Journal of Ethnopharmacology 137 (2011) 1337–1344 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Phytochemical analysis, antioxidant and anti-inflammatory activities of Phyllanthus simplex Hemendra S. Chouhan, Sushil K. Singh ∗ Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi 221005, India a r t i c l e i n f o Article history: Received 28 December 2010 Received in revised form 2 July 2011 Accepted 30 July 2011 Available online 5 August 2011 Keywords: Carragennan-induced paw edema LPS Total antioxidant capacity Total phenol content a b s t r a c t Ethnopharmacological relevance: Phyllanthus simplex (Family: Euphorbiacae) is widely used in traditional medicines for treatment of various diseases including inflammation. Materials and methods: Petroleum ether extract (PSPE) and ethanol extract (PSEE) of the whole plant of Phyllanthus simplex were characterized for their total phenolics, tannins and flavonoids content. These extracts were standardized by HPTLC using phyllanthin and gallic acid respectively as markers. Antioxidant activity of extracts was evaluated by the DPPH, hydroxyl and superoxide radicals scavenging assay. The total antioxidant capacity of extracts was determined. Anti-inflammatory activity was evaluated by their effect on nitric oxide (NO) production in isolated rat peritoneal macrophages; carragennan-induced paw edema and formation of cotton pellet-induced granuloma in rats. Results: Abundance of phenolics was found in PSEE. Phyllanthin and gallic acid content in PSPE and PSEE were found to be 14.5 and 0.65% (w/w) respectively. PSEE showed concentration dependent significant scavenging of DPPH, hydroxyl and superoxide radicals with IC50 values 102.219, 171.485 and 24.73 ␮g/ml respectively. PSEE significantly inhibited NO production in isolated rat peritoneum macrophages. Moreover, it also exhibited significant inhibition of carragennan-induced paw edema (58.48 ± 0.028%, p < 0.001, at 6 h, 200 mg/kg oral dose) and cotton pellet-induced granuloma formation (45.671 ± 0.712%, p < 0.001, at 200 mg/kg oral dose). Anti-inflammatory activity of PSEE was found to be comparable to diclofenac sodium. Conclusions: Significant antioxidant and anti-inflammatory activities were found in PSEE which may be attributed to its high phenolic content. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The harmful effects of free radicals on human health are well recognized, and are being continuously investigated. Free radical species viz. hydroxyl, nitric oxide (NO), superoxide work in an intricate way in the biological systems and their overproduction, deleteriously affect the membrane lipids, cellular proteins and enzymes, i.e. oxidative stress. This oxidative stress causes cell death and eventually leads to inflammatory disorders, cancer, diabetes, etc. However, antioxidants are helpful in protecting cells from such oxidative damage (Marx, 1987; Aruoma, 1998). Synthetic compounds viz. butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are well known antioxidants but are carcinogenic (Ito et al., 1983). Natural products from the plant sources are relatively safe and have immense potential to display overwhelming biological activities which depend upon their nature, structure ∗ Corresponding author. Tel.: +91 542 6702736; fax: +91 542 2316428. E-mail address: sksingh.phe@itbhu.ac.in (S.K. Singh). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.069 and interaction with other molecules in the assortment (Djeridane et al., 2006). Phyllanthus simplex Retz. (Family: Euphorbiacae) is used in traditional medicine for the treatment of diarrhea, jaundice, gonorrhea, hyperglycemia, liver disease, mammary abscess, febrifuge, itching, pruritis including inflammation. Phyllanthus simplex is also reported to be used as astringent, diuretic, hepatoprotective and cathartic (Nadkarni and Nadkarni, 1976; Chopra et al., 1980; Prakash et al., 1995; Kirtikar and Basu, 2000). In our previous studies, we have found that ethanol extract of Phyllanthus simplex possess good lipid peroxidation inhibitory activity (Kumar et al., 2007) and is effective in normalizing the antioxidative enzymes viz. superoxide dismutase, catalase and glutathione peroxidase in liver and kidney tissues of alloxan induced diabetic mouse (Shabeer et al., 2009). However, there is no report available on anti-inflammatory activity of Phyllanthus simplex. The aim of this study was to analyze the plant for its anti-inflammatory activity to support the claim of its use in treatment of inflammation by traditional healers. Further, assessment of its antioxidant activity was undertaken to strongly support its anti-inflammatory activity, as free radicals are known mediator/aggravator of inflammation. Plant 1338 H.S. Chouhan, S.K. Singh / Journal of Ethnopharmacology 137 (2011) 1337–1344 phenolics, tannins and flavonoids are known to act as powerful antioxidant; hence it is worthwhile to estimate their total content in the plant extract in view of their possible role in antioxidant and anti-inflammatory activity. All the compounds and organic solvents were purchased from Merck unless otherwise stated. