Food Sci. Biotechnol. 21(5): 1421-1431 (2012) DOI 10.1007/s10068-012-0187-x RESEARCH ARTICLE Evaluation of Antioxidant and Pharmacological Properties of Psychotria nilgiriensis Deb & Gang Murugaiyan Iniyavan, Devadoss Sangeetha, Shanmugam Saravanan, and Thangaraj Parimelazhagan Received: 4 April 2012 / Revised: 8 May 2012 / Accepted: 15 May 2012 / Published Online: 31 October 2012 © KoSFoST and Springer 2012 Abstract The purpose of the present study was to investigate the antioxidant and pharmacological properties of fruit, stem, and leaf extract of Psychotria nilgiriensis. Acetone extract of P. nilgiriensis fruit was found to have highest total phenolics (505.74 mg GAE/g extract), tannin (460.78 mg GAE/g extract), and flavonoid (67.78 mg RE/ g extract) content. In vitro antioxidant studies revealed that the acetone extract of fruit posses significant antioxidant activity in DPPH radical scavenging, ABTS•+, and ferric reducing antioxidant power (FRAP) assays. In vivo studies revealed that P. nilgiriensis fruit (400 mg/kg) showed good analgesic activity in hot plate method (72%), acetic acid induced writhing test (53%) and also showed significant in carrageenan induced inflammation (73.54%). GC/MS analysis of fruit showed the presence of sesquiterpenes. The study highlights the significant medicinal value of the plant. Keywords: antioxidant, analgesic, anti-inflammatory, Psychotria nilgiriensis Introduction During the last few decades there has been an increasing interest in the study of medicinal plants and their traditional use in different parts of the world. The chemical novelty and diversity associated with medicinal plant products are higher than that of any other source. Hence, it is accepted that plants are useful in their crude or advanced form of Murugaiyan Iniyavan, Devadoss Sangeetha, Shanmugam Saravanan, Thangaraj Parimelazhagan (
) Bioprospecting Laboratory, Department of Botany, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India Tel: +919750006025; Fax: E-mail: drparimel@gmail.com drugs. Medicinal plants are known to contain innumerable biologically active compounds. A free radical is any atom (e.g., oxygen, nitrogen) with at least 1 unpaired electron in the outermost shell, and is capable of independent existence. The interest in reactive oxygen species (ROS) on recent years in biology and medicine is evident because of their strong relationship with many common and life threatening human diseases. ROS are implicated in the development of many chronic disorders, such as cancer, diabetes mellitus, atherosclerosis, nephritis, rheumatism, cardiovascular diseases, gastrointestinal tract disorders, and inflammatory injury. Antioxidants are vital substances that play an important role in disease prevention and protecting the body from damage caused from free radical-induced oxidative stress owing to their abilities to remove free radicals. Earlier studies reported that plants have potent antioxidant and represent an important source of natural antioxidants. The identification of new effective antioxidants is a topic of interest; several plant extracts or secondary metabolites have shown potential to protect against oxidant-induced damage (1). Inflammation is the tissue reaction to infection, irritation, or foreign substance. It is a part of the host defense mechanisms that are known to be involved in the inflammatory reactions such as release of histamine, bradykinin, and prostaglandins. The development of nonsteroids in overcoming human sufferings such as rheumatoid arthritis has evoked much interest in the extensive search for new drugs with this property (2). Carrageenan induced paw edema is widely used for determining the acute phase of inflammation. The genus Psychotria of Rubiaceae family contains medicinally valuable indole alkaloids namely psychotridine and brachycerine. These alkaloids are widely used to cure problems in central nerves system of human. Ethnobotanical and chemotaxonomical studies on species of Psychotria 1422 resulted in the discovery of a set of novel bioactive monoterpinoid indole alkaloids (MIAs), some of them with clear pharmaceutical potential. Few reports have been published on antioxidant activities of the crude extracts or compounds isolated from Psychotria. Tender fruit of Psychotria nilgiriensis (commonly called as odai kaapi patchilai in Tamil) is consumed along with honey for its action against rheumatism. It is being used by Kanikkar tribes of Kalakad, Mundanthurai at Tirunelveli district, and Irula tribes of Thottabeta at Nilgiris district, Tamilnadu, India (3). Even though the plant has been reported for the medicinal property and uses, very limited reports are available regarding the phytochemical and pharmacological aspects. Therefore, this study was designed to analyze radical scavenging activity and pharmacological property of different solvent extraction from the P. nilgiriensis fruit, stem, and leaves. Materials and Method Collection of plant materials The fresh plant parts of Psychotria nilgiriensis were collected from Thottabetta, Nilgiris, Tamil Nadu during the month of February-April, 2011. The collected plant material was identified and its authenticity was confirmed by comparing the voucher specimen at the herbarium of Botanical survey of India, Southern circle Coimbatore, Tamil Nadu. Freshly collected plant material was cleaned to remove adhering dust and then dried under shade. Fruit, stem, and leaves of the samples were separated and