In Vitro Antioxidant Activity of Leaf Extracts of Alangium Salvifolium (L.F.) Wang (Alangiaceae)

Bioscience Discovery, 5(1):74-81, Jan. 2014 © RUT Printer and Publisher (http://jbsd.in) ISSN: 2229-3469 (Print); ISSN: 2231-024X (Online) Received: 07-12-2013, Revised: 22-12-2013, Accepted: 01-01-2014e

Full Length Article

In vitro antioxidant activity of leaf extracts of Alangium salvifolium (L.f.) Wang (Alangiaceae)

Sakthidevi G, Mohan V R and Jeeva S1

Ethnopharmacology unit, Research Department of Botany, V.O.Chidambaram College, Tuticorin-628008, Tamil Nadu. 1Department of Botany, Scott Christian College (Autonomous), Nagercoil-629003, Kanyakumari, Tamil Nadu.

ABSTRACT In vitro antioxidant activity of petroleum ether, benzene, ethyl acetate, methanol and ethanol extracts of leaf of Alangium salvifolium have been tested using various antioxidant model system viz, DPPH, hydroxyl, superoxide, ABTS and reducing power. The methanol extract of leaf showed potent DPPH, ABTS radical cation scavenging activities. Ethanol extract of leaf showed strong hydroxyl, superoxide radical scavenging activities. Methanol extract of Alangium salvifolium showed the highest reducing ability. This study indicates significant free radical scavenging potential of Alangium salvifolium which can be exploited for the treatment of various free radical mediated ailments.

Key words: In vitro antioxidant activity, Alangium salvifolium, flavonoid, DPPH, ABTS