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was procured from Sigma–Aldrich (India). l(+)-Ascorbic acid (AA) was acquired from Sisco Research Laboratory Pvt. Ltd. (India). Double distilled water was used throughout the experiment. mobile phase vapor for 30 min. Plates were removed from the chamber and dried completely in the air at room temperature, scanned at 260 and 520 nm (absorption/reflectance mode) before and after the spray of anisaldehyde-H2 SO4 using CAMAG TLC scanner 3 (6 mm × 0.45 mm slit dimensions; 40 mm/s scanning speed) supplied with CAMAG software (WinCATS, version 1.4.5.2027). For calibration, 1–5 ␮l marker compound solutions (prepared by dissolving 1 mg each phyllanthin and gallic acid) were separately applied on precoated HPTLC plate. Test solutions of phyllanthin and gallic acid were chromatographed as explained for plates A and B respectively. Linear regression equation was obtained from the calibration graph plotted between peak area and concentration of marker compounds. Presence of marker compound(s) in the extracts was confirmed by overlapping spectrum of peak of corresponding Rf and quantified using linear regression equation. 2.2. Animals 2.6. Estimation of total sugar content (TSC) Wistar albino rats (120–150 g) of either sex were procured from the central animal house of Institute of Medical Sciences, Banaras Hindu University, Varanasi (Reg. No. 542/02/ab/CPCSEA) and were acclimatized in the laboratory condition at 12 h light/dark cycle for 15 days. Rats were allowed to have free access of water and standard diet; and were fasted overnight before the experiment. Approval from Institutional Ethical Committee was taken for the commencement of animal experimental studies. Guidelines for the care of laboratory animals and the investigation of experimental pain in conscious animals had been followed during the experiment (Zimmerman, 1983). TSC of PSPE and PSEE was determined by the method as described by Dubois et al. (1956). Briefly, 2 ml extract was mixed with 1 ml of 5% phenol and 5 ml of conc. H2 SO4 in a test tube, and incubated for 30 min at room temperature. Absorbance of mixture was measured at 490 nm wavelength against the blank. Standard curve of glucose was prepared similarly by taking solution of 12.5, 25, 50, 75 and 100 ␮g/ml concentrations. Results were expressed as glucose equivalent (GE) in mg/g dry weight of extract. 2. Materials and methods 2.1. General 2.3. Plant materials Whole plants of Phyllanthus simplex were collected from the campus of Banaras Hindu University, Varanasi in July 2009 and were identified morphologically by Prof. N.K. Dubey, Department of Botany, Banaras Hindu University, Varanasi. A voucher specimen (PCRL-43) was deposited in the Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi for future reference. Air and shade dried plant material, which comprises of leaves, aerial parts and roots of the plant in the ratio of 1:3:1 (by weight), was pulverized to coarse crude powder and stored in an air tight container at room temperature till the extraction. 2.4. Preparation of extracts Pulverized plant material (1.0 kg) was extracted successively by soxhlation with petroleum ether (fraction collected at 60–80 ◦ C) and ethanol. Petroleum ether extract (PSPE) and ethanol extract (PSEE) were concentrated under vacuum (yield 7.6 and 12.4%, w/w respectively) and stored separately in air tight containers in refrigerator till use. 2.5. HPTLC fingerprint analysis Samples for the HPTLC study were prepared by dissolving 30 mg of PSPE and PSEE in 1 ml of chloroform and methanol respectively. HPTLC was performed on 20 cm × 20 cm precoated Silica gel 60 F254 HPTLC plate (Merck) where 5 ␮l of PSPE and PSEE samples were applied to separate plates (A and B) as bands 5 mm wide, 10 mm apart, 10 mm from the bottom edge, starting 10 mm from the side edge of the plate, by means of a CAMAG automatic TLC sampler (Linomat V). The plates were developed to a distance of 80 mm with hexane–ethyl acetate 7.5:2.5 and ethyl acetate–formic acid–water 44:3:3 as the mobile phase for plates A and B