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Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 2, 2005, pp. 105-109 Anti-Nociceptive and Anti-Inflammatory Activity of Araucaria bidwillii Hook KFH NAZEER AHAMED, VENKATESAN KUMAR, SUNDARARAJAN RAJA, KAKALI MUKHERJEE and PULOK KUMAR MUKHERJEE School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata (N.A., V.K., K.M., P.K.M.); Survey of Medicinal Plant Unit, Govt Arts College Campus, Ooty (S.R.); India. Address correspondence to: Dr. Pulok K Mukherjee, School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. Tele fax: +91 (33) 24146046. E-mail: pulokm@yahoo.com Received July 12, 2005; Revised September 3, 2005; Accepted September 4, 2005 Code Number: pt05023 ABSTRACT The effect of alcoholic extracts of leaf from Araucaria bidwillii Hook. [ABH] (Family: Araucariaceae) was evaluated in experimental models of pain and inflammation. Oral administration of 100, 200 and 300 mg/kg of leaf extracts of ABH were used for the above study. The leaf extract at 300 and 200 mg/kg showed significant reduction in acetic acid induced writhings in mice with a maximum effect of 65.1% re-duction at 300 mg/kg dose. In hot plate method the percentage of pain inhibition was found to be 81.69% and 66.1% with both the tested dose of the leaf extract respectively. The effect produced by the alcoholic extract at the highest dose was comparable to that of acetyl salicylic acid at 100 mg/kg (91.52%). The alcoholic extracts of A. bidwillii showed significant inhibition in carrageenan (18.61%, 32.12% and 45.64%) and serotonin (32.81%, 38.68% and 40.75%) induced hind paw oedema in rats at 100, 200 and 300 mg/kg of the ABH extract respectively. The anti-inflammatory effects showed by the extract were comparable to that of standard indomethacin 5 mg/kg (68.51% and 63.28%). The results suggest that the anti-inflammatory and analgesic effect of the extracts as claimed in folklore medicine, which may be me-diated via both peripheral and central mechanisms. Keywords: Araucaria bidwillii Hook, Ethanol extract, Inflammation, Anti-nociceptive, Paw oedema A scientific evaluation of herbs according to their traditional methods of use in various diseases manage-ment can incorporate into the complementary and alter-native medicine (CAM) system elsewhere. The plant Araucaria bidwillii Hook. (Family: Araucariaceae) popularly known as monkey-puzzle is a evergreen plant distributed in high altitudes hills areas of Western Ghats of Nilgiris and northern part of Himalayan regions in India [ 1 , 2 ]. The leaves and oleoresins of this plant is traditionally used to treat pain, inflammation and mental stress in thoda tribes of Nilgiris district, Tamil Nadu [ 0 ]. The decoction of the oleoresins is traditionally used in Thailand for treatment of insomnia [ 4 ]. Anti-ulcer and anti-pyretic activity has been reported for leaf and oleo-resins of this plant [ 5 - 7 ]. The genus Araucaria is rich in biflavones and many of them have been isolated from the leaves. In order to evaluate the pharmacological basis for the use of A. bidwillii Hook. in folk medicine for treatment of pain and inflammation, the present study was designed to investigate the antinociceptive and anti-inflammatory effect of alcoholic extract of the leaves on inflammation induced edema and pain behavior in animals. MATERIALS AND METHODS Plant Material The fresh leaves of A. bidwillii were collected from the Botanical Garden, Udhagamandalam, Nilgiris Dis-trict, Tamil Nadu. The plant materials were authenti-cated at survey of medicinal plants and collection unit, Government of India, Nilgiris District. A voucher specimen (JUNPSL 2002-01) of this plant material has been retained in the School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata. Preparation of Extracts The shade dried, coarse powdered leaves of A. bid-willii were extracted separately in the soxhlet extraction apparatus using ethanol (95%). The resultant alcoholic extract was concentrated using rotary vacuum evapora-tor. The extracts were then freeze-dried and stored in vacuum desiccator (yield of the leaf extract was 13.16% w/w). Phytochemical Character of the Extract The hydroalcoholic extracts of leaf was subjected to qualitative analysis where the presence of major phyto-constituents like flavanoids were found. The leaf extract was chromatographed on precoated silica gel GF254 plate using mixture of toluene: ethyl acetate: formic acid: water (35:50:10:5) as mobile phase. Silica gel plate was first observed under UV 254 nm, UV 366 nm and then sprayed with natural product – polyethylene glycol reagent (NP-PEG) to detect the presence of the flavonoids [ 8 , 9 ]. A number of various yellow–green spots were observed with Rf value between 0.16-0.60. This characteristic spots are in accordance with the ear-lier phytochemical investigation of Araucaria bidwillii Hook, in which several biflavones have been reported to be present in the leaf extracts [ 10 , 11 ]. Further isolation of the various phytoconstituents from leaf is