Biotech, India) for 48 h. The extract was filtered with Whatman No. 1 filter paper. The filtrate of aqueous extract was quickly frozen at - 50 C and dried for 48 h using a vacuum freeze dryer (Christ alpha 1-2 / LD plus, Germany) to produce a yield of 8.87 % of dry extract. The resulting extract was reconstituted with distilled water to produce the desired concentrations and used for further analysis (Jayakumar et al., 2009; Gulcin et al., 2011). 25

4 2.3. Phytochemical screening The dry extract was used for the phytochemical screening of compounds, namely flavonoids, alkaloids, saponins, steroids and reducing sugars (Harborne, 1998). Approximately 0.2 g of the extract was dissolved in 2 ml of methanol and heated. A chip of magnesium metal was added to the mixture, followed by the addition of a few drops of concentrated hydrochloric acid. The formation of a red colour was indicative of the presence of flavonoids. Approximately 0.5 g of the extract was dissolved in 3 ml of chloroform, and a few drops of filtered concentrated sulphuric acid were carefully added to the filtrate to form a lower layer. A reddish-brown colour at the interface was a positive indicator for the presence of steroids. A 2-ml aliquot of the extract was treated with Dragendorff's reagent to test for the presence of alkaloids. Approximately 1 ml of alcoholic extract was diluted separately with 20 ml of distilled water and shaken in a graduated cylinder for 15 minutes. A 1-cm layer of foam indicated the presence of saponins; 2 ml of TCA was added to 1 ml of extract, and the formation of a yellow-to-red precipitate showed the presence of terpenoids. A total of 5 ml of Benedict s solution was added to 1 ml of the extract and heated in a boiling water bath. A red, yellow or green precipitate indicated the presence of reducing sugars Determination of total phenolics The amount of total phenolics in the aqueous leaf extract of M. emarginata was determined with the Folin-Ciocalteu reagent (Singleton et al., 1999), With some modifications, 100 µl of Folin-Ciocalteu reagent and 200 µl of Na 2 CO 3 (2% w/v) were added to 100 µl of the plant extract solution (1 mg/ml). The resulting mixture was incubated at 45 C with shaking at 200 rpm for 15 min. The absorbance of the samples was measured at 765 nm using a UV-visible spectrophotometer. The total phenolics were expressed as mg/g of gallic acid equivalents (GAE). GA of different concentrations (0 to 10 µg/ml) was dissolved in methanol, and a linear standard graph 26

5 was obtained by plotting the concentration of GA along the X-axis and absorbance along the Y-axis. All experiments were performed in triplicate Estimation of total flavonoids A modified aluminium chloride method was used for determining the total flavonoid content in the plant extract (Shen et al., 2009). A total of 100 µl of sample (1 mg/ml) was mixed with 600 µl of methanol, 40 µl of 10% aluminium chloride, 40 µl of 1 M potassium acetate and 1320 µl of distilled water, and the tubes were kept at room temperature for 30 min. The absorbance of the reaction mixture was measured at 420 nm with a UV-visible spectrophotometer. The total flavonoid content was obtained from extrapolation of the calibration curve, which was made by preparing various concentrations of a quercetin solution (0 to 20 µg/ml) in methanol. The concentrations of total flavonoids were expressed in µg/ml of plant extract quercetin equivalent (QE) Determination of reducing power The reducing power of the aqueous extract was determined by the method of Oyaizu (1986). A mixture containing 2.5 ml of 0.2 M phosphate buffer (ph 6.6) and 2.5 ml of K 3 Fe(CN) 6 (1% w/v) was added to 1.0 ml of the various concentration of plant extract dissolved in distilled water (200 to 1000 µg/ml). The resulting mixture was incubated at 50 C for 20 min, followed by the addition of 2.5 ml of TCA (10% w/v). The mixture was centrifuged at 5000 rpm for 10 min and to the 2.5 ml of supernatant, an additional 2.5 ml of distilled water and 0.5 ml of FeCl 3 (0.1%, w/v) were added. The absorbance was then measured at 700 nm against a blank sample. The BHT was used as a standard to determine the relative reducing power of M. emarginata , 2-Diphenyl-1-Picrylhydrazyl (DPPH) assay The antioxidant activity of the plant extract was measured as described previously Kikuzaki and Nakatani, (1993). A total of 1 ml of mm DPPH 27

