Arun. P. V et al. / Journal of Pharmacy Research 2011,4(6),1796-1799 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Antioxidant potential of paniculatum Arun. P. V, Sachin. S, P.Suganyadevi* P.G and Research Department of Biotechnology, Dr. Mahalingam Centre for Research and Development, NGM College, Pollachi 642001 Tamilnadu. Received on: 11-02-2011; Revised on: 16-03-2011; Accepted on:21-04-2011 ABSTRACT Clerodendrum paniculatum belongs to verbenacae family. Ethno medical importance of various of Clerodendrum has been reported in various indigeneous systems of medicines and folk medicines. It is used as a medicine for the treatment of sore eyes, urinary tract, gonorrhea and kidney problems. Also pharmacological properties of have not yet been extensively studied, it is therefore thought worthwhile to study about the unrevealed properties of this . The leaf and root of plant are used for preparing extraction using methanol as a solvent. On using solvent extraction phytochemical screening was analyzed, it reveals the presence of flavanoids, tannins, cardio glycosides, saponins, and terpenoids like compounds. Antioxidant analysis was studied by enzymatic and non enzymatic methods, which showed that the plant are good antioxidants. Further analysis was to carried out antimicrobial, anticancer and DNA damaging protecting activity of methanolic extract of Clerodendrum paniculatum. This study investigate about various biological activities of C. paniculatum which can be used as conventional therapy for various disease.