dried in shade for 16-25 days. The dried plant samples were powdered and used for further studies. Chemicals DPPH, ABTS, Trolox, linoleic acid, FolinCiocalteu reagent, ammonium thiocyanate, aluminum chloride, thiobarbituric acid (TBA), TPTZ, polyvinyl polypyrrolidone (PVPP), ferrous ammonium sulfate, EDTA disodium salt, metaphosphoric acid, 2,6-dichoroindophenols (DIP). Chemicals are purchased from Sigma-Aldrich (St. Louis, MO, USA), Merck and Himedia (Mumbai, India). All other reagents used were of analytical grade. Successive solvent extraction The air dried powdered plant materials of P. nilgiriensis were extracted separately in Soxhlet extractor successively with petroleum ether, chloroform, acetone, and methanol. Each time before extracting with the next solvent, the material was dried in hot air oven below 40oC. Finally, the material was macerated using hot water with occasional stirring for 48 h and the water extract was filtered. The different solvent extracts were concentrated by rotary vacuum evaporator (RE300; Yamato, Tokyo, Japan) and then air dried. The Iniyavan et al. dried extract obtained with each solvent was weighed. The percentage of yield was expressed in terms of air dried weight of plant material. The extracts thus obtained were used directly for the estimation of total phenolics and also for the assessment of antioxidant potential through various biochemical assays. The extracts were freeze dried and stored in desiccators until further analysis. Determination of total phenolic and tannin contents The total phenolics of the plant extracts were determined by Folin-Ciocalteu method. Using the same extract the tannins were estimated after treatment with PVPP. The amount of total phenolics and tannins were calculated in gallic acid equivalents (GAE) as described by Siddhuraju and Becker (4). Estimation of flavonoids Flavonoid content of a plant material was measured by the aluminum chloride colorimetric assay (5). Samples were analyzed in triplicates and the amounts of flavonoids were expressed in rutin equivalents (RE). Determination of ascorbic acid (vitamin C) The ascorbic acid was determined using the DIP method with simple modification (6). The concentrations of the ascorbic acid in the sample were determined through comparison with the absorbance of standard ascorbic acid at different concentration. In vitro antioxidant assays DPPH radical scavenging activity: DPPH radical scavenging activity was assessed according to modified method of Blios (7) was used to determine the free radical scavenging activities of plant extract. In this method, a commercially available and stable free radical DPPH soluble in methanol was used. Sample extracts at various concentrations were taken and the volume was adjusted to 100 µL with methanol. Four mL of 0.1 mM freshly prepared methanolic DPPH solution was added. The reaction mixture was vortexed well and kept at room temperature for 20 min. The resulting solutions were read at 517 nm using a UV spectrophotometer (UV 1800; Shimadzu, Kyoto, Japan). Methanol was used as the blank. Negative control was without any inhibitor or extract. Commercial antioxidants butylhydroxy anisole (BHA), butylhydroxy toluene (BHT), quercetin, and rutin dissolved in methanol were used as the positive control under the same assay conditions in all tests. The free radical scavenging activities of the samples were expressed as IC50 value (i.e., the concentration of the sample required to inhibit 50% of DPPH concentration). ABTS•+ scavenging activity: The total antioxidant activity of the samples was measured by ABTS radical cation decolorization assay according to the method of Re et al. Phytochemical and Pharmacological Properties of P. nilgiriensis (8). Seven mM ABTS aqueous solution was added with 2.4 mM potassium persulfate and this mixture was incubated at dark for 12-16 h to produce ABTS•+. Prior to assay, this solution was diluted in ethanol (about 1:89 v/v) and equilibrated at 25oC to give an absorbance of 0.700 ±0.02 at 734 nm. About 1 mL of diluted ABTS solution was added to about 30 µL sample solution and 10 µL of Trolox (final concentration 0-15 µmol) in ethanol. One mL of diluted ABTS solution was mixed with 30 µL of ethanol served as the negative control. All the test tubes were vortexed well and incubated exactly for 30 min at room temperature. After incubation the absorbance of samples and standards (BHT and rutin) were measured at 734 nm against the ethanol blank. The results were expressed as the concentration of Trolox having equivalent antioxidant activity expressed as µmol/g sample extracts Assay of superoxide radical scavenging activity: The assay was based on the capacity of various extracts to inhibit formazan formation by scavenging the superoxide radicals generated in riboflavin-light-NBT (nitroblue tetrazolium) system (9). Each 3 mL reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 20 µg riboflavin, 12 mM EDTA, 0.1 mg NBT, and 100 µL sample solution. Reaction was started by illuminating the reaction mixture with sample extract for 90 s. Immediately after illumination the absorbance was measured at 590 nm. The entire reaction assembly was enclosed in a box lined with aluminum foil. Identical tubes with reaction mixture kept in the dark served as blanks. The percentage inhibition of superoxide anion generation was calculated. % Inhibition =[(control OD–sample OD)/control OD]×100 Determination of metal chelating activity: The chelation of