The Neuropharmacological evaluation of ocimum kenyense, ocimum kilimandscharicum and ocimum masaiense species found in Kenya
The genus ocimum consists of 35 species of aromatic annual perennial herbs and shrubs in the family Lamiaceae. The members of this genus find extensive application in traditional medicine systems all over the world. This study screened organic extracts prepared from three species indigenous to Kenya; Ocimum kilimandscharicum, Ocimum masaiense and Ocimum kenyense for analgesic activity. The most potent extract from the screening experiments then underwent bioassay guided fractionation procedures in an attempt to isolate the chemical moiety (-ies) responsible for the antinociceptive activity. Organic extracts were prepared from the various plant parts of these species that is the root, stem and leaf by soxhlet extraction using ethanol, chloroform and 1: 1 chloroform/ethanol mixture as the extraction solvents. The resulting extracts were then evaporated to dryness using a rotor evaporator. The antinocicptive activities of these extracts were then assayed in the Tail Flick Test (radiant form). The experimental data was analyzed statistically with Kruksall-Wallis non-parametric test using GraphPad Prism® statistical software with p<O.05 being set as the level of significance. Extracts with the most potent antinociceptive activity were then sleeted for evaluation in the Formalin Test and for fractionation. Various receptor agonists/ blockers were administered together with the extract in the Formalin Test in an attempt to elucidate the mechanism(s) of the antinociceptive action of the extract. The extract underwent fractionation using the liquid-solid adsorption column chromatography technique using dichloromethane, ethyl acetate and methanol as the eluting solvents. The resulting fractions were combined on the basis of TLC records. The antinociceptive activity of the resulting fractions was assayed in the Tail Flick Test. The most potent non polar fractions underwent further fractionation using a combination of Preparative TLC and Liquid-solid adsorption chromatography techniques until pure compounds were yielded. The antinociceptive activity of the pure compounds was assayed in the Tail Flick Test and their structure elucidated using a combination of Proton NMR and GC-MS. Twenty one of the twenty five extracts screened showed significant antinociceptive activity in the Tail Flick Test. The chloroform/ethanol extract of Ocimum masatense roots possessed the most potent analgesic activity at the 100mg/kg dose level (12.6 ± 2.61 seconds) in this test (p=0.0041). It also showed significant antinociceptive activity in the Formalin Test (p<O.OOOl). Atropine (p= 0.0009) significantly enhanced the antinociceptive effects of the extract in the early phase of the Formalin Test but had no significant effects in the late phase. Ketamine (p= 0.0005) and Naloxone (p=0.005) significantly inhibited but did not abolish the antinociceptive effects of the extract in the first phase but had no significant effects in the late phase of the Formalin Test. Capsaicin had a significant inhibitory effect but did not abolish the antinociceptive effect of the extract in both phases of the Formalin Test (p= 0.006 and p= 0.0001). Fractionation of ten (10) grams of dichloromethane/methanol extract of Ocimum masaiense roots using Dichloromethane, ethyl acetate and methanol as the successive elution solvents yielded six fractions on the basis of TLC patterns. All the fractions showed significant antinociceptive activity in the Tail Flick Test (p< 0.05). Fractionation of fractions I, II and III using Hexane and hexane; dichloromethane mixtures (Hexane 90%, DCM 10%) yielded two compounds (Ia and Ib) in sizable quantities of 3g and 2g respectively. These compounds showed significant antinociceptive activity in the Tail Flick Test (p<0.0003 and p<0.006 respectively). Compound Ia was identified as betulinic acid while the structure of compound Ib could not be conclusively determined. It is therefore concluded that; 1. Organic extracts prepared from various plant parts from three Ocimum species indigenous to Kenya that is Ocimum masaiense, Ocimum kilimandscharicum and Ocimum kenyense possessed statistically significant antinociceptive activity in the Tail Flick Test. This validates the traditional uses of Ocimum kilimandscharicum and Ocimum kenyense as analgesics/antirheumatics. 2. Betulinic acid a compound with known analgesic activity was isolated from Ocimum masaiense; the first time this compound has been isolated from an Ocimum species.