NTRODUCTION Currently available synthetic antioxidants Reactive Oxygen Species (ROS) such as like butylated hydroxyl anisole (BHA), butylated superoxide anion, hydroxyl radical and hydrogen hydroxyl toluene (BHT), tertiary butylated peroxide play a crucial role in the development of hydroquinone and gallic acid esters have been various ailments such as arthritis, asthma, suspected to cause or prompt negative health dementia, mongolism, carcinoma and Parkinson’s effects. Hence, strong restrictions have been disease. The free radicals in the human body are placed on their application and there is a trend to generated through aerobic respiration or from substitute them with naturally occurring exogenous sources (Narayanaswamy and antioxidants. Recently, there has been an upsurge Balakrishnan, 2011). Many oxidative stress related of interest in the therapeutic potentials of diseases are as an outcome of accumulation of free medicinal plants as antioxidants in reducing such radicals in the body. Antioxidant compounds may free radical induced tissue injury. Many plant function as free radical scavengers, complexes of extracts and phytochemicals have shown to have pro-oxidant metals, reducing agents and quenches free radical scavenging properties but generally of singlet oxygen formation (Satheeshkumar et al., there is still a demand to find more information 2010). Antioxidants are considered as a promising concerning the antioxidant potential of plant therapeutic approach as they may be playing species. neuroprotective (preventing apoptosis) and Alangium salvifolium (L.f.) Wang (Family: neurodegenerative roles. The main characteristic of Alangiaceae) commonly known as Alangi in Tamil an antioxidant is its ability to trap free radicals was found distributed in South India. The leaves of (Mon et al., 2011). Alangium salvifolium are used as astringent, http://biosciencediscovery.com 74 ISSN: 2231-024X (Online) Sakthidevi et al., laxative, refrigerant and used to treat rheumatism, content was expressed as gram of gallic equivalents leprosy, syphilis and asthma (Kijima et al., 1992). per 100 gram of dry weight (g100g-1DW) of the The root bark is used as purgative, astringent, plant samples. anthelmintic, antipyretic, expectorant, anti- Estimation of Flavonoids inflammatory, emetic, diaphoretic, anticancer, The flavonoids content was determined according antimicrobial and antitumor agents (Ali et al., to Eom et al., (2007). An aliquot of 0.5ml of sample 1983; Rao et al., 1999 and Anonymous, 1992). The (1mg/mL) was mixed with 0.1ml of 10% aluminium root is used as hypotensive agent, anthelmintic and chloride and 0.1ml of potassium acetate (1M). In used in the treatment of biliousness, inflammation this mixture, 4.3ml of 80% methanol was added to and snakebite. The bark shows antitubercular make 5mL volume. This mixture was vortexed and activity. The fruits are used as laxative, refrigerant, the absorbance was measured emetic and antiphlegmatic agent. As there is no spectrophotometrically at 415nm. The value of detailed report on in vitro antioxidant activity using optical density was used to calculate the total different solvents in the leaves of this plant, the flavonoid content present in the sample. present study was carried out to develop in vitro DPPH radical scavenging activity antioxidant data on the leaves which is essential for The DPPH is a stable free radical and is widely used its pharmacological studies. to assess the radical scavenging activity of antioxidant component. This method is based on MATERIALS AND METHODS the reduction of DPPH in methanol solution in the Collection of plant sample presence of a hydrogen donating antioxidant due Leaves of Alangium salvifolium (L.f.) Wang was to the formation of the non radical form DPPH-H collected from Kottaram, Kanyakumari District, (Shen et al., 2010). Tamil Nadu. With the help of local flora, voucher The free radical scavenging activity of all the specimens were identified and preserved in the extracts was evaluated by 1, 1-diphenyl-2-picryl- Ethnopharmacology Unit, Research Department of hydrazyl (DPPH) according to the previously Botany, V.O.Chidambaram College, Tuticorin, Tamil reported method (Shen et al., 2010). Briefly, an Nadu for further references. 0.1mM solution of DPPH in methanol was Plant sample extraction prepared, and 1mL of this solution was added to 3 Leaf of A. salvifolium was cleaned, shade dried and ml of the solution of all extracts in methanol at pulverized to powder in a mechanical grinder. different concentration (50,100,200, Required quantity of powder was weighed and 400&800μg/mL).The the absorbance was transferred to Stoppard flask and treated with measured at 517 nm using a UV-VIS petroleum ether, benzene, ethyl acetate, methanol spectrophotometer (Genesys 10S UV: Thermo and ethanol until the powder is fully immersed. electron corporation). Ascorbic acid was used as The flask was shaken every hour for the first six the reference. Lower absorbance values of reaction hours and then it was kept aside and again shaken mixture indicate higher free radical scavenging after 24hours. This process was repeated for three activity. The capability to scavenging the DPPH days and then the extract was filtered. The extract radical was calculated by using the following was collected and evaporated to dryness by using formula. vacuum distillation unit. The final extracts thus DPPH scavenging effect (% inhibition) = {(A0 – obtained were used for in vitro antioxidants A1)/A0)*100} activity. The methanol extract was used for the Where, A0 is the absorbance of the control estimation of total phenolic and flavonoids. reaction, and A1 is the absorbance in presence of Estimation of total phenolic content all of the extract samples and reference. All the Total phenolic content was estimated using the tests were performed in triplicates and the results Folin-Ciocalteu method (Lachman et al., 2000). were averaged. Samples (100µL) were mixed thoroughly with 2 ml Hydroxyl radical scavenging activity of 2% Na2CO3. After 2 min. 100 µL of Folin- The scavenging capacity for hydroxyl radical was Ciocalteu reagent was added to the mixture. The measured according to the modified method of resulting mixture was allowed to stand at room Halliwell et al. (1987). Stock solutions of EDTA temperature for 30 min and the absorbance was (1mM), FeCl3 (10mM), Ascorbic Acid (1mM), H2O2 measured at 743 nm against a blank. Total phenolic http://biosciencediscovery.com 75 ISSN: 2231-024X (Online) Bioscience Discovery, 5(1):74-81, Jan. 2014 ISSN: 2229-3469 (Print) (10mM) and Deoxyribose (10 mM), were prepared temperature for 12-16 h before use. The ABTS + in distilled deionized water. The assay was Solution were diluted with ethanol to an performed by adding 0.1mL EDTA , 0.01mL of absorbance of 0.70+0.02 at 734 nm. After addition

