MATERIALS AND METHODS

Plant collection: The C. brevipes plant was collected on 20th January, 2009 from a dry farmland at Lalupon village in Ibadan, Oyo state, Nigeria. The plant was authenticated by Mr. Ugbogu O.A. and Mr. Soyewo L.T. of Plant Taxonomy Department, Forestry Research Institute Ibadan, Nigeria (FRIN). A voucher specimen FHI NO 108450 was deposited at FRIN herbarium.

Plant extraction: The plant was air dried and crushed with mechanical crusher. The grinded material was hermetically sealed in plastic for use. The plant sample (2000 g) was subjected to cold extraction. It was soaked in 99% ethanol for a week. The ethanol extract was then collected and concentrated using rotary evaporator at 35°C to give ethanol residue. The ethanol residue (150 g) was then macerated with n-hexane, chloroform and EtOAc successively. Each of the extracts were collected separately and concentrated to give hexane, chloroform and EtOAc residues respectively using rotary evaporator at 35°C.

Antimycobacterial susceptibility test: This was carried out at the TB Research Laboratory, The Zankli Medical Centre (ZMC), Abuja, Nigeria. The agar dilution method using the egg enriched Lowenstein-Jansen (LJ) medium was used as reported by the International Union Against Tuberculosis LJ Medium, 2007 (Nwachukwu et al., 2009). This was done in a safety cabinet Lamilplus 7. Isoniazid, ethanbutol, dihydrostreptomycin and rifampicin were used as standard control drugs at final drug concentration of 0.2, 2.0, 8.0 and 40.0 μg mL^-1, respectively in the LJ. The phytodrugs were screened at 5 mg mL^-1. The slopes were stored at 4°C prior to inoculation. Well characterized clinical isolates (MTB 050 and MTB 303) of Mycobacterium tuberculosis which are positive to NO₃ reduction, negative catalyst labile tests and shows the presence of serpentinous cords on Zn smear. The isolates obtained from the ZMC were diluted in sterile distilled water to 10^-2 and 10^-4. This corresponds to 1.0 and 0.5 Mcfarland, respectively. 10 μL of each of the 10^-2 and 10^-4 innoculum concentrations were inoculated using a micropipette on separate standard drugs, phytodrugs and negative control LJ slant media and incubated for six weeks at 37°C. Inoculated media were checked after 3 days for contamination.

Phytochemical methods: Preliminary phytochemical tests for alkaloids, steroids, tannins, anthraquinone, resins, carbohydrate, glycosides, saponins, phenolics and flavonoids were carried out on the extracts. The methods were based on those reported by Trease and Evans (1989) and Sofowora (1993).

RESULTS AND DISCUSSION

Phytochemical test: The result of phytochemical screening revealed some differences in the constituents of the three extracts tested distributed in varying degrees (Table 1). Results obtained from the qualitative phytochemical tests carried out on the fractionated extracts revealed that the extracts contained a wide array of phytochemicals which include carbohydrate, tannins, flavonoids, alkaloids, sterols, resins and phenols. The absence of glycosides, saponins and anthraquinones was also observed. It is a fact that the phytochemical constituent can help one to speculate on the medicinal value of the stem bark. Flavonoids, Tannins, saponins and alkaloids have been reported to have pronounced physiological effect particularly on the nervous system. The presence of these phytochemicals in the C. brivipes extracts suggests that the plant is pharmacologically active, supporting the claim by traditional healers. The result supported the reported phytochemical components indicating presence of alkaloids and flavonoids in the Coffea genus (Simkin et al., 2008; Koshiro et al., 2006; Andrade et al., 1998). Phytochemical results of each extracts also suggested that carbohydrate, alkaloids, tanins, sterols, flavonoids, resins and phenols can be isolated from C. brivipes plant.

Table 1:	Phytochemical screening of the extract of C. brivipes
+: Present; -: Not present

Antimycrobacterial susceptibility test: Table 2 shows that there was no growth of MTB 050 observed against Isoniazid (0.2 μg mL^-1), Dihydrostreptomycin (8.0 μg mL^-1), Ethanbutol (2.0 μg mL^-1), Hexane extract (5 mg mL^-1), Ethyl acetate extract (5 mg mL^-1) after 6-weeks of incubation. These results suggested that the drugs and extracts are active against the MTB 050 isolate. The MTB 050 was not susceptible (after 6-weeks of incubation) to rifampicin because bacterial colonies growth observed was in the range of 200-500 and 20-100 for 10^-2 and 10^-4 inoculums concentrations, respectively. The chloroform extract (5 mg mL^-1) showed partial activity against MTB 050 because at it was not active at high inoculums concentration.

The results of antimycrobacterial susceptibility screening (Table 3) depicted the following: Hexane extract show activity at 5 mg mL^-1 against both MTB 303 at 10^-2 and 10^-4 innoculum concentrations; chloroform fraction show activity at 5 mg mL^-1 against MTB 303 at both innoculum (10^-2 and 10^-4) while ethylacetate fraction show activity at 5 mg mL^-1 against MTB 303 at low innoculum (10^-4).

Table 2:	Antimycobacterial susceptibility result for MTB 050 of the extract of C. brivipes
-: No growth after 6 weeks incubation at 37°C, +: 1-19 colonies growth after 6 weeks incubation at 37°C, +1: 20-100 colonies growth after 6 weeks incubation at 37°C, +2: 100-200 colonies growth after 6 weeks incubation at 37°C, +3: 200-500 colonies growth after 6 weeks incubation at 37°C, 4+: >500 colonies growth (confluent growth) after 6weeks incubation at 37°C

Table 3:	Antimycobacterial susceptibility result for MTB 303 of the extract of C. brivipes
-: No growth after 6 weeks incubation at 37°C ,+: 1-19 colonies growth after 6 weeks incubation at 37°C, +1: 20-100 colonies growth after 6 weeks incubation at 37°C, +2: 100-200 colonies growth after 6 weeks incubation at 37°C, +3: 200-500 colonies growth after 6 weeks incubation at 37°C, 4+: >500 colonies growth (confluent growth) after 6weeks incubation at 37°C

MTB 050 strains show resistance to rifampicin activity at both innoculum whereas hexane and ethylacetate fractions exhibited good bacterialcidal activity at 5 mg mL^-1 against this rifampicin resistance strain, which serve as a good replacement for rifampicin as an anti-Tb drug. Other tested standard drugs exhibited good bactericidal activity on the two strains at both innoculum except rifampicin that show no activity on MTB 050 strain at both innoculum.

The result of the phytochemical and antimycrobacterial studies suggested the presence of active anti-Tb agent(s) from C. brivipes plant, a contribution to the development of new pharmaceuticals which can serve as good replacement for the existing Tb-drugs. The presence of these compounds may have accounted for the bacterialcidal activity of the various fractions of C. brivipes extract against Mycobacterium tuberculosis strains. Phenolic compounds, alkaloid and saponins are known to exhibit antibacterial activity and antioxidant activity (Ayoola et al., 2008; Hwang et al., 2001; Claus et al., 1990).

CONCLUSION

The active phyto-compound(s) in this present study can be isolated from various fractions of C. brivipes plant paving way for the discovery of novel drugs to combat the rifampicin resistant mycobacterial strain as indicated. The advantage of the extract of C. brivipes plant over the rifampicin and the possible mode of action calls for further work.

ACKNOWLEDGMENT

The authors wish to thank the management of the Zankli Medical Centre, Abuja for making it possible for us to have access to the facilities at their TB research laboratory. We also wish to thank ISESCO-COMSTECH for given IAO a research grant (No. 3189).