respectively, in a CAMAG twin-trough chamber previously saturated with 2.7. Estimation of total phenolics (TPC) and total tannins content (TTC) TPC of PSPE and PSEE was determined by the method described by Vernon et al. (1999). Briefly, 1 ml extract and 1 ml Folin-Ciocalteu Reagent (FCR) were added to 9 ml of water in to a 25 ml volumetric flask. Mixtures were vortexed and set aside for 5 min. Sodium carbonate solution (7%, w/v, 10 ml) was added to the mixtures, diluted with water to 25 ml and were incubated for 1.5 h at room temperature. Absorbance of mixtures was then measured at 765 nm against the blank. TPC was expressed as gallic acid equivalent (GAE) in mg/g dry weight of extract. Non-tannin phenolics were estimated by precipitating tannins of extracts with gelatin (Makkar et al., 1993). Gelatin (200 mg) was mixed with 2.0 ml of water and 2.0 ml of extract and allowed to stand for 15 min at 4 ◦ C followed by vortex and resulting mixture was filtered through Whatman filter paper no. 1. The filtrate (150 ␮l) was diluted with water (up to 1.0 ml) and then non-tannin phenolics were estimated by the procedure similar to that of TPC estimation. TTC of PSPE and PSEE was determined by subtracting non-tannin phenolics from total phenolics content. 2.8. Estimation of total flavonoids content Total flavonoids content of PSPE and PSEE was determined by the method of Kumaran and Karunakaran (2006). PSEE (1 ml, 0.1%, w/v in ethanol), AlCl3 solution (1 ml, 2%, w/v in ethanol) and a drop of acetic acid were mixed and the volume was made to 25 ml in a volumetric flask with water. Reaction mixture was incubated for 40 min at room temperature and absorbance was measured at 415 nm against the blank. The blank was prepared in similar manner without AlCl3 . Similarly, absorbance of PSEE and rutin solution (0.5%, w/v in ethanol, as standard) was measured. Total flavonoids content (TFC) was expressed as rutin equivalents (RE) and calculated by using the following formula: X= Am0 A0 m (1) H.S. Chouhan, S.K. Singh / Journal of Ethnopharmacology 137 (2011) 1337–1344 where X = flavonoid content of extract (mg/g); A = absorbance of extract; A0 = absorbance of rutin; m = weight of extract; m0 = weight of rutin in solution. 1339 were determined. AA was used as a standard and experiment was performed in triplicate. 2.9. Evaluation of total antioxidant capacity (TAC) 2.13. Isolation of rat peritoneal macrophages and in vitro effect on NO production in activated macrophages TAC of PSPE and PSEE was determined by phosphomolybdenum assay (Prieto et al., 1999). Briefly, 0.1 ml extract and 1 ml phosphomolybdenum reagent (28 mM sodium phosphate and 4 mM ammonium molybdate in 0.6 M sulphuric acid) were mixed in capped test tubes and were incubated at 95 ◦ C for 90 min on a water bath. Test tubes were removed from the water bath, cooled at room temperature and absorbance of reaction mixtures was measured at 695 nm. Similarly, series of reaction mixture were made using different concentrations of ascorbic acid (AA) and their absorbance was measured. Calibration graph was plotted between absorbance and concentrations of AA. TAC of extracts was calculated from the graph and expressed as milligrams of ascorbic acid equivalent (AAE) per gram of dry weight of extract. Rats were anaesthetized with diethyl ether and 10 ml of chilled Ca and Mg free-phosphate buffer saline (PBS, pH 7.4) was injected in the peritoneal cavity; and abdomen was massaged for 5 min. The peritoneal fluid was then aspirated out, centrifuged at 1500 rpm for 10 min and cell pellets were washed three times with PBS. The pellets were suspended in 1 ml RPMI media and viable cells were counted by trypan blue exclusion method using hemocytometer. Macrophages (1 × 105 cells/well) were treated with different concentrations of extract (100–500 ␮g/ml) and exposed to lipopolysaccharide (1 ␮g/ml) in 96 well plate; and incubated for next 16 h. Supernatant of culture medium was pipetted out and collected in another plate. Accumulated NO radical in the culture supernatant was estimated by griess reagent (Batkhuu et al., 2002). 2.10. Evaluation of DPPH radical scavenging activity DPPH radical scavenging activity of PSPE and PSEE was evaluated based on the method given by Gordon et al. (2001). Briefly, 0.5 ml DPPH solution (0.05%, w/v in methanol) was mixed with different concentrations (25, 50, 75, 100 and 200 ␮g/ml) of extract in capped test tubes and incubated at room temperature for 30 min. After incubation, absorbance of mixtures was measured at 517 nm against the blank. DPPH radical scavenging activity was calculated by using the following formula: % inhibition = 1 − A  e Ac × 100 (2) where Ae and Ac are absorbance of extract and control respectively. IC50 value (concentration of extract required to scavenge 50% of radicals) was determined by a graph plotted between percentage inhibition and concentration. AA was used as a standard and experiment was performed in triplicate. 