ferrous ions was estimated by method of Dinis et al. (10). Briefly, 50 µL of 2 mM FeCl2 was added to 1 mL of different concentrations of the extract (50, 100, 150, 200, and 250 µg/µL). A 0.2 mL of 5 mM ferrozine was added in the test tubes to initiate the reaction. The mixture was vigorously shaken and left to stand at room temperature. After 10 min, the absorbance was read spectrophotometrically at 562 nm. One mL of deionised water, instead of sample, was used as a control. All the reagents without addition of sample extract were used as negative control. EDTA (20 mg/mL) was used as standard. The results were expressed as mg EDTA equivalent/g extract. Ferrous reducing antioxidant power assay (FRAP): The antioxidant capacities of extracts were estimated according to the procedure described by Pulido et al. (11). The FRAP reagent contained 2.5 mL of 20 µmol/L TPTZ solution in 40 µmol/L HCl, 2.5 mL of 20 µmol/L FeCl3·6H2O, and 25 mL of 0.3 mol/L acetate buffer (pH 3.6). Freshly prepared FRAP reagent was incubated at 37oC. To the 90 µL of 1423 distilled water and 30 µL of test sample or methanol (for the reagent blank), 900 µL of FRAP reagent was added. The prepared test samples and reagent blank were again incubated in water bath at 37oC for 30 min. At the end of incubation, readings of colored product (ferrous tripyridyltriazine complex) were taken immediately at 593 nm. Lipid peroxidation inhibitory activity: The lipid peroxidation inhibitory activity of the extracts was determined according to the method of Duh et al. (12). Briefly, egg lecithin was homogenized (1%, w/v) in 10 mM phosphate buffer (pH 7.4) and blend well with blender machine to ensure proper liposome formation. Test samples of different concentrations (0.1-0.9 mg/mL) were added to liposome mixture (1 mL); the control was without test sample. Lipid peroxidation was induced by adding FeCl2 (10 µL, 400 mM) and Lascorbic acid (10 µL, 200 mM). After incubation for 1 h at 37oC the reaction was stopped by adding HCl (2 mL, 0.25 N) containing trichloroacetic acid (150 mg/mL) and TBA (3.75 mg/mL). The reaction mixture was subsequently boiled for 15 min, cooled, centrifuged at 1,000×g for 15 min and the absorbance of the supernatant was measured at 532 nm and compared with that of BHA and ascorbic acid (10 mg/mL). Percentage of radical scavenging activity was calculated using the following formula: % Inhibition =[(Abscontrol –(Abssample –Abssample blank/Abscontrol]×100 In vivo studies Selection of animal’s: Swiss albino mice weighing 20-30 g and Wistar albino rats of 200-250 g were used for the pharmacological studies. They were housed in a clean polypropylene cage and maintained under standard laboratory conditions (temperature 25±3ºC with dark/light cycle 12/ 12 h; 35-60 humidity). They were fed with standard pellet diet (VRK Nutritional solutions, Sangli, Maharastra) and water ad libitum. The studies were carried out at Nandha College of Pharmacy and Research Institute, Perundurai, Tamil Nadu, India. The experimental protocol was subjected to the scrutiny of the Institutional Animal Ethics Committee, and was cleared by same before beginning the experiment (688/02/C/CPCSEA). Acute toxicity: Acute oral toxicity study was performed according to acute toxic class method. Swiss albino mice (n=6) of either sex selected by random sampling technique were used for acute toxicity study. The animals were kept fasting for overnight providing only water, after which extracts were administered by gavages at different doses from 50 to 2,000 mg/kg BW, which were increased progressively so that each dose was 50% higher than the preceding (13). The treated animals were free to access water ad libitum. Observations were made at 2, 4, 8, up to 48 h for treatment related behavioral changes like apathy, reduced locomotor behavior. If mortality was observed in 1424 Iniyavan et al. 4 out of 6 animals, then the dose administered was assigned as toxic dose. If mortality was observed in 2 animals, then the same dose was repeated again to confirm the toxic dose. Analgesic activity Hot plate method: The hotplate method was used to measure response latencies according to the method described by Eddy and Leimbach (14) with minor modification. Animals of either sex were randomly selected and divided into 6 groups consisting of 5 mice in each group. Each group received a particular treatment i.e., control (untreated), standard (diclofenac sodium 10 mg/kg), and the acetone extract of fruit and stem (200 and 400 mg/kg). The animals were positioned on Eddy’s hot plate kept at a temperature of 55±0.5oC. The time taken by the animals to lick the fore or hind paw or jump out of the place was taken as the reaction time. The latency was recorded at the time of 0 (just before any treatment) and 30, 60, 120, and 240 min after oral administration of samples and intraperitoneal administration of standard. A latency period of 15 s was observed to avoid damage to the paw. The percentage thermal pain stimulus relief or protection was determined by applying the formula: % Protection against thermal pain stimulus =[(test mean–control mean)/test mean]×100 Acetic acid-induced writhing in mice: The test was performed as described by Koster et al. (15). Swiss albino mice of either sex were divided into 6 groups of 5 mice each. Group I served as control (acetic acid 1.0 mL/kg i.p.) while group II (positive control) was administrated with standard drug- aspirin at a dose of 150 mg/kg. Based on the acute