FeCl3,0.1mL H2O2, 0.36mL of deoxyribose, 1.0mL of of 100μL of sample or trolox standard to 3.9 mL of the extract of different concentration diluted ABTS+ solution, absorbance was measured (50,100,200,400 &800μg/mL) dissolved in distilled at 734 nm by Genesys 10S UV-VIS (Thermo water,0.33mL of phosphate buffer (50mM , pH scientific) exactly after 6 minutes. Results were 7.9), 0.1mL of ascorbic acid in sequence . The expressed as trolox equivalent antioxidant capacity mixture was then incubated at 370c for 1 hour. (TEAC). 1.0mL portion of the incubated mixture was mixed ABTS radical cation activity = {(A0 –A1)/A0)*100} with 1.0mL of 10%TCA and 1.0mL of 0.5% TBA (in Where, A0 is the absorbance of the control 0.025M NaOH containing 0.025% BHA) to develop reaction, and A1 is the absorbance in presence of the pink chromogen measured at 532nm. The all of the extract samples and reference. All the hydroxyl radical scavenging activity of the extract is tests were performed in triplicates and the results reported as % inhibition of deoxyribose were averaged. degradation is calculated by using the following Reducing Power equation The reducing power of the extract was determined

Hydroxyl radical scavenging activity= {(A0 – by the method of Kumar and Hemalatha (2011). 1.0 A1)/A0)*100} mL of solution containing 50,100,200,400 Where, A0 is the absorbance of the control &800μg/mL of extract was mixed with sodium reaction, and A1 is the absorbance in presence of phosphate buffer (5.0 mL, 0.2 M, pH6.6) and all of the extract samples and reference. All the potassium ferricyanide (5.0 mL, 1.0%): The mixture tests were performed in triplicates and the results was incubated at 50oC for 20 minutes. Then 5mL of were averaged. 10% trichloroacetic acid was added and centrifuged Superoxide radical scavenging activity at 980 g (10 minutes at 5oC) in a refrigerator The superoxide anion scavenging activity was centrifuge. The upper layer of the solution (5.0 mL) measured as described by Srinivasan et al. (2007). was diluted with 5.0 mL of distilled water and ferric The superoxide anion radicals were generated in chloride and absorbance read at 700 nm. The 3.0 ml of Tris – HCL buffer (16 mM, pH 8.0), experiment was performed thrice and results were containing 0.5 mL of NBT (0.3mM), 0.5 ml NADH averaged. (0.936mM) solution, 1.0 mL extract of different Statistical analysis concentration (125,250,500 &1000μg/ml), and 0.5 Antioxidant activities like DPPH radical scavenging mL Tris – HCl buffer (16mM, pH 8.0). The reaction activity, hydroxyl radical scavenging activity, was started by adding 0.5 mL PMS solution superoxide radical activity, ABTS radical cation (0.12mM) to the mixture, incubated at 25oC for 5 scavenging activity and reducing powers were min and the absorbance was measured at 560 nm estimated in triplicate determinations. Data were against a blank sample, ascorbic acid. The analyzed using the statistical analysis system SPSS percentage inhibition was calculated by using the (SPSS software for windows release 17.5; SPSS Inc., following equation Chicago IL, USA) Estimates of mean, standard error Superoxide radical scavenging activity= for aforesaid parameters were calculated.

{(A0 –A1)/A0)*100} Where, A0 is the absorbance of the control RESULTS reaction, and A1 is the absorbance in presence of Total phenolic content and total flavonoid content all of the extract samples and reference. All the The total phenolic and total flavonoid content of tests were performed in triplicates and the results the methanol extract of Alangium salvifolium leaf were averaged. were found to be 0.12g 100g -1 and 0.54g 100g-1. Antioxidant Activity by Radical Cation (ABTS. +) DPPH radical scavenging activity ABTS assay was based on the slightly modified DPPH radical scavenging activity of petroleum method of Huang et al (2011). ABTS radical cation ether, benzene, ethyl acetate, methanol and (ABTS+) was produced by reacting 7mM ABTS ethanol extracts of A. salvifolium leaf were shown solution with 2.45 mM potassium persulphate and in Figure1. allowing the mixture to stand in the dark at room http://biosciencediscovery.com 76 ISSN: 2231-024X (Online) Sakthidevi G et al.,

