Antioxidant Activity of the plant Andrographis paniculata (Invitro)

Chapter 4 Antioxidant Activity of the plant Andrographis paniculata (Invitro) 4.1 INTRODUCTION Antioxidants prevents or repairs the cells against reactive oxygen species, reduces damage caused by free radicals such as oxygen, peroxides, superoxide, hydroxyl radicals and peroxyl radicals, which leads to chain reaction and finally it ends up into cell damage (Mattson and Cheng, 2006). Natural antioxidants prevents body tissues from cell damage, protects degenerative diseases such as cancer, ischema, atherosclerosis, cardiovascular diseases which plays a key role in maintenance of health and prevention (Uddin et al., 2008; Jayasri et al., 2009). Antioxidants exert their activity by scavenging that forms stable radicles which persist reaction with the electrons from the molecules in the sudden surrounding environment. They may damage crucial bio molecules in all membranes, if these radicals are not scavenged effectively in time. Mitochondria and the DNA results in abnormalities leading to diseased conditions present in lipids and proteins (Uddin et al., 2008). Thus, free radicals are involved in a number of diseases like muscular dystrophy, disease that caused through heredity, cataract, damages liver cells, insulin deficiency, gastric diseases, reproduction problem, rheumatoid arthritis, neural diseases etc., in humans (Chen et al., 2006; Uddin et al., 2008). Sufficient amount of antioxidants were not produced in the human body, which is essential for preventing oxidative stress. Natural antioxidant defences removes free radicals that are generated in the body, such as glutathione or catalases (Sen, 1995). The deficiency had to be compensated by using natural exogenous antioxidants, as a chemopreventive mechanism in plants (Madsen and Bertelsen., 1995; Rice Evans et al., 1997; Diplock et al., 1998; Cai and Sun, 2003). In human body, many plants serve to control the levels of anti-oxidants. In phytochemicals, the free radicals generated in the body as health protective have been recorded in biological activities that are responsible for prevention of many diseases (Hertog and Feskens, 1993; Anderson and Teuber, 2001). Many dietary polyphenolic constituents that 57

are derived from plants is necessary for good health, reduces the damage of cellular biomolecules (Rice-Evans and Miller, 1997; Jayasri et al., 2009). Methanol extract of Cinnamon contains a number of antioxidant compounds which can effectively scavenge reactive oxygen species including superoxide anions and hydroxyl radicals as well as other free radicals invitro. The fruit of Cinnamon, an unconditional part of the plant, contains a good amount of phenolic antioxidants which may protect against mutagenesis to counteract the damaging effects of free radicals. Antioxidants are often added to foods to prevent the radical chain reactions of oxidation, and they act by inhibiting the initiation and propagation step leading to the termination of the reaction and delay the oxidation process. Due to safety concerns of synthetic compounds, food industries have focused on finding natural antioxidants to replace synthetic compounds. In addition, there is growing trend in consumer preferences for natural antioxidants, all of which has given more impetus to explore natural sources of antioxidants. In the present investigation, among the four plants, Andrographis paniculata showed good activity against both antidiabetic and antioxidant properties, hence this plant is chosen for further studies. The study is aimed at understanding the plant s role as an antioxidant. Antioxidants are gaining importance due to the increase in the reactive oxygen species generated in our cells due to modern lifestyle. Along with being an anti-diabetic drug, if the extracts are found to be potent antioxidants, their pharmacological value would greatly increase. The plants were extracted with different solvents based on polarity. The antioxidant property of each of the extracted solvents were analyzed in different assays such as DPPH, Total phenolic content,h 2 O 2 radical scavenging activity, β carotene bleaching activity,total reducing power. 4.2 MATERIALS AND METHODS 4.2.1 PLANT COLLECTION Fresh leaves of the plant were collected from Vellore District, Tamil Nadu, India. Plant material were carefully washed with tap water, rinsed with distilled water and dried in air for 1hr. After that it was processed into small particles and dried at 26 degree celcius for one week. They were again grinded into fine powder and stored at room temperature. 58

