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. 2014 Apr 3;11(3):200-9.
doi: 10.4314/ajtcam.v11i3.28. eCollection 2014.

Phytochemical investigation and anti-microbial activity of Clausena anisata (Willd), Hook

Nicholas Agyepong  1 , Christian Agyare  1 , Martin Adarkwa-Yiadom  2 , Stephen Yao Gbedema  1
Affiliations

Phytochemical investigation and anti-microbial activity of Clausena anisata (Willd), Hook

Nicholas Agyepong et al. Afr J Tradit Complement Altern Med. .

Abstract

Background: Clausena anisata belongs to the family Rutaceae, a shrub widely used in West Africa for the treatment of bacterial and fungal infections of the skin including boils, ringworm and eczema. The study was designed to evaluate the antimicrobial activity and phytochemical screening of ethanol leaf extract of C. anisata (CLE).

Method: Antimicrobial activity of CLE was investigated using agar well diffusion and micro-dilution methods against four Gram-positive bacteria (Bacillus substilis NCTC 10073, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus thuringiensis ATCC 13838) and two Gram-negative bacteria (Pseudomonas aeruginosa ATCC 4853, Proteus vulgaris ATCC 4175) and a clinical isolate of Candida albicans.

Results: CLE was active against all test organisms with minimum inhibitory concentration (MIC), range of 0.5 to 7.0 mg/mL against Gram-positive bacteria, 2.5 to 1.0 mg/mL against Gram-negative bacteria and 5.5mg/mL against C. albicans. The MICs of the methanol fraction of CLE were 0.6 mg to 5.0/mL and 1.0 to 3.0 mg/mL for Gram-positive and Gram-negative bacteria respectively. Chloroform fraction had MIC of 3.0 to 7.5 mg/mL and 2.0 to 6.5 mg/mL for Gram-positive and Gram-negative bacteria, respectively and petroleum ether fraction had 4.5 to 8.0 mg/mL for Gram-positive and Gram-negative bacteria. The CLE exhibited static action against all test organisms within a range of 0.5 to 22.0 mg/mL. Phytochemical screening of C. anisata revealed the presence of tannins, flavonoids, steroids, saponins, glycosides and alkaloids. HPLC finger-printing of the CLE and its fractions were determined.

Conclusion: These results may justify the medicinal uses of C. anisata for the treatment of microbial infections.

Keywords: Phytochemical screening; antibacterial; antifungal; static action.

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Figures

Figure 2
Figure 2
HPLC chromatogram of the ethanol leaf extract (CLE) at λ 254 nm.
Figue 3
Figue 3
HPLC chromatogram of the methanol fraction of the CLE at λ 254 nm.
Figure 4
Figure 4
HPLC chromatogram of the chloroform fraction of CLE at λ 254 nm.
Figure 5
Figure 5
HPLC chromatogram of the petroleum ether fraction CLE at λ 254 nm.
Figure 6
Figure 6
The TLC chromatogram of ethanol extract and fractions. Thin layer chromatogram of crude ethanol extract (CR), methanol (M), petroleum ether (P) and chloroform (C) fractions visualised under UV (254 and 365nm), day light and sprayed with anisaldehyde reagent. Six (6) prominent bands were revealed under the UV light of the ethanol crude extract. The petroleum ether, chloroform, and methanol fractions showed 6, 5 and 3 spots respectively under the same condition. This is due to the maximum absorbance of the compound.
Figure 7
Figure 7
Bio-autography of ethnol extract and fractions of C. anisata. The bio-autography showed zones of growth inhibition against S. aureus (A) and C. albicans (B). The zones of bacteria growth inhibition were visible as yellow clear areas against purple background after spraying with MTT. Crude ethanol leaf extracts (CR), methanol (M), petroleum ether (P) and chloroform (C) fractions.
Figure 8
Figure 8
Mean zones of inhibition of different fractions (20mg/ml) of ethanol leaf extract of C. anisata . The mean zones of growth inhibition was determined from three independent result (n=3) for all test organisms. Test organisms: E. faecalis (EF), B. subtilis (BS), S. aureus (SA) P. aeruginosa (PA), P. vulgaris (PV), B. thurigiensis (BT) and C. albicans (CA).
Figure 9
Figure 9
Survival of E. faecalis in extract at different concentrations of C. anisata within 24 h. Control: Broth culture of E. faecalis without the extract.
Figure 10
Figure 10
Survival of P. aeruginosa in extract at different concentrations of C. anisata within 24 h. Control: Broth culture of P. aeruginosa without the extract.
Figure 11
Figure 11
Survival of C. albicans in extract at different concentrations of C. anisata within 24 h. Control: Broth culture of C. albicans without the extract.
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