50 100 200 400 800 120 50 100 200 400 800 140 120 100

100

80 80 60 60 40

activity (%) activity 40 activity (%) 20

20 Hydroxyl radical scavenging radical Hydroxyl

DPPH radicalscavenging 0 0 Petroleum Benzene Ethyl Methanol Ethanol Ascorbic Petroleum Benzene Ethyl Methanol Ethanol Ascorbic Ether Acetate acid Ether Acetate acid Concentration (µg/mL) Concentration (µg/mL)

Figure 1: DPPH radical scavenging activity of different solvent Figure 2: Hydroxyl radical scavenging activity of different extracts of leaf of Alangium salvifolium solvent extracts of leaf of Alangium salvifolium

120 50 100 200 400 800 120 50 100 200 400 800 100 100 80 80

40 (%) 40

20 20

Superoxide dismutase dismutase (%) activity Superoxide 0 Petroleum Benzene Ethyl Methanol Ethanol Ascorbic ABTS cation radical scavenging activity scavenging radical cation ABTS Petroleum Benzene Ethyl Methanol Ethanol Trolox Ether Acetate acid Ether Acetate Concentration (µg/mL) Concentration (μg/mL)

Figure 3: Superoxide radical scavenging activity of different Figure 4: ABTS radical cation scavenging activity of different solvent extracts of leaf of Alangium salvifolium solvent extracts of leaf of Alangium salvifolium

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0.5

0.4

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Reducing power(OD)activity 0 PetroleumEther Benzene Ethyl Acetate Methanol Ethanol Ascorbic acid Concentration (μg/mL)

Figure 5: Reducing power ability of different solvents extract of leaf of Alangium salvifolium

The scavenging effect increases with the concentration methanol extract of A. salvifolium concentration of standard and samples. Among the leaf possessed 104.13% scavenging activity of solvent tested, methanol extract exhibited highest DPPH. DPPH radical scavenging activity. At 800µg/mL

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Hydroxyl radical scavenging activity IC50 value Hydroxyl radical scavenging activity of petroleum IC 50 values of petroleum ether extract of A. ether, benzene, ethyl acetate, methanol and salvifolium leaf and standard ascorbic acid for ethanol extract of A. salvifolium leaf were DPPH, hydroxyl, superoxide scavenging and trolox presented in figure 2. Ethanol extract showed very for ABTS radical cation scavenging were found to potent activity. At 800µg/mL concentration, be 17.15µg/mL and 20.78µg/ml; 21.14µg/mL and ethanol extract of A. salvifolium leaf possessed 21.96µg/mL; 20.87µg/mL and 23.24µg/mL and

119.33% scavenging activity on hydroxyl radical. 21.62µg/mL and 22.15µg/mL respectively. IC 50 Superoxide radical scavenging activity values of benzene extract of A. salvifolium leaf and The A. salvifolium leaf extracts were subjected to standard ascorbic acid for DPPH, hydroxyl, the superoxide radical scavenging activity and the superoxide scavenging and trolox for ABTS radical results were shown in figure 3. It indicates that cation scavenging were found to be 16.34µg/mL ethanol extract of A. salvifolium leaf (800µg/mL) and 20.78µg/mL; 17.03µg/mL and 21.96µg/ml; exhibited the maximum superoxide radical 18.93µg/mL and 23.24µg/mL and 18.73µg/mL and scavenging activity of 103.84% which is higher than 22.15µg/mL respectively. IC50 values of ethyl the standard ascorbic acid whose scavenging effect acetate extract of A. salvifolium leaf and standard is 101.23%. ascorbic acid for DPPH, hydroxyl, superoxide trolox ABTS radical cation scavengiing activity for ABTS radical cation scavenging were found to The A. salvifolium leaf extracts were subjected to be 13.47µg/mL and 20.78µg/Ll; 15.43µg/mL and ABTS radical cation scavenging activity and the 21.96µg/ml; 17.53µg/mL and 23.24µg/mL and results were presented in figure 4. The methanol 22.42µg/mL and 22.15µg/mL respectively. IC50 extract exhibited potent ABTS radical cation values of methanol extract of A. salvifolium leaf scavenging activity in concentration dependent and standard ascorbic acid for DPPH, hydroxyl, manner. At 800µg/mL concentration, A. salvifolium superoxide scavenging and trolox for ABTS radical leaf extracts possessed 106.87% scavenging activity cation scavenging were found to be 23.63µg/mL on ABTS which is slightly higher than the standard and 20.78µg/ml; 22.83µg/mL and 21.96µg/ml; trolox whose scavenging activity is 98.34%. 23.13µg/mL and 23.24µg/mL and 22.98µg/mL and

