Pharmaceutical Biology ie ev rR ee rP Fo " # ! % !& " #' ! ! ( " ) * ) !+ & !+ " ) * ) !+ ! " # ! ! $ " ) * ) ! $ % !+ $" ) * ) !+ $ ! $ " #' * ! ! , " #' * & ! /. 0! 1 1 ! 23! . ( $ 344 454! ! w ! $ 6 7 ! ) ! ! - ./ !. ly On ( % # ) ! URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu ! Page 1 of 34 Evaluation of an ethnomedicinal formulation containing Semecarpus kurzii and Hernandia peltata used for the management of inflammation Sonali Das2, Hemanta Mukherjee1, SK Milan Ahmed3, Pallab K. Haldar3, Asit B. Mandal2, Ambikesh Mahapatra4, Pulok K Mukherjee3, Sekhar Chakraborty1 and Debprasad Chattopadhyay1*, 1 ICMR Virus Unit, I.D. & B.G. Hospital, GB 4, 1ST Floor, 57, Dr. Suresh C Banerjee Road, rP Fo Beliaghata, Kolkata 700010, India 2 Directorate of Seed Research, Kushmaur, P.O.Kaithauli, Maunath Bhanjan 275101, U.P. India ee 3 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India 4 Department of Chemistry, Jadavpur University, Kolkata, India Dr. D. Chattopadhyay M.Sc., Ph.D. ie ev Corresponding Author* rR Fellow, British Soc Antimicrob Chemother (England) Assistant Director (Scientist) w ICMR Virus Unit Calcutta On ID & BG Hospital, GB 4, First Floor 57 Dr Suresh C Banerjee Road ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology Beliaghata, Kolkata 700 010, India Tel: 0091 33 2353 7425/7424 [Telefax]; 9433331293 (Mob) E. Mail: debprasadc@yahoo.co.in; debprasadc@gmail.com Keywords: Ethnomedicines, Antiinflammatory activity, Analgesic activity, Traditional use. 1 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology Abstract Context: Scientific validation of an ethnomedicinal formulation containing Semecarpus kurzii leaves (SKL) and Hernandia peltata stem-bark (HPB), traditionally used in ailments related to inflammation, pain, fever and skin problems. Objective: To validate in vivo and in vitro analgesic and antiinflammatory activities of methanol extract (ME) of SKL, HPB and their combination. rP Fo Materials and methods: The analgesic activity was tested by acetic acid induced writhing reflex and tail flick methods in Swiss albino mice, while the anti-inflammatory activity was studied by carrageenan and dextran induced paw edema, and cotton pellet-induced granuloma in Wistar rats at doses of 0-500 mg/kg body weight. The in vivo vascular permeability, in ee vitro membrane stabilizing activity in human RBC, and denaturation of egg protein were rR examined to know the possible mode of action. Results: Significant (p<0.01) analgesic and antiinflammatory activity was observed in ev combination of SKL and HPB extracts at 250mg/kg each. Moreover, the SKL extract alone significantly inhibit acetic acid-induced vascular permeability (64.4%) at 500mg/kg, while in ie combination (250mg/kg each) the inhibition was 69.49% (P<0.01). Furthermore, SKL in w combination with HPB (at 0.25 mg/ml each) could prevent RBC haemolysis (61.91%) and inhibition of protein denaturation (76.52%) like indomethacin. On Discussion and Conclusion: The SKL and HPB extracts alone (500mg/kg) and in combination (250mg/kg each) had significant analgesic and antiinflammatory activity, ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 2 of 34 probably by inhibiting the release of certain inflammatory mediators and membrane stabilization due to flavonoids, triterpene and related phytochemicals present in the extracts. Thus, this combination may provide the scientific rationale of its folk use. 2 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 3 of 34 Introduction The use of traditional medicines and medicinal plants for primary health care has been widely observed in most developing countries (UNESCO, 1996), including India. India has been blessed with four riches biodiversity zones, and one such richest tropical biodiversity zone is the Andaman & Nicobar Island, situated about 1200 km away from the mainland India. The tropical rain forest of Andaman & Nicobar Islands harbour about 2500 angiospermic plants, rP Fo of which 52 are used as medicaments by its aboriginal populations viz. Great Andamanese, Onge, Jarawas and Sentinels of Andaman group of Islands, and Nicobarese and Shompen of the Nicobar group of Islands. These tribes used diverse herbal formulations containing several endemic or extra-Indian plant species as time tested remedies (Dagar & Dagar, 1991) ee for ailments ranging from cancer to common cold. Furthermore, many plant species of these rR islands possess unique bioactive compounds due to congenial environment which bear scope for new drug development. ev An ethnomedicinal formulation containing Semecarpus kurzii leaf (SKL) and Hernandia ie peltata stem bark (HPB) at 1:1 ratio was used by Onge, Nicobarese and Shompen tribes of w Andaman and Nicobar Islands for fever, pain, and skin ailments (Mandal et al., 2000). S. kurzii (Anacardiaceae), locally known as Bara Bhilawa, is an endemic species to South On Andaman Islands (Mandal et al., 2000), whose leaves are used for treating skin ailments and pain (Bhargava, 1983; Chakraborty & Vasudeva Rao, 1988; Dagar & Dagar, 1991); resin for ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology painful wound, fever, allergy, and helminthic infections (Das et al., 2006). On the other hand, H. peltata (Hernandiaceae) locally known as jack box is a littoral tree of the seashores, used to treat wounds, tumour, headache, epilepsy and fits (Bhargava, 1983; Chakraborty & Vasudeva Rao, 1988; Dagar & Dagar, 1991). Until now there were no pharmacological studies on these ethnomedicinal remedy of Andaman and Nicobar Islands, either alone or in 3 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology formulation. Thus, the present study was undertaken to evaluate the in vivo analgesic and antiinflammatory efficacies and possible mode of