Key words: Phytochemical analysis, total phenolic assay, antioxidant-enzymatic assay.non enzymatic assay INTRODUCTION Clerodendrum paniculatum belongs to verbenacae family. The genus Clerodendrum is Total phenolic assay widely distributed in tropical and sub tropical regions of the world. It is the largest genus Total phenolic compounds in root and leaf samples of Clerodendrum paniculatum were in verbenacae family. The first description for genus was given by Linnaeus in 1753. It quantified by using Folin-ciocalteu’s method(25). 50 µl of Folin-ciocalteu’s reagent (50% was Adanson who changed the latin name the latin name Clerodendron tonits to Greek v/v) were added to 10µl of sample extract. It was incubated for 5 min. After incubation 50µl form “ clerodendron”. In Greek ‘klero’ means “chance’’ and ‘dendron’ means “tree”. The of 20 % (w/v) sodium carbonate and water was added to final volume of 400 µl. Blank was common name of Clerodendrum paniculatum is Pagoda flower. The origin of the plant is prepared by replacing the reagent by water to correct for interfering compounds. After 30 in Asian countries. Ethno medical importance of various species of Clerodendrum genus min of incubation, the absorbance was measured using spectrophotometer at 760 nm. has been reported in various indigenous systems of medicines and folk medicines. The Total phenol contents were expressed in terms of gallic acid equivalent (gm/100g of dry plant has got immense medical importance which is used for treatment for inflammation mass), which is used as a reference compound (2,27). ulcer, vitrated vata, pitta wound and skin disease. A decoction of root is used as tonic for aches and pains. It is used as a medicine for the treatment of sore eyes, urinary tract, Antioxidant Assay gonorrhea and kidney problems (1,29). In the past few years interest in the study of The antioxidant activity of plant extracts were determined by both non enzymatic and antioxidant activity of plant extract has been increased due to the fact that reactive oxygen enzymatic assays. All the assays were carried out in triplicates and average values were species is responsible for various diseases. So now a days antioxidants are added to considered. variety of foods to prevent free radicals the inhibition of free radicals generation can serve as facile model for evaluating the activity of anticancerous agents(35). Understanding that NON ENZYMATIC ASSAYS there has been increasing demand to evaluate the antioxidant and anticancer properties of bioactive compounds from plant origin rather than to look at synthetic options. The DPPH radical-scavenging activity objective of this work is to evaluate the phytochemical analysis and antioxidant properties The antioxidant activity was determined using DPPH radical scavenging activity was of methanolic extract of leaf and root of C.paniculatum as a part of exploration of new and assesed according to the method of Larrauri, et al (3) with some modifications. Various novel bio-active compounds. concentrations of test compounds in methanol were mixed with a methanolic solution having a final DPPH radical concentration of 0.1 mM. The mixture was shaken vigorously MATERIALS AND METHODS and left to stand for 30 min in the dark. Scavenging capacity was measured spectrophoto- metrically at 517 nm. In the experiment L-ascorbic acid was used as a positive control. Experimental details DPPH-scavenging activity (%) = [1-(absorbance of the sample-absorbance of blank)/ absorbance of the control] ×100 Plant Materials The fresh leaf and root of Clerodendrum paniculatum plants were collected from the local Hydroxyl radical scavenging activity areas of Calicut ,Kerala and stored in deep freezer at -20°C for further analysis The hydroxyl radical scavenging activity was determined according to the methods described by Jayaprakash et al (4) 0.1 ml of Clerodendrum paniculatum extracts of leaf Extraction of plant material and root was taken in different test tubes. 1.0 ml of iron-EDTA solution (0.1% ferrous The plant parts of leaves and root were separated and were allowed to shade dried. The ammonium sulfate and0.26% EDTA), 0.5 ml of DMSO (0.85% v/v in 0.1 M Phosphate dried leaves and roots were powdered in a blender to obtain a coarse powder. 10gm of each buffer, pH 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 ml samples were taken and mixed with 100ml of methanol in a separate round bottomed flask of 0.22% ascorbic acid. Test tubes were capped tightly and heated on a water bath at 80- 0 and kept as air tight for 48 hours and was shaken frequently for uniform mixing of 90 c for 15 min. The reaction was terminated by the addition of 1 ml of ice cold TCA (17.5 powdered samples distribution. Then the solutions were filtered through whattman No.1 %w/v). 3 ml of Nash reagent (75 g of ammonium acetate, 3 ml of glacial acetic acid, and filter paper and extracts of each sample were stored. This clear extract was used for 2 ml of acetyl acetone were mixed and raised to 1 L with distilled water was added to all preliminary screening for bioactive compounds and further investigation for potential of the tubes and left at room temperature for 15 min for the color development. The antioxidant properties. intensity of the yellow color formed was measured spectrophotometrically at 412 nm against the reagent blank. The percentage of hydroxyl radical scavenging activity is Preliminary screening calculated by using the formula: The presence of bio- active compounds was screened for saponins, flavanoids, terpenoids, % of hydroxyl radical scavenging activity=1-absorbance of sample/absorbance of saponins, glycosides and tannins.(17) blank×100 Determination of superoxide anion radical-Scavenging activity Superoxide radicals were generated by the method of Ginnopolites and Ries (5) with some *Corresponding author. modifications all solutions were prepared in 0.05 M phosphate buffer (pH 7.8). The photo P.Suganya devi induced reactions were performed in aluminium foil-lined box with two 30W fluorescent lamps. The distance between the reaction solution and the lamp was adjusted until the Dr. Mahalingam Centre for Research and Developement intensity of illumination reached about 4000 lux. A 30µL aliquot of various concentra- P.G. Department of Biotechnology,N.G.M.College, tions of test compounds was mixed with 3ml of reaction buffer solution (1.3 mm ribofla- Pollachi, Coimbatore, Tamilnadu () – 642001 vin, 13

Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1796-1799 Arun. P. V et al. / Journal of Pharmacy Research 2011,4(6),1796-1799 mM methionine, 63 µM nitro blue tetrazolium and 100µM EDTA, pH 7.8). The reaction Assay of glucose-6-phosphate dehydrogenase activity solution was illuminated for 15 min at 25 º C. The reaction mixture, without sample, was The enzyme was assayed by the method of Balinsky and Bernstein (13). The solution used as a control. The scavenging activity was calculated as follows: scavenging activity containing 0.1ml each of 0.1M Tris-HCL BUFFER PH8.2),0.2 Mm NADP and 0.1M