Reducing power 22.15µg/mL respectively. IC50 values of ethanol Figure 5 showed the reducing ability of different extract of A. salvifolium leaf and standard ascorbic solvent extracts of A. salvifolium leaf compared to acid for DPPH, hydroxyl, superoxide scavenging and ascorbic acid. The results clearly indicate that the trolox for ABTS radical cation scavenging were reducing power of the A. salvifolium leaf extracts found to be 22.91µg/mL and 20.78µg/ml; increased with the standard ascorbic acid. Among 23.94µg/mL and 21.96µg/mL; 23.43µg/mL and the solvent tested, methanol extract exhibited 23.24µg/mL and 21.06µg/mL and 22.15µg/mL higher reducing activity. respectively (Table 1).

Table 1: IC50 values of different solvent extracts of Alangium salvifolium leaf

Superoxide Hydroxyl Different solvent extracts DPPH assay dismutase ABTS assay assay activity Petroleum ether 17.15 21.14 23.24 21.62 Benzene 16.34 17.03 23.43 18.73 Ethyl acetate 13.47 15.43 23.13 22.42 Methanol 23.63 22.83 17.53 22.98 Ethanol 22.91 23.94 18.93 21.06 Standard (Ascorbic acid) 20.78 21.96 20.87 - Standard (Trolox) - - - 22.15