2.11. Evaluation of hydroxyl radical-scavenging activity by phenanthroline-Fe(II) oxidation assay Hydroxyl radical scavenging activity of PSPE and PSEE was evaluated by the method of Jin et al. (1996). Briefly, 1,10phenanthroline (200 ␮l, 3.75 mM), FeSO4 (200 ␮l, 3.75 mM) and H2 O2 (400 ␮l, 0.5%, v/v) were taken in test tubes containing different concentrations of extract (400 ␮l, prepared in 0.76 mol/L in pH 7.4 phosphate buffer), mixed, incubated for 1 h at 37 ◦ C and absorbance of resulting mixture was measured at 532 nm. Hydroxyl radical scavenging activity of extracts was determined by using Eq. (2) and IC50 values were determined. AA was used as a standard and experiment was performed in triplicate. 2.12. Superoxide radical scavenging activity Superoxide radical scavenging activity of PSPE and PSEE was evaluated by the method of Kuda and Ikemori (2009) with slight modification. PMS (0.1 mM, 0.1 ml), NBT (1 mM, 0.1 ml) and different concentrations of extract (25–200 ␮g/ml) were mixed and volume of mixture was made up to 0.9 ml with 0.05 M KH2 PO4 buffer (pH 7.4). The superoxide radicals in reaction mixture were generated by the addition of 0.1 ml 2 mM NADH. Reaction mixture was then incubated at 25 ◦ C for 10 min, and absorbance of mixture was measured at 570 nm. Superoxide scavenging activity of extracts was determined by using Eq. (2) and the IC50 values 2.14. Evaluation of anti-inflammatory activity by carrageenan induced rat paw edema model The anti-inflammatory activity of PSPE and PSEE was determined by carrageenan induced rat paw edema model (Winyard and Willoughby, 2003). Rats were randomly divided into six groups (A–F) containing six rats in each group. Paw edema was induced by the injection of 0.1 ml carrageenan suspension (in normal saline, 1%, w/v) in sub-plantar region of right hind paw of rats. Group A (control), received an oral dose of sodium caboxymethylcellulose solution (1 ml, 1%, w/v); Group B, given an oral dose of diclofenac sodium (50 mg/kg) and served as standard; Groups C–F were test groups. Groups C and D were administered 100 and 200 mg/kg oral dose of PSPE respectively and Groups E and F received 100 and 200 mg/kg oral dose of PSEE respectively. Volume of right hind paw of rats was measured by using plethysmometer 30 min before carrageenan injection, and 1 h interval up to 6 h, then at 12 and 24 h after carrageenan injection. Reduction of edema volume with respect to control is the measure of anti-inflammatory activity as well as efficacy of the drug/extract. 2.15. Evaluation of anti-inflammatory activity by cotton pellet-induced granuloma formation model Rats were divided into six groups (A–F) containing six rats in each group and were anaesthetized with diethyl ether. Sterile cotton pellet (10 mg) was then implanted in the shaved axilla region of rats using small incision. Group A (control), received an oral dose of sodium caboxymethylcellulose solution (1 ml, 1%, w/v); Group B, administered an oral dose of diclofenac sodium (50 mg/kg) and served as standard; Groups C–F were test groups. Groups C and D were given 100 and 200 mg/kg oral dose of PSPE respectively and Groups E and F were given 100 and 200 mg/kg oral dose of PSEE respectively. All groups received treatment for seven consecutive days starting from the day of implantation. Cotton pellets were removed on the eighth day after anaesthetizing rats with diethyl ether and made free from extraneous tissues. Pellets were dried at 60 ◦ C for 24 h and weighed to determine mean weight of granuloma tissue formed (Winyard and Willoughby, 2003). 2.16. Statistical analysis All results were expressed as mean ± SEM of three parallel measurements. The data was subjected to one-way analysis of variance 1340 H.S. Chouhan, S.K. Singh / Journal of Ethnopharmacology 137 (2011) 1337–1344 Fig. 1. HPTLC chromatogram obtained from (a) phyllathin (b) petroleum ether extract of Phyllanthus simplex (c) gallic acid and (d) ethanol extract of Phyllanthus simplex. (ANOVA) followed by Tukey multiple comparison test. Results were processed by the MS Excel computer programme (2007). 