toxicity studies, the doses for acetone extract of P. nilgiriensis fruit and stem were decided to be 200 and 400 mg/kg and was given to the Group III, IV, V, and VI respectively. Thirty min later all the groups were treated with 0.75% acetic acid at the dose of 0.1 mL/10 g BW. Mice were placed in individual cages. The numbers of abdominal contractions were counted 5 min after acetic acid injection for a period of 10 min. Percentage inhibition of writhing was obtained using the formula: Inhibition (%) mean no. of writhings – mean no. of writhings     (control) (test) = ----------------------------------------------------------------------------------------------------------------------- ×100 mean no. of writhings (control) Anti-inflammatory study Carrageenan-induced hind paw edema: The antiinflammatory property of acetone extract of P. nilgiriensis fruit and stem were studied against carrageenan-induced acute paw edema as described by Ghosh (16). The animals were divided into 6 groups of 6 animals each and were fasted for a period of 24 h prior to the study. Group 1 as control; Group 2 received indomethacin 10 mg/kg suspended in 1% sodium carboxymethyl cellulose (SCC) as standard. Group 3 and 4 were treated with 200 and 400 mg/kg of acetone extracts of P. nilgiriensis fruit, Group 5 and 6 were treated with 200 and 400 mg/kg of acetone extracts of P. nilgiriensis stem suspended in 1% SCC. Oedema was induced by injecting 0.1 mL of 1% solution of carrageenan in saline into the subplantar region of the right hind paw of the rats. The vehicle, extracts and the standard drugs were administered 60 min prior to the injection of the phlogestic agent. The volumes of edema of the injected and the contralateral paws were measured at 1, 2, 3, 4, and 5 h after the induction of inflammation. The inflammation was measured by using an electronic vernier caliper (CD-6 CSX; Digimatic caliper, Mitutoyo, Japan) and calculates the percentage of paw oedema inhibition. GC/MS analysis The GC/MS analysis of the P. nilgiriensis acetone extract was carried out at the South India Textile Research Association (SITRA), Coimbatore. The GC was carried out by using Thermo-GC Trace Ultra Version 5.0 equipment with running time of 38.94 min and the MS was carried out by using Thermo MS-DSQ II equipment (Thermo Fisher Scientific Inc., Waltham, MA, USA). Auto sampler and GC interfaced to a MS instrument employing the following conditions: column TR 5-MS capillary standard non-polar column (30 M, i.d.: 0.25 mm, FILM: 0.25 µm) composed of 5% phenyl polysilphenyle-siloxane; helium (99.999%) was used as carrier gas at a constant flow of 1 mL/min; and an injection volume of 0.5 EI was employed (split ratio of 10:1) injector temperature 250oC; ion-source temperature 280oC. The MS of the unknown component was compared with the spectrum of the known components stored in the National Institute of Standard Technology (NIST) library. The name, molecular weight and structure of the components of the test materials were ascertained. Statistical analyses All the experiments were done in triplicates and the results were expressed as mean±standard deviation (SD). The data were statistically analyzed using one way analysis of variance (ANOVA) followed by Duncan’s test for antioxidant studies and by Dunnet’s t-test for analgesic and anti-inflammatory studies. Mean values were considered statistically significant when p<0.05, <0.01, and <0.001 Results and Discussion Extract yield percentage The acetone extract of fruit showed higher amount of yield 16.30%. The yield of leaf Phytochemical and Pharmacological Properties of P. nilgiriensis (13.6%) and stem (10.4%) were high in methanol extract. On the other hand, petroleum ether extract of stem (1.05%) showed lower percentage compare to the other solvents. Total phenolics, tannins, flavonoids, and ascorbic acid Total phenolics, tannins, flavonoids, and ascorbic acid content of various solvent extracts of P. nilgiriensis plant parts were showed in Table 1. The highest levels of total phenolic (505.74 mg GAE/g extract) and tannin (460.78 mg GAE/g extract) contents were found in acetone extract of P. nilgiriensis fruit when compared to other extracts. The petroleum ether extract of fruit showed very poor content of phenols (25.91 mg GAE/g extract) and tannin (1.54 mg GAE/g extract). In our study, phenolics and tannin content of P. nilgiriensis fruit was showed a significant positive correlation with the antioxidant activity of the plant extracts. The enrichment of phenolic compounds within plant extracts is correlated with their enhanced antioxidant activity. A strong relationship between total phenolic content and antioxidant activity in fruits, vegetables, and grain products has been reported by Dorman et al. (17). Recently, it has been reported that the high molecular weight phenolics such as tannins have more ability to reduce or scavenge free radicals. Tannins interfere with iron absorption through a complex formation with iron when it is in the gastrointestinal lumen which decreases the bioavailability of iron. The role of flavonoids as antioxidants has been well established and there have been numerous reports on structure-activity relationship in the last decade. The highest levels of avonoid contents were found in acetone extract of P. nilgiriensis fruit (67.78±5.03 mg RE/g extract), while avonoid contents of petroleum ether extract 1425 of fruit were very low (1.52±0.09 mg RE/g extract). Many avonoid are