6 prepared in methanol was mixed with 1.0 ml of aqueous extract ranging from 20 to 100 µg/ml. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance was measured spectrophotometrically at 517 nm. The scavenging ability of the plant extract was calculated using the following equation: DPPH Scavenging activity (%) = [(A c A s )] / (A c )] 100 (2.7) Where A c is the absorbance of DPPH + methanol; A s is the absorbance of DPPH radical + sample (i.e. standard or plant extract) ABTS scavenging activity The ABTS scavenging activity of the plant extract was performed using the method described by Re et al., (1999). The working solution was prepared by mixing stock solutions of 7 mm ABTS and 2.4 mm potassium persulphate in equal amounts and allowing them to react for 12 h at room temperature in the dark. The resulting solution was later diluted with distilled water, and the absorbance read at 734 nm using a UV-visible spectrophotometer. A total of 1 ml of freshly prepared ABTS solution was added to 1 ml of the plant extract, the reaction mixture was vortexed for 10 seconds and the absorbance was measured at 734 nm after 6 min. The above protocol was used for the standard BHT of various concentrations (20 to 100 µg/ml). The percentage of the extract s ABTS scavenging inhibition activity was calculated and compared with that of BHT. The percentage of the ABTS scavenging inhibition was calculated from the equation in section

7 2.9. Scavenging activity of superoxide anion The superoxide anion scavenging activity of the extract was determined by the method of Yen and Chen (1995). The reaction mixture consists of 1 ml Nitroblue tetrazolium (NBT) and 1 ml of plant extract (20 to 100 µg/ml), 1 ml of 60 µm potassium metabisulphite (PMS) (prepared in phosphate buffer 0.1 M, ph 7.4) and 1 ml of NADH (in phosphate buffer) was incubated at 25 C for 5 min, the absorbance was read at 560 nm. The percent of scavenging inhibition of superoxide radical was calculated from the above mentioned same equation (2.7) Determination of inhibitory activity on lipid peroxidation A total of 0.1 ml of Swiss Albino rat liver homogenate (100 mg of rat liver tissue in 1 ml of 50 mm phosphate buffer, ph 7.0) was homogenised and centrifuged at 10,000 rpm for 15 min at 4 C, and the supernatant was used for analysis. Then, 100 μl of 0.16 mm ferrous ammonium sulphate, 100 μl of 30 mm KCl, and different concentrations of extract (100 to1000 μg/ml) were incubated for 1 h at 37 C. The lipid peroxide formed was estimated by measuring thiobarbituric acid-reacting substances (TBARS) (Ohkawa et al., 1979). A total of 0.4 ml of the incubation mixture was treated with 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 0.8% TBA and 1.5 ml of 20% glacial acetic acid, ph 3.5. The volume of the mixture was brought up to 4 ml with distilled water, and the reaction mixture was kept in a water bath at 100 C for 1 h. After cooling, 1 ml of distilled water and 5 ml of an n-butanol and pyridine mixture (15:1, v/v) were added, and the solution was stirred vigorously. After centrifugation, the absorbance of the organic layer was measured at 532 nm. The percentage of lipid peroxidation inhibition was determined by comparing the results of the test compounds (treated with the M. emarginata extract) with those of the control (not treated with the M. emarginata extract). BHT was used as a standard. The percentage of the lipid peroxide scavenging ability of the extract was calculated by the formula in section

8 3. RESULTS AND DISCUSSION 3.1. Phytochemicals The phytochemical screening of the aqueous extract of M. emarginata indicated the presence of six main classes of compounds: flavonoids, steroids, alkaloids, saponins, terpenoids and reducing sugars (Table 2.1). While some of the paper suggests alkaloids are absent in M. emarginata (Bhatt Mehul et al., 2010), but our qualitative assay for alkaloids shows the presence of alkaloids in M. emarginata. The total phenolics content of the aqueous leaf extract was mg of gallic acid equivalents/g of aqueous extract with reference to gallic acid standard curve (y=0.101x+0.114, R² = 0.999). The total flavonoids content of the plant were mg of quercetin equivalents/g of aqueous extract with reference to quercetin standard curve (y=0.093x+0.047, R² = 0.994). Generally, Most of the bioactive compounds fall under the classification of above mentioned phytochemicals, which possess antioxidant activities and fluorescence quenching potential. Table 2.1. The Phytochemical screening of M. emarginata extract revealed the presence of following compounds Phytochemical Screening Flavonoids Steroids Alkaloids Saponin Terpenoids Reducing sugars ++ = moderate amount Presence = trace amount 30

9 3.2. Determination of reducing power The reducing capacity of an aqueous extract is a major indicator of antioxidant activity, and the efficiency of certain antioxidants can be correlated with their reducing power (Jayaprakasha et al., 2000). The reducing power of the M. emarginata aqueous extract was compared with a standard BHT at 700 nm and was found to increase proportionally to the extract concentration (Figure 2.2). The reducing power of 1 mg/ml M. emarginata extract had an optical density of , which was comparable to that of the standard BHT with an optical density of The reducing power of medicinal M. emarginata might be due to its hydrogen donating ability, as reported by Shimada et al, (1992). M. emarginata contains high amounts of reductones, which could react with free radicals to stabilise and terminate radical chain reactions. Figure 2.2. Reducing power of Merremia emarginata aqueous extract compared with butylated hydroxytoluene (BHT). Each value is expressed as mean ± standard deviation (n=3) 31