3. Results and discussion Plants of genus Phyllanthus, which comprises of about 600 species, are distributed throughout tropical and subtropical countries. These plants are traditionally used to cure diseases having inflammation as pathophysiological factor. However, only a few plant species viz. Phyllanthus amarus, Phyllanthus niruri, Phyllanthus urinaria, etc. have been pharmacologically investigated for their anti-inflammatory activity (Vormisto et al., 1997; Raphael and Kuttan, 2003; Kiemer et al., 2003; Rao et al., 2006; Fang et al., 2008). Phyllanthus simplex, belonging to this genus, is widely used for treatment of inflammation in Indian traditional system of medicine (Nadkarni and Nadkarni, 1976; Chopra et al., 1980; Prakash et al., 1995; Kirtikar and Basu, 2000). The leaf juice of the plant is used to treat the inflamed eyes (Eusebio and Umali, 2004). However, these claims are not substantiated through scientific studies demonstrating the anti-inflammatory activity. The pharmacological activity of a plant extract is largely dependant upon its composition, nature, and the structure of constituent(s) present in the extract. Therefore, the extracts were standardized for the total content of some biologically important constituents before the appraisal of anti-inflammatory and antioxidant activities. 3.1. Phytochemical evaluation 3.1.1. Phytochemical analysis and HPTLC fingerprint Plants of genus Phyllanthus are rich source of lignans and hydrolysable phenols such as phyllanthin and gallic acid (Dhalwal et al., 2006; Kiadó, 2010) which are key components, responsible for antioxidant activity (Krithika et al., 2009; Chirdchupunseree and Pramyothin, 2010; Luo et al., 2011). Preliminary phytochemical analysis of extracts revealed the abundance of lignans, steroids, teriterpenes in PSPE, and phenolics and glycosides in PSEE. Moreover, PSPE has been found to be enriched with lignans, particularly phyllanthin, whereas abundance of phenolics viz. gallic acid was found in the PSEE. Therefore, we standardized the extracts (PSPE and PSEE) by HPTLC using phyllanthin and gallic acid respectively as marker compounds. Phyllanthin was detected in PSPE at Rf = 0.19 (hexane–ethylacetate, 7.5:2.5) by direct comparison of its chromatogram and its amount was found to be 14.5% (w/w). Similarly, gallic acid was detected in PSEE at Rf = 0.82 (ethyl acetate–formic acid–water, 44:3:3) and its amount was found to be 0.65% (w/w) (Fig. 1). 3.1.2. Total phenolics, tannins and flavonoids content Plant phenolics, tannins and flavanoids represent major groups of plant constituents that work predominantly as powerful antioxidants or scavenger of free radicals. They play beneficial role in human health and cure/prevent ailments such as inflammatory H.S. Chouhan, S.K. Singh / Journal of Ethnopharmacology 137 (2011) 1337–1344 1341 disorders, cancer and diabetes which occur due to the deregulation of free radicals generation in the cells (Conner and Grisham, 1996; Haslam, 1996; Croft, 1998; Middleton et al., 2000). The total content of sugars, phenolics, tannins and flavonoinds in the petroleum ether and ethanol extracts of Phyllanthus simplex was estimated keeping above in view. TSC of extract was estimated spectrophotometrically by measuring color intensity (at 490 nm) of orange yellow product formed as a result of chemical reaction between free reducing groups of sugar with phenol in the presence H2 SO4 (Dubois et al., 1956). Results suggested the presence of significantly high (p < 0.05) TSC in PSEE (484.0 ± 2.898 mg GE/g) than PSPE (235.583 ± 0.794 mg GE/g) as determined by the standard curve of glucose (y = 0.002x + 0.014; r2 = 0.985). Reduction of FCR into a blue-color complex by the phenolics was used to estimate TPC. In this assay, intensity of the colored complex formed is equal to amount of phenolics used in the reduction of FCR and decrease in the color intensity was measured spectrophotometrically at 750 nm (Vernon et al., 1999). TPC was found to be significantly more in PSEE (108.556 ± 0.128 mg GAE/g; p < 0.05) than PSPE (11.556 ± 0.064 mg GAE/g), and was estimated by using calibration curve of gallic acid (y = 0.009x + 0.024; r2 = 0.999). Gelatin adsorbed tannins were also determined by the FCR and were found to be 3.85% and 39.04% of TPC of the PSPE and PSEE respectively. In another experiment, spectrophotometric quantification of TFC was carried out which utilizes measurement of color intensity of flavonoid–aluminum complex at 415 nm (Kumaran and Karunakaran, 2006). PSEE (126.182 ± 4.233 mg RE/g) was found to contain significantly higher amount of flavonoids (p < 0.05) than that of PSPE (58.336 ± 7.981 mg RE/g). 