found to be strong antioxidants capable of effectively scavenging the ROS because of their phenolic hydroxyl groups (18). The results revealed that the acetone extract of fruit contain more avonoid than other parts of the same plant. It is well known that vitamin C has been considered by some scientists as a ‘universal panacea’ due to its broad biochemical and pharmacological functions. Vitamin C acting as a highly effective antioxidant it can protect indispensable molecules in the body from damage by free radicals and ROS. More recently, vitamin C as potent tyrosinase inhibitor has been studied in detail by Zeng et al. (19). The results suggested that the acetone extract of P. nilgiriensis fruit have maximum amount of vitamin C content (4.15%). Moreover, fruits contain vitamin C which expresses antioxidant activity. In vitro antioxidant assays DPPH assay: In the DPPH assay, the antioxidants were able to reduce the stable DPPH radical to the yellow colored diphenyl-picrylhydrazine. The free radical-scavenging activities of different parts of P. nilgiriensis samples along with standards such as α-tocopherol, rutin, BHA, and BHT were determined by the DPPH radical scavenging assay. The highest free radical scavenging activity was exerted by acetone extract of fruit (20.0 µg/mL). Chloroform extract of leaf was found to be least radical scavenging activity (847.50 µg/mL) (Fig. 1). The relatively stable DPPH radical has been widely used to examine the ability of compounds to act as free radical scavengers or hydrogen donors and thus to evaluate the antioxidant activity. Acetone extract of the P. nilgiriensis of fruit (20.0 µg/mL), Table 1. Total phenolics, tannin, flavonoids, and vitamin C content of P. nilgiriensis 1) Tannins (mg GAE/g extract) Flavonoids (mg RE/g extract) Vitamin C (g/100 g) 25.91±1.351) 77.31±5.88 0505.74±82.38* 0225.91±12.34* 48.04±6.12 1.54±0.64 30.81±4.250 460.78±80.70* 182.63±15.00* 5.18±6.73 1.52±0.09 2.22±0.38 67.78±5.03* 5.33±0.12 3.24±0.03 1.19±0.06 1.33±0.02 04.15±0.03* 1.73±0.04 02.64±0.10* Stem Petroleum ether Chloroform Acetone Methanol Hot water 50.70±5.84 97.62±2.34 0216.11±16.57* 178.71±8.66* 62.18±4.39 15.83±4.210 21.57±6.790 150.14±15.86* 129.27±8.02*0 20.31±6.010 20.24±1.99* 6.25±0.14 21.05±1.96* 5.50±0.12 3.00±0.17 1.27±0.01 1.32±0.01 1.33±0.01 1.81±0.01 02.18±0.01* Leaf Petroleum ether Chloroform Acetone Methanol Hot water 57.84±8.92 71.71±3.10 0469.33±50.92* 66.11±5.13 56.16±2.31 26.75±7.950 20.17±8.280 408.54±51.81* 8.82±5.93 14.99±3.180 10.50±0.390 10.21±0.220 7.71±0.28 11.09±0.530 2.83±0.410 1.32±0.01 02.09±0.04* 02.89±0.01* 02.05±0.00* 1.94±0.59 Plant sample Solvent Fruit Petroleum ether Chloroform Acetone Methanol Hot water Total phenolics (mg GAE/g extract) 1) Values are mean of triplicate determination (n=3)±SD; Statistically significant at *p<0.05 1426 Iniyavan et al. Fig. 1. DPPH radical scavenging activities of P. nilgiriensis. Values are mean of triplicate determination (n=3)±SD; Statistically significant at *p<0.05 stem (30.90 µg/mL), and leaf (103.30 µg/mL) showed spectacular value in DPPH assay than the other solvent extracts. The acetone extract of plant parts were possibly contained some substrates, which were electron donors and could react with free radicals to convert them to more stable products and terminate the radical chain reaction. IC50 value of fruit extract (20.0 µg/mL) was unconditionally significant than the standards α-tocopherol, rutin, and BHT. Phenolic content profile of this plant may be endorsed to scavenge the free radicals. The antioxidant potential of the plant to scavenge free radicals was ensured in the DPPH assay. ABTS radical scavenging activity: The Trolox equivalents antioxidant capacity (TEAC) was measured using the improved ABTS•+ radical decolorization assay; one of the most frequently employed methods for antioxidant capacity, which measures the ability of a compound to scavenge ABTS•+ radical. Hagerman et al. (20) have reported that the high molecular weight phenolics (tannins) have more ability to quench free radicals (ABTS•+) and that effectiveness depends on the molecular weight, the number of aromatic rings and nature of hydroxyl groups substitution than the specific functional groups. ABTS•+ assay is an excellent tool to determine the antioxidant activity of hydrogendonating antioxidants (scavenging aqueous phase radicals) and of chain breaking antioxidants (scavenging lipid peroxyl radicals). Acetone extract of P. nilgiriensis fruit showed significant result (41,343.51 µmol TE/g extract) against ABTS free radical. Root (35,437.30 µmol TE/g extract), stem (7,451.96 µmol TE/g extract), and leaf (7,296.71 µmol TE/g extract) extracts also confirm moderate result in radical scavenging and ensure their place in free radical scavenging (Table 2). Petroleum ether extract of leaves (189.00 µmol TE/g extract) failed to produce considerable result against ABTS•+. Low polarity of the solvent may be reason for the poor result in petroleum either extracts. Higher concentrations of extract were more effective in quenching free radicals in the system (21). Earlier reports of Psychotria species showed tremendous result in ABTS assay the values obtained were meagerly equivalent to antioxidant standard Trolox. Superoxide radical scavenging activity: Superoxide anion plays an important role in formation of ROS. Although superoxide is a relatively weak oxidant, it decomposes to form stronger ROS, such as singlet oxygen and hydroxyl