action of these two plants alone and in combination (1:1), as used by the ethnic community. Materials and methods Plant materials, preparation of extract(s) and traditional formulation rP Fo The SKL and HPB were collected from rain forest of South Andaman and Great Nicobar Islands, in August, December and April of 2007- 2008 to rule out the seasonal variation. The plants were identified and authenticated by Dr Sreekumar, Botanical Survey of India (BSI), Port Blair, and the voucher specimens (No. ABM 071BSI/PB: 2008 and ABM 072BSI/PB: ee 2008) were deposited in the BSI, Port Blair. The plant parts were shade dried, separately rR powdered, passed through 40-mesh sieve and stored for future use. The powdered materials (200 g) of respective plant parts were extracted with 95% methanol for 72 h at room ev temperature (Arunachalam et al., 2002). The whole extract was separately collected, repeatedly filtered (Whatman No. 1 filter paper), and solvent evaporated to dryness under ie reduced pressure in an Eyela Rotary Evaporator (Japan) at 40-45°C. The concentrated w extracts of SKL (yield 8.9 ± 0.13%) and HPB (9.15 ± 0.11%) were stored in a desiccator for further study. A weighted amount of the dried extract(s) were dissolved in 2% (w/v) Tween- On 80 and diluted in sterile distilled water; while the formulation was made by mixing the dried extract of SKL and HPB (1:1). The formulation was dissolved in 2% (w/v) Tween-80 diluted ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 34 in sterile distilled water for pharmacological studies, whenever required. Standardization of the extract(s) was done by physicochemical constant, fluorescence analysis and phytochemical tests (WHO, 1998; Arunachalam et al., 2009). Physicochemical parameters like total ash, acid insoluble ash content, water content and the property of 4 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 5 of 34 powdered drug in different solutions under UV312 nm and visible light was determined following WHO Guideline, 1998. Preliminary phytochemical studies are performed by standard methods (Chhabra et al., 1984; Arunachalam et al., 2009) to know the chemical groups present within the extracts. HPLC instrumentation and experimental conditions rP Fo HPLC analysis was done by the method of Kontogianni et al., 2009, using a Shimadzu liquid chromatograph consisting of binary LC‐20AD pumps, coupled with a SPD-M20A photodiode array detector. Injection was done through a Rheodyne 7725 injector equipped with a 20 L loop, and a reverse phase C18, 250 X 4.6 mm, 5µm, Zorbax column (Agilent, USA). ee Both the column and HPLC system were kept at ambient (25°C) conditions. The mobile rR phase was 1% acetic acid in HPLC grade water: methanol (10:90) for SKL and 1% acetic acid in HPLC grade water: methanol (35:65) for HPB with the flow rate of 1 ml/min. The ev absorbance was monitored at 210 nm with the injection volume of 20 L. ie The stock solutions were prepared by dissolving 10 mg of pure ursolic acid and chlorogenic w acid (Sigma, USA) separately in 10 ml of methanol. All the samples were filtered through 0.45 m membrane, and 20 l of this sample solution was injected into the HPLC system for On analysis. Identification of Ursolic acid and Chlorogenic acid was based on their retention time and comparison of their UV spectra. Quantitative analysis was achieved by five point ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology calibration curves for Ursolic acid and Chlorogenic acid, at concentrations of 5–1000 g/ml and from the equation Y= 6987.2 X + 60941 for ursolic acid and Y= 6977.3 + 60943 for Chlorogenic acid respectively, where Y represents the area of the extract and X represents the concentration of either Ursolic acid or Chlorogenic acid. The regression coefficient values were 0.9985 for Ursolic acid and 0.9945 for Chlorogenic acid. Real samples were diluted 5 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology accordingly to fit the dynamic linear range of the regression line, when necessary. All measurements were performed in triplicates. Animals Healthy inbreed Swiss albino male mice (20 ± 2 g) and adult Wistar male rats (150-180 g) were housed at 23 ± 4° C, of relative humidity 60-70% in the animal house facility of the rP Fo Pharmaceutical Technology Department, Jadavpur University, Kolkata and were fed with standard pellet diet and water ad libitum. All the animals were acclimatized for one- week before the experiments, and all experiments were carried out according to the Institutional Animal Ethics committee 07.07.2008). (Permission No. JU/Pharm/007/2008; dated ev rR Drugs and chemicals guidelines ee Paracetamol, morphine sulfate, diclofenac disodium, carrageenan, dextran powder and all other analytical grade chemicals were purchased from the respective manufacturers. w ie Acute toxicity study The acute toxicity of SKL and HPB extracts alone and in combination was conducted on 13 On groups of mice (n=6) at oral (p.o.) doses of (200-2000) mg/kg body weight including a control group. While the same in Wistar rats at 200, 500, 1000 and 2000, using 9 groups of ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 6 of 34 animals (n=6). Simultaneously, the acute toxicity of SKL and HPB extracts was tested in Wistar Rats at 200, 500, 1000 and 2000 mg/kg body weight orally along with a control group. The animals were observed for toxic symptoms and death within 24h (6, 12, 18 and 24 h) and then daily for next 14 days (OECD, 2000). The study showed neither any observable toxic effects (agility, muscular tonus, tremors, convulsions, problem in breathing, and water or 6 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 7 of 34 food intake) nor any death following treatment, and hence, the procedure was repeated up to 