under pro-gress in our laboratory. Chemicals and Drugs Serotonin, and carrageenan were purchased from Sigma Chemical Co., St. Louis, MO. Acetylsalicylic acid, indomethacin, carboxy methyl cellulose and acetic acid were purchased from Ranbaxy laboratories, New Delhi, India. Other reagents were of analytical grade and were procured form SISCO Research Laboratories Pvt Ltd, Mumbai, India. Test Samples and Standards Suspension of the ethanol extract of the leaf (AEL) of A. bidwillii was prepared in sodium carboxy methyl cellulose (CMC, 0.3%) using distilled water. Acetylsali-cylic acid (100 mg/kg) and indomethacin (5 mg/kg) were used as standards. Gastric administration of all drugs was accomplished via oral gavage. Test Animals Wister rats (200–250 g) and albino mice (20–25 g) of either sex were used in this investigation. Animals were maintained under standard environmental condi-tions and had free access to feed and water ad libitum. Experiments on animals were performed based on ani-mal ethics guidelines of Institutional Animal Ethics Committee. Albino mice (n=6, per group) were used for anti-nociceptive activity and Wister rats (n=6, per group) were used for anti-inflammatory screening and divided into five different groups. First group served as control animals they were treated with 0.3% CMC. Three groups of animals were treated with the alcoholic leaf extract (AEL) at three different doses (100, 200 and 300 mg/kg). Fifth group of animals were treated with standard drugs. The standard drugs acetyl salicylic acid 100 mg/kg (anti-nociceptive activity) or indomethacin 5 mg/kg b.wt (anti-inflammatory activity) were used. Acute Toxicity Study in Mice Acute toxicity was carried out using 50% mortality till 24 h following oral administration of extracts in Swiss albino mice and the LD50 was calculated [ 12 ]. Animals were divided into different groups. The control animal group received the vehicle (0.3% CMC) while the test group treated with various graded dose of leaf extract orally. Animals were observed individually after dosing. Observation includes mortality and gross behav-iors like body positions, locomotion, rearing, tremors, gait was observed. The effect of leaf extract on passiv-ity, grip strength, pain response, stereotypy, vocaliza-tion, righting reflex, were also assessed [ 13 ]. Anti-Nociceptive Activity Hot Plate Method. Albino mice were placed in alu-minum hot plate kept at a temperature of 55 ± 0.5 °C for a maximum time of 30 s [ 14 ]. Reaction time was re-corded when animals licked their fore, hind paws and jumped at before 0, 15, 30, 45 and 60 min and after oral administration of AEL (100, 200 and 300 mg/kg). Ace-tyl salicylic acid 100 mg/kg was used as a reference drug. Acetic Acid-Induced Writhing Test. Antinocicep-tive response of the extract AEL (100, 200 and 300 mg/kg) was assessed by counting number of writhes (constriction of abdomen, turning of trunk and extension of hind legs) induced by 1% acetic acid solution (1mL: 100 g) in mice [ 15 ]. Number of writhes per animal was counted during 30 min test period, beginning 3 min after the injection of acetic acid. Acetyl salicylic acid 100 mg/kg b.wt was used as a reference drug. Anti-Inflammatory Activity Carrageenan Induced Rat Paw Oedema. Oedema was induced by subplanter injection of 0.1 mL of 1 % freshly prepared suspension of carrageenan into the right hind paws of the rats of all groups. The volume of the injected paws and contra-lateral paws were meas-ured at 1, 2, 3, 4 and 5 hours intervals using Plethys-mometer [ 16 ]. The AEL (100, 200 and 300 mg/kg) ex-tracts was administered to three groups of animal and remaining two groups of animals received 0.3% CMC (Control 10 mL/kg) and indomethacin 5 mg/kg as stan-dard drug respectively. Serotonin Induced Rat Paw Oedema. The paw oedema was induced in the right hind paw by sub planter injection of 0.05 mL of 1 % freshly prepared solution of serotonin [ 16 ]. The volume of injected paws and contra-lateral paws were measured at 1, 2, 3, 4 and 5 hours intervals using plethysmometer. AEL (100, 200 and 300mg/kg) extracts was administered to three groups of animal and remaining groups of animals re-ceived 0.3% CMC (Control 10 mL/kg) and indometha-cin 5 mg/kg (Standard) respectively for assessing com-parative pharmacological significance. Statistical Analysis Results are reported as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). If the overall p-Value was found statistically significant (p < 0.05), further comparisons among groups were made according to post hoc Tukey’s test. All statistical analyses were performed using SPSS statistical version 8 software package (SPSS® Inc., USA). RESULTS Effect of Hydro Alcoholic Extracts of ABH on Acute Toxicity Test in Mice Animal treated with 5000 mg/kg of alcoholic extract of leaf (AEL) was observed for 24 hrs and showed no changes in behavior, a fact indicating low toxicity of the AEL tested at various dose levels. Hot Plate Test Fig 1 shows the results of the hot plate test. Three doses of extracts of