radicals, which initiate peroxidation of lipids. Acetone extracts of P. nilgiriensis effectively scavenged superoxide in a concentration-dependent manner. The acetone extract of leaf (54.90%), fruit (32.97%), and stem (16.91%) has good superoxide radical scavenging activity (Table 2). Further, superoxides are also known to indirectly initiate lipid peroxidation as a result of H2O2 formation, creating precursors of hydroxyl radicals. These results clearly suggest that the antioxidant activity of P. nilgiriensis is also related to its ability to scavenge superoxides. Metal chelating: Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the result that the red color of the complexes decreases. The Fe2+ chelating activity of extracts are shown in Table 2. Ferrozine can quantitatively form complex with Fe2+. In the presence of other chelating agents, the complex formation is disrupted which results in the decreased intensity of the red color of the complex. Measurement of the rate of color reduction therefore allows estimation of the chelating activity of the coexisting chelator. In this assay, both P. nilgiriensis extract and EDTA interfered with the formation of ferrous and ferrozine complex suggesting that it has chelating activity Phytochemical and Pharmacological Properties of P. nilgiriensis 1427 Table 2. ABTS, metal chelating, FRAP, superoxide radical scavenging, and lipid peroxidation activities of P. nilgiriensis Metal chelating FRAP activity (mg EDTA (mmol Fe (II)/mg equivalents/g extract) extract) Plant sample Solvent ABTS (µM Trolox equivalent/g extract) Fruit Petroleum ether Chloroform Acetone Methanol Hot water 173.47±14.221) 712.12±26.04 41,343.51±58.46*0, 4,775.60±88.27*, 1,427.62±74.92*, 3.95±2.45 3.60±3.25 65.70±1.698 29.43±1.750 50.46±2.15* 85.56±1.92 129.21±11.76 4,713.33±2.89*0, 1,411.48±1.28*0, 516.35±13.04 4.52±1.15 8.48±0.79 36.94±2.47* 23.64±2.42* 17.32±1.530 7.87±2.97 35.39±3.420 060.67±11.24* 31.65±4.51* 12.36±2.970 Stem Petroleum ether Chloroform Acetone Methanol Hot water 139.05±4.680 242.32±9.570 7,451.96±65.09*, 1,287.56±20.46*, 413.10±18.22 4.10±1.75 9.98±0.75 66.67±0.92* 33.49±2.160 35.76±1.440 101.75±2.620 161.90±4.760 1,490.56±6.31*0, 859.26±3.39* 316.98±0.990 6.27±2.58 6.33±1.96 16.91±4.40* 32.97±3.56* 14.64±2.830 21.35±13.98 25.28±7.040 052.25±10.30* 30.34±7.04* 14.04±4.240 Leaf Petroleum ether Chloroform Acetone Methanol Hot water 189.00±15.47 255.82±3.090 7,296.71±23.38*, 1,277.43±29.66*, 404.32±15.33 4.76±2.32 6.08±1.06 51.32±0.09* 30.95±3.200 45.34±0.84* 111.75±0.270 167.14±2.470 1,314.44±0.96*0, 757.04±5.48* 270.48±0.480 3.35±1.46 8.80±3.03 54.90±3.78* 38.05±3.15* 14.90±2.040 14.79±0.320 17.42±3.930 69.66±3.51* 36.52±5.87* 10.30±1.970 1) Superoxide radical Lipid peroxidation scavenging (%) (%) activity activity 1) Values are mean of triplicate determination (n = 3)±SD; Statistically significant at *p<0.05 and captures ferrous ion before ferrozine. Especially the acetone extracts of stem (66.67 mg EDTA/g extract), fruit (65.70 mg EDTA/g extract), and leaf (51.32 mg EDTA/g extract) showing the higher values of ion chelating capacity. At the same time petroleum ether extract of fruit was had very poor metal chelating activity (3.60 mg EDTA/g extract) when compared to other parts of the plant. While the other extracts showed the metal chelating activity ranging from 50.46-3.60 mg EDTA/g. Metal chelating capacity was significant since the extract reduced the concentration of the catalyzing transition metal in lipid peroxidation. It was reported that chelating agents, which form δ-bonds with a metal, are effective as secondary antioxidants because they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion (22). Results reveal that P. nilgiriensis plant extracts has an effective capacity for iron binding, suggesting that its act as antioxidant. FRAP assay: The FRAP assay measures the antioxidant effect of any substance in the reaction medium as reducing ability. FRAP assay was used by several authors for the assessment of antioxidant activity of various samples. Halvorsen et al. (23) suggested most of the secondary metabolites are redox-active compounds that will be picked up by the FRAP assay. Antioxidative activity has been proposed to be related to reducing power. Therefore, the antioxidant potential of P. nilgiriensis fruit, stem, and leaves were estimated for their ability to reduce TPTZ-Fe (III) complex to TPTZ-Fe (II). The ferric reducing ability of the acetone extracts of P. nilgiriensis revealed excellent FRAP activity. The acetone extract of fruit resulted greater value (4,713.33 µmol Fe (II)/mg extract) of ferric reducing power and the petroleum ether extract of fruit resulted least value (85.56 µmol Fe (II)/mg extract) (Table 2). Ferric reducing antioxidant power of the plant showed greater variability according to the part used and solvent. The results on reducing powers demonstrate the electron donor properties of P. nilgiriensis extracts thereby neutralizing free radicals by forming stable products. Lipid peroxidation: Lipid peroxidation was an oxidative deterioration process of polyunsaturated fatty acids which is induced by radical. One of the degradation products of lipid peroxidation is malondialdehyde which causes cell damage can form a pink color chromogen with TBA acid. The antioxidant compounds present in the extract scavenged the hydroxyl radicals generated in the Fenton reaction in the egg yolk. Among different extracts P. nilgiriensis the acetone