5.0 g/kg. On the basis of toxicity data the dose regimen for further tests were selected as 400 and 500 mg/kg of extracts alone and 250 mg/kg (each) in combination. Analgesic activity Acetic acid-induced writhing tests rP Fo The writhing test was performed according to the method of Koster et al., 1959. Briefly, Swiss albino mice of either sex were divided into seven groups of six animals each. The first group served as control; the second group received paracetamol (50 mg/kg), while the 3rd to 6th group received SKL and HPB extract at 400 and 500 mg/kg respectively, and the last ee group was administered with a combination of SKL and HPB (250 mg/kg each) as i.p. After rR 30 min of drug administration, the animals were orally fed with 1% v/v fresh acetic acid solution (10 ml/kg), and immediately counted the numbers of writhing or stretches ev (abdominal contraction, trunk twist response and extension of hind limbs) for 10 min. A reduction in the writhing number compared to the control was considered as evidence of ie analgesia (Pradeepa et al., 2012). The percentage inhibition of writhing was calculated as: % w Inhibition= C-T/C X 100. Where C is the mean number of writhes produced by the control group and T represents the mean number of writhes produced by the test groups. ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology Tail flick test Swiss albino mice of either sex weighing 20 ± 2g were divided into seven groups of five animals each. The tail of each mouse was placed on the nichrome wire of an analgesiometer (Techno Lab, Lucknow, India) and the time taken by the animal to withdraw (flick) its tail from the hot wire was taken as a reaction time. The extract of SKL, and HPB at 400 and 500 mg/kg and the combination of SKL and HPB (250 mg/kg each) were injected i.p., using 7 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology morphine sulphate (5 mg/kg) as standard drug. Analgesic activity was measured after 30 min of administration of test and standard drug (Das et al., 2011) and the percentage inhibition was calculated by the same formula as above. Anti-inflammatory activity Carrageenan-induced rat paw edema (acute model) rP Fo Wister male rats were divided into seven groups of six animals (n=6) each. The extracts of SKL and HPB at 400 and 500 mg/kg body weight were administered orally to the animals of groups I to IV, while Vth group received a combination of SKL and HPB (250 mg/kg each), 60 min prior to carrageenan injection. The VIth group serves as vehicle control, and the VIIth ee group received diclofenac disodium (20 mg/kg) as drug control, for assessing comparative rR pharmacological significance. Edema was induced by sub-plantar injection of 0.05 ml of fresh carrageenan suspension (1% w/v) in normal saline into the right hind paw of each rat ev (Winter et al., 1962). The paw volume (linear circumference) was measured at 0 h and at 1 to 5 h after carrageenan injection by a plethysmometer (Oyemitan et al., 2008). The ie antiinflammatory activity was evaluated using the ratio of the changes in paw diameter in w treated and untreated group by the formula: Anti-inflammatory activity (%) = (1- D/ C) X 100; Where D is the change in paw diameter in treated group and C is the change in paw diameter in untreated group. Dextran-induced rat paw edema (sub-acute model) ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 8 of 34 The hind paw edema in each rat was induced by sub-plantar injection of 0.1 ml of 1% dextran solution (Das et al., 2011) in the right foot. Paw volumes were measured 30 min before and after dextran injection. The treatment of extracts (test), control (vehicle), and standard drug 8 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 9 of 34 was the same as described for carrageenan model. The percentage inhibition of edema was calculated by the method of Arunachalam et al., 2002. Cotton pellet induced granuloma (chronic model) The rats were divided into seven groups of six animals each, and the animals were anaesthetized after shaving of the fur. Sterile preweighed cotton pellets (10 mg) were rP Fo implanted in the axilla region of each rat through a single needle incision (D’Arcy et al., 1960). The extracts of SKL and HPB were administered orally at 400 and 500 mg/kg to the groups’ I - IV, and a combination of SKL and HPB (250 mg/kg each) to group V. The group VI serves as vehicle control, while group VII received diclofenac disodium (20 mg/kg), for ee consecutive seven days from the first day of implantation. On 8th day the animals were rR anaesthetized, cotton pellets were removed surgically and made free from extraneous tissues. To obtain constant weight, the pellets were incubated at 370C for 24 h and dried at 600 C. The ev granuloma formation was measured by the increase in dry weight of the pellets (Winter & Porter, 1957). Acetic acid-induced vascular permeability in mice w ie The vascular permeability was tested by the method of Whittle (1964). Briefly, mice were On divided into seven groups, six per group (n=6). Group I served as vehicle control, groups II V were treated orally with 400 and 500 mg/kg of SKL and HPB extract; group VI with a ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology combination of SKL and HPB (250 mg/kg each), and group VII received indomethacin (20 mg/kg). One hour after the treatment, 0.2 ml of 0.2% Evans’ blue in normal saline was injected intravenously to each mouse through tail vein and 30 min later, each mouse was injected i.p. with 0.2 ml of 0.6% acetic acid in normal saline. After 1 h, the animals were sacrificed, and the abdominal cavity was exposed and washed with 5 ml normal saline to 9 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology collect the content in a sterile test tube. The