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Antioxidant and free radical scavenging activity of Spondias pinnata

Antioxidant and free radical scavenging activity of Spondias pinnata Background: Many diseases are associated with oxidative stress caused by free radicals. Current research is directed towards finding naturally-occurring antioxidants of plant origin. The aim of the present study was to evaluate the in vitro antioxidant activities of Spondias pinnata stem bark extract. Methods: A 70% methanol extract of Spondias pinnata stem bark was studied in vitro for total antioxidant activity, for scavenging of hydroxyl radicals, superoxide anions, nitric oxide, hydrogen peroxide, peroxynitrite, singlet oxygen and hypochlorous acid, and for iron chelating capacity, reducing power, and phenolic and flavonoid contents. Results: The extract showed total antioxidant activity with a trolox equivalent antioxidant concentration (TEAC) value of 0.78 ± 0.02. The IC values for scavenging of free radicals were 112.18 ± 3.27 μg/ml, 13.46 ± 0.66 μg/ml and 24.48 ± 2.31 μg/ml for hydroxyl, superoxide and nitric oxide, respectively. The IC for hydrogen peroxide scavenging was 44.74 ± 25.61 mg/ml. For the peroxynitrite, singlet oxygen and hypochlorous acid scavenging activities the IC values were 716.32 ± 32.25 μg/ml, 58.07 ± 5.36 μg/ml and 127.99 ± 6.26 μg/ml, respectively. The extract was found to be a potent iron chelator with IC = 66.54 ± 0.84 μg/ml. The reducing power was increased with increasing amounts of extract. The plant extract (100 mg) yielded 91.47 ± 0.004 mg/ ml gallic acid-equivalent phenolic content and 350.5 ± 0.004 mg/ml quercetin-equivalent flavonoid content. Conclusion: The present study provides evidence that a 70% methanol extract of Spondias pinnata stem bark is a potential source of natural antioxidants. and inflammatory diseases [1,2]. All human cells protect Background It is increasingly being realized that many of today's dis- themselves against free radical damage by enzymes such eases are due to the "oxidative stress" that results from an as superoxide dismutase (SOD) and catalase, or com- imbalance between formation and neutralization of pro- pounds such as ascorbic acid, tocopherol and glutathione oxidants. Oxidative stress is initiated by free radicals, [3]. Sometimes these protective mechanisms are dis- which seek stability through electron pairing with biolog- rupted by various pathological processes, and antioxidant ical macromolecules such as proteins, lipids and DNA in supplements are vital to combat oxidative damage. healthy human cells and cause protein and DNA damage Recently, much attention has been directed towards the along with lipid peroxidation. These changes contribute development of ethnomedicines with strong antioxidant to cancer, atherosclerosis, cardiovascular diseases, ageing properties but low cytotoxicities. Page 1 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Spondias pinnata (Linn. f.) Kurz (Family – Anacardiaceae) from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbitu- is a deciduous tree distributed in India, Sri Lanka and ric acid (TBA) was obtained from Loba Chemie, Mumbai, South-East Asian countries. In India it is commonly seen India. Sodium nitrite was obtained from Qualigens Fine in the deciduous to semi-evergreen forests of the Western Chemicals, Mumbai, India. Ghats. The genus Spondias includes 17 described species, 7 of which are native to the neotropics and about 10 are Plant material native to tropical Asia. The phytochemistry of this plant The bark of the S. pinnata plant was collected from the has been studied [4]. The gum exudate of the species has Bankura district of West Bengal, India and authenticated been found to contain acidic polysaccharides [5]. A crude through the Central Research Institute of Ayurveda, Kolk- extract of S. pinnata has been reported to show antibacte- ata, India. rial activity [6]. In ethnomedicine, equal quantities of bark juice of S. pinnata and Syzygium cumuni are prescribed Extraction as a remedy for dysentery [7]. An aqueous extract of this The stem bark of S. pinnata was dried at room temperature plant inhibits the citrus canker of lime [8]. However, there for 7 days, finely powdered and used for extraction. The has been no report on the antioxidant properties of this powder (100 g) was mixed with 500 ml methanol:water species. (7:3) using a magnetic stirrer for 15 hours, then the mix- ture was centrifuged at 2850 × g and the supernatant was The objective of the present study was to evaluate the anti- decanted. The pellet was mixed again with 500 ml metha- oxidant potential and free radical scavenging activity of a nol-water and the entire extraction process was repeated. 70% methanol extract of S. pinnata. The extract was exam- The supernatants collected from the two phases were ined for different reactive oxygen species (ROS) scaveng- mixed in a round bottom flask and concentrated under ing activities including hydroxyl, superoxide, nitric oxide, reduced pressure in a rotary evaporator. The concentrated hydrogen peroxide, peroxynitrite, singlet oxygen and extract was then lyophilized. The residue was kept at - hypochlorous acid, and for phenol and flavonoid con- 20°C for future use. tents, iron chelating capacity and total antioxidant activ- ity. Total antioxidant activity .+ The ability of the test sample to scavenge ABTS radical .+ cation was compared to trolox standard [9]. The ABTS Methods Chemicals radical cation was pregenerated by mixing 7 mM ABTS 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) stock solution with 2.45 mM potassium persulfate (final (ABTS) was obtained from Roche Diagnostics, Man- concentration) and incubating for 12–16 h in the dark at nheim, Germany. 6-hydroxy-2,5,7,8-tetramethychroman- room temperature until the reaction was complete and .+ 2-carboxylic acid (Trolox) was obtained from Fluka, the absorbance was stable. The absorbance of the ABTS Buchs, Switzerland. Potassium persulfate (K S O ), ethyl- solution was equilibrated to 0.70 (± 0.02) by diluting 2 2 8 enediamine tetraacetic acid (EDTA), ascorbic acid, 2- with water at room temperature, then 1 ml was mixed deoxy-2-ribose, trichloroacetic acid (TCA), mannitol, with 10 μl of the test sample (0.05–10 mg/ml) and the nitro blue tetrazolium (NBT), reduced nicotinamide ade- absorbance was measured at 734 nm after 6 min. All nine dinucleotide (NADH), phenazine methosulfate experiments were repeated six times. The percentage inhi- (PMS), sodium nitroprusside (SNP), sulfanilamide, naph- bition of absorbance was calculated and plotted as a func- thylethylenediamine dihydrochloride (NED), L-histidine, tion of the concentration of standard and sample to lipoic acid, sodium pyruvate, quercetin and ferrozine were determine the trolox equivalent antioxidant concentra- obtained from Sisco Research Laboratories Pvt. Ltd, Mum- tion (TEAC). To calculate the TEAC, the gradient of the bai, India. Hydrogen peroxide, potassium hexacyanofer- plot for the sample was divided by the gradient of the plot rate, Folin-Ciocalteu reagent, sodium carbonate for trolox. (Na CO ), butylated hydroxytoluene (BHT), sodium 2 3 hypochlorite (NaOCl), aluminium chloride (AlCl ), Hydroxyl radical scavenging ammonium iron (II) sulfate hexahydrate This was assayed as described by Elizabeth and Rao [10] ((NH ) Fe(SO ) 6H O), potassium nitrite (KNO ), N, N- with a slight modification. The assay is based on quantifi- 4 2 4 2 2 2 dimethyl-4-nitrosoaniline and xylenol orange were cation of the degradation product of 2-deoxyribose by obtained from Merck, Mumbai, India. Gallic acid and cur- condensation with TBA. Hydroxyl radical was generated 3+ cumin were obtained from MP Biomedicals, France. Fer- by the Fe -ascorbate-EDTA-H O system (the Fenton 2 2 rous sulfate and catalase were obtained from HiMedia reaction). The reaction mixture contained, in a final vol- Laboratories Pvt. Ltd, Mumbai, India. Evans Blue was pur- ume of 1 ml, 2-deoxy-2-ribose (2.8 mM); KH PO -KOH 2 4 chased from BDH, England. Manganese dioxide was buffer (20 mM, pH 7.4); FeCl (100 μM); EDTA (100 μM); obtained from SD Fine Chemicals, Mumbai, India. Dieth- H O (1.0 mM); ascorbic acid (100 μM) and various con- 2 2 ylene-triamine-pentaacetic acid (DTPA) was obtained centrations (0–200 μg/ml) of the test sample or reference Page 2 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 compound. After incubation for 1 h at 37°C, 0.5 ml of the bated at room temperature for 30 min. The absorbance of reaction mixture was added to 1 ml 2.8% TCA, then 1 ml the ferric-xylenol orange complex was measured at 560 nm. All tests were carried out six times and sodium pyru- 1% aqueous TBA was added and the mixture was incu- bated at 90°C for 15 min to develop the color. After cool- vate was used as the reference compound [14]. ing, the absorbance was measured at 532 nm against an appropriate blank solution. All tests were performed six Peroxynitrite scavenging . - times. Mannitol, a classical OH scavenger, was used as a Peroxynitrite (ONOO ) was synthesized by the method positive control. Percentage inhibition was evaluated by described by Beckman et al. [15]. An acidic solution (0.6 comparing the test and blank solutions. M HCl) of 5 ml H O (0.7 M) was mixed with 5 ml 0.6 M 2 2 KNO on an ice bath for 1 s and 5 ml of ice-cold 1.2 M Superoxide radical scavenging NaOH was