Anti-inflammatory effect of methanolic extract of Gmelina arborea bark and its fractions against carrageenan induced paw oedema in rats

Abstract Gmelina arborea has been traditionally used for treatment of abdominal pain, burning sensation and as stomachic. The anti-inflammatory effect of aqueous and methanol extract of this plant has been studied but detailed investigations on anti-inflammatory activity of G. arborea stem bark are still not available. Therefore, the present study was designed to evaluate the anti-inflammatory activity of methanol extract and its fractions using carrageenan induced paw oedema model. Methanol extract at the dose of 500 mg/kg and its ethyl acetate fraction at 50 mg/kg showed significant reduction in paw oedema in comparison with standard drug. Ethyl acetate fraction was subjected to column chromatography which resulted in isolation of a new flavonoid (GM-01). Anti-inflammatory effect of methanol extract and its fractions can be attributed to the presence of the flavonoid GM-01. Further studies are underway for evaluation of anti-inflammatory effect of GM-01 and identification of active constituents present in ethyl acetate fraction.


General
The bark was obtained from Madurai district of Tamil Nadu in August 2011. The taxonomic identity of the plant was confirmed by Mr. Ram Prasad, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar and a voucher (# S.R.BotSci/96) has been deposited in department herbarium. Carrageenan (Sigma Chemicals, St. Louis, USA) and diclofenac sodium injection (Novartis India Ltd., Bombay) were used in the present study. All other solvents and reagents were of analytical grade. Melting point was determined with melting point apparatus. An IR spectrum was recorded using KBr pellets on Shimadzu 8400 S FT-IR Spectrophotometer. Mass spectra E 1 and ESI-methods were recorded on Shimadzu GCMS-QP-2000A. The 1 H-NMR was recorded on a 300 MHz FT-NMR and chemical shifts were reported in δ values using tetramethylsilane as internal standard.

Preparation and fractionation of methanol extract
Methanol extract was prepared by subjecting powdered stem bark to successive soxhlet extraction for not less than 48 hours using petroleum ether (60-80 o C), chloroform, methanol and water. Each extract was concentrated by distilling off the solvent using rotary evaporator and evaporated to dryness on the water-bath. Forty grams of the methanol extract was suspended uniformly in 100mL water and extracted with hexane. Further fractionation was done using ethyl acetate (3x150 mL) and butanol (3x150 mL). Ethyl acetate fraction (1g) was subjected to column chromatography using silica gel (# 60-120). A total of 300 fractions, each of 25 ml, were collected and pooled on the basis of TLC profile to get nine fractions F 1 -F 9 . F 8 was eluted using mobile phase hexane: ethyl acetate (98:2) and it showed a single spot of yellow color on TLC which led to isolation of GM-01. Characterization of GM-01 was done by spectroscopic analysis which revealed that the phytoconstituent belongs to flavonoid family.

Animal study
Wistar rats (100-150 g) were obtained from Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. They were fed a standard rodent diet with water ad libitum. The protocol was duly approved by the Institutional Animal Ethics committee (16/1009/BT) of Guru Nanak Dev University, Amritsar. Carrageenan was used to induce inflammation (oedema) in paw. The linear paw circumference was measured at hourly interval for 4 h using digital vernier calliper. The animals were divided into nine groups, each containing 6 rats. The doses of the methanol extract for animal study was selected based on the literature survey and in previous study, similar doses of 250 and 500mg/kg were given to animals for evaluating the anti-inflammatory activity. The doses of ethyl acetate and butanol fractions were selected based on their yield. The percentage (%) inhibition of oedema was calculated using formula:- Where T t = Thickness of rat paw after giving standard/extract; T o = Thickness of rat paw after injecting carrageenan

Statistical analysis
The data obtained from different groups were statistically analysed using one way analysis of variance followed by Tukey's test. Results were expressed as Mean ± SEM. p<0.05 was considered to be statistically significant. Instat software version 3 (Graph Pad Software Inc., San Diego) was used to carry out all statistical analyses.

Compound Isolation
Melting   Values are expressed as mean ± SEM, statistical analysis was performed by one way ANOVA followed by Tukey's test. a p < 0.05 vs control, b p < 0.05 vs standard. Figure S1. TLC of ethyl acetate fraction