3.2. Assessment of in vitro antioxidant activity Although antioxidant activity is present in all plants, it is still important to investigate the antioxidant property of Phyllanthus simplex because reactive oxygen species viz. OH, NO and superoxide radicals have key roles in pathogenesis of inflammatory disorders and scavenging of these free radicals by antioxidants can lessen inflammation (Conner and Grisham, 1996). Therefore, the antioxidant property of plant extracts was investigated by determining their total antioxidant capacity; DPPH, OH and superoxide free radical scavenging activity in this experiment so as to find out possible relationship between antioxidant and anti-inflammatory activity. 3.2.1. Total antioxidant capacity Antioxidants have been found to exhibit their activity by various mechanisms at different stages of oxidation reaction. Phosphomolybdenum assay, initially developed by Prieto et al. (1999) for the measurement of antioxidant activity of vitamin E, was used for the evaluation of total antioxidant capacity of PSPE and PSEE. Molybdenum (Mo)(IV) of ammonium molybadate gets converted into Mo(V) by reducing species of the extract and forms a green colored complex with phosphate ion present in the reaction mixture. The color intensity of this complex corresponds to the amount of complex formed. Findings of this study revealed nearly same TAC in PSPE and PSEE (206.111 ± 1.925 and 256.667 ± 1.667 mg AAE/g) which may be attributed to their high phenolics content. The total antioxidant capacity of PSEE was found to be nearly same to that of the whole plant methanolic extract of Phyllanthus amarus, Phyllanthus maderaspatensis and Phyllanthus virgatus while it was less than that of methanolic extract of Phyllanthus debilis and Phyllanthus urinaria (Kumaran and Karunakaran, 2006). 3.2.2. DPPH radical scavenging activity DPPH radical scavenging assay is widely employed and preferred for the measurement of antioxidant activity, because of its imperviousness to the side and enzymatic reactions. DPPH in Fig. 2. Effect of petroleum ether and ethanol extract of Phyllanthus simplex on the in vitro scavenging activity of (a) DPPH free radicals (b) hydroxyl free radicals (c) superoxide free radicals. methanol occurs as free radical and showed pink colored solution which becomes faint after acquiring proton(s) from the antioxidant(s) (Gordon et al., 2001). Hence, measurement of reduction in the color intensity of methanolic DPPH solution may be used to evaluate the antioxidants’ strength to donate proton. Fig. 2a demonstrated significant (p < 0.05) DPPH radical scavenging activity of PSPE and PSEE; the IC50 values were found 190.556 and 102.219 ␮g/ml respectively for PSPE and PSEE. These values are found to be higher than that of the aqueous and methanolic extracts of leaves of Phyllanthus niruri (Sabir and Rocha, 2008) but it is less than the value (7.6 ␮g/ml) obtained for ethanolic extract of leaves of Phyllanthus muellerianus; and aqueous and methanolic extracts obtained from the leaves (15.3 and 9.1 ␮g/ml respectively) and fruit parts (32.6 and 14.5 ␮g/ml respectively) of Phyllanthus niruri (Agyare et al., 2009). The DPPH radical scavenging activity of PSEE seems to be better than that observed in the methanolic extract of Phyllanthus amarus, Phyllanthus maderaspatensis, Phyllanthus virgatus, Phyllanthus debilis and Phyllanthus urinaria; as 10 time higher 1342 H.S. Chouhan, S.K. Singh / Journal of Ethnopharmacology 137 (2011) 1337–1344 Table 1 Effect of petroleum ether and ethanol extract of Phyllanthus simplex on the volume of carrageenan-induced paw edema (in ml). Time interval (h) 1 2 3 4 5 6 12 24 Control 1.26 1.48 1.57 1.60 1.57 1.53 1.56 1.53 ± ± ± ± ± ± ± ± Diclofenac sodium, 50 mg/kg 0.023 0.020 0.025 0.015 0.012 0.013 0.014 0.013 0.87 0.95 0.95 0.84 0.69 0.47 0.12 0.06 ± ± ± ± ± ± ± ± * 0.013 0.013* 0.013* 0.010* 0.011* 0.012* 0.023* 0.023* PSEE PSPE 100 mg/kg 200 mg/kg 100 mg/kg 200 mg/kg 1.26 1.46 1.53 1.47 1.38 1.26 1.13 1.05 1.26 1.42 1.46 1.29 1.20 1.07 0.87 0.72 1.23 1.40 1.36 1.21 1.04 0.87 0.59 0.45 1.20 1.34 1.20 1.08 0.83 0.64 0.25 0.16 ± ± ± ± ± ± ± ± 0.016 0.023 0.032 0.018* 0.019** 0.024* 0.021* 0.019* ± ± ± ± ± ± ± ± 0.019 0.028 0.029 0.022* 0.045* 0.021* 0.027* 0.028* ± ± ± ± ± ± ± ± 0.031 0.031 0.029* 0.021* 0.035* 0.030* 0.030* 0.033* ± ± ± ± ± ± ± ± 0.025 0.019** 0.032* 0.026* 0.041* 0.028* 0.030* 0.030* All the values are expressed as mean ± S.E.M (n = 6). * p < 0.001 when compared with control group. ** p < 0.01 when compared with control group. concentration of DPPH solution was used to evaluate the activity of PSEE (Kumaran and Karunakaran, 2006). 