http://biosciencediscovery.com 78 ISSN: 2231-024X (Online) Sakthidevi G et al., DISCUSSION: The antioxidant properties of the scavenging activity. In the present study methanol different solvent extracts of A. salvifolium were extract exhibited more DPPH radical scavenging significantly corroborated by the phytochemical activity with IC50 value 23.63µg/mL compared to constituents of the extracts. ascorbic acid (20.78µg/ml). From the result, in the Phenolic compounds are known as powerful chain present study a dose dependent relationship in the breaking antioxidants and they are very important DPPH radical scavenging activity. plant constituents because of their scavenging Hydroxyl radical is the most deleterious ability, which is due to their hydroxyl groups and reactive among the ROS and it bears the (Shahidi and Wanasundara, 1992). Total phenolic shortest half life compared with other free radicals. assay by using Folin-Ciocalteu reagent is a simple The oxygen derived hydroxyl radicals along with convenient and reducible method. It is employed the added transition metal ion (Fe2+) causes routinely in studying phenolic antioxidants. degradation of deoxyribose into malodialdehyde Flavonoids are a group of polyphenolic compounds, which produces a pink chromogen with which exhibit several biological effects such as thiobarbituric acid (Halliwell, 1994). The extracts antiinflammatory, antihepatotoxic, antiulcer, when added to the reaction mixture, scavenged antiallergic, antiviral and anticancer activities the hydroxyl radicals and prevented the (Umamaheswari and Chatterjee, 2008). They are degradation of deoxyribose. In the present study capable of effectively scavenging the reactive O2 IC50 values were found to be 23.94µg/mL and species because of their phenolic hydroxyl groups 21.96µg/mL respectively for methanol extract of A. and so they are potent antioxidants also (Cao et al., salvifolium leaf and ascorbic acid. The hydroxyl 1997). In view of their wide pharmacological and radical scavenging activity may be due to the biological actions, they have a greater therapeutic presence of various phytochemicals including potential. The presence of high phenolic and polyphenols and flavonoids in A. salvifolium leaf flavonoid content in the different solvent extracts extracts. Superoxide anions are the most common of A. salvifolium has contributed directly to the free radicals in vitro and are generated in a variety antioxidant activity by neutralizing the free of biological systems, either by auto-oxidation radicals. Free radical is a molecule with an unpaired processes or by enzymes. The concentration of electron and is involved in bacterial and parasitic superoxide increases under conditions of oxidative infection, lung damage, inflammation, reperfusion stress and related situations (Lee et al., 2002). injury, cardiovascular disorders, atherosclerosis, Moreover, superoxide anions produce other kinds aging and neoplastic diseases (Jamuna et al., 2012). of cell damaging free radicals and oxidizing agents They are also involved in autoimmune disorder like (Hu and Kitts, 2000). Therefore, A. salvifolium leaf rheumatoid arthritis, etc. Our results demonstrated was undertaken to test whether it has scavenging that the different solvent extracts of leaf of A. activity of superoxide anions. The IC50 values were salvifolium possess free radical scavenging activity found to be 23.43µg/mL and 23.24µg/mL with different in vitro models like DPPH, hydroxyl respectively for methanol extract of A. salvifolium radical scavenging activity, superoxide radical leaf and ascorbic acid. The results clearly indicate scavenging activity, ABTS radical cation scavenging that the A. salvifolium leaf extracts have a activity and reducing power activity assays. noticeable effect as scavenging superoxide radical. The result of DPPH scavenging activity, in Another screening method for antioxidant this study indicates that the plant was potently activity is the ABTS radical cation decolourization active. This suggests that the plant extract contain assay. This assay is widely used to assess the compounds that are capable of donating hydrogen antioxidant capacity. The present investigation, this to a free radical in order to remove odd electron method showed results the quite similar to those which is responsible for radical’s reactivity (Dharani obtained in the DPPH reaction. Of the successively et al., 2011). Among five extracts, methanol extract extracted A. salvifolium leaf with different solvents, showed maximum scavenging activity followed by the methanol extract possessed potent ABTS ethanol extract. However this study provides a scavenging activity. The IC50 values of ABTS were definite report about the free radical scavenging found to be 22.98µg/mL and 22.15µg/mL capacity of A. salvifolium leaf, since the antioxidant respectively for methanol extract of A. salvifolium activity of a drug may depend on the free radical leaf and trolox.

http://biosciencediscovery.com 79 ISSN: 2231-024X (Online) Bioscience Discovery, 5(1):74-81, Jan. 2014 ISSN: 2229-3469 (Print) Here, the A. salvifolium leaf extracts, ABTS radical and active oxygen. J. Am. Oil Chem. Soc., 75: radical cation scavenging activity showed a direct 455-461. role of its phenolic compounds in free radical Duh PD, Tu YY and Yen, 1999. Antioxidant activity scavenging. of the aqueous extract of harng Jyur Reducing power activity is often used to (Chrysanthemum morifolium Ramat.) Lebensmittel- evaluate the ability of natural antioxidant to Wissenschaft and Technol., 32: 269-277. donate electron (Yildirim et al., 2000 and Dorman Eom SH, Cheng WJ, Hyoung JP, Kim EH, Chung MI, et al., 2003). Many reports have revealed that Kim MJ, Yu C and Cho DH, 2007. Far infra red ray there is a direct correlation between antioxidant irradiation stimulates antioxidant activity in Vitis activities and reducing power of certain plant flexuosa Thunb. Berries. Kor. J. Med. Crop. 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How to Cite this Article: Sakthidevi G, MohanVR and Jeeva S, 2014. In vitro antioxidant activity of leaf extracts of Alangium salvifolium (L.f.) Wang (Alangiaceae). Biosci. Disc., 5(1):74-81.

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