content was centrifuged to eliminate contaminants, and the absorbance (A) of the solution was measured by a spectrophotometer at 590 nm. The vascular permeability effects, expressed as the absorbance (A590), represented the total amount of dye leaked into the intraperitoneal cavity. Membrane stabilizing activity rP Fo This was assessed by hypotonic solution-induced human erythrocyte haemolysis (Shinde et al., 1999). Whole blood collected from a healthy volunteer (DC) in a heparinized tube was washed thrice with isotonic buffer (154 mM NaCl in 10 mM sodium phosphate buffer, pH 7.4) for 10 min at 3000 g. The test sample consisted of RBC (0.50 ml) in 5 ml hypotonic ee solution (50 mM NaCl in 10 mM sodium phosphate buffer saline, pH 7.4) containing the rR extracts (0.2 - 1.0 mg/ml) alone and in combination, or indomethacin (0.1 mg/ml). The control tube contain RBC (0.5 ml) mixed with hypotonic-buffered saline. The mixtures were ev incubated for 10 min at room temperature, centrifuged (3000 g for 10 min) and the absorbance of the supernatant was measured at A540 nm. The percentage inhibition of ie haemolysis or membrane stabilization, tested in triplicate, was calculated as % Inhibition of w haemolysis = 100 x {OD1 - OD2/ OD1}, Where OD1 is the Optical density of hypotonic buffer saline and OD2 is the Optical density of test sample in hypotonic solution. ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 10 of 34 Effect on in vitro protein denaturation Test solution (1ml) containing 100-1000 g/ml of extracts, alone or in combination or indomethacin (100 g/ml) was mixed with 1 ml of egg albumin (1 mM) and incubated at 27 ± 1° C for 15 min. The mixture was then placed at 70° C in a water bath for 10 min to induce denaturation of albumin. After cooling, the turbidity was measured at 590 nm (Mizushima, 1966) and the average of three sets was taken. The percentage inhibition of denaturation was 10 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 11 of 34 calculated relative to the control as % inhibition of denaturation = (Abs of control - Abs of treated)/Abs of control X 100 (Ramalingam et al., 2010). Statistical Analysis The results were expressed as mean, mean ± S.D. and SEM (Standard Eror Mean). The significance between the two groups was evaluated by Student’s t-test, and the significance of difference among the various treated and control group were analyzed by one-way Anova rP Fo followed by Dunnett’s t-test (Woodson, 1987). Results In this study analgesic and anti-inflammatory activity of ME of SKL and HPB and their ee combination (1:1), used as ethnomedicinal formulation, was tested by in vivo and in vitro screening methods. ev rR Standardization of sample and physicochemical study of extract The ME of plant parts collected in different session showed that the post rainy session ie samples (August) had the minimum amount of water, total ash and acid insoluble ash content, w with maximum yield (Table 1), and thus selected for further study. Under visible and UV312 nm light the colour of powdered SKL extracts was yellowish to light brown; while it was grey On to light brown in HPB extract. Preliminary phytochemical screening of the SKL extract revealed the presence of flavonoids, triterpene, tannins, saponins and glycosides; while HPB extract contains alkaloid, flavonoid, steroid and tannin. ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology The quantification by HPLC analysis revealed that, ursolic acid (triterpene) in the ME of SKL was 0.39 % (w/w) while the amount of chlorogenic acid in HPB extract was 0.46 % (w/w). The ursolic acid in SKL and chlorogenic acid in HPB extract is unknown as one of the 11 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology main principle of the plants, hence we have quantified the leaf extract of S. kurzii for ursolic acid and bark extract of H .peltata for chlorogenic acid as marker compound (pure Ursolic acid and chlorogenic acid, Sigma). Specimen HPLC chromatograms of the standard ursolic acid and ME of SKL are represented in Figure 1 (a and b), while standard chlorogenic acid and ME of HPB are represented in Figure 1 (c and d). rP Fo Acute toxicity Toxicity study conducted over 14 days showed no observable toxic effects (convulsion, ataxia, agility, dyspnoea, water or food intake, diarrhoea or diuresis) or any death, signifying that both SKL and HPB extracts possess good safety profile. However, slightly reduced ee motor activity, ataxia and hyperventilation was observed in mice at 5 g/kg orally. The median rR lethal dose (LD50) of SKL and HPB extract(s) was found to be 3.6 g/kg and 3.2 g/kg respectively in mice. In rats, the LD50 of SKL and HPB extract(s) was 4.0 g/kg and 3.6 g/kg ev orally, respectively. Thus, the dose regimen for further study was selected as 400 and 500 mg/kg alone, and 250 mg/kg (each extract) in combination. w ie Analgesic activity: On Effect of acetic acid induced writhing test The results of acetic acid induced writhing test with SKL and HPB extracts (400 and 500 mg/kg), and their combination (250 mg/ml each) i.p. presented in Table 2, showed that the ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 12 of 34 inhibition of writhing reflexes was 59.87% at 400 mg/kg and 64.70% at 500 mg/kg of SKL. While it was 61.66% at 400 mg/kg, and 73.08% at 500 mg/kg of HPB extract (p < 0.01). Interestingly, the combination of SKL and HPB extracts (250 mg/kg each) produced maximum inhibition (78.04%), which was nearly similar to the paracetemol treated group (81.95%). 