added. Excess H O was removed by treatment 2 2 This activity was measured by the reduction of NBT with granular MnO prewashed with 1.2 M NaOH and the according to a previously reported method [11]. The non- reaction mixture was left overnight at -20°C. Peroxynitrite enzymatic phenazine methosulfate-nicotinamide adenine solution was collected from the top of the frozen mixture dinucleotide (PMS/NADH) system generates superoxide and the concentration was measured spectrophotometri- -1 -1 radicals, which reduce nitro blue tetrazolium (NBT) to a cally at 302 nm (ε = 1670 M cm ). purple formazan. The 1 ml reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT An Evans Blue bleaching assay was used to measure perox- (50 μM), PMS (15 μM) and various concentrations (0–20 ynitrite scavenging activity. The assay was performed by a μg/ml) of sample solution. After incubation for 5 min at standard method [16] with a slight modification. The ambient temperature, the absorbance at 562 nm was reaction mixture contained 50 mM phosphate buffer (pH measured against an appropriate blank to determine the 7.4), 0.1 mM DTPA, 90 mM NaCl, 5 mM KCl, 12.5 μM quantity of formazan generated. All tests were performed Evans Blue, various doses of plant extract (0–200 μg/ml) six times. Quercetin was used as positive control. and 1 mM peroxynitrite in a final volume of 1 ml. After incubation at 25°C for 30 min the absorbance was meas- Nitric oxide radical scavenging ured at 611 nm. The percentage scavenging of ONOO was At physiological pH, nitric oxide generated from aqueous calculated by comparing the results of the test and blank sodium nitroprusside (SNP) solution interacts with oxy- samples. All tests were performed six times. Gallic acid gen to produce nitrite ions, which may be quantified by was used as the reference compound. the Griess Illosvoy reaction [12]. The reaction mixture Singlet oxygen scavenging contained 10 mM SNP, phosphate buffered saline (pH 7.4) and various doses (0–70 μg/ml) of the test solution The production of singlet oxygen ( O ) was determined in a final volume of 3 ml. After incubation for 150 min at by monitoring N, N-dimethyl-4-nitrosoaniline (RNO) 25°C, 1 ml sulfanilamide (0.33% in 20% glacial acetic bleaching, using a previously reported spectrophotomet- acid) was added to 0.5 ml of the incubated solution and ric method [17,18]. Singlet oxygen was generated by a allowed to stand for 5 min. Then 1 ml of napthylethylen- reaction between NaOCl and H O , and the bleaching of 2 2 ediamine dihydrochloride (NED) (0.1% w/v) was added RNO was monitored at 440 nm. The reaction mixture and the mixture was incubated for 30 min at 25°C. The contained 45 mM phosphate buffer (pH 7.1), 50 mM O , 50 mM histidine, 10 μM RNO and pink chromophore generated during diazotization of NaOCl, 50 mM H 2 2 nitrite ions with sulphanilamide and subsequent cou- various concentrations (0–200 μg/ml) of sample in a final pling with NED was measured spectrophotometrically at volume of 2 ml. It was incubated at 30°C for 40 min and 540 nm against a blank sample. All tests were performed the decrease in RNO absorbance was measured at 440 nm. six times. Curcumin was used as a standard. The scavenging activity of sample was compared with that of lipoic acid, used as a reference compound. All tests were Hydrogen peroxide scavenging performed six times. This activity was determined according to a previously Hypochlorous acid scavenging described method [13] with minor changes. An aliquot of 50 mM H O and various concentrations (0–2 mg/ml) of Hypochlorous acid (HOCl) was prepared immediately 2 2 samples were mixed (1:1 v/v) and incubated for 30 min at before the experiment by adjusting the pH of a 10% (v/v) room temperature. After incubation, 90 μl of the H O - solution of NaOCl to 6.2 with 0.6 M H SO , and the con- 2 2 2 4 sample solution was mixed with 10 μl HPLC-grade meth- centration of HOCl was determined by measuring the anol and 0.9 ml FOX reagent was added (prepared in absorbance at 235 nm using the molar extinction coeffi- -1 -1 advance by mixing 9 volumes of 4.4 mM BHT in HPLC- cient of 100 M cm . The assay was carried out as grade methanol with 1 volume of 1 mM xylenol orange described by Aruoma and Halliwell [19] with minor and 2.56 mM ammonium ferrous sulfate in 0.25 M changes. The scavenging activity was evaluated by measur- H SO ). The reaction mixture was then vortexed and incu- ing the decrease in absorbance of catalase at 404 nm. The 2 4 Page 3 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 reaction mixture contained, in a final volume of 1 ml, 50 was added to 0.3 ml distilled water followed by NaNO mM phosphate buffer (pH 6.8), catalase (7.2 μM), HOCl (0.03 ml, 5%). After 5 min at 25°C, AlCl (0.03 ml, 10%) (8.4 mM) and increasing concentrations (0–100 μg/ml) was added. After a further 5 min, the reaction mixture was of plant extract. The assay mixture was incubated at 25°C treated with 0.2 ml 1 mM NaOH. Finally, the reaction for 20 min and the absorbance was measured against an mixture was diluted to 1 ml with water and the absorb- appropriate blank. All tests were performed six times. ance was measured at 510 nm. All tests were performed six Ascorbic acid, a potent HOCl scavenger, was used as a ref- times. The flavonoid content was calculated from a quer- erence [20]. cetin standard curve. 2+ Fe chelation Statistical analysis The ferrous ion chelating activity was evaluated by a All data are given as the mean ± SD of six measurements. standard method [21] with minor changes. The reaction Statistical analysis was performed using KyPlot version was carried out in HEPES buffer (20 mM, pH 7.2). Briefly, 2.0 beta 15 (32 bit). The IC values were calculated by the various concentrations (0–120 μg/ml) of plant extract formula Y = 100*A1/(X + A1), where A1 = IC , Y = were added to 12.5 μM ferrous sulfate solution and the response (Y = 100% when X = 0), X = inhibitory concen- reaction was initiated by the addition of ferrozine (75 tration. The IC values were compared by paired t tests. p μM). The mixture was shaken vigorously and incubated < 0.05 was considered significant. for 20 min at room temperature, then the absorbance was measured at 562 nm. All tests were performed six times. Results EDTA was used as a positive control. Total antioxidant activity The total antioxidant activity of the extract was calculated .+ Reducing power from the decolorization of ABTS , which was measured 3+ The Fe -reducing power of the extract was determined by spectrophotometrically at 734 nm. Interaction with the the method of Oyaizu [22] with a slight modification. Dif- extract or standard trolox suppressed the absorbance of .+ ferent concentrations (0.0–0.4 mg/ml) of the extract (0.5 the ABTS radical cation and the results, expressed as per- ml) were mixed with 0.5 ml phosphate buffer (0.2 M, pH centage inhibition of absorbance, are shown in figure 1(a) 6.6) and 0.5 ml potassium hexacyanoferrate (0.1%), fol- and figure 1(b), respectively. The TEAC value of the extract lowed by incubation at 50°C in a water bath for 20 min. was 0.78 ± 0.02. After incubation, 0.5 ml of TCA (10%) was added to ter- minate the reaction. The upper portion of the solution (1 Hydroxyl radical scavenging ml) was mixed with 1 ml distilled water, and 0.1 ml FeCl This assay shows the abilities of the extract and standard solution (0.01%) was added. The reaction mixture was mannitol to inhibit hydroxyl radical-mediated deoxyri- 3+ left for 10 min at room temperature and the absorbance bose degradation in an Fe -EDTA-ascorbic acid and H O 2 2 was measured at 700 nm against an appropriate blank reaction mixture. The results are shown in figure 2. The solution. All tests were performed six times. A higher IC values (Table 1) of the extract and standard in this absorbance of the reaction mixture indicated greater assay were 112.18 ± 3.27 μg/ml and 571.45 ± 20.12 μg/ reducing power. Butylated hydroxytoluene (BHT) was ml, respectively. The IC value of the extract was less than used as a positive control. that of the standard. At 200 μg/ml, the percentage inhibi- tion values were 53.7% and 23% for S. pinnata and man- Determination of total phenolic content nitol, respectively. Total phenolic content was determined using Folin-Cio- calteu (FC) reagent according to the method of Singleton Superoxide radical scavenging and Rossi [23] with a slight modification. Briefly, the The superoxide radicals generated from dissolved oxygen plant extract (0.1 ml) was mixed with 0.75 ml of FC rea- by PMS-NADH coupling can be measured by their ability gent (previously diluted 1000-fold with distilled water) to reduce NBT. The decrease in absorbance at 560 nm with and incubated for 5 min at 22°C, then 0.06% Na CO the plant extract and the reference compound quercetin 2 3 solution was added. After incubation at 22°C for 90 min, indicates their abilities to quench superoxide radicals in the absorbance was measured at 725 nm. All tests were the reaction mixture. As shown in figure 3, the IC values performed six times. The phenolic content was evaluated (Table 1) of the plant extract and quercetin on superoxide from a gallic acid standard curve. scavenging activity were 13.46 ± 0.66 μg/ml and 42.06 ± 1.35 μg/ml, respectively. The IC value of the extract was Determination of total flavonoid content less than that of the standard. At 20 μg/ml, the percentage The total