4.2.2 PLANT PREPARATION AND EXTRACTION BY DIRECT EXTRACTION METHOD Sequencing extracts of the plants were prepared by successive maceration of the fine powder (10g) for 2 days at room temperature with 100ml of various solvents on an orbital shaker. The solvents used were n-hexane, Ethyl Acetate, Chloroform and Methanol respectively. The final extracts obtained were filtered and the filtrates were subjected to lyophilisation (Shimadzu Analytical (India) Pvt.Ltd., Mumbai, India) to obtain powdered extracts which were used for the following assays. 4.3 DETERMINATION OF ANTIOXIDANT ACTIVITY 4.3.1 2,2-DIPHENYL-2-PICRYLHYDRAZYL FREE RADICAL SCAVENGING ACTIVITY (DPPH) One ml of four concentrations (25, 50, 75 and 100mg/ml) of each extract of the plant dissolved in methanol was added to 1ml of DPPH (0.16mM in methanol). 1ml DPPH in methanol with 1ml pure methanol was used as a control. The reaction mixtures were mixed and kept in the darkness at room temperature for half an hour. Their absorbance was then read at 517nm (Farhan et al., 2012).The ability of plant extracts in DPPH scavenging activity was calculated using the following equation Where, Absorbance control (DPPH Solution without extract) and Absorbance sample (DPPH Solution with extract). Comparison of the results with positive control, ascorbic acid was done. The percentage inhibition was plotted against the sample extract concentration in order to calculate the IC 50 values, which is the concentration (µg/ml) of extract that causes 50% loss of DPPH activity. 4.3.2 HYDROGEN PEROXIDE RADICAL SCAVENGING ACTIVITY One ml of extract at four concentrations 25, 50, 75,100 mg/ml was taken in 4 test tubes. 0.6 ml of hydrogen peroxide (40mM) was added with phosphate buffer. The samples were incubated at 30 0 C for 10 minutes (Ruch et al.,1989). After incubation, absorbance 59

was determined at 230 nm against phosphate buffer as a blank (Wettasinghe et al., 1997). This was done in replicate. The percentage H 2 O 2 scavenged was calculated using the formula: Where A 1 = absorbance of the H 2 O 2 without extract. A 2 = absorbance of the sample with extract. 4.3.3 TOTAL PHENOLIC CONTENT: One ml aliquots of extracts (100 mg/l) were added to a Erlenmeyer flask containing nine ml of water. The mixture was added with1 ml of Folin-Ciocalteu s reagent and vortexed. After 5 min, 10 ml of 7% sodium carbonate was added to the mixture and incubated for 90 min at 25 C. Absorbance of the sample against reagent blank was determined at 750 nm. Using standard curve, a reagent blank was prepared and the amount of phenolic compound in the extract was determined. Results of the plant extracts was then calculated based on the equation below and expressed as mg gallic acid equivalent (GAE)/g fresh weight (Anyasor et al., 2010) Where C = total content of phenolic compound in gallic acid equivalent (GAE)/g, c = the concentration of gallic acid established from the calibration curve (μg/ml) V= volume of extract (ml) m = weight of the crude plant extract (g). 4.3.4 β - CAROTENE BLEACHING ACTIVITY To 3ml of β-carotene solution (5mg of β carotene/50ml of chloroform) 40mg of linoleic acid and 400mg of Tween 20 was added. Then, this mixture was vortexed well. To the dried mixture, 100ml of distilled water was added in order to form β-carotene linoleic acid emulsion. 1ml of solvent extract was taken in 4 test tubes and 1.5ml of emulsion was 60