extract of leaf (69.66%), fruit (60.67%), and stem (52.25%) showed maximum activity (Table 2). The acetone extract of leaf showed significant activity compared to other extracts and positive control (ascorbic acid and BHA). High lipid peroxidation inhibitions showed by acetone extract could be related to the presence of phenolic compound, which has been shown to be correlated to the antioxidant activity of natural plant product (24). This inhibition of lipid peroxidation may either be due to chelation of Fe ion or by scavenging of the free radicals. In vivo study Acute toxicity: The acetone extract of fruit and stem of P. nilgiriensis was evaluated for its acute toxicity in rats. The extract did not alter the general behavior and failed to produce any mortality even at highest dose of 2,000 mg/kg. 1428 Iniyavan et al. Table 3. Analgesic activity of P. nilgiriensis fruit and stem using hot plate method Reaction time (s) Group Control (water) Pentazocine Acetone extract of fruit Acetone extract of stem Dose (mg/kg) 1 mL 10 200 400 200 400 1) Inhibition (%) Time after drug administration (min) Initial 1) 1.25±0.25 1.50±0.29 1.25±0.25 01.5±0.29 1.75±0.25 02.0±0.41 30 60 2.3±0.25 04.0±0.41* 3.25±0.25* 03.0±0.41* 3.75±0.25* 03.5±0.29* 2.3±0.25 006.3±0.63** 05.0±0.71* 5.5±0.5* 5.75±0.48* 04.75±0.25** 120 240 2.5±0.2900 2.00±0.4100 07.3±0.48*** 8.00±0.41*** 6.5±0.96** 6.75±0.25** 07.0±0.71*** 7.25±1.25*** 5.75±0.25**0 5.5±1.19** 6.75±0.25*** 006.5±0.65*** NA 75.248 70.83 72.02 51.79 69.11 1) Values are expressed as mean±SEM (n=6); Significantly different at *p<0.05, **p<0.01, ***p<0.001 when compared to control In acute toxicity study, the extract was found to be safe at 2,000 mg/kg even after 48 h, and revealed that this plant might be well thought-out as a wide harmless one. Analgesic activity/Hot plate method: The hot plate model has been found to be suitable for the evaluation of centrally but not of peripherally acting analgesics. This model measures a complex response to a non-inflammatory and acute nociceptive input. In this analgesic testing model, pentazocine significantly prolonged the reaction time of animals with relatively extended duration of stimulation, confirming centrally mediated activity. Acetone extract of P. nilgiriensis fruit and stem showed significant analgesic activity at 200 and 400 mg/kg (Table 3). Analgesic activity of the latter dose was often compared with the standard drug Pentazocine. At the dose level 400 mg/kg and 240 min reaction time fruit extract showed higher of analgesic activity 7.25±1.25 (72%), than the stem extract 6.5±0.65 (69%). The present ndings of the hot plate test indicate significant analgesic effect of acetone extract of P. nilgiriensis fruit and stem through central mechanism. Several Psychotria species are used medicinally for pain related purpose. Amador et al. (25) reported that leaves and flowers of Psychotria colorata were showed very significant level of analgesic activity in hot plate method. Many of the earlier studies on Psychotria species proved their analgesic ability. Acetic acid-induced writhing test: Acetic acid-induced writhing responses in rats were used to examine the analgesic effect. This method is not only simple and reliable but also affords rapid evaluation of peripheral type of analgesic action. In this test, the animals react with characteristic stretching behavior, which is called writhing. The oral administration of acetone extract of P. nilgiriensis stem and fruit depicted a dose dependent analgesic activity and the values±SEM for the extract are shown in Table 4. Injection of acetic acid into the control mice resulted in 78.0±1.08 writhes. Pretreatment with acetone extract of P. nilgiriensis fruit and stem at doses of 200 and 400 mg/kg reduced the number of writhes to 21.00±0.63 (64.20% inhibition) and 33.75±2.16 (53% inhibition), respectively. Interestingly, the extract dose of 400 mg/kg registered Table 4. Analgesic activity of P. nilgiriensis fruit and stem using acetic acid-induced method Group Control (water) Aspirin Acetone extract of fruit Acetone extract of stem Dose (mg/kg) Number of writhes Inhibition (%) 1 mL 150 200 400 200 400 78.0±1.081) 36.25±2.06*0 45.25±6.5500 32.25±6.70*0 0.48±6.650 033.75±2.16*** NA 53 35 54 32 53 1) Values are expressed as mean±SEM (n=6); Significantly different at *p<0.05, ***p<0.001 when compared to control higher levels of analgesic activity than the standard drug aspirin (29.00±1.36 writhes; 50.57% inhibition) at a dose of 130 mg/kg. It was also observed that the onset of writhing was delayed and duration of writhing was shortened with extract pretreatment. It was found that the acetone extracts of P. nilgiriensis fruit and stem significantly (p<0.05) inhibited the acetic acid-induced writhing response and potentiated the analgesic activity of aspirin as well. The abdominal constriction is related to the sensitization of nociceptive receptors to prostaglandins. In a previous study reported that extract of P. colorata have possess high amount of analgesic activity (26). It is, therefore, possible that fruit and stem of P. nilgiriensis produced analgesic effect probably due to the inhibition of synthesis or action of prostaglandins. Anti-inflammatory activity Carrageenan-induced paw oedema in rats: Carrageenaninduced paw edema as an in vivo model for inflammation has been frequently used to assess the anti-inflammatory effect of natural products. Carrageenan-induced paw edema model is thought to