flavonoid content was determined with alumin- inhibition of the plant extract was 55.2% whereas that of ium chloride (AlCl ) according to a known method [24] quercetin was 29.6%. using quercetin as a standard. The plant extract (0.1 ml) Page 4 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Total antiox Figure 1 idant activity Total antioxidant activity. Total antioxidant activity of plant extract and trolox. Effect of (a) Spondias pinnata extract and (b) reference compound trolox on decolorization of ABTS radical cation. The percentage inhibition was plotted against the con- centration of sample. All data are expressed as mean ± S.D. (n = 6). Nitric oxide radical scavenging inhibition of the plant extract was 61.2% whereas that of S. pinnata extract also caused a moderate dose-dependent curcumin was 44.1%. inhibition of nitric oxide with an IC (Table 1) of 24.48 ± 2.31 μg/ml (figure 4). Curcumin was used as a reference Hydrogen peroxide scavenging Hydrogen peroxide scavenging was assayed by the FOX compound and 90.82 ± 4.75 μg/ml curcumin was needed for 50% inhibition. The IC value of the extract was less reagent method [14]. Figure 5 shows that the plant extract than that of the standard. At 70 μg/ml, the percentage is a very poor scavenger of H O (IC = 44.74 ± 25.61 mg/ 2 2 50 ml) compared to standard sodium pyruvate (IC = 3.24 ± 0.3 mg/ml). The IC value (Table 1) of the extract was greater than that of the standard. At a concentration of 2 mg/ml, the scavenging percentages were 6.5% and 57.7% for S. pinnata and sodium pyruvate, respectively. Peroxynitrite scavenging Figure 6 shows that the peroxynitrite scavenging activity of the plant extract was concentration-dependent. The calcu- was 716.32 ± 32.25 μg/ml, which was lower lated IC than that of the reference compound gallic acid (IC = 876.24 ± 56.96 μg/ml) (Table 1), indicating that the sam- ple is more potent scavenger of peroxynitrite than gallic acid. At 200 μg/ml, the scavenging percentages were 22.3% and 15.8% for S. pinnata and gallic acid, respec- tively. Singlet oxygen scavenging H Figure 2 ydroxyl radical scavenging assay S. pinnata extract was an effective scavenger of singlet oxy- Hydroxyl radical scavenging assay. Hydroxyl radical gen (figure 7) and this activity was comparable to that of scavenging activities of the Spondias pinnata extract and the lipoic acid. The IC value (Table 1) of the test sample was reference compound mannitol. The data represent the per- 58.07 ± 5.36 μg/ml whereas that of lipoic acid was 46.15 centage inhibition of deoxyribose degradation. The results ± 1.16 μg/ml. The IC value of the extract was higher than are mean ± S.D. of six parallel measurements. ***p < 0.001 vs that of the reference compound. At 200 μg/ml, the per- 0 μg/ml. IC = 112.18 ± 3.27 μg/ml. The IC value of the 50 50 standard is 571.45 ± 20.12 μg/ml. centage scavenging of the plant extract was 73.3% whereas that of lipoic acid was 75.3%. Page 5 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Table 1: Scavenging of reactive oxygen species and iron chelating activity (IC values) of Spondias pinnata and reference compounds Activity Extract/Reference IC (#) Hydroxyl radical (OH ) scavenging Spondias pinnata 112.18 ± 3.27 (6) Mannitol 571.45 ± 20.12 (6)*** .- Superoxide anion (O ) scavenging Spondias pinnata 13.46 ± 0.66 (6) Quercetin 42.06 ± 1.35 (6)*** Nitric oxide radical (NO) scavenging Spondias pinnata 24.48 ± 2.31 (6) Curcumin 90.82 ± 4.75 (6)*** Hydrogen peroxide (H O ) scavenging Spondias pinnata 44.74 ± 25.61 (6) 2 2 Sodium pyruvate 3.24 ± 0.30 (6) * Peroxynitrite (ONOO ) scavenging Spondias pinnata 716.32 ± 32.25 (6) Gallic acid 876.24 ± 56.96 (6)*** Singlet oxygen ( O ) scavenging Spondias pinnata 58.07 ± 5.36 (6) Lipoic acid 46.15 ± 1.16 (6) ** Hypochlorous acid (HOCl) scavenging Spondias pinnata 127.99 ± 6.26 (6) Ascorbic acid 235.95 ± 5.75 (6)*** Iron Chelating Spondias pinnata 66.54 ± 0.84 (6) EDTA 1.27 ± 0.05 (6)*** # Units of IC for all activities are μg/ml, except H O scavenging, where the units are mg/ml. Data are expressed as mean ± S.D. Data in 50 2 2 parenthesis indicate number of independent assays. EDTA, Ethylenediamine tetraacetic acid. * p < 0.05. ** p < 0.01. *** p < 0.001 vs Spondias pinnata. Hypochlorous acid scavenging the plant extract was 49.2% whereas that of ascorbic acid Figure 8 shows the dose-dependent hypochlorous acid was 34.6%. scavenging activity of S. pinnata extract compared to that 2+ of ascorbic acid. The results indicate that the extract scav- Fe chelation 2+ enged hypochlorous acid more efficiently (IC = 127.99 Ferrozine produces a violet complex with Fe . In the pres- ± 6.26 μg/ml) than ascorbic acid (IC = 235.95 ± 5.75 μg/ ence of a chelating agent, complex formation is inter- ml) (Table 1). At 100 μg/ml, the percentage scavenging of rupted and as a result the violet color of the complex is Superoxide radical Figure 3 scavenging assay Nitric oxide radical scavenging assay Figure 4 Superoxide radical scavenging assay. Scavenging effect Nitric oxide radical scavenging assay. The nitric oxide of Spondias pinnata plant extract and the standard quercetin radical scavenging activity of Spondias pinnata extract and the on superoxide radical. The data represent the percentage standard curcumin. The data represent the percentage nitric superoxide radical inhibition. All data are expressed as mean oxide inhibition. Each value represents mean ± S.D. (n = 6). ± S.D. (n = 6). ***p < 0.001 vs 0 μg/ml. IC = 13.46 ± 0.66 ***p < 0.001 vs 0 μg/ml. IC = 24.48 ± 2.31 μg/ml. The IC 50 50 50 μg/ml. The IC value of the standard is 42.06 ± 1.35 μg/ml. value of the standard is 90.82 ± 4.75 μg/ml. Page 6 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Figure 5 H O scavenging assay Singlet oxyg Figure 7 en scavenging assay 2 2 H O scavenging assay. Effects of Spondias pinnata plant 2 2 Singlet oxygen scavenging assay. Effects of Spondias pin- extract and the standard sodium pyruvate on the scavenging nata plant extract and the standard lipoic acid on the scav- of H O . The data represent the percentage H O scaveng- enging of singlet oxygen. The results are mean ± S.D. of six 2 2 2 2 ing. All data are expressed as mean ± S.D. (n = 6). **p < 0.01 parallel measurements. ***p < 0.001 vs 0 μg/ml. IC = 58.07 and ***p < 0.001 vs 0 mg/ml. IC = 44.74 ± 25.61 mg/ml. The 50 ± 5.36 μg/ml. The IC value of the standard is 46.15 ± 1.16 IC value of the standard is 3.24 ± 0.3 mg/ml. 50 μg/ml. decreased. The results [figure 9(a) and figure 9(b)] dem- Reducing power 2+ 3+ 2+ onstrated that formation of the ferrozine-Fe complex is As illustrated in figure 10, Fe was transformed to Fe in inhibited in the presence of the test and reference com- the presence of S. pinnata extract and the reference com- pounds. The IC values (Table 1) of the plant extract and pound BHT to measure the reductive capability. At 0.1 EDTA were 66.54 ± 0.84 μg/ml and 1.27 ± 0.05 μg/ml, mg/ml, the absorbances of the plant extract and BHT were respectively. At 120 μg/ml, the percentage inhibition of 0.32 and 0.02, respectively, while at 0.4 mg/ml, the the plant extract was 51.8% whereas at 45 μg/ml that of absorbances of both extract and BHT were almost the EDTA was 99.5%. same. This result indicates that maximum activity is Peroxynitrite a Figure 6 nion scavenging assay HO Figure 8 Cl scavenging assay Peroxynitrite anion scavenging assay. The peroxynitrite HOCl scavenging assay. Hypochlorous acid scavenging anion scavenging activity of Spondias pinnata plant extract and activities of Spondias pinnata plant extract and the standard the standard gallic acid. Each value represents mean ± S.D. (n ascorbic acid. All data are expressed as mean ± S.D. (n = 6). = 6). ***p < 0.001 vs 0 μg/ml. IC = 716.32 ± 32.25 μg/ml. **p < 0.01 and ***p < 0.001 vs 0 μg/ml. IC = 127.99 ± 6.26 50 50 The IC value of the standard is 876.24 ± 56.96 μg/ml. μg/ml. The IC value of the standard is 235.95 ± 5.75 μg/ml. 50 50 Page 7 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 .