12 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 13 of 34 Effect of tail flick induced test The results of tail flick test, presented in Table 2, showed that the SKL extract had reaction time of 9.25 sec (57.24%) at 400 mg/kg and 7.67 sec (64.53%) at 500 mg/kg i.p doses. While the reaction times in vehicle control and morphine sulfate (5 mg/kg) group was 21.63 sec and rP Fo 5.20 sec (75.95%), respectively. The HPB extracts at 400 mg/kg showed reaction time as 9.11 sec with 57.88% inhibition; while at 500 mg/kg doses it was 6.51 sec (69.90% inhibition). Thus, significant (P < 0.01) activity was noted with both the extracts at 500 mg/kg alone and in combination of SKL and HPB (250 mg/kg each), with maximum ee inhibition of 71.70% at 6.12 sec, close to morphine (75.95%). Antiinflammatory activity ev rR Effect of carrageenan induced paw edema The results of antiinflammatory activity of SKL and HPB extracts and their combination, ie presented in Table 3, showed that SKL at 500 mg/kg exhibited 65.08% inhibition of paw w edema; while at 400 mg/kg the inhibition was 56.94%. Here the standard drug diclofenac disodium (20 mg/kg) produced 74.23% inhibition of edema volume after 3 h of treatment. On The HPB extract exhibited 43.05% inhibition at 400 mg/kg and 52.20% at 500 mg/kg, respectively. However, the combination of SKL and HPB extracts (at 250 mg/kg each) ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology produced highest inhibition (72.54%) which is highly significant (P < 0.01), compared to the drug control group. Effect of dextran induced paw edema In dextran-induced paw edema test the maximum inhibition (74.36 %) of edema swelling was noted with 20 mg/kg of diclofenac disodium, followed by SKL extract at 400 mg/kg 13 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology (55.37%) and 500 mg/kg (64.24%), respectively. However, HPB extract showed significant (P < 0.05) inhibition (50.63%) of paw edema at 500 mg/kg dose only. Here again, the combination of SKL and HPB extracts (250 mg/kg each) revealed highest inhibition (72.15%). All these data are significant (P < 0.01) with respect to the control groups, indicating the possible antiinflammatory activity of these extracts (Table 3). rP Fo Effect of cotton pellet induced granuloma The results presented in Figure 2 showed that SKL and HPB extracts significantly inhibited the granuloma weight. The SKL extract produced 54.40% inhibition at 500 mg/kg (p < 0.01) compared to diclofenac disodium (60.77%) at 20 mg/kg (p < 0.05). However, 49% ee inhibitions of granuloma weight was recorded with 500 mg/kg HPB extract, while the rR combination of SKL and HPB extracts produced 57.55% inhibition, nearly equal to diclofenac disodium. ev Inhibition of acetic acid-induced vascular permeability in mice ie Effect of the oral administration of SKL and HPB extracts (400 and 500 mg/kg) on acetic w acid-induced vascular permeability in mice was presented in Figure 3. Results showed that SKL at 400 and 500 mg/kg inhibited 54.23% and 64.4% vascular permeability, compared to On indomethacin (70.33%). While HPB extract at 400 and 500 mg/kg produced 42.37% and 52.54% inhibition of vascular permeability. Here again the highest inhibiton (69.49%) was ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 14 of 34 recorded when both the extracts were combined. All these data are significant at 5% level (P < 0.05). Membrane stabilizing activity To validate the in vivo data further we conducted the non-specific membrane stabilization study with both the extracts alone and in combination. The results presented in Table 4, 14 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 15 of 34 showed that the SKL and HPB extracts can protect the erythrocyte membrane against lysis induced by hypotonic solution. Both extracts at 0.5 mg/ml significantly (P < 0.01) protect the erythrocyte membrane (59.45% and 54.79% inhibition) against lysis, compared to indomethacin (63.69% inhibition) at 0.1 mg/ml. While the combination of SKL and HPB achieved 61.91% protection in half the concentration (0.25 mg/ml) of each extract, which is highly significant (p < 0.01) and nearly equal to the standard drug. rP Fo Inhibition of protein denaturation The inhibitory effect of SKL and HPB extracts on protein denaturation, presented in Figure 4, showed that both the extracts (200 and 400 g/ml) moderately inhibited (34.64 - 51.37%) the ee denaturation of egg albumin. However, at 800 g/ml extracts produced 65.64% and 63.80% rR inhibition of egg albumin denaturation, compared to indomethacin (86.36%) at 100 g/ml. Here again the combination of SKL and HPB (400 µg/ml each), produced 76.52% inhibition ev (P < 0.01), similar to indomethacin (86.36%). ie Discussion w We have studied the in vivo analgesic and antiinfammatory activity along with the vascular permeability, in vitro membrane stabilization of RBC and inhibition of protein denaturation On of the standardized extracts of two ethnomedicinal plants S. kurzii and H. peltata, alone and in combination (as formulation), used by some ethnic community of Andaman and Nicobar ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology Islands, India. The importance of this study lies on their ethnomedicinal use and endangered nature of one of its component S. kurzii, which have not been validated yet. The HPLC analysis of the ME of both the plants showed the presence of ursolic acid and chlorogenic acid as one of the component, and both these compounds are known to have analgesic and antiinflammatory activity (Liu, 1995; Azza et al., 2011). Nevertheless our findings for the 15 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology first time scientifically validated this combination for analgesic and antiinflammatory activity, which is probably due to several group of compounds including chlorogenic acid and ursolic acid. The analgesic activity tested by acetic acid- induced writhing model, indicated that the numbers of writhing movements were significantly less in the mice treated