Analgesic activities of fractions of Stereospermum kunthianum stem bark.
E Omogbai, F Abiodun, S Okpo, C Poh
Keywords
fractions, pain, stem bark., stereospermum kunthianum
Citation
E Omogbai, F Abiodun, S Okpo, C Poh. Analgesic activities of fractions of Stereospermum kunthianum stem bark.. The Internet Journal of Pharmacology. 2009 Volume 8 Number 1.
Abstract
Introduction
Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in term of such damage 1. Many pathological disorders are accompanied with painful conditions. Man from time immemorial has used various substances from animals, plants and minerals sources to alleviate pain. Many rural dwellers of the world dependent largely on herbs for the treatment of painful conditions because medicinal herbs constitute indispensable components of traditional medicine practice due to low cost, easy access and ancestral experience 2.
Materials And Methods
Plant collection and preparation:
The plant was sought for based on its uses in human traditional medicine by most rural dwellers in Africa. Fresh stem bark of
Extraction and chromatographic analyses:
The powdered plant (500 g) was extracted with methanol (4x5Lx48 h) by maceration at room temperature. The extract was evaporated to dryness to yield a residue (80 g) which was subjected to vacuum liquid chromatography over silica gel F254 using gradient solvent systems n-C6H14 : CHCl3 and CHCl3 : CH3OH, gradient up to 100 % CH3OH as eluting solvents to obtain 19 fractions. The 19 fractions were subjected to repeated thin layer chromatography (TLC) using MeOH: CHCl3 (3 : 1) as solvent system to obtain fractions with similar spots and Rf –values which were then bulked into 3 fractions [ A(27.36g), B( 41.40g) and C(10.23 g)]. Each of the fractions (A, B, and C) was evaluated for analgesic activity.
The fractions (A, B and C) were further subjected to column chromatography as previously described 8, 9. The column was eluted with solvents of increasing polarity consisting of n-C6H14: CHCl3 (9 : 1) and CHCl3 : CH3OH (1 : 4) mixtures. The column chromatography of fractions A, B and C yielded 99, 75 and 85 fractions, respectively, which were subjected to thin layer chromatographic analyses and subsequently bulked based on the Rf – values and TLC profile, to further produced 5, 6, and 4 fractions respectively, with yields as follows:
A [J(0.21g); K(1.24g); L(9.20g); M(5.39g); N(1.17g)]; B[Q(1.08g); R(1.70g); S(7.39g); T(6.82g);V(6.70g); W(6.19g)], and C[U(0.25g); X(0.50g); Y(3.36g); Z(2.30g)]. Fractions L, S and Y were evaluated for analgesic activities.
Animals
The use of animals for analgesic experiments was according to international guidelines and approved experimental protocols. Swiss mice of either sex obtained from the Animal House unit of the Department of Pharmacology & Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria were used. The animals maintained under standard laboratory conditions (12 h light and dark cycles) had free access to standard chow (Bendel Feeds and Flour Mills, Plc. Ewu, Nigeria) and drinking water.
Mouse writhing assay
The acetic acid-induced abdominal writhing test was performed according to the procedure described previously 10. Mice were randomly allocated into groups of five animals per group. The animals received normal saline (10 ml/kg, i.p), acetylsalicylic acid (100 mg/kg, sc. suspended in 5% tragacanth in normal saline), or each of the vacuum liquid chromatography fractions A, B, and C (100, 200 or 400 mg/kg, i.p.), thirty minutes before intraperitoneal injection of acetic acid (10 ml/kg, 1% v/v in normal saline). The animals were observed for writhes which consisted of constriction of the abdominal muscles together with stretching of the hind limbs. The writhes were cumulatively counted for 30 minutes following acetic acid injection and the analgesic effect determined as described 10.
Formalin test in mice
The method of Dubuisson and Dennis was used 11. Mice were randomly allotted into groups of five animals each. The animals were given normal saline (10 ml/kg i.p), morphine (10 mg/kg, i.p) or each of the column chromatography fractions L, S and Y (100, 200 or 400 mg/kg, i.p.), thirty minutes before injection of twenty microlitres of 1% formalin into the right hind paw of mice. The behavioral responses of nociception including biting and licking of the injected paw were noted and the time spent was recorded for up to 30 minutes. The first 5 minutes was considered as the early phase (neurogenic phase) and the last 15 minutes as the late phase (inflammatory phase) of the nociceptive response. Analgesic effect was expressed as a reduction in the time spent in licking or biting of the injected paw.
Statistical Analysis
Data were presented as mean ± SEM and analyzed using the Student’s t-test. Results were considered significant at p<0.05, p<0.0001. Pain inhibition is expressed as simple percentage.
Results
The present study was designed to assess the analgesic activity of the fractions of
Values are mean ± SEM. *P<0.0001 significantly different from the normal saline treated animals, student’s t-test (n=5). Inhibition of pain is expressed as a percentage in parentheses.
Figure 1
Values are mean ± SEM, **P <0.0001 significantly differently from the normal saline treated animals, Student’s t-test (n = 5). Pain inhibition is presented as percentage.
Figure 2
Values are mean ± SEM,*P<0.05, **p <0.0001, significantly differently from the normal saline treated animals, Student’s t-test (n = 5). Pain inhibition is presented as percentage.
Figure 3
Values are mean ±SEM, *P<0.05,**P <0.0001, significantly differently from the normal saline treated animals, Student t-test ( n = 5). Pain inhibition is presented as percentage.
Figure 4
Discussion
The results of the present study demonstrated that the fractions of
Conclusion
It can be concluded that the fractionation of
Acknowledgement
The authors are grateful for the Niger Delta University Postgraduate Fellowship to one of us (Ching FP).