+ ABTS is a blue chromophore produced by the reaction between ABTS and potassium persulfate. Addition of the plant extract to this pre-formed radical cation reduced it to ABTS in a concentration-dependent manner. The results were compared with those obtained using trolox and the TEAC value demonstrates that the extract is a potent anti- oxidant. Hydroxyl radicals are the major active oxygen species causing lipid peroxidation and enormous biological dam- 2+ Fe Figure 9 chelation assay 2+ age [31]. They were produced in this study by incubating Fe chelation assay. Effects of (a) Spondias pinnata plant 2+ ferric-EDTA with ascorbic acid and H O at pH 7.4, and extract and (b) standard EDTA on ferrozine-Fe complex 2 2 reacted with 2-deoxy-2-ribose to generate a malondialde- formation. The data are expressed as percentage inhibition of chromogen formation. The results are mean ± S.D. of six hyde (MDA)-like product. This compound forms a pink parallel measurements. ***p < 0.001 vs 0 μg/ml. IC = 66.54 chromogen upon heating with TBA at low pH [32]. When ± 0.84 μg/ml. The IC value of the standard is 1.27 ± 0.05 S. pinnata extract was added to the reaction mixture, it μg/ml. removed the hydroxyl radicals from the sugar and pre- vented the reaction. The IC value indicates that the plant extract is a better hydroxyl radical scavenger than the shown at a lower dose by the extract (0.1 mg/ml) than by standard mannitol. BHT. Superoxide anion is also very harmful to cellular compo- Determination of total phenolic content nents [33]. Robak and Glyglewski [34] reported that flavo- Phenolic compounds may contribute directly to antioxi- noids are effective antioxidants mainly because they dative action. The total phenolic content was 91.47 ± scavenge superoxide anions. As shown in figure 3, the 0.004 mg/ml gallic acid equivalent per 100 mg plant superoxide radical scavenging activities of the plant extract. extract and the reference compound are increased mark- edly with increasing concentrations. The results suggest Determination of total flavonoid content that the plant extract is a more potent scavenger of super- The total flavonoid content of the 70% methanolic extract oxide radical than the standard quercetin. of S. pinnata was 350.5 ± 0.004 mg/ml quercetin equiva- lent per 100 mg plant extract. It is well known that nitric oxide has an important role in various inflammatory processes. Sustained levels of pro- Discussion duction of this radical are directly toxic to tissues and con- In living systems, free radicals are constantly generated tribute to the vascular collapse associated with septic and they can cause extensive damage to tissues and bio- shock, whereas chronic expression of nitric oxide radical molecules leading to various disease conditions, espe- is associated with various carcinomas and inflammatory cially degenerative diseases, and extensive lysis [25]. Many conditions including juvenile diabetes, multiple sclerosis, synthetic drugs protect against oxidative damage but they arthritis and ulcerative colitis [35]. The toxicity of NO have adverse side effects. An alternative solution to the increases greatly when it reacts with superoxide radical, problem is to consume natural antioxidants from food forming the highly reactive peroxynitrite anion (ONOO ) supplements and traditional medicines [26,27]. Recently, [36]. The nitric oxide generated from sodium nitroprus- many natural antioxidants have been isolated from differ- side reacts with oxygen to form nitrite. The extract inhibits ent plant materials [28,29]. The antioxidant and polyphe- nitrite formation by directly competing with oxygen in the nol contents of S. pinnata have also been determined by reaction with nitric oxide. The present study proved that Chuyen et al. [30], using concentrated leaf extract. The the extract studied has more potent nitric oxide scaveng- antioxidant activity has been studied by the inhibition of ing activity than the standard curcumin. ascorbic acid-induced lipid peroxidation and Catechin has been used as the standard for phenolic content, Hydrogen peroxide is a weak oxidizing agent that inacti- whereas in the present study, the antioxidant capacity of vates a few enzymes directly, usually by oxidation of bark extract was measured by an improved ABTS radical essential thiol (-SH) groups. It can cross cell membranes cation decolorization assay and gallic acid was used as the rapidly; once inside the cell, it can probably react with 2+ 2+ standard for phenolic content measurement; both studies Fe and possibly Cu ions to form hydroxyl radicals and showed promising results. Therefore, it is clear that both this may be the origin of many of its toxic effects [37]. the leaf and stem bark extracts of the plant have good anti- From the results, it appeared that the H O scavenging 2 2 oxidant activities as well as high polyphenolic contents. Page 8 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 erate lipid peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can perpetuate the chain reaction [42]. Metal chelating capac- ity is significant since it reduces the concentration of the transition metal that catalyzes lipid peroxidation [43]. According to the results, the plant extract is not as good as the standard EDTA; but the decrease in concentration- dependent color formation in the presence of the extract indicates that it has iron chelating activity. The reducing capacity of a compound may serve as a sig- nificant indicator of its potential antioxidant activity. However, the activities of antioxidants have been attrib- uted to various mechanisms such as prevention of chain initiation, decomposition of peroxides, reducing capacity and radical scavenging [44]. As shown in figure 7, the Reducing power Figure 10 assay reducing power of the plant extract was compared with Reducing power assay. The reductive abilities of Spondias the standard BHT and found to be superior. pinnata extract and the standard BHT. The absorbance (A ) was plotted against concentration of sample. Each value rep- The results indicate that S. pinnata plant extract contains resents mean ± S.D. (n = 6). *** p < 0.001 vs 0 mg/ml. significant amounts of flavonoids and phenolic com- pounds. Both these classes of compounds have good anti- oxidant potential and their effects on human nutrition activity of the plant extract is negligible compared to that and health are considerable. The mechanism of action of of the standard sodium pyruvate. flavonoids is through scavenging or chelation [45]. Phe- nolic compounds are also very important plant constitu- ) is relatively stable compared to Peroxynitrite (ONOO ents because their hydroxyl groups confer scavenging other free radicals but once protonated it forms the highly ability [44]. reactive peroxynitrous acid (ONOOH) [38]. Generation of excess ONOO leads to oxidative damage and tissue Conclusion injury [39]. Peroxynitrite bleaches Evans Blue by oxidizing On the basis of the results obtained in the present study, it. According to the present results, the plant extract inhib- it is concluded that a 70% methanolic extract of Spondias its Evans Blue bleaching by scavenging peroxynitrite and pinnata bark, which contains large amounts of flavonoids its activity is greater than that of the reference gallic acid. and phenolic compounds, exhibits high antioxidant and free radical scavenging activities. It also chelates iron and Singlet oxygen is generated in the skin by ultraviolet radi- has reducing power. These in vitro assays indicate that this ation. It is a high energy form of oxygen and is known as plant extract is a significant source of natural antioxidant, one of the ROS. Singlet oxygen induces hyperoxidation which might be helpful in preventing the progress of var- and oxygen cytotxicity and decreases antioxidative activity ious oxidative stresses. However, the components respon- [40]. The present study indicates that the S. pinnata extract sible for the antioxidative activity are currently unclear. has good scavenging activity for singlet oxygen but is not Therefore, further investigation is needed to isolate and as efficient as the standard lipoic acid. identify the antioxidant compounds present in the plant extract. Furthermore, the in vivo antioxidant activity of this At sites of inflammation, the oxidation of Cl ions by the extract needs to be assessed prior to clinical use. neutrophil enzyme myeloperoxidase results in the pro- duction of another harmful ROS, hypochlorous acid [41]. Competing interests HOCl has the ability to inactivate the antioxidant enzyme The authors declare that they have no competing interests. catalase through breakdown of the heme prosthetic group. Catalase inactivation is inhibited in the presence of Authors' contributions the extract, signifying its HOCl scavenging activity. The BH: Performed the study. results suggest that S. pinnata is a more efficient scavenger than the standard ascorbic acid. SB: Design, analysis and acquisition of data. Iron can stimulate lipid peroxidation by the Fenton reac- NM: Supervised the study design and drafted the manu- 2+ 3+ - . tion (H O + Fe = Fe + OH + OH ) and can also accel- script. 2 2 Page 9 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 23. Singleton VL, Rossi JA: Colorimetry of total phenolics with Acknowledgements phosphomolybdic-phosphotungstic acid reagents. Am J Enol Mr. B. Hazra is grateful to the Indian Council of Medical Research, Govern- Vitic 1965, 16:144-158. ment of India for providing fellowship. The authors would like to thank Mr. 24. Zhishen J, Mengcheng T, Jianming W: The determination of flavo- Rhitajit Sarkar, Dr. Sourav Mandal and Mr. Ranjit Das for assistance. noid content in mulberry and their scavenging effects on superoxide radicals. Food Chem 1999, 64:555-559. 25. Halliwell B, Gutteridge JM: Free radicals in biology and medicine Oxford: References Oxford University Press; 1998. 1. Braca A, Sortino C, Politi M, Morelli I, Mendez J: Antioxidant activ- 26. Yazdanparast R, Ardestani A: In vitro antioxidant and free radical ity of flavonoids from Licania licaniaeflora. J Ethnopharmacol scavenging activity of Cyperus rotundus. J Med Food 2007, 2002, 79:379-381. 10:667-674. 2. Maxwell SR: Prospects for the use of antioxidant therapies. 27. Yazdanparast R, Bahramikias S, Ardestani A: Nasturtium oficinale Drugs 1995, 49:345-361. reduces oxidative stress and enhances antioxidant capacity 3. Niki E, Shimaski H, Mino M: Antioxidantism-free radical and bio- in hypercholesterolaemic rats. 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Pedraza-Chaverrí J, Arriaga-Noblecía G, Medina-Campos ON: dioprotective effects, and dietary sources. J Nutr Biochem 1996, Hypochlorous acid scavenging capacity of garlic. Phytother Res 7:66-76. 2007, 21:884-888. 21. Haro-Vicente JF, Martinez-Gracia C, Ros G: Optimization of in Pre-publication history vitro measurement of available iron from different fortifi- cants in citric fruit juices. Food Chem 2006, 98:639-648. The pre-publication history for this paper can be accessed 22. Oyaizu M: Studies on products of browning reactions: antioxi- here: dant activities of products of browning reaction prepared from glucose amine. Jap J Nutr 1986, 44:307-315. http://www.biomedcentral.com/1472-6882/8/63/prepub Page 10 of 10 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Complementary and Alternative Medicine Springer Journals