with SKL and rP Fo HPB extracts alone and in combination, comparable to untreated group. The effect of the extracts when compared to paracetemol, suggests that both the extracts might have peripheral analgesic effect. Furthermore, the analgesic effect tested by the tail flick test was comparable to that of morphine treated control, suggesting central analgesic effect. rR ee Formation of edema by a complex array of enzyme activation, mediator release, cell migration, extravasations of fluid, tissue breakdown and repair is one of the cardinal sign of ev inflammation (Vane & Bolting, 1995). We used the most acceptable carrageenan induced paw edema model, as carrageenan helps to release the chemical mediators in an orderly and ie biphasic manner (Di Rosa, 1972; Sur et al., 2002), where the vasoactive histamine and 5HT w (bradykinin and serotonin) released in the early phase and prostaglandins (kinin) in the late phase (Heller et al., 1998), resulting in increased vascular permeability, leading to the On accumulation of fluid in the tissues to form edema (White, 1999). On the other hand, dextranmediated inflammation was reduced probably due to antihistaminic effects of the extracts, as ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 16 of 34 dextran cause inflammation through the release of histamine and serotonin (Ghosh et al., 1963). Carrageenan- and dextran-induced paw edema test revealed that SKL and HPB extracts possesses significant anti-inflammatory activity at 400 and 500 mg/kg oral dose, while the combination of both the extract (at 250 mg/kg) showed much better activity (p<0.01) at 3 h, comparable to diclofenac disodium. Thus, the ability of SKL and HPB 16 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 17 of 34 extracts alone or in combination to reduce the edema volume, suggests that the phytochemicals of these extracts, including ursolic acid and chlorogenic acid, may block the release of some mediators like histamine, bradykinin, serotonin and prostaglandins. Furthermore, the decrease in the cotton-pellet induced granuloma weight by the extracts is due to the inhibition of proliferative phage of inflammation (Rajavel et al., 2009), as the inflammatory response induced by the cotton pellet can modulate the release of mediators rP Fo leading to the tissue proliferation and granuloma formation (Stoeck et al., 2004). We use the acetic acid induced vascular permeability test, a capillary permeability assay, to confirm the antiinflammatory potential of the tested extracts. The acetic acid cause dilation of ee blood vessels and increased permeability through the release of histamine, prostaglandins and rR leukotrienes by stimulating mast cell (Brown & Roberts, 2006). During inflammation these mediators increase vascular permeability, while acetic acid cause an immediate sustained ev reaction over 24 h and its inhibition by the test extracts alone or in combination, suggests that SKL and HPB may effectively suppress the exudative phase of acute inflammation. w ie Furthermore, the non-specific RBC membrane stabilization test showed the protective effect on hypotonic saline induced RBC lysis, an index of antiinflammatory activity (Oyedapo & On Famurewa, 1995). When RBC is exposed to hypotonic medium, it undergoes haemolysis and oxidation of haemoglobin (Augusto et al., 1982; Ferrali et al., 1992) due to excessive ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology accumulation of fluid within the cell. Such injury renders the RBC more susceptible to secondary damage through free radical induced lipid peroxidation (Ferrali et al., 1992). This is consistent with the observation that the breakdown of bio-membranes induces free radical generation, which in turn enhance cellular damage (Maxwell, 1995). Thus, compounds with membrane-stabilizing property can protect cell membrane against injurious substances (Liu et 17 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology al., 1992; Perenz et al., 1995; Shinde et al., 1999) by interfering the release of phospholipases to trigger the formation of inflammatory mediators (Aitadafoun et al., 1996). Here the significant (p<0.01) membrane stabilizing activity of SKL and HPB extracts alone and in combination, suggests that the extracts might inhibit the release of chemical mediators in inflammatory events. rP Fo Moreover, we have studied the denaturation of proteins, as it is a well known cause of inflammation (Mizushima, 1966), and several anti-inflammatory drugs inhibit thermally induced protein denaturation dose dependently (Grant et al., 1970). Here, the ability of the tested extracts to bring down thermal denaturation of protein is possibly a contributing factor ee for its anti-inflammatory activity, probably due to the presence of bioactive flavonoids and rR related compounds like chlorogenic acid and ursolic acid, as reported with other plants (Middleton et al., 2000; Havsteen, 2002). ev The contemporary literature revealed that the Malaysian species of H. peltata Meisn, used by ie the Samoan healers, has anti-hypertensive (Singh, 1986; Dittmar, 1991), antitumor (Pernet, w 1971), piscicide (Nishino & Mitsui, 1973) and anti-arteriosclerosis (Ebel & Roth, 1987) activity. Chemically H. peltata Meisn of Malaysia is reported to contain benzylisoquinoline On alkaloid hebridamine (Chalandre et al., 1986) that inhibit chloroquine-resistant Plasmodium falciparum (Angerhofer et al., 1999), and artherosclerosis (Lakshmi et al., 2009). Moreover ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 18 of 34 the lignans like podophyllotoxins (deoxypodophyllotoxin and deoxypicropodophyllin) isolated from H. peltata Meisn have anticancer activity (Pettit et al., 2004) while hernanol, benzopyran, epiaschantin, deoxypodorhizone inhibit