be a biphasic event. In early phase histamine and serotonin are released, while in the late phase prostaglandins, proteases and lysozymes are released (27). Carrageenan-induced paw edema remained even 4 h after its injection into the sub plantar region of rat paw. Indomethacin as a reference standard drug inhibited Phytochemical and Pharmacological Properties of P. nilgiriensis 1429 Table 5. Anti-inflammatory activity of P. nilgiriensis fruit and stem using carrageenan-induced paw edema in rats Group Control (water) Indomethacin Acetone extract of fruit Acetone extract of stem Oedema induced by carrageenan (mm) Dose (mg/kg) 1 mL 10 200 400 200 400 0h 1) 1) 4.86±0.32 5.08±0.17 4.98±0.10 4.96±0.14 5.22±0.06 5.10±0.21 1h 2h 3h 4h 5.49±0.11 5.21±0.15 5.83±0.16 5.84±0.20 5.67±0.36 5.25±0.20 5.92±0.32 5.26±0.12 5.48±0.19 5.16±0.12 5.12±0.38 5.15±0.29 6.53±0.18 05.44±0.10* 5.62±0.19 05.17±0.13* 05.05±0.15* 05.17±0.21* 6.96±0.21 05.28±0.19* 6.25±0.11 05.36±0.18* 5.89±0.16 05.45±0.12* 1) Values are expressed as mean±SEM (n=6); Significantly different at *p<0.05, when compared to control Fig. 2. GC/MS chromatogram of acetone extract of P. nilgiriensis fruit. the edema formation due to carrageenan to an extent of (5.28±0.1 mm at 4 h) at the dose of 10 mg/kg. The acetone extract of P. nilgiriensis fruit and stem significantly inhibited edema formation in rats (p<0.05) in a dose dependant manner. The acetone extracts at the dose of 400 mg/kg inhibited edema formation to the extent of (5.45±0.12 and 5.36±0.18 mm, respectively at 4 h) and the edema was found to be reduced to 1.51±0.12 mm (Table 5). The results obtained indicated that acetone extracts of fruit and stem has significantly (p<0.05) inhibited the formation of rat paw edema, both in early and the late phases. Researchers demonstrated that inflammatory effect induced by carrageenan is associated with free radical. The carrageenan induced inflammatory response has been linked to neutrophil inltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide, and hydroxyl radicals, as well as the release of other neutrophil-derived mediators (28). Oral pretreatment of animals with acetone extracts of fruit and stem has resulted in a significant inhibition of carrageenanevoked hind paw edema because these extracts have also showed renounced activity in in vitro antioxidant assays. GC/MS analysis GC/MS chromatogram of acetone extract of P. nilgiriensis fruit along with their retention time (RT) are shown in the Fig. 2. Major phytocomponents present in the P. nilgiriensis fruit along with molecular formula, molecular weight, peak area, and structure were presented in the Table 6. The GC/MS chromatogram of acetone extract of P. nilgiriensis fruit showed the presence of several active principle compounds. More than 6 peaks were identified from acetone extract of fruit at retention time of 38.94 min. The chromatogram obtained from GC has shown the phytochemical strength of the P. nilgiriensis extract. The sesquiterpinoid compounds are more specific to the Rubiaceae family. In GC/MS analysis of P. nilgiriensis acetone extract of fruit, a sesquiterpinoids quinines compound known as Nakijiquinones B was present in this plant which is commonly known for its fungicidal property. Nakijiquinones, the only natural product known to selectively inhibit the Her-2/Neu protooncogene which is associated with cancer treatment (29). 3, 4-Epoxy-7-octen2-one is the compound present in this plant which can act as antioxidant, hypocholesterolemic and anti-inflammatory agent. 9-Octadecenoic acid (Z)-(CAS) oleic acid with 1430 Iniyavan et al. Table 6. GC/MS analytical report for major phytoconstituents in acetone extract of P. nilgiriensis Name of the compound 2-Hydroxy-2,N-dimethyloctanoic acid amide octanamide, 2-hydroxy-N,2dimethyl- (CAS) Retention Molecular time (min) weight 16.84 % of Area 187 Structure Uses Pharmaceutical raw materials & food additives 0.40 C10H21NO2 (Z)-2-Methylhex-4-en-3-yl Nphenylcarbamate 20.87 233 2.15 C14H19NO2 9-Octadecenoic acid (Z)- (CAS), oleic acid 28.90 282 Central nervous system stimulant used in the treatment of Narcolepsy in adults and attention deficit disorder in children Antioxidant and antiinflammatory activity 2.05 C18H34O2 3,4-Epoxy-7-octen-2-one 32.39 140 Antioxidant, anti-inflammatory and hypocholesterolemic agent 12.54 C8H12O2 2-Hydroxypyrrolo[2,1-a] isoquinoline3-carboxylic acid ethyl ester 34.33 255 Anti-inflammatory, antioxidant and antiviral 4.08 C26H28O2Sn Nakijiquinone B 36.86 443 Anti-diabetic and antiinflammatory activity 53.68 C26H37NO antioxidant and anti-inflammatory property. Oleic acid may hinder the progression of adrenoleukodystrophy (ALD), a fatal disease that affects the brain and adrenal glands. Oleic acid may be responsible for the hypotensive (blood pressure reducing) effects of olive oil (30). Several other compounds were also depicted through GC/MS chromatogram. They are also having notable medicinal property. The GC/MS analysis profile has also confirmed the anti inflammatory and analgesic properties of P. nilgiriensis. According to the data derived from the present study, P. nilgiriensis extracts was found to be an effective antioxidant in different in vitro assays when compared to standard antioxidant compounds. In all the in vitro and in vivo studies and GC/MS analysis, acetone extract of fruit showed notable radical scavenging, analgesic and anti inflammatory properties. 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