Antioxidant and free radical scavenging activity of Spondias pinnata

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Springer Journals
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Copyright © 2008 by Hazra et al; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Complementary & Alternative Medicine; Internal Medicine; Chiropractic Medicine
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1472-6882
DOI
10.1186/1472-6882-8-63
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19068130
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Abstract

Background: Many diseases are associated with oxidative stress caused by free radicals. Current research is directed towards finding naturally-occurring antioxidants of plant origin. The aim of the present study was to evaluate the in vitro antioxidant activities of Spondias pinnata stem bark extract. Methods: A 70% methanol extract of Spondias pinnata stem bark was studied in vitro for total antioxidant activity, for scavenging of hydroxyl radicals, superoxide anions, nitric oxide, hydrogen peroxide, peroxynitrite, singlet oxygen and hypochlorous acid, and for iron chelating capacity, reducing power, and phenolic and flavonoid contents. Results: The extract showed total antioxidant activity with a trolox equivalent antioxidant concentration (TEAC) value of 0.78 ± 0.02. The IC values for scavenging of free radicals were 112.18 ± 3.27 μg/ml, 13.46 ± 0.66 μg/ml and 24.48 ± 2.31 μg/ml for hydroxyl, superoxide and nitric oxide, respectively. The IC for hydrogen peroxide scavenging was 44.74 ± 25.61 mg/ml. For the peroxynitrite, singlet oxygen and hypochlorous acid scavenging activities the IC values were 716.32 ± 32.25 μg/ml, 58.07 ± 5.36 μg/ml and 127.99 ± 6.26 μg/ml, respectively. The extract was found to be a potent iron chelator with IC = 66.54 ± 0.84 μg/ml. The reducing power was increased with increasing amounts of extract. The plant extract (100 mg) yielded 91.47 ± 0.004 mg/ ml gallic acid-equivalent phenolic content and 350.5 ± 0.004 mg/ml quercetin-equivalent flavonoid content. Conclusion: The present study provides evidence that a 70% methanol extract of Spondias pinnata stem bark is a potential source of natural antioxidants. and inflammatory diseases [1,2]. All human cells protect Background It is increasingly being realized that many of today's dis- themselves against free radical damage by enzymes such eases are due to the "oxidative stress" that results from an as superoxide dismutase (SOD) and catalase, or com- imbalance between formation and neutralization of pro- pounds such as ascorbic acid, tocopherol and glutathione oxidants. Oxidative stress is initiated by free radicals, [3]. Sometimes these protective mechanisms are dis- which seek stability through electron pairing with biolog- rupted by various pathological processes, and antioxidant ical macromolecules such as proteins, lipids and DNA in supplements are vital to combat oxidative damage. healthy human cells and cause protein and DNA damage Recently, much attention has been directed towards the along with lipid peroxidation. These changes contribute development of ethnomedicines with strong antioxidant to cancer, atherosclerosis, cardiovascular diseases, ageing properties but low cytotoxicities. Page 1 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Spondias pinnata (Linn. f.) Kurz (Family – Anacardiaceae) from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbitu- is a deciduous tree distributed in India, Sri Lanka and ric acid (TBA) was obtained from Loba Chemie, Mumbai, South-East Asian countries. In India it is commonly seen India. Sodium nitrite was obtained from Qualigens Fine in the deciduous to semi-evergreen forests of the Western Chemicals, Mumbai, India. Ghats. The genus Spondias includes 17 described species, 7 of which are native to the neotropics and about 10 are Plant material native to tropical Asia. The phytochemistry of this plant The bark of the S. pinnata plant was collected from the has been studied [4]. The gum exudate of the species has Bankura district of West Bengal, India and authenticated been found to contain acidic polysaccharides [5]. A crude through the Central Research Institute of Ayurveda, Kolk- extract of S. pinnata has been reported to show antibacte- ata, India. rial activity [6]. In ethnomedicine, equal quantities of bark juice of S. pinnata and Syzygium cumuni are prescribed Extraction as a remedy for dysentery [7]. An aqueous extract of this The stem bark of S. pinnata was dried at room temperature plant inhibits the citrus canker of lime [8]. However, there for 7 days, finely powdered and used for extraction. The has been no report on the antioxidant properties of this powder (100 g) was mixed with 500 ml methanol:water species. (7:3) using a magnetic stirrer for 15 hours, then the mix- ture was centrifuged at 2850 × g and the supernatant was The objective of the present study was to evaluate the anti- decanted. The pellet was mixed again with 500 ml metha- oxidant potential and free radical scavenging activity of a nol-water and the entire extraction process was repeated. 70% methanol extract of S. pinnata. The extract was exam- The supernatants collected from the two phases were ined for different reactive oxygen species (ROS) scaveng- mixed in a round bottom flask and concentrated under ing activities including hydroxyl, superoxide, nitric oxide, reduced pressure in a rotary evaporator. The concentrated hydrogen peroxide, peroxynitrite, singlet oxygen and extract was then lyophilized. The residue was kept at - hypochlorous acid, and for phenol and flavonoid con- 20°C for future use. tents, iron chelating capacity and total antioxidant activ- ity. Total antioxidant activity .+ The ability of the test sample to scavenge ABTS radical .+ cation was compared to trolox standard [9]. The ABTS Methods Chemicals radical cation was pregenerated by mixing 7 mM ABTS 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) stock solution with 2.45 mM potassium persulfate (final (ABTS) was obtained from Roche Diagnostics, Man- concentration) and incubating for 12–16 h in the dark at nheim, Germany. 6-hydroxy-2,5,7,8-tetramethychroman- room temperature until the reaction was complete and .+ 2-carboxylic acid (Trolox) was obtained from Fluka, the absorbance was stable. The absorbance of the ABTS Buchs, Switzerland. Potassium persulfate (K S O ), ethyl- solution was equilibrated to 0.70 (± 0.02) by diluting 2 2 8 enediamine tetraacetic acid (EDTA), ascorbic acid, 2- with water at room temperature, then 1 ml was mixed deoxy-2-ribose, trichloroacetic acid (TCA), mannitol, with 10 μl of the test sample (0.05–10 mg/ml) and the nitro blue tetrazolium (NBT), reduced nicotinamide ade- absorbance was measured at 734 nm after 6 min. All nine dinucleotide (NADH), phenazine methosulfate experiments were repeated six times. The percentage inhi- (PMS), sodium nitroprusside (SNP), sulfanilamide, naph- bition of absorbance was calculated and plotted as a func- thylethylenediamine dihydrochloride (NED), L-histidine, tion of the concentration of standard and sample to lipoic acid, sodium pyruvate, quercetin and ferrozine were determine the trolox equivalent antioxidant concentra- obtained from Sisco Research Laboratories Pvt. Ltd, Mum- tion (TEAC). To calculate the TEAC, the gradient of the bai, India. Hydrogen peroxide, potassium hexacyanofer- plot for the sample was divided by the gradient of the plot rate, Folin-Ciocalteu reagent, sodium carbonate for trolox. (Na CO ), butylated hydroxytoluene (BHT), sodium 2 3 hypochlorite (NaOCl), aluminium chloride (AlCl ), Hydroxyl radical scavenging ammonium iron (II) sulfate hexahydrate This was assayed as described by Elizabeth and Rao [10] ((NH ) Fe(SO ) 6H O), potassium nitrite (KNO ), N, N- with a slight modification. The assay is based on quantifi- 4 2 4 2 2 2 dimethyl-4-nitrosoaniline and xylenol orange were cation of the degradation product of 2-deoxyribose by obtained from Merck, Mumbai, India. Gallic acid and cur- condensation with TBA. Hydroxyl radical was generated 3+ cumin were obtained from MP Biomedicals, France. Fer- by the Fe -ascorbate-EDTA-H O system (the Fenton 2 2 rous sulfate and catalase were obtained from HiMedia reaction). The reaction mixture contained, in a final vol- Laboratories Pvt. Ltd, Mumbai, India. Evans Blue was pur- ume of 1 ml, 2-deoxy-2-ribose (2.8 mM); KH PO -KOH 2 4 chased from BDH, England. Manganese dioxide was buffer (20 mM, pH 7.4); FeCl (100 μM); EDTA (100 μM); obtained from SD Fine Chemicals, Mumbai, India. Dieth- H O (1.0 mM); ascorbic acid (100 μM) and various con- 2 2 ylene-triamine-pentaacetic acid (DTPA) was obtained centrations (0–200 μg/ml) of the test sample or reference Page 2 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 compound. After