Neisseria gonorrhoeae (Pettit et al., 2004). Interestingly another plant of the formulation, S. kurzii, is reported to contain 9hydroxy-cis-12-enoic (isoricinoleic) acid and fatty acids (Farooqui et al., 1985). However, the 18 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 19 of 34 phytochemical study of this formulation revealed the presence of flavonoids, tannins, saponins, glycosides, alkaloid and steroid, and the HPLC revealed the presence of ursolic acid and chlorogenic acid as one of their compounds. Conclusion In conclusion, this study suggests that the traditional formulations of SKL and HPB in rP Fo combination (250 mg/kg each) may offer beneficial effects in the management of inflammatory conditions, as both the extracts significantly inhibited writhing reflexes, paw oedema, vascular permeability, membrane-stabilization and protein denaturation, probably due to the flavonoids like chlorogenic acid, triterpenes like ursolic acid and related ee phytophores present in both the extracts. Further studies involving the purification of the rR chemical constituents and the investigations on biochemical pathways are required to establish the exact mechanism of its action. However, these findings endorsed the vernacular ev medicinal use of both these unique plants to treat ailment related to inflammatory conditions. ie Acknowledgements w Financial support from Central Agricultural Research Institute is highly acknowledged. Declaration of interest statement There is no conflict of interest in any form between the authors. ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology 19 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology References Aitadafoun M, Mounieri C, Heyman SF, Binistic C, Bon C, Godhold J. (1996). 4Alkoxybenzamides as new potent phosholipase A2 inhibitors. Biochem Pharm, 51, 737742. Angerhofer CK, Guinaudeau H, Wongpanich V, Pezzuto JM, Cordell GA. (1999). Antiplasmodial and cytotoxic activity of natural bisbenzylisoquinoline alkaloids. J Nat rP Fo Prod, 62, 59-66. Augusto O, Kunze KL, Montellano PR. (1982). Nphenylprotoporphyrin formation in the haemogolobin-phenylhydrazine reaction. J Bio Chem, 257, 6231-6241. Arunachalam G, Chattopadhyay D, Mandal AB, Sur TK, Mandal SC. (2002). Evaluation of ee antiinflammatory activity of Alstonia macrophylla wall ex A.DC. leaf extract. Phytomedicine, 9, 632-635. rR Arunachalam G, Bag P, Chattopadhyay D. (2009). Phytochemical and phytotherapeutic ev evaluation of Mallotus peltatus (Geist.) Muell. Arg. var acuminatus and Alstonia macrophylla Wall ex A. DC: Two ethnomedicine of Andaman Islands, India. J ie Pharmacog Phytother, 1, 01-13. w Azza EM, Yieldez B, Mahmoud K, Abdullatif M, (2011). Chlorogenic acid as potential antiinflammatory analgesic agent: an investigation of the possible role of nitrogen-based On radicals in rats. International Journal of Pharmacology & Toxicology Science, 1, 24-33. Bhargava N. (1983). Ethnobotanical studies of the tribes of Andaman and Nicobar Islands, ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 20 of 34 India. I. Onge. Econ Bot. 37, 110-119. Brown JN, Roberts J. (2006). Histamine, bradykinin, and their antagonists, In: Gilman AG, Hardman JG, Limbird LE, Goodman and Gilman’s the Pharmacological Basis of Therapeutics, 11th ed. McGraw Hill Co: New York. 645-667. 20 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 21 of 34 Chakraborty T, Vasudeva Rao MK. (1988). Ethnobotanical studies of the Shompen of Great Nicobar Islands. J Econ Taxon Bot, 12(1), 43-54. Chalandre MC, Cabalion P, Guinaudeau H. (1986). Étude des Hernandiacées. XII. Dimères aporphine–benzylisoquinoléine originaux isolés de Hernandia peltata. Canadian J Chem, 64, 123-126. Chhabra SC, Viso FC, Mshin EN. (1984). Phytochemical screening of Tanzanian medicinal rP Fo plants. J Ethnopharmacol, 11, 157-179. D’Arcy PD, Howard EM, Muggleton PW, Townsend SB. (1960). The antiinflammatory action of griseofulvin in experimental animals. J Pharm Pharmacol, 12, 659-665 Dagar HS, Dagar JC. (1991). Plant folk medicines among Nicobarese of Katchal Island, ee India. Econ Bot, 45, 114-119. rR Das S, Haldar PK , Pramanik G, Panda SP, Bera S. 2011. Evaluation of Analgesic and AntiInflammatory Activity of Diospyros Cordifolia Extract, Afr J Trad Comp and Alt Med, 8 (1), 11-14. ev Das S, Sheeja TE, Mandal AB. (2006). Ethnomedicinal uses of certain plants from Bay ie Islands. Indian J Traditional Knowledge, 5(2), 207-211. w Dittmar A. (1991). The effectiveness of Hernandia species (Hernandiaceae) in traditional Samoan medicine and accordining to scientific analysis. J Ethnopharmacol, 33, 243-251. On Di Rosa M. (1972). Biological properties of carrageenan. J Pharm Pharmacol, 24, 89-102. Ebel S, Roth H. (1987). Lexikon der Pharmazie. Thieme, Stuttgart, 10, 704S, 217. ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology Farooqi JA, Jamal S, Ahmad I. (1985). Isoricinoleic acid in Semecarpus kurzii seed oil. J American Oil Chemists' Society, 62(12), 1702-1703. Ferrali M, Signorni C, Ciccoli L, Comporti M. (1992). Iron release and membrane damage in erythrocytes exposed to oxidizing agents, phenylhydrazine, divicine and isouramil. Biochem J, 285, 295-301. 21 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology Ghosh MN, Banerjee RH, Mukherjee SK. (1963). Capillary permeability increasing property of hyaluronidase in rat. Indian J Physiol Pharmacol, 7, 17-21. Grant NH, Alburn HE, Kryzanauskas C. (1970). Stabilization of serum albumin by antiinflammatory drugs. Biochem Pharmacol, 19, 715-722. Havsteen BH. (2002). The biochemistry and medical significance of the flavonoids. Pharmacol Therapeut, 96(2-3), 67-202. rP Fo Heller A, Koch T, Schmeck J, Acker VK. (1998). Lipid mediators in inflammatory disorders. Drugs, 55, 487-496. Kontogianni VG, Exarchou V, Troganis A, Gerothanassis LP. (2009). Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts ee using two-dimensional nuclear magnetic resonance spectroscopy-Comparison with HPLC rR methods. Analytica Chimica Acta, 635(2), 188–195. Koster R, Anderson M, De Beer EJ. (1959). Acetic acid for analgesics screening. Federation Proceed, 18, 412-417. ev Lakshmi V, Pandey K, Mishra SK, Srivastava S, Mishra M, Agarwal SK. (2009). An ie Overview of family Hernandiaceae. Records of Nat Prod, 3(1), 1-22. w Liu GT, Zhang TM, Wang BE, Wang YW. (1992). Protective action of seven natural phenolic compounds against peroxidative damage to biomembranes. Biochem Pharmacol, On 43, 147-152. Liu J. (1995). Pharmacology of oleanolic acid and ursolic acid. J Ethnopharmacol, 49 (2), ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 34 57-68. Mandal AB, Chattopadhyay D, Coomar T. (2000). Rare and endangered flowering plants of Bay Islands with special reference to endemics and extra Indian Taxa. Indian Forester, 126 (4), 389 -396. Maxwell SRJ. (1995). Prospects for the use of anti-oxidant therapies. Drugs, 49, 345-361. 22 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 23 of 34 Middleton E, Kandaswami C, Theoharides TC. (2000). The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol Rev, 52, 673-751. Mizushima Y. (1966). Screening test for anti-rheumatic drugs. Lancet, 2, 443. Nishino C, Mitsui T. (1973). Lignans from Hernandia ovigera. Tethedron Let, 4, 335-338. OECD Guideline 425 (2000). Acute Oral Toxicity. Environmental Health and Safety rP Fo Monograph series on Testing and Assessment No. 24. Oyemitan IA, Iwalewa EO, Akanmu MA and Olugbad TA. (2008). Antinociceptive and antiinflammatory effects of essential oil of Dennettia Tripentala G. Baker (Annonaceae) in Rodents, AJTCAM, 5(4), 355-362. ee Oyedapo OO, Famurewa AJ. (1995). Anti-protease and membrane stabilizing activities of rR extracts of Fagra zanthoxiloides, Olax subscropioides and Tetrapleura tetraptera. Internat J Pharmacog, 33, 65-69. ev Pernet R. (1971). Revue des Hernandiacees. Planta Med, 20, 314-319. Perez RM, Perez S, Zavala MA, Salazar M. (1995). Anti-inflammatory activity of the bark of ie Hippocratea excelsa. J Ethnopharmacol, 47, 85-90. w Pettit GR, Meng Y, Gearing RP, Herald DL, Pettit RK, Doubek DL, Chapuis J-C, Tacket LP. (2004). Antineoplastic agents. 522. Hernandia peltata (Malaysia) and Hernandia On nymphaeifolia (Republic of Maldives). J Nat Prod, 67(2), 214-220. Pradeepa K, Krishna V, Venkatesh, Girish Kumar K, Santosh Kumar SR, Joy H Hoskeri, ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology Gnanesh AU. 2012. Antinociceptive activity of Delonix elata leaf extract, Asian Pac J Trop Biomed S229-S231 Rajavel R, Sivakumar T, Jagadeeswaran M, Malliga P. 2009. Evaluation of analgesic and anti-inflammatory activities of Oscillatoria willei in experimental animal models. J Med Plants Res, 3(7), 533-537. 23 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology Ramalingam R, Bindhu Madhabi B, Ravinder Nath A, Duganath N, Udaya Sri E, Banji D. (2010). In vitro antidenaturation and antibacterial activity of Zizyphus oenoplia. Der Pharmacia Lettre, 2(1), 87-93. Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf VO. (1999). Membrane stabilizing activity- a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoter, 70, 251-257. rP Fo Singh YN. (1986). Traditional medicine in Fiji: Some herbal folk cures used by Fiji Indians. J Ethnopharmacol, 15, 57-88. Stoeck M, Riedel R, Hochhaus G, Ha¨ fner D, Masso J, Schmidt B, Hatzelmann A, Marx D, Bundschuh D. 2004. In Vitro and in Vivo Anti-Inflammatory Activity of the New ee Glucocorticoid Ciclesonide. J Pharmacol Exp Therap, 309(1), 249–258. rR Sur TK, Pandit S, Bhattacharya DK, Ashok Kr CK, Mohanlakshmi S, Chattopadhyay D, Mandal SC. (2002). Studies on anti-inflammatory activity of Betula alnoides bark. Phytother Res, 16(7), 669-671. ev UNESCO, 1996. Culture and health, orientation texts-world decade for cultural development ie 1998-1997. Document CLT/DEC/PRO-1996, Paris, France, pp: 129. w Vane JR, Bolting RM. (1995). New insights into the mode of action of anti-inflammatory drugs. Inflammation Res, 44, 1-10. On Wagner H, Bladt S. (1996). A Thin Layer Chromatography Atlas. Second ed. Verlag, Berlin, Heidelberg, Germany, Plant Drug Analysis. (WRONGLY CITED) ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 34 White M. (1999). Mediators of inflammation and inflammatory process. J Allergy Clin Immunol, 103, 5378-5381. Whittle BA. (1964). The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics. Brit J Pharmacol, 22, 246–253. 24 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Page 25 of 34 Macroscopic and Microscopic Examination: Quality Control methods for medicinal plant materials, WHO, Geneva, (1998). Winter CA, Risley EA, Nuss GW, (1962). Carrageenan induced oedema in hind paw of rat as assay for antiinflamatory drugs. Exp Biol Med, 111, 544-547. (In Correct Jour Name---) Winter CA, Porter CC. (1957). Effect of alteration in side chain upon anti-inflammatory and liver glycogen activities in hydrocortisone esters. J Americ Pharmacol Soc, 46, 515. rP Fo Woodson RF. (1987). Statistical Methods for the Analysis of Biomedical Data, Probalility and Mathematical Statistics. UK: Wiley, Chichester, 315-316. w ie ev rR ee ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology 25 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu Pharmaceutical Biology !" w ie ev rR ee rP Fo ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 26 of 34 URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu w ie ev rR ee rP Fo ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pharmaceutical Biology !" 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