3.2.3. Hydroxyl radical scavenging activity Reactive oxygen species viz. hydroxyl and superoxide free radical have been implicated in the etiology of many diseases and conditions, and eventually resulted in the cell injury/death. In the biological systems, cells’ injury causes leakage of transition metal ions viz. Fe++ ion from the cellular protein which further generates hydroxyl radicals by the process ‘Fenton chemistry’. NBT (Kuda and Ikemori, 2009). Superoxide radical scavenging activity of PSPE and PSEE is presented in Fig. 2c which shows concentration dependant activity. The IC50 values for PSPE and PSEE were found as 33.351 and 24.730 ␮g/ml respectively. The activity of PSEE was found to be comparable to that of ascorbic acid (IC50 = 18.927 ␮g/ml). Further, superoxide radical scavenging activity of PSEE was found to be better than the activity reported for the methanolic extract of whole plant of Phyllanthus amarus, Phyllanthus maderaspatensis, Phyllanthus virgatus, Phyllanthus debilis and Phyllanthus urinaria (Kumaran and Karunakaran, 2006). Fe++ + H2 O2 → Fe+++ + OH + OH• 3.3. Assessment of anti-inflammatory activity Above reaction is a cardinal mechanism for the generation of hydroxyl radical in cells. However, presence of antioxidant prevents the cellular damage either by quenching of OH• radicals or by chelating transition metal ion (Aruoma, 1998). PhenanthrolineFe(II) oxidation assay utilizes the above mechanism for the in vitro production of OH• radicals and is widely employed for the evaluation of hydroxyl radical scavenging activity of antioxidant(s) (Jin et al., 1996). Antioxidant(s) inhibit redox conversion of phenthroline-Fe(II) to Fe(III) form by the scavenging of OH• radicals. The former complex exhibited maximum absorbance at 532 nm. Hence, drop in the absorbance of reaction mixture due to presence of antioxidant(s) may be used to express its hydroxyl radical scavenging activity. Our finding suggests that Phyllanthus simplex possess good in vitro hydroxyl radical scavenging activity and higher activity was found in PSEE than PSPE (Fig. 2b), and IC50 values for PSEE and PSPE were 171.485 and 350.328 ␮g/ml respectively. Moreover, hydroxyl radical scavenging activity of PSEE was comparable with ascorbic acid (IC50 = 102.925 ␮g/ml). 3.2.4. Superoxide radical scavenging activity Activated phagocytes viz. monocytes, macrophages, eosinophils, and neutrophils, in biological systems, are involved in the production of superoxide radicals which are implicated in process like killing bacteria by phagocytes and their amount in the system is regulated by enzymes such as superoxide dismutases and catalase. Any disturbance in this regulation process results in oxidative stress eventually causing cell death and/or disease. However, presence of antioxidants is helpful in restoring the normal balance between scavenging of superoxide radicals and their production; therefore, antioxidants may be helpful in preventing diseases (Marx, 1987; Aruoma, 1998). Hence, we evaluated antioxidant potential of PSPE and PSEE by determining their in vitro superoxide scavenging activity. The experiment was based on spectrophotometric determination of color intensity of reduced NBT at 560 nm formed due to the action of superoxide radicals. Presence of plant extract quenches the generated superoxide radicals and hence, diminishes the color intensity of reduced Inflammation is a phenomenon which involves several factors working in an intricate system and characterized by external manifestation like redness and swelling of skin (Winyard and Willoughby, 2003). Macrophages and neutrophils have been found to play important role, and inflict acute and chronic inflammation. These cells are triggered at the site of inflammation and release many inflammatory mediators including NO radicals. These radicals are involved in further initiation and maintenance of inflammatory events that leads to conversion of acute phase to the chronic phase of inflammation (Batkhuu et al., 2002). Inhibition of NO radical production by the macrophages is an important target, and could be used as in vitro model for the evaluation of anti-inflammatory activity. Further, in vivo evaluation of antiinflammatory activity can be done by inducing inflammation in animals by many methods. Rat paw edema model is frequently used as an acute inflammation model whereas subcutaneous implan- Fig. 3. Effect of petroleum ether and ethanol extract of Phyllanthus simplex on nitric oxide production in LPS-stimulated in isolated macrophages from rat peritoneum. Accumulated Nitrites (a stable NO metabolite) level in the cell culture media was measured by the griess