incubation for 1 h at 37°C, 0.5 ml of the bated at room temperature for 30 min. The absorbance of reaction mixture was added to 1 ml 2.8% TCA, then 1 ml the ferric-xylenol orange complex was measured at 560 nm. All tests were carried out six times and sodium pyru- 1% aqueous TBA was added and the mixture was incu- bated at 90°C for 15 min to develop the color. After cool- vate was used as the reference compound [14]. ing, the absorbance was measured at 532 nm against an appropriate blank solution. All tests were performed six Peroxynitrite scavenging . - times. Mannitol, a classical OH scavenger, was used as a Peroxynitrite (ONOO ) was synthesized by the method positive control. Percentage inhibition was evaluated by described by Beckman et al. [15]. An acidic solution (0.6 comparing the test and blank solutions. M HCl) of 5 ml H O (0.7 M) was mixed with 5 ml 0.6 M 2 2 KNO on an ice bath for 1 s and 5 ml of ice-cold 1.2 M Superoxide radical scavenging NaOH was added. Excess H O was removed by treatment 2 2 This activity was measured by the reduction of NBT with granular MnO prewashed with 1.2 M NaOH and the according to a previously reported method [11]. The non- reaction mixture was left overnight at -20°C. Peroxynitrite enzymatic phenazine methosulfate-nicotinamide adenine solution was collected from the top of the frozen mixture dinucleotide (PMS/NADH) system generates superoxide and the concentration was measured spectrophotometri- -1 -1 radicals, which reduce nitro blue tetrazolium (NBT) to a cally at 302 nm (ε = 1670 M cm ). purple formazan. The 1 ml reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT An Evans Blue bleaching assay was used to measure perox- (50 μM), PMS (15 μM) and various concentrations (0–20 ynitrite scavenging activity. The assay was performed by a μg/ml) of sample solution. After incubation for 5 min at standard method [16] with a slight modification. The ambient temperature, the absorbance at 562 nm was reaction mixture contained 50 mM phosphate buffer (pH measured against an appropriate blank to determine the 7.4), 0.1 mM DTPA, 90 mM NaCl, 5 mM KCl, 12.5 μM quantity of formazan generated. All tests were performed Evans Blue, various doses of plant extract (0–200 μg/ml) six times. Quercetin was used as positive control. and 1 mM peroxynitrite in a final volume of 1 ml. After incubation at 25°C for 30 min the absorbance was meas- Nitric oxide radical scavenging ured at 611 nm. The percentage scavenging of ONOO was At physiological pH, nitric oxide generated from aqueous calculated by comparing the results of the test and blank sodium nitroprusside (SNP) solution interacts with oxy- samples. All tests were performed six times. Gallic acid gen to produce nitrite ions, which may be quantified by was used as the reference compound. the Griess Illosvoy reaction [12]. The reaction mixture Singlet oxygen scavenging contained 10 mM SNP, phosphate buffered saline (pH 7.4) and various doses (0–70 μg/ml) of the test solution The production of singlet oxygen ( O ) was determined in a final volume of 3 ml. After incubation for 150 min at by monitoring N, N-dimethyl-4-nitrosoaniline (RNO) 25°C, 1 ml sulfanilamide (0.33% in 20% glacial acetic bleaching, using a previously reported spectrophotomet- acid) was added to 0.5 ml of the incubated solution and ric method [17,18]. Singlet oxygen was generated by a allowed to stand for 5 min. Then 1 ml of napthylethylen- reaction between NaOCl and H O , and the bleaching of 2 2 ediamine dihydrochloride (NED) (0.1% w/v) was added RNO was monitored at 440 nm. The reaction mixture and the mixture was incubated for 30 min at 25°C. The contained 45 mM phosphate buffer (pH 7.1), 50 mM O , 50 mM histidine, 10 μM RNO and pink chromophore generated during diazotization of NaOCl, 50 mM H 2 2 nitrite ions with sulphanilamide and subsequent cou- various concentrations (0–200 μg/ml) of sample in a final pling with NED was measured spectrophotometrically at volume of 2 ml. It was incubated at 30°C for 40 min and 540 nm against a blank sample. All tests were performed the decrease in RNO absorbance was measured at 440 nm. six times. Curcumin was used as a standard. The scavenging activity of sample was compared with that of lipoic acid, used as a reference compound. All tests were Hydrogen peroxide scavenging performed six times. This activity was determined according to a previously Hypochlorous acid scavenging described method [13] with minor changes. An aliquot of 50 mM H O and various concentrations (0–2 mg/ml) of Hypochlorous acid (HOCl) was prepared immediately 2 2 samples were mixed (1:1 v/v) and incubated for 30 min at before the experiment by adjusting the pH of a 10% (v/v) room temperature. After incubation, 90 μl of the H O - solution of NaOCl to 6.2 with 0.6 M H SO , and the con- 2 2 2 4 sample solution was mixed with 10 μl HPLC-grade meth- centration of HOCl was determined by measuring the anol and 0.9 ml FOX reagent was added (prepared in absorbance at 235 nm using the molar extinction coeffi- -1 -1 advance by mixing 9 volumes of 4.4 mM BHT in HPLC- cient of 100 M cm . The assay was carried out as grade methanol with 1 volume of 1 mM xylenol orange described by Aruoma and Halliwell [19] with minor and 2.56 mM ammonium ferrous sulfate in 0.25 M changes. The scavenging activity was evaluated by measur- H SO ). The reaction mixture was then vortexed and incu- ing the decrease in absorbance of catalase at 404 nm. The 2 4 Page 3 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 reaction mixture contained, in a final volume of 1 ml, 50 was added to 0.3 ml distilled water followed by NaNO mM phosphate buffer (pH 6.8), catalase (7.2 μM), HOCl (0.03 ml, 5%). After 5 min at 25°C, AlCl (0.03 ml, 10%) (8.4 mM) and increasing concentrations (0–100 μg/ml) was added. After a further 5 min, the reaction mixture was of plant extract. The assay mixture was incubated at 25°C treated with 0.2 ml 1 mM NaOH. Finally, the reaction for 20 min and the absorbance was measured against an mixture was diluted to 1 ml with water and the absorb- appropriate blank. All tests were performed six times. ance was measured at 510 nm. All tests were performed six Ascorbic acid, a potent HOCl scavenger, was used as a ref- times. The flavonoid content was calculated from a quer- erence [20]. cetin standard curve. 2+ Fe chelation Statistical analysis The ferrous ion chelating activity was evaluated by a All data are given as the mean ± SD of six measurements. standard method [21] with minor changes. The reaction Statistical analysis was performed using KyPlot version was carried out in HEPES buffer (20 mM, pH 7.2). Briefly, 2.0 beta 15 (32 bit). The IC values were calculated by the various concentrations (0–120 μg/ml) of plant extract formula Y = 100*A1/(X + A1), where A1 = IC , Y = were added to 12.5 μM ferrous sulfate solution and the response (Y = 100% when X = 0), X = inhibitory concen- reaction was initiated by the addition of ferrozine (75 tration. The IC values were compared by paired t tests. p μM). The mixture was shaken vigorously and incubated < 0.05 was considered significant. for 20 min at room temperature, then the absorbance was measured at 562 nm. All tests were performed six times. Results EDTA was used as a positive control. Total antioxidant activity The total antioxidant activity of the extract was calculated .+ Reducing power from the decolorization of ABTS , which was measured 3+ The Fe -reducing power of the extract was determined by spectrophotometrically at 734 nm. Interaction with the the method of Oyaizu [22] with a slight modification. Dif- extract or standard trolox suppressed the absorbance of .+ ferent concentrations (0.0–0.4 mg/ml) of the extract (0.5 the ABTS radical cation and the results, expressed as per- ml) were mixed with 0.5 ml phosphate buffer (0.2 M, pH centage inhibition of absorbance, are shown in figure 1(a) 6.6) and 0.5 ml potassium hexacyanoferrate (0.1%), fol- and figure 1(b), respectively. The TEAC value of the extract lowed by incubation at 50°C in a water bath for 20 min. was 0.78 ± 0.02. After incubation, 0.5 ml of TCA (10%) was added to ter- minate the reaction. The upper portion of the solution (1 Hydroxyl radical scavenging ml) was mixed with 1 ml distilled water, and 0.1 ml FeCl This assay shows the abilities of the extract and standard solution (0.01%) was added. The reaction mixture was mannitol to inhibit hydroxyl radical-mediated deoxyri- 3+ left for 10 min at room temperature and the absorbance bose degradation in an Fe -EDTA-ascorbic acid and H O 2 2 was measured at 700 nm against an appropriate blank reaction mixture. The results are shown in figure 2. The solution. All tests were performed six times. A higher IC values (Table 1) of the extract and standard in this absorbance of the reaction mixture indicated greater assay were 112.18 ± 3.27 μg/ml and 571.45 ± 20.12 μg/ reducing power. Butylated hydroxytoluene (BHT) was ml, respectively. The IC value of the extract was less than used as a positive control. that of the standard. At 200 μg/ml, the percentage inhibi- tion values were 53.7% and 23% for S. pinnata and man- Determination of total phenolic content nitol, respectively. Total phenolic content was determined using Folin-Cio- calteu (FC) reagent according to the method of Singleton Superoxide radical scavenging and Rossi [23] with a slight modification. Briefly, the The superoxide