Collection of plant materials
Barks of M. nagi (MN) were purchased from a local market. The plant was identified and authenticated by S. Kitchlu, Indian institute of integrative medicine (CSIR), Jammu, India. A voucher specimen (SU/DPS/Herb/32) of the same has been deposited in the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot for future reference.
Preparation of plant extract
Barks were dried in shade, moderately ground by electric grinder and subjected to soxhlet extraction using ethyl acetate and later solvent was evaporated at reduced pressure to afford ethyl acetate extract (yield-5.2% w/w). M. nagi aqueous extract was obtained by boiling fresh powder in distilled water (100° C) and later by evaporating water from the decanted portion under reduced pressure (yield-29.4% w/w). The extracts were stored in refrigerator and prepared freshly in sodium carboxy methyl cellulose (SCMC) solution just before the experiments.
Carrageenan induced rat paw edema
The rats were divided into six groups containing five rats in each group. Acute inflammation was induced16
by 0.1 ml of 1.0% of λ-carrageenan in normal saline (0.9% w/v NaCl) was injected to the sub plantar region of left hind paw. The extract was administered once to the rats 1 hr before λ-carrageenan injection.
Different groups were treated as follows:
Group I: λ -Carrageenan + Vehicle
Group II: λ -Carrageenan + Aspirin (100 mg/kg. P.o.)
Group III: λ -Carrageenan + Ethyl acetate extract (100 mg/kg. P.o.)
Group IV: λ -Carrageenan + Ethyl acetate extract (200 mg/kg. P.o.)
Group V: λ -Carrageenan + Aqueous extract (100 mg/kg. P.o.)
Group VI: λ -Carrageenan + Aqueous extract (200 mg/kg. P.o.)
The paw volume was measured at 3 hrs after λ -carrageenan injection with the help of plethysmometer. The anti inflammatory activity was evaluated based on the ration of the changes in paw diameter in treated and untreated groups as per the formula given below:
Anti inflammatory activity (%) = [1-(Vt/Vc)] × 100
Vtand Vc are edema volume in drug treated and control groups respectively.
Male Wistar albino rats (250-300 g) were subjected to carrageenan and histamine rat paw edema induced inflammation tests (n=5, in each group). All the animals were housed in groups in polypropylene cages and placed in climate controlled central animal house having temperature 22 ± 2°C, relative humidity 60 ± 5%, and a 12 h light/dark cycle (lights on at 08:00 h and off at 20:00 h). The animals were fed standard pellet diet (Amrut, Pranav Agro Industries Ltd, India) and water ad libitum. All the protocols were approved (approval no-SU/DPS/IAEC/1005) by Institutional Animal Ethics Committee (IAEC) of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India.
Administration of drugs
Aspirin was dissolved in distilled water. While ethyl acetate and aqueous extract were prepared as suspension in distilled water using 0.5% SCMC as the suspending agent. Animals were assigned to different treatment groups (n=5, in each group). The control group received 0.5% SCMC, (1 ml/kg, p.o.), whereas test groups received ethyl acetate and aqueous extract. All drugs, ethyl acetate and aqueous extract were prepared before 1.00hr of experimentation. All the doses of extracts were administered orally.
Acute toxicity study
The acute toxicity study was performed as per the method described by Litchfield and Wilcoxon (1949) and LD50 was calculated accordingly. Briefly, the Ethyl acetate and aqueous extract in the dose range of 10-1600 mg/kg were administered orally to different groups of mice (n = 10). The animals were examined every 30 min up to a period of 3 h and then, occasionally for additional period of 4 h; finally, overnight mortality was recorded. All tests on rats were performed at three dose levels 100 and 200 mg/kg, p.o. body weight corresponding to 10 and 20% of LD50 value (1000 mg/kg, i.p.), respectively.
All the data were expressed as mean ± SEM from five animals. The data obtained was analyzed using the one-way ANOVA followed by Dunnett Multiple Comparisons Test for determining the level of significance and p < 0.05 was considered statistically significant.