(%) = (1-absorbance of the sample/absorbance) ×100 (Absorbance of samples calcu- MgCl2 was taken in a cuvette along with 0.5ml of water and suitable aliquots of enzyme lated with five replicates and its average taken) extract. The reaction was initiated by the addition of 0.1ml of 6mM glucose-6- phosphate and 0.D. increase was measured at 340nm. The activity of the enzymes is expressed in Determination of reducing power terms of units/mg protein, in which one unit is equal to the amount of the enzymes that The reducing power was determined according to the method of Oyaizu (6). 1 ml of plant brought about an increase in O.D. of 0.01/min. extract solution (final concentration 100-500 mg/l) was mixed with 2.5 ml phosphate buffer (0.2M, pH 6.6) and 2.5 ml potassium ferricyanide (10g/l), then mixture was RESULTS AND DISCUSSION incubated at 50°C for 20 minutes. Two and one-half, 2.5 ml of trichloroacetic acid (100g/ l) was added to the mixture,which was then centrifuged at 3000 rpm for 10 min. Finally, Preliminary screening for bio-active compounds 2.5 ml of the supernatant solution was mixed with 2.5 ml of distilled water and 0.5 ml On preliminary screening the methanol extracts of C.paniculatum showed the presence of Ferric chloride (1g/l) and absorbance measured at 700nm in UV-Visible spectophotometer flavonoids, steroids, triterpenoids, phenols, cardioglycosides and anthraquinones like (Geneysis-5 UV-Visible spectrophotometer). Ascorbic acid was used as standard and compounds(16,17) phosphate buffer used as blank solution. The absorbance of the final reaction mixture of two parallel experiments was expressed as mean ± standard deviation. Increased absor- Presence of flavonoids bance of the reaction mixture indicates stronger reducing power. a. Pink or red colour shows the presence of flavonoids b. Formation of brown colour confirms the presence of flavonoids Determination of inhibitory effect on deoxyribose degradation Inhibitory effect of root and leaf sample on deoxy ribose degradation was determined by Presence of steroids measuring the reaction activity between either antioxidants and hydroxyl radicals (referred The upper layer turns red reveals presence of steroid and sterols compound in the extracts to as non-site specific scavenging assay or antioxidants and iron ions referred to as (site specific scavenging assay) described by Lee,Kim,and Jang (7). For the non site specific Presence of triterpenoids scavenging assay,0.1 ml aliquote of different concentration of test sample was mixed with Appearance of red to violet color indicates the presence of triterpenoids.

1 ml of reaction buffer (100µM FeCl2 , 104µM EDTA , 1.5 mM H2O2 2.5 mM deoxy ribose and 100µ M L-ascorbic acid , Ph7.4 ) and incubated for 1h at 37°C. A 1 ml aliquot Presence of cardioglycosides of 0.5% 2- thiobarbituric acid in 0.025 M NaOH and 1 ml of 2.8% trichloroacetic acid Presence of reddish brown color at the interface indicates the presence of cardioglycasides. were added to the mixture and it was heated for 30 min at 80°C. The mixture was cooled on ice and the absorbance was measured at 532 nm .Site specific scavenging activity, Test for anthraquinones which represented the ability of test sample to chelate iron and interfere with hydroxyl A.The presence of red or violet colour in the lower phase indicates the presence of free radical generation was measured using the same reaction buffer with out EDTA. Percent anthroquinones. inhibition of deoxyribose degradation was calculated as (1-absorbance of sample / B.Development of red or violet color in the lower phase indicates the presence of absorbance of control)x 100 anthroquinone derivatives in the extract.