reaction assay. H.S. Chouhan, S.K. Singh / Journal of Ethnopharmacology 137 (2011) 1337–1344 Fig. 4. Effect of petroleum ether and ethanol extract of Phyllanthus simplex on cotton pellet-induced granuloma formation. tation of biomaterial is a good example of chronic inflammatory model (Winyard and Willoughby, 2003). We investigated the antiinflammatory effects of PSPE and PSEE by evaluating their effect on NO radical production in isolated macrophages from rat peritoneal (in vitro method) and by carragennan-induced paw edema and cotton pellet-induced granuloma formation in rat models (in vivo methods). Our findings of in vitro study suggested that the antiinflammatory activity of PSPE and PSEE was mediated by their inhibitory action on NO production in a dose dependant manner (p < 0.05) (Fig. 3). Plant phenolics are known to exhibit protective role in chronic inflammatory diseases which are linked with excessive generation of NO (Huang et al., 2011). Thus, phenolics of Phyllanthus simplex may be responsible for its anti-inflammatory activity by controlling NO production. The presence of phyllanthin and gallic acid in PSPE and PSEE respectively were found to have an effect on the NO production in the earlier studies (Fang et al., 2008; Kolodziej et al., 2008). Therefore, these compounds might play an important role in demonstrating anti-inflammatory activity of the plant. Table 1 and Fig. 4 summarize the results of anti-inflammatory effect of PSPE and PSEE evaluated in the carragennan-induced paw edema rat model and cotton pellet-induced granuloma formation in rat model respectively. Injection of carrageenan causes biphasic inflammation i.e. early phase (0–3 h) and late phase (after 3 h) in rat. In the early phase, inflammatory reactions are due to the secretion of pro-inflammatory mediators’ viz. histamines, 5-hydroxytryptamine and bradykinins, whereas, prostaglandins and leucotrienes synthesis mediate the late phase inflammation (Singh et al., 2009). Study demonstrated that PSEE displays a dose dependant reduction of edema volume both in early and late hours of carrageenan inflammation. However, maximum effect (p > 00.1) was observed in the late hours of carrageenan inflammation (58.48 ± 0.028 and 89.35 ± 0.03% inhibition at 6 and 24 h respectively, at 200 mg/kg oral dose) and the effect was comparable to that of diclofenac sodium (69.13 ± 0.012 and 95.87 ± 0.023% inhibition at 6 and 24 h respectively) used as a standard. These findings suggest that the anti-inflammatory activity of Phyllanthus simplex extract may be due to the inhibition of the synthesis/release of these inflammatory mediators predominantly by prostaglandin. These results are in good agreement with the previous reports on anti-inflammatory activity of the other species of genus Phyllanthus (Kiemer et al., 2003; Raphael and Kuttan, 2003; Kassuya et al., 2005). Further, pretreatment with PSEE exhibited significant reduction in the granuloma formation demonstrating its potential to 1343 cure chronic inflammation. Inflammatory events occurring during the implant of cotton pellet are characterized by infiltration of macrophage, leucocytes, neutrophils; formations of fibroblast cells; accumulation of proteins and fluid proliferation (Ismail et al., 1997; Vilela et al., 2010); and hence forms the proliferative phase of inflammation. The study suggested that the potential of PSEE (45.671 ± 0.712%, p < 0.001, at 200 mg/kg oral dose) in treating chronic inflammation may be due to its effect on proliferative component of inflammation. It has been recognized that free radicals are involved in inflammation (Aruoma, 1998). There are many evidences of relationship between free radical scavenging and anti-inflammatory activities and hence, antioxidants play beneficial role in the treatment of inflammatory diseases (Conner and Grisham, 1996). PSPE and PSEE are found to display both antioxidant and anti-inflammatory activities which support the existence of above relationship. Therefore, our findings vindicate the claims of traditional healers for the use of Phyllanthus simplex in the treatment of inflammation, and conditions associated with inflammation. 4. Conclusions Our studies clearly bring out that the ethanolic extract of the whole plant of Phyllanthus simplex possess better antioxidant and anti-inflammatory activities, attributed to its higher phenolics content, than its petroleum ether extract; and also validates its ethnomedicinal use. Acknowledgement H.S.C. is thankful to the UGC, New Delhi for the award of research fellowship. 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