radicals generated from dissolved oxygen plant extract (0.1 ml) was mixed with 0.75 ml of FC rea- by PMS-NADH coupling can be measured by their ability gent (previously diluted 1000-fold with distilled water) to reduce NBT. The decrease in absorbance at 560 nm with and incubated for 5 min at 22°C, then 0.06% Na CO the plant extract and the reference compound quercetin 2 3 solution was added. After incubation at 22°C for 90 min, indicates their abilities to quench superoxide radicals in the absorbance was measured at 725 nm. All tests were the reaction mixture. As shown in figure 3, the IC values performed six times. The phenolic content was evaluated (Table 1) of the plant extract and quercetin on superoxide from a gallic acid standard curve. scavenging activity were 13.46 ± 0.66 μg/ml and 42.06 ± 1.35 μg/ml, respectively. The IC value of the extract was Determination of total flavonoid content less than that of the standard. At 20 μg/ml, the percentage The total flavonoid content was determined with alumin- inhibition of the plant extract was 55.2% whereas that of ium chloride (AlCl ) according to a known method [24] quercetin was 29.6%. using quercetin as a standard. The plant extract (0.1 ml) Page 4 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Total antiox Figure 1 idant activity Total antioxidant activity. Total antioxidant activity of plant extract and trolox. Effect of (a) Spondias pinnata extract and (b) reference compound trolox on decolorization of ABTS radical cation. The percentage inhibition was plotted against the con- centration of sample. All data are expressed as mean ± S.D. (n = 6). Nitric oxide radical scavenging inhibition of the plant extract was 61.2% whereas that of S. pinnata extract also caused a moderate dose-dependent curcumin was 44.1%. inhibition of nitric oxide with an IC (Table 1) of 24.48 ± 2.31 μg/ml (figure 4). Curcumin was used as a reference Hydrogen peroxide scavenging Hydrogen peroxide scavenging was assayed by the FOX compound and 90.82 ± 4.75 μg/ml curcumin was needed for 50% inhibition. The IC value of the extract was less reagent method [14]. Figure 5 shows that the plant extract than that of the standard. At 70 μg/ml, the percentage is a very poor scavenger of H O (IC = 44.74 ± 25.61 mg/ 2 2 50 ml) compared to standard sodium pyruvate (IC = 3.24 ± 0.3 mg/ml). The IC value (Table 1) of the extract was greater than that of the standard. At a concentration of 2 mg/ml, the scavenging percentages were 6.5% and 57.7% for S. pinnata and sodium pyruvate, respectively. Peroxynitrite scavenging Figure 6 shows that the peroxynitrite scavenging activity of the plant extract was concentration-dependent. The calcu- was 716.32 ± 32.25 μg/ml, which was lower lated IC than that of the reference compound gallic acid (IC = 876.24 ± 56.96 μg/ml) (Table 1), indicating that the sam- ple is more potent scavenger of peroxynitrite than gallic acid. At 200 μg/ml, the scavenging percentages were 22.3% and 15.8% for S. pinnata and gallic acid, respec- tively. Singlet oxygen scavenging H Figure 2 ydroxyl radical scavenging assay S. pinnata extract was an effective scavenger of singlet oxy- Hydroxyl radical scavenging assay. Hydroxyl radical gen (figure 7) and this activity was comparable to that of scavenging activities of the Spondias pinnata extract and the lipoic acid. The IC value (Table 1) of the test sample was reference compound mannitol. The data represent the per- 58.07 ± 5.36 μg/ml whereas that of lipoic acid was 46.15 centage inhibition of deoxyribose degradation. The results ± 1.16 μg/ml. The IC value of the extract was higher than are mean ± S.D. of six parallel measurements. ***p < 0.001 vs that of the reference compound. At 200 μg/ml, the per- 0 μg/ml. IC = 112.18 ± 3.27 μg/ml. The IC value of the 50 50 standard is 571.45 ± 20.12 μg/ml. centage scavenging of the plant extract was 73.3% whereas that of lipoic acid was 75.3%. Page 5 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Table 1: Scavenging of reactive oxygen species and iron chelating activity (IC values) of Spondias pinnata and reference compounds Activity Extract/Reference IC (#) Hydroxyl radical (OH ) scavenging Spondias pinnata 112.18 ± 3.27 (6) Mannitol 571.45 ± 20.12 (6)*** .- Superoxide anion (O ) scavenging Spondias pinnata 13.46 ± 0.66 (6) Quercetin 42.06 ± 1.35 (6)*** Nitric oxide radical (NO) scavenging Spondias pinnata 24.48 ± 2.31 (6) Curcumin 90.82 ± 4.75 (6)*** Hydrogen peroxide (H O ) scavenging Spondias pinnata 44.74 ± 25.61 (6) 2 2 Sodium pyruvate 3.24 ± 0.30 (6) * Peroxynitrite (ONOO ) scavenging Spondias pinnata 716.32 ± 32.25 (6) Gallic acid 876.24 ± 56.96 (6)*** Singlet oxygen ( O ) scavenging Spondias pinnata 58.07 ± 5.36 (6) Lipoic acid 46.15 ± 1.16 (6) ** Hypochlorous acid (HOCl) scavenging Spondias pinnata 127.99 ± 6.26 (6) Ascorbic acid 235.95 ± 5.75 (6)*** Iron Chelating Spondias pinnata 66.54 ± 0.84 (6) EDTA 1.27 ± 0.05 (6)*** # Units of IC for all activities are μg/ml, except H O scavenging, where the units are mg/ml. Data are expressed as mean ± S.D. Data in 50 2 2 parenthesis indicate number of independent assays. EDTA, Ethylenediamine tetraacetic acid. * p < 0.05. ** p < 0.01. *** p < 0.001 vs Spondias pinnata. Hypochlorous acid scavenging the plant extract was 49.2% whereas that of ascorbic acid Figure 8 shows the dose-dependent hypochlorous acid was 34.6%. scavenging activity of S. pinnata extract compared to that 2+ of ascorbic acid. The results indicate that the extract scav- Fe chelation 2+ enged hypochlorous acid more efficiently (IC = 127.99 Ferrozine produces a violet complex with Fe . In the pres- ± 6.26 μg/ml) than ascorbic acid (IC = 235.95 ± 5.75 μg/ ence of a chelating agent, complex formation is inter- ml) (Table 1). At 100 μg/ml, the percentage scavenging of rupted and as a result the violet color of the complex is Superoxide radical Figure 3 scavenging assay Nitric oxide radical scavenging assay Figure 4 Superoxide radical scavenging assay. Scavenging effect Nitric oxide radical scavenging assay. The nitric oxide of Spondias pinnata plant extract and the standard quercetin radical scavenging activity of Spondias pinnata extract and the on superoxide radical. The data represent the percentage standard curcumin. The data represent the percentage nitric superoxide radical inhibition. All data are expressed as mean oxide inhibition. Each value represents mean ± S.D. (n = 6). ± S.D. (n = 6). ***p < 0.001 vs 0 μg/ml. IC = 13.46 ± 0.66 ***p < 0.001 vs 0 μg/ml. IC = 24.48 ± 2.31 μg/ml. The IC 50 50 50 μg/ml. The IC value of the standard is 42.06 ± 1.35 μg/ml. value of the standard is 90.82 ± 4.75 μg/ml. Page 6 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 Figure 5 H O scavenging assay Singlet oxyg Figure 7 en scavenging assay 2 2 H O scavenging assay. Effects of Spondias pinnata plant 2 2 Singlet oxygen scavenging assay. Effects of Spondias pin- extract and the standard sodium pyruvate on the scavenging nata plant extract and the standard lipoic acid on the scav- of H O . The data represent the percentage H O scaveng- enging of singlet oxygen. The results are mean ± S.D. of six 2 2 2 2 ing. All data are expressed as mean ± S.D. (n = 6). **p < 0.01 parallel measurements. ***p < 0.001 vs 0 μg/ml. IC = 58.07 and ***p < 0.001 vs 0 mg/ml. IC = 44.74 ± 25.61 mg/ml. The 50 ± 5.36 μg/ml. The IC value of the standard is 46.15 ± 1.16 IC value of the standard is 3.24 ± 0.3 mg/ml. 50 μg/ml. decreased. The results [figure 9(a) and figure 9(b)] dem- Reducing power 2+ 3+ 2+ onstrated that formation of the ferrozine-Fe complex is As illustrated in figure 10, Fe was transformed to Fe in inhibited in the presence of the test and reference com- the presence of S. pinnata extract and the reference com- pounds. The IC values (Table 1) of the plant extract and pound BHT to measure the reductive capability. At 0.1 EDTA were 66.54 ± 0.84 μg/ml and 1.27 ± 0.05 μg/ml, mg/ml, the absorbances of the plant extract and BHT were respectively. At 120 μg/ml, the percentage inhibition of 0.32 and 0.02, respectively, while at 0.4 mg/ml, the the plant extract was 51.8% whereas at 45 μg/ml that of absorbances of both extract and BHT were almost the EDTA was 99.5%. same. This result indicates that maximum activity is Peroxynitrite a Figure 6 nion scavenging assay HO Figure 8 Cl scavenging assay Peroxynitrite anion scavenging assay. The peroxynitrite HOCl scavenging assay. Hypochlorous acid scavenging anion scavenging activity of Spondias pinnata plant extract and activities of Spondias pinnata plant extract and the standard the standard gallic acid. Each value represents mean ± S.D. (n ascorbic acid. All data are expressed as mean ± S.D. (n = 6). = 6). ***p < 0.001 vs 0 μg/ml. IC = 716.32 ± 32.25 μg/ml. **p < 0.01 and ***p < 0.001 vs 0 μg/ml. IC = 127.99 ± 6.26 50 50 The IC value of the standard is 876.24 ± 56.96 μg/ml. μg/ml. The IC value of the standard is 235.95 ± 5.75 μg/ml. 50 50 Page 7 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 .