ENZYMATIC ASSAY Total phenolic assay Phenolic compounds are an important group of secondary metabolites, which are synthe- Superoxide dismutase (SOD) sized by plants due to plant adaptation in response to biotic and abiotic stresses (infection, The superoxide dismutase activity was determined according to the procedure of Das et al water stress, cold stress, high visible light) (14).It has been already reported that there is (8).In this method 1.4 ml aliquots of reaction mixture (comprising 1.11 ml of 50 mM a strong correlation between total polyphenols content and antioxidant activity, In this phosphate buffer of pH 7.4 , 0.075 ml of 20 mM L-methionine 0.04 ml of 1% (v/v )Triton study, for the first time, the quantification of total phenolic compounds of C.paniculatum X-100 0.075 ml of 10 mM Hydroxylamine hydrochloride and 0.1 ml of 50 mM EDTA) leaf and root was done So far as plant phenolics constitute one of the major groups of was added to 100 µi of the sample extract and incubated at 30°C for 5 minutes . 80µl of compounds acting as primary antioxidants or free radical terminators, it was reasonable to 50 µM riboflavin was then added and the tubes were exposed for 10 min to 200 W Philips determine their total amount in the selected plant extracts (15). In this study, the content fluorescent lamps. After the exposure of the time 1ml of Greiss reagent (mixture of equal of total phenols in methanolic extracts expressed in gallic acid equivalents (GAE) varied volume of 1% sulphanilamide in 5% phosphoric acid ) was added and the absorbance of between leaf and root extracts are 3.52 g/100g and 4.08 g/100g respectively (Fig-1). The the colour formed was measured at 543 nm One unit of enzyme activity is 50% inhibition phenols contain hydroxyls that are responsible for the radical scavenging effect mainly due of the rate of autooxidation of pyrogallol asdetermined by change in absorbance/min at to redox properties (16). According to our study, the high phenolic content in of 420 nm.The activity of SOD is expressed as units/mg protein. C.paniculatum can explain its high free radical scavenging activity and these findings suggest that total polyphenols content is a good predictor of in vitro antioxidant activity. Assay of Catalase activity (17) Catalase activity was assayed by the method of Aebe ( 9)with some modifications.The enzymes extract (0.5 ml) was added to the reaction mixture containing 1ml of 0.01 M 4.2 phosphate buffer (PH-7.0), 0.5 ml of (0.2 M hydrogen peroxide. 0.4ml water and incu- bated for different time period. The reaction was terminated by the addition of 2ml of acid 4.1 reagent (dichromate /acetic acid mixture) which was prepared by mixing 5% potassium 4 dichromate with glacial acetic acid (1:3 by volume). To the control the enzyme was added after the addition of acid reagent. All the tubes were heated for 10 minutes and the 3.9 leaf absorbance was read at 610nm. Catalase activity was expressed in terms of µmoles of 3.8 hydrogen peroxide consumed/min/mg protein. root 3.7 Assay of Peroxidase activity 3.6

The assay was carried out by the method of Addy and Goodman (10). The reaction Expressed in g/100g mixture consisted of 3ml of buffered pyrogallol (0.005 M pyrogallol in 0.1M phosphate 3.5 buffer (pH7.0) and 0.5ml of 1% hydrogen peroxide. To this added 0.1ml enzymes 3.4 extracted and O.D change was measured at 430nm for every 30 seconds for 2 minutes.The peroxidase activity was calculated using an extinction coefficient of oxidized pyrogallol 3.3 (4.5 liters/mol). 3.2