10 3.3. 2,2-diphenyl-1-Picrylhydrazyl (DPPH) assay M. emarginata extract was treated with DPPH, which is reacted with methanol or absolute ethanol to yield purple DPPH radicals. Antioxidants, such as polyphenolics and flavonoids, in the sample might have scavenged DPPH radicals, resulting in a decrease in the observed intensity of purple (Blois, 1958). Table 2.2 shows the inhibition of the DPPH radical-scavenging activity of the ME extract (IC 50 =86.5 µg/ml) compared with the control BHT (IC 50 =23.4 µg/ml). The ME extract had a high DPPH radical inhibitory capacity compared with those previously reported for other Indian leafy vegetables, namely Asteracantha longifolia Nees, Bacopa monnieri (Linn.) Pennell, Bauhinia racemosa Lam, Centella asiatica (Linn.) Urban, Chenopodium album Linn, Enhydra fluctuans Lour, Ipomoea reptans (Linn.) Poir, Moringa oleifera Lam, yctanthes arbortristis Linn, Paederia foetida Linn, and Trigonella foenumgraecum Linn (Figure 2.3) (Dasgupta and De, 2007). Table 2.2. The scavenging activity of aqueous extract of Merremia emarginata and BHT IC 50 value* Name of antioxidant assay Plants extract (µg/ml) BHT (µg/ml) DPPH radical scavenging ABTS radical scavenging Superoxide anion radical scavenging *Amount of plant extract and BHT required for 50% inhibition of DPPH radical 32

11 Figure 2.3. Inhibitory effect of Merremia emarginata aqueous extract on DPPH radicals compared with butylated hydroxytoluene (BHT) 3.4. ABTS scavenging activity The antioxidant ability of the ME extract was compared with that of the standard BHT using the ABTS method, as this is a well-accepted method for the determination of the antioxidant activity of hydrogen-donating antioxidants. The aqueous extract (100 µg/ml) inhibited the blue colour by 70.5%. Table 2.2 depicts the ABTS radical-scavenging activity of the ME extract (IC 50 = 30.1 µg/ml) compared with that of the BHT control (IC 50 =27.2 µg/ml) (Figure 2.4). Subhasree et al, (2009) reported some Indian (Trigonella foenum-graecum, Centella asiatica, Sauropus androgynus and Pisonia alba) green leafy vegetables ABTS antioxidant capability. This was carried out by measuring the ability of methanol extracts of these plants. 33

12 Figure 2.4. Inhibitory effect of Merremia emarginata aqueous extract on ABTS radicals compared with butylated hydroxytoluene (BHT) 3.5. Scavenging activity of superoxide anion The superoxide radical is known to be very harmful to cellular components since it is a precursor of more reactive oxygen species (Gulcin et al., 2010). In the present study, the plant extract was found to be a prominent scavenger of superoxide radicals. BHT is a commercial antioxidant, which was used as a positive control for comparative study (Figure 2.5). The IC 50 values were found to be 40.3 µg/ml and 18.1 µg/ml for ME plant aqueous extract and BHT respectively (Table 2.2). 34

13 Figure 2.5. Scavenging effect of Merremia emarginata aqueous extract on superoxide radicals compared with butylated hydroxytoluene (BHT) 3.6. Determination of inhibitory activity on lipid peroxidation Lipid peroxidation is oxidative degradation of membrane lipids, the process leads to cause oxidative degradation of lipids, because the unsaturated fatty acid molecules are not very stable, they will easily react with reactive oxygen molecule (ROS), so thereby form lipid peroxide radical, it may cause cellular damage including DNA repair (Halliwell and Gutteridge, 1984). In the present investigation, M. emarginata plant extract was evaluated for inhibitory activity of lipid peroxidation. Both M. emarginata extract and BHT standard inhibited lipid peroxidation in a concentration dependent manner. Figure 2.6 shows the inhibition of lipid peroxidation activity of M. emarginata and commercial antioxidant BHT. The IC 50 value for M. emarginata extract and BHT are 325 µg/ml and 100 µg/ml respectively, against lipid peroxidation. Some of the Indian green leafy vegetables (Trigonella foenum-graecum, Centella asiatica, Sauropus androgynus and Pisonia alba) lipid peroxidation activity 35

14 shows similar IC 50 values (Subhasree et al., 2009). These in vitro antioxidant assay results obtained show a fairly constant high percentage inhibition of M. emarginata. Figure 2.6. Inhibitory effect of Merremia emarginata aqueous extract on lipid peroxidation radicals compared to butylated hydroxytoluene (BHT). Each value is expressed as mean ± standard deviation (n=3) 4. CONCLUSION The results and discussion of present study conclude that the aqueous extract of M. emarginata consists of considerable quantity of total phenolics, flavonoids compounds and the phytochemicals screening indicated presence alkaloids, steroids, saponin, phenolics, flavonoids, reducing sugars and terpenoids. It exhibited high antioxidant and free radical scavenging activities. The in vitro assays like DPPH, ABTS, superoxide anion scavenging activity and inhibitory activity of lipid peroxidation indicates that the M. emarginata plant extract is a significant source of natural antioxidant. 36