A. bidwillii increased the reaction time in a dose-dependent manner to the thermal stimulus. The highest nociception inhibition of thermal stimu-lus was exhibited at a higher dose of the extracts 300 mg/kg of leaf AEL (81.69%), which is comparable to the acetylsalicylic acid (91.52%). Acetic Acid-Induced Writhing Test Dose dependent antinoceceptive effect was noted with the extract at the tested dose levels ( Fig 2 ). Maxi-mum percentage of inhibition of writhing response ex-hibited by the AEL extract at 300 mg/kg was 65.1%, while the same at 200 and 100 mg/kg showed 54.64 and 30.8% reduction in acetic acid induced writhing response respectively, which was comparable to that of standard acetylsalicylic acid (100 mg/kg) that caused 54.08% pain inhibition. Carrageenan-Induced Edema Test Effect of the extracts and reference drug on paw edema induced by carrageenan, has been shown in Fig 3 . Paw edema in rats reached its peak at 4 hrs after car-rageenan administration. Administration of various doses of AEL produced a significant inhibition of the oedema at the end of 3 hrs with carrageenan administra-tion. Maximum percentage of inhibition of oedema ex-hibited by the AEL extract at 300 mg/kg was 45.64%. This effect was comparable to that standard indometha-cin. Serotonin Induced Rat Paw Oedema The results of serotonin induced rat paw oedema test were reported in Fig 4 . It was observed that leaf extracts of A. bidwillii exerted a significant edema reduction from the first hour and remained along the time. Ad-ministration of AEL at different doses produced signifi-cant inhibition (p < 0.05) of oedema at the end of 3 hr with serotonin administration. However higher dose 200 and 300 mg/kg of AEL extracts exhibited maximum inhibition of paw edema (38.68 and 40.75%) respec-tively as compared to that of control group. DISCUSSION Pain and inflammation is associated with many pathophysiology of various clinical conditions like ar-thritis, cancer and vascular diseases [ 18 - 20 ]. A number of natural products are used in various traditional medi-cal systems to treat relief of symptoms from pain and inflammation. The AEL extract demonstrated significant antinociceptive activity at two different dose lev-els in various animal models of pain. In hot plate test, nociceptive reaction towards thermal stimuli in mice is a well-validated model for detection of opiate analgesic as well as several types of analgesic drugs from spinal ori-gin [ 21 ]. Acetic acid-induced writhing has been used as a model of chemonociception induced pain, which in-creases PGE2 and PGF2 peripherally [ 22 ]. Thus the anti-nociceptive activity shown by AEL extracts in hot plate and acetic acid induced writhing test indicate that alcoholic extracts of leaf might possess centrally and peripherally mediated anti-nociceptive properties. It is well known that the carrageenan-induced paw oedema is characterized by a biphasic event, with involvement of different inflammatory mediators: in first phase (dur-ing the first 2 h after carrageenan injection), chemical mediators such as histamine and serotonin play a role, while in second phase (3–5 h after carrageenan injec-tion) kinin and prostaglandin are also involved [ 23 ]. Our results revealed that administration of AEL inhibited the edema starting from the first hour and during all phases of inflammation, which is probably inhibition of differ-ent aspects and chemical mediators of inflammation. The effects of alcoholic extract of ABH in inflammation process induced by serotonin suggest that they act by affecting a time-delayed system in a similar fashion to glucocorticoids. The ability of the extracts to suppress abdominal writhes, increase pain threshold latency, inhibition of the phases of carrageenan as well as suppression of the serotonin induced inflammation confirm the analgesic and anti-inflammatory properties of the extract. The chemical analysis of the leaf extract showed the presence of biflavones as the major constituents. These findings justify traditional use of this plant in the treatment of pain and other inflammatory conditions and validate its claim of being used for the said purpose in folklore medicine. It can be concluded that alcoholic extracts of ABH possesses analgesic and anti- inflam-matory properties, which are probably mediated via inhibition of prostaglandin synthesis as well as central inhibitory mechanisms which may be of potential bene-fit for the management of pain and inflammatory disorders. ACKNOWLEDGEMENT We are thankful to Department of Science and Technology (DST), Govt of India, New Delhi for the necessary financial assistance to Dr. [Mrs] Kakali Muk-herjee through Fast Track Project grant. Thanks are also due to Dabur Research Foundation, India for providing the fellowship to Mr. KFH Nazeer Ahamed. Authors are thankful to Dr. V. Ram Sunder, Manager, Botanical Garden, Udhagamandalam, Nilgiris District, Tamil Nadu for his help in collection of the plant material. REFERENCES
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