Assay of ascorbate oxidase activity Figure 1: Total phenolic assay of C.paniculatum extracts Assay of ascorbate oxidase activity was carried out according to the procedure of Vines, and Oberbacher,(11). The sample was homogenized {1:5(w/v)} with phosphate buffer (0.1 M PH 6.5) and centrifuged at 3000g for 15 minutes at 5°C. The supernatant obtained NON ENZYMATIC ANTIOXIDANT ASSAYS was used as enzymes source. To 3.0ml of the substrate solution (8.8mg ascorbic acid in 300 ml phosphate buffer, PH 5.6).0.1ml of the enzymes extract was added and the absor- DPPH radical-scavenging activity bance change at 265nm was measured for every 30second for a period of 5 minutes. One In the present study several free radical scavenging activities of Clerodendrum paniculatum enzyme unit is equivalent to 0.01 O.D. changes per min. (14) extracts of leaf and root were evaluated by DPPH scavenging assay (18,33) . Methanolic leaves and root extracts of C.paniculatum has got profound antioxidant activity. DPPH

Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1796-1799 Arun. P. V et al. / Journal of Pharmacy Research 2011,4(6),1796-1799 antioxidant assay is based on the ability of DPPH a stable free radical, to decolorize in the and non site specific assay. Using the same concentration greater antioxidant activity was presence of antioxidants. The DPPH radical contains an odd electron, which is respon- observed in site specific than non site specific. In site specific assay the leaf extract shown sible for the absorbance at 517 nm and also for visible deep purple color. When DPPH 97.62% and root 72.52% (Fig-3). In non site specific leaf and root shows 65%and 52% accepts an electron donated by an antioxidant compound, the DPPH is decolorized which respectively. This implies that the extract inhibited deoxyribose. degradation mainly by can be quantitatively measured from the changes in absorbance The methanol leaves and chelating the metal ions rather than by scavenging hydroxyl radical directly. root extract of C.paniculatum exhibited a significant dose dependent inhibition of DPPH activity. In this study, C.paniculatum shows high inhibition of DPPH activity shows that 120 54.25% in leaf extract and where as 94% in root extract (Fig2). The results of DPPH-free radical scavenging assay suggest that the root extracts are more capable of scavenging free 100 radicals leaf 100 80 90 root 60 80 70 leaf 40

60 root % of Acidity 20 50 40 0

% of Acidity 30 Non site specific Site specific

20 Figure 3: Deoxyribose degradation assay of C.paniculatum 10 0 ENZYMATIC ANTIOXIDANT ASSAY DPPH RP HR SAR The inevitable generation of Reactive oxygen species in biological systems and oxidative damage is counterpoised by an array of enzymatic defense system. The level of antioxidant Figure 2: Non Enzymatic assay of of C.paniculatum extracts (DPPH-2,2-diphenyl-1- enzymes assessed in C.paniculatum are collectively represented in the table1. picrylhydrazyl, RP-Reducing power,HR-Hydroxy radical,SAR-Superoxide anion radical) Catalase activity The catalase activity was observed to be maximum in C.paniculatum (20.53 U/g tissue).

Reducing power The activity of these enzymes is to degrade H2O2 to non toxic form. So in our results the The reducing ability of a compound generally depends on the presence of reductants which activity of the enzyme was found to be high which detoxify more H2O2 molecule. It was have been exhibited antioxidative potential by breaking the free radical chain, donating a reported that in plants the antioxidant enzymes namely catalase have been shown to hydrogen atom. (19). The presence of reductants (i.e. antioxidants) in C.paniculatum leaf increase when subjected to stress condition (23). and root extracts causes the reduction of the Fe 3+ ferricyanide complex to the ferrous form. Therefore, the Fe 2+ can be monitored by measuring the formation of Perl’s Prussian blue Peroxidase activity at 700 nm.The reducing power of C.paniculatum leaves were 81.2% and roots were The peroxidase activity was higher than ascorbate oxidase in C.paniculatum (12U/g 60.8% which shows that extracts was very potent and the power of the extract was tissue) Table 1. The activity has been shown increased when subjected to stress condi- increased with quantity of sample tions. (24)