added. Then, the mixture was incubated in water bath at 50ºC for 60 minutes. This was done in replicate. Finally, the absorbance was read at 470nm (Ruch et al., 1989). This was done in triplicates. The final activity of Andrographis paniculata extract was calculated by using the following equation: Where DR C = Degradation rate of the control, DR S = Degradation rate of the sample [(a/b)/60] a=initial absorbance of the sample, b=absorbance after 60 min of incubation. 4.3.5 REDUCING POWER Four concentrations (25, 50, 75 and 100 mg/ml) of A. paniculata solvent extracts were added with 2.5ml of phosphate buffer and 2.5 ml of potassium ferricyanide. Then, the mixture was kept in the incubator at 50 0 C for 30 minutes. 2.5 ml of trichloroacetic acid was added and the mixture was kept for centrifugation at 3000 rpm for 10 minutes. 2.5 ml of supernatant solution was taken and added with 2.5 ml of distilled water and 0.5 ml of ferric chloride. Standard used are L-ascorbic acid. Finally, the absorbance of all samples was studied at 700 nm. This was done in triplicates (Yildirim et al., 2001). 4.4 STATISTICAL ANALYSIS Statistical analyses were done using MS-Excel 2010. The standard errors in all the analyses were found to be less than or equal to 5% of the mean values. 4.5 RESULTS AND DISCUSSION Plants are potential sources of natural antioxidants. Environmental stresses may trigger the oxidative stress in plants, generating the formation of reactive oxygen species (ROS), leading to cellular damage, metabolic disorders, and senescence processes (Zhang et al., 2006). Indeed, ROS can react with biological molecules, that leads to cell damage (Kong et al., 2003). Plants produce immunity to protect themselves from the harmful effect of oxidative stress (Bartoli et al., 1999). As in abiotic stress, growth regulators also will results in the production of Reactive Oxygen Species, which activates the immunity of 61

antioxidant enzymes. Generation of Reactive Oxygen Species causes cellular damage by activating the chain reaction (Nayyar et al., 2006). ROS scavenging is one among the common defences response in cell signalling and homeostasis. ROS scavenging depends on the detoxification mechanism generated during mitochondrial oxidative mechanism as well as in cellular response (Jaleel et al., 2007). 4.5.1 DPPH RADICAL SCAVENGING ASSAY The results for the antioxidant activity are given in Table 4.1. The antioxidant activity for the methanolic extract was found to be the highest. In all cases except the methanolic extract, the antioxidant activity decreased till a concentration of 75 mg/ml, after which, a sharp increase was seen at 100 mg/ml. And hence the activity was found to be highest at a concentration of 100 mg/ml. The DPPH activity increased with concentration in case of the methanol extract. These results have been graphically represented in Figure 4.1. The higher DPPH activity of methanolic extract can be featured to its highest phenolic compound content. This is because flavonoids, the primary compounds responsible for antioxidant activity of plants are phenolic compounds (Pietta et al., 2000). 62

% Antioxidant Activity (DPPH) Table 4.1: Percentage antioxidant activity of the 4 extracts at 4 concentrations. DPPH Concentration(mg/ml): Hexane Ethyl Chloroform Methanol Extract Acetate 25 5.84±0.34 34.6±0.28 23.06±0.18 45.02±0.24 50 9.32±0.28 32.4±0.14 16.04±0.12 43.14±0.29 75 3.26±0.14 12.8±0.19 11.34±0.16 46.28±0.03 100 10.09±0.16 35.8±0.24 24.26±0.18 49.04±0.12 The values represent Mean±SEM(%) for sets of three values. 60 50 40 30 20 10 0 25 50 75 100 Concentration (mg/ml) Hexane Ethyl Acetate Chloroform Methanol Figure 4.1: Percentage antioxidant activity of the 4 extracts at 4 concentrations. 63

4.5.2 HYDROGEN PEROXIDE RADICAL SCAVENGING ACTIVITY The scavenging activity of the methanol extract was found to be the highest, followed by chloroform and ethyl acetate, respectively. This has special significance due to the fact that H 2 O 2 is a potent oxidant. It has a strong tendency to oxidize DNA in the cells, causing mutations. Reactive agents of H 2 O 2 may sometimes even cause cellular damage due to the generation of free hydroxyl radicals inside the cell. The H 2 O 2 radical scavenging activity is attributed to primary antioxidants, which in case of plants would be phenolic (Dorman et al., 2003).Results of the H 2 O 2 radical scavenging activity are described in Table 4.2. Thus, the higher activity of the methanol extract is most likely due to this. The results are graphically depicted in Figure 4.2. Table 4.2: Percentage H2O2 radical scavenging activity of the 4 extracts at 4 concentrations H 2 O 2 Radical Scavenging Activity Concentration(mg/ml): Hexane Ethyl Chloroform Methanol Extract Acetate 25 13.02±0.86 28.1±0.24 46.1±0.42 50.1±0.68 50 24.08±0.92 32.2±1.4 49.5±0.85 61.8±1.2 75 26.09±0.24 46.8±1.26 56.1±1.36 72.4±1.68 100 28.23±1.63 59.1±2.01 62.4±1.88 75.6±1.78 The values represent Mean±SEM(%) for sets of three values. 64