+ ABTS is a blue chromophore produced by the reaction between ABTS and potassium persulfate. Addition of the plant extract to this pre-formed radical cation reduced it to ABTS in a concentration-dependent manner. The results were compared with those obtained using trolox and the TEAC value demonstrates that the extract is a potent anti- oxidant. Hydroxyl radicals are the major active oxygen species causing lipid peroxidation and enormous biological dam- 2+ Fe Figure 9 chelation assay 2+ age [31]. They were produced in this study by incubating Fe chelation assay. Effects of (a) Spondias pinnata plant 2+ ferric-EDTA with ascorbic acid and H O at pH 7.4, and extract and (b) standard EDTA on ferrozine-Fe complex 2 2 reacted with 2-deoxy-2-ribose to generate a malondialde- formation. The data are expressed as percentage inhibition of chromogen formation. The results are mean ± S.D. of six hyde (MDA)-like product. This compound forms a pink parallel measurements. ***p < 0.001 vs 0 μg/ml. IC = 66.54 chromogen upon heating with TBA at low pH [32]. When ± 0.84 μg/ml. The IC value of the standard is 1.27 ± 0.05 S. pinnata extract was added to the reaction mixture, it μg/ml. removed the hydroxyl radicals from the sugar and pre- vented the reaction. The IC value indicates that the plant extract is a better hydroxyl radical scavenger than the shown at a lower dose by the extract (0.1 mg/ml) than by standard mannitol. BHT. Superoxide anion is also very harmful to cellular compo- Determination of total phenolic content nents [33]. Robak and Glyglewski [34] reported that flavo- Phenolic compounds may contribute directly to antioxi- noids are effective antioxidants mainly because they dative action. The total phenolic content was 91.47 ± scavenge superoxide anions. As shown in figure 3, the 0.004 mg/ml gallic acid equivalent per 100 mg plant superoxide radical scavenging activities of the plant extract. extract and the reference compound are increased mark- edly with increasing concentrations. The results suggest Determination of total flavonoid content that the plant extract is a more potent scavenger of super- The total flavonoid content of the 70% methanolic extract oxide radical than the standard quercetin. of S. pinnata was 350.5 ± 0.004 mg/ml quercetin equiva- lent per 100 mg plant extract. It is well known that nitric oxide has an important role in various inflammatory processes. Sustained levels of pro- Discussion duction of this radical are directly toxic to tissues and con- In living systems, free radicals are constantly generated tribute to the vascular collapse associated with septic and they can cause extensive damage to tissues and bio- shock, whereas chronic expression of nitric oxide radical molecules leading to various disease conditions, espe- is associated with various carcinomas and inflammatory cially degenerative diseases, and extensive lysis [25]. Many conditions including juvenile diabetes, multiple sclerosis, synthetic drugs protect against oxidative damage but they arthritis and ulcerative colitis [35]. The toxicity of NO have adverse side effects. An alternative solution to the increases greatly when it reacts with superoxide radical, problem is to consume natural antioxidants from food forming the highly reactive peroxynitrite anion (ONOO ) supplements and traditional medicines [26,27]. Recently, [36]. The nitric oxide generated from sodium nitroprus- many natural antioxidants have been isolated from differ- side reacts with oxygen to form nitrite. The extract inhibits ent plant materials [28,29]. The antioxidant and polyphe- nitrite formation by directly competing with oxygen in the nol contents of S. pinnata have also been determined by reaction with nitric oxide. The present study proved that Chuyen et al. [30], using concentrated leaf extract. The the extract studied has more potent nitric oxide scaveng- antioxidant activity has been studied by the inhibition of ing activity than the standard curcumin. ascorbic acid-induced lipid peroxidation and Catechin has been used as the standard for phenolic content, Hydrogen peroxide is a weak oxidizing agent that inacti- whereas in the present study, the antioxidant capacity of vates a few enzymes directly, usually by oxidation of bark extract was measured by an improved ABTS radical essential thiol (-SH) groups. It can cross cell membranes cation decolorization assay and gallic acid was used as the rapidly; once inside the cell, it can probably react with 2+ 2+ standard for phenolic content measurement; both studies Fe and possibly Cu ions to form hydroxyl radicals and showed promising results. Therefore, it is clear that both this may be the origin of many of its toxic effects [37]. the leaf and stem bark extracts of the plant have good anti- From the results, it appeared that the H O scavenging 2 2 oxidant activities as well as high polyphenolic contents. Page 8 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 erate lipid peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can perpetuate the chain reaction [42]. Metal chelating capac- ity is significant since it reduces the concentration of the transition metal that catalyzes lipid peroxidation [43]. According to the results, the plant extract is not as good as the standard EDTA; but the decrease in concentration- dependent color formation in the presence of the extract indicates that it has iron chelating activity. The reducing capacity of a compound may serve as a sig- nificant indicator of its potential antioxidant activity. However, the activities of antioxidants have been attrib- uted to various mechanisms such as prevention of chain initiation, decomposition of peroxides, reducing capacity and radical scavenging [44]. As shown in figure 7, the Reducing power Figure 10 assay reducing power of the plant extract was compared with Reducing power assay. The reductive abilities of Spondias the standard BHT and found to be superior. pinnata extract and the standard BHT. The absorbance (A ) was plotted against concentration of sample. Each value rep- The results indicate that S. pinnata plant extract contains resents mean ± S.D. (n = 6). *** p < 0.001 vs 0 mg/ml. significant amounts of flavonoids and phenolic com- pounds. Both these classes of compounds have good anti- oxidant potential and their effects on human nutrition activity of the plant extract is negligible compared to that and health are considerable. The mechanism of action of of the standard sodium pyruvate. flavonoids is through scavenging or chelation [45]. Phe- nolic compounds are also very important plant constitu- ) is relatively stable compared to Peroxynitrite (ONOO ents because their hydroxyl groups confer scavenging other free radicals but once protonated it forms the highly ability [44]. reactive peroxynitrous acid (ONOOH) [38]. Generation of excess ONOO leads to oxidative damage and tissue Conclusion injury [39]. Peroxynitrite bleaches Evans Blue by oxidizing On the basis of the results obtained in the present study, it. According to the present results, the plant extract inhib- it is concluded that a 70% methanolic extract of Spondias its Evans Blue bleaching by scavenging peroxynitrite and pinnata bark, which contains large amounts of flavonoids its activity is greater than that of the reference gallic acid. and phenolic compounds, exhibits high antioxidant and free radical scavenging activities. It also chelates iron and Singlet oxygen is generated in the skin by ultraviolet radi- has reducing power. These in vitro assays indicate that this ation. It is a high energy form of oxygen and is known as plant extract is a significant source of natural antioxidant, one of the ROS. Singlet oxygen induces hyperoxidation which might be helpful in preventing the progress of var- and oxygen cytotxicity and decreases antioxidative activity ious oxidative stresses. However, the components respon- [40]. The present study indicates that the S. pinnata extract sible for the antioxidative activity are currently unclear. has good scavenging activity for singlet oxygen but is not Therefore, further investigation is needed to isolate and as efficient as the standard lipoic acid. identify the antioxidant compounds present in the plant extract. Furthermore, the in vivo antioxidant activity of this At sites of inflammation, the oxidation of Cl ions by the extract needs to be assessed prior to clinical use. neutrophil enzyme myeloperoxidase results in the pro- duction of another harmful ROS, hypochlorous acid [41]. Competing interests HOCl has the ability to inactivate the antioxidant enzyme The authors declare that they have no competing interests. catalase through breakdown of the heme prosthetic group. Catalase inactivation is inhibited in the presence of Authors' contributions the extract, signifying its HOCl scavenging activity. The BH: Performed the study. results suggest that S. pinnata is a more efficient scavenger than the standard ascorbic acid. SB: Design, analysis and acquisition of data. Iron can stimulate lipid peroxidation by the Fenton reac- NM: Supervised the study design and drafted the manu- 2+ 3+ - . tion (H O + Fe = Fe + OH + OH ) and can also accel- script. 2 2 Page 9 of 10 (page number not for citation purposes) BMC Complementary and Alternative Medicine 2008, 8:63 http://www.biomedcentral.com/1472-6882/8/63 23. Singleton VL, Rossi JA: Colorimetry of total phenolics with Acknowledgements phosphomolybdic-phosphotungstic acid reagents. Am J Enol Mr. B. Hazra is grateful to the Indian Council of Medical Research, Govern- Vitic 1965, 16:144-158. ment of India for providing fellowship. The authors would like to thank Mr. 24. 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BMC Complementary and Alternative MedicineSpringer Journals

Published: Dec 9, 2008

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