Hydroxy radical scavenging assay Table:1 Shows the different antioxidants in C.paniculatum The hydroxyl radical is an extremely reactive free radical formed in biological system and Assays Values has been implicated it is the major active oxygen centered radical formed from the reaction Super oxide dismutase 12 U/g tissue of various hydroperoxides with transition metal ions which is capable of damaging almost Peroxidase 12 U/g tissue every molecule found in living system causing lipid peroxidation and biological damage Ascorbate oxidase 13 U/g tissue (20,33). The scavenging of hydroxyl radical maximum scavenging effect was found to be Catalase 20.53 U/g tissue 77% in leaf extracts of C.paniculatum where as in root shows 66% (Fig-2 ) The Glucose -6- phosphate dehydrogenase 14 U/g tissue inhibition effect of C.paniculatum leaf shows 77% were found to be (BHT=99.8% and Superoxide dismutase activity ascorbic acid=99.7%) indicating more radical scavenging activity than the root 66% this The superoxide dismutase activity was observed to be minimum in C.paniculatum (12 ability of the extracts shows to quench hydroxyl radicals seems to be a good scavenger of U/g tissues) (25) reported that the superoxide dismutase activity was maximum activity active oxygen species thus reducing the rate of chain reaction. may be due to the total phenol content. So as the SOD activity was minimum this may be due to the total phenol content which is correlating with our results also. So, as the Superoxide anion radical scavenging activity SOD activity is higher which means they can scavenge more superoxide ion. C.paniculatum The formation of superoxide radical leads to a cascade formation of other ROS in the cell. showed a definite, positive effect on wound healing with a significant increase in the level Endogenously, superoxides could be produced in large amounts by various metabolic and of two powerful antioxidant enzymes, superoxide dismutase and catalyses. Superoxide physiological processes. The superoxide radical indirectly initiates lipid oxidation as a dismutase catalyse the one electron dismutation of superoxide into hydrogen peroxide and result of superoxide and hydrogen peroxide serving as precursors of singlet oxygen and oxygen (26) hydroxyl radicals (21). Therefore, in the present investigation, it was considered impor- tant to characterize the scavenging ability of C.paniculatum extracts against the superox- Ascorbate oxidase activity ide anion. In the PMS–NADH–NBT system, the superoxide radical derived from The ascorbate oxidase activity was higher in C.paniculatum (13U/g tissue) which means dissolved oxygen by PMS–NADH coupling reaction reduces NBT. The decrease in they have higher capacity to degrade ascorbate. Ascorbate has been found in the chloro- absorbance at 560 nm with antioxidants indicates the consumption of superoxide radicals plast, cystol, vacuoles and extra cellular compartments of the plant cells and shown to in the reaction mixture In this study, the scavenging of superoxide radical maximum effect function as a reductant for many free radicals(12,24). was found to be 63% in leaf of C.paniculatum where as in root shows 78% Glucose – 6 - phosphate dehydrogenase activity Deoxy ribose degradation activity Glucose-6- phosphate dehydrogenase (14 U/g tissues) was also higher. Since NADPH is Hydroxy radical can be formed by fenton reaction in the presence of reduced transition an ultimate reducing equivalent utilized by dehydro ascorbate reductase to maintain the metals such as metals such as Fe2+ and H O which is known to be the most reactive of all 2 2 balance between dehydro ascorbate and ascorbate through GSH-GSSG system. This has the reduced forms of dioxygen and is thought to initiat cell damage in vivo (22). To been compensated with increase in glucose 6 phosphate dehyerogenase activity in the determine whether the methanolic extracts of C.paniculatum reduce hydroxyl radical same which is correlating with our results (24,37). generation by chelating metal ions or by directly scavenging hydroxyl radicals, the effect 3+ of the methanolic extracts on hydroxyl radicals generated by Fe were analysed by CONCLUSION determining the degree of deoxyribose degradation. The inhibitory effect of hydroxyl It has been reported that reactive oxygen species contribute to various pathophysiological radical induced by the deoxyribose degradation of C. paniculatum was both site specific condition and endogenous defense mechanisms have evolved to offer protection in these

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Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1796-1799