Antioxidant Activity (H2O2) 80 70 60 50 40 30 20 10 0 25 50 75 100 Concentration (mg/ml) Hexane Ethyl Acetate Chloroform Methanol Figure 4.2:Percentage radical scavenging activity of the 4 extracts at 4 concentrations. 4.5.3 TOTAL PHENOLIC CONTENT The results for the total phenolic content are given in Table 4.3. As inferred from the table, the methanol extract had the highest total phenolic content. This further implies that the methanol extract contains the maximum number of tannins and flavonoids, since these are phenolic compounds. Table 4.3: Total phenolic content of the extracts in Gallic Acid Equivalents/gram. Extract Total Phenolics (GAE/g) n-hexane 0.35±0.0175 Ethyl Acetate 0.33±0.0165 Chloroform 0.51±0.0255 Methanol 0.82±0.0410 The values represent Mean±SEM for sets of three values. 65

4.5.4 β CAROTENE BLEACHING ACTIVITY β carotene bleaching activity is again an indirect measure of the antioxidant activity of a plant extract. β carotene gets bleached by hydro peroxides like H 2 O 2 (Khare et al., 2012).In the presence of antioxidants, this bleaching is greatly reduced due to the neutralizing effect of the antioxidants on the hydro peroxides. The results of the β carotene bleaching activity are given in Table 4.4. Again, the methanol extract showed the lowest amount of β carotene, and hence, the highest concentration of antioxidants. This can be correlated to the results of the antioxidant activity and total phenolic content. The results are graphically depicted in Figure 4.3. Table 4.4: β carotene bleaching activity of the extracts. Concentration(mg/ml): Hexane Ethyl Chloroform Methanol Extract Acetate 25 67.24±0.14 45.03±0.16 54.00±0.18 74.08±0.12 50 54.32±0.18 43.12±0.18 56.13±2.00 76.02±0.24 75 68.14±1.29 48.07±0.29 58.12±1.04 78.04±1.02 100 72.32±0.14 52.01±0.24 60.03±1.02 82.06±0.14 The values represent Mean±SEM(%) for sets of three values. 66

% β Carotene 100 80 60 40 20 0 25 50 75 100 Concentration (mg/ml) Hexane Ethyl Acetate Chloroform Methanol Figure 4.3: β carotene bleaching activity of the 4 extracts at 4 concentrations.. 4.5.5 REDUCING POWER The ferric reducing assay measures the ability of an antioxidant to reduce a reactive oxygen species against that species oxidative power (Saklani et al., 2011). This is important to make the reactive oxygen species more stable and unreactive. The results are tabulated in Table 4.5 and graphically represented in Figure 4.4. As can be observed from the graph, the methanol extract showed a reducing power nearly equal to that of L Ascorbic Acid. The chloroform extract showed a very good reducing power at lower concentrations. However, at higher concentrations, its reducing power was lesser than that of the methanol extracts. 67

Total Reducing Power Table 4.5: Total reducing power of the 4 extracts at 4 concentrations. Concentration Hexane Ethyl Chlorofor Methanol Ascorbic (mg/ml) Acetate m Acid Extract 25 0.12±0.006 0.16±0.008 0.24±0.012 0.14±0.007 0.11±0.005 50 0.14±0.007 0.29±0.014 0.34±0.017 0.32±0.016 0.25±0.012 75 0.24±0.012 0.34±0.017 0.42±0.021 0.44±0.022 0.46±0.023 100 0.32±0.016 0.42±0.021 0.68±0.034 0.72±0.036 0.74±0.037 The values represent Mean±SEM for sets of three values. 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 25 50 75 100 Concentration (mg/ml) Hexane Ethyl Acetate Chloroform Methanol Ascorbic Acid Figure 4.4: Total reducing power of the 4 extracts at 4 concentrations. 68

4.6 CONCLUSION Based on the above experiments, we can conclude that methanol extracts of A. paniculata are the most effective antioxidants. These could find potential application in today s urban lifestyle which increases our exposure to various harmful oxidants. These findings indicate that in addition to its anti-diabetic effect, A. paniculata has a potent antioxidant activity. 69