Anti-mycobacterium activity
Tuberculosis is ranked second after HIV-AIDS as the leading cause of death worldwide [
29]. Although Western methods of healing may be preferred by modernized individuals, the traditional medicines still serve as a primary health care system preferred in developing countries. The results for both the anti-mycobacterial and cytotoxic activity of fractions, isolated compounds and derivatives from
Curtisia dentata are shown in Table 1. The methanol fraction was the most active with minimum inhibitory concentration (MIC) of 22.2 μg/ml; this is followed by acetone extracts with minimum inhibitory concentration of 44.2 μg/ml. The chloroform fraction and ethanol extracts had MIC values of >50 mg/ml against the selected
Mycobacterium strain. The implication on fractional extracts in the current study is that the compounds responsible for the activity in the plant extracts may have been more soluble, abundantly and highly distributed between the methanol and acetone as moderately polar solvents. The isolated compounds had MIC values of greater than 50 μg/ml. On the contrary, ursolic acid purchased from Sigma Aldrich (Germany) revealed a potent MIC values ranging from10 to 20 μg/ml against the same
Mycobacterium species in the resazurin assay [
30]. Besides differences in terms of assays and agar medium used in maintaining the organism, the level of purity of the compound may also play a role in the different results obtained. A mixture of oleanoleic acid and ursolic acid revealed an MIC value of 62.5 μg/ml against
M. tuberculosis while a mixture of lupeol, β-amyrin and alpha amyrenone revealed an MIC value of 312.25 μg/ml [
31].
In the current study, the fractions revealed better inhibition of
M. tuberculosis compared to the isolated compounds. Contrarily, [
32] reported the isolated compounds to possess more anti-tubercular activity compared to fractions.
In the current study, ursolic acid acetate (UAA) and betulinic acid acetate (BAA) revealed the most potent anti-tubercular activity compared to both the fractions and the isolated compounds, revealing the MIC values of 3.4 and 19.8 μg/ml respectively. These results are in accordance with those of [
33], which revealed that the derivatives possess much higher anti-tubercular activity compared to the parental compound isolated from plant materials. The anti-tubercular activity observed in the current work is much greater on derivatives followed by fractions and then isolated parent compounds. The outer cell wall of the
Mycobacterium is unique because it possess the lipid rich bilayer that consist of mycolic acid- high molecular weight fatty acids which contains 60 to 90 carbon atoms with a basic β-hydroxyl-α-alkyl branched structure [
34]. For that reason, in the quest to find new drugs, the focus should be on the plant materials with potential to inhibit mycolic acid and this may well explain the lengthy treatment given to patients. However, we still need to further assess the anti-mycobacterial activity of the active plant materials from
C. dentata and further study the possible mode of action of such extracts, compounds and derivatives.
Earlier, our research group investigated the antimicrobial potential of leaf extracts from
C. dentata against organisms that may cause sexually transmitted infections and opportunists isolated from immunocompromised HIV patient [
35‐
37]. The acetone extract had the lowest MIC value of 0.01 mg/ml against
C. albicans while ethanol extract had an MIC value of 0.10 mg/ml against
M. hominis. Furthermore, the diethyl ether extract had MIC values of 3.13 mg/ml against
Escherichia coli,
Proteus mirailis and
Moraxella catarrhalis, thereby validating the use of the plant species in the treatment of sexually transmitted and related urinary tract infections. In the antioxidant assay, the acetone extract had up to 52% inhibition of DPPH at 1 mg/100 ml.
Assuming the MIC and IC50 to be at 50 and 300 μg/ml respectively, the selectivity index (SI) of the isolated compounds and derivatives was calculated. Betulinic acid and β-sitosterol have a potent SI value of 6 in both cell lines. The safety margin of the two compounds is better guaranteed compared to that of lupeol and ursolic acid.
In the cytotoxicity studies, the selected derivatives and isolated compounds exhibited some varying degrees of toxicity (Table
1). Generally, the isolated compounds were not toxic to cell lines used in the current study. Lupeol had an IC
50 of 278.8 and 289.4 μg/ml against HEK 293 and HepG2 respectively. However, other authors only refer to the IC
50 of 100 μg/ml as potentially toxic to cell lines [
38]. Moreover, the American National Cancer Institute (NCI) refer to an IC
50 of less than 30 μg/ml to be toxic after an incubation period of 72 h [
39], while others refer to IC
50 of greater than 20 μg/ml as toxic [
40].
Table 1
Anti-mycobacterial activity of extracts, compounds and derivatives from C. dentata
Fractions | Acetone | 44.2 | | | | |
Ethanol | >50 | | | | |
Chloroform | >50 | | | | |
Methanol | 22.2 | | | | |
Isolated compounds | β-sitosterol | >50 | >300 | >300 | 6 | 6 |
Betulinic acid | >50 | >300 | >300 | 6 | 6 |
Ursolic acid | >50 | 122.4 | >300 | 2.45 | 6 |
Lupeol | >50 | 278.8 | 289.4 | 5.58 | 5.79 |
Derivatives | Betulinic acid acetate | 19.8 | 357.80 ± 2.14 | 358.20 ± 2.23 | 18.1 | 18.1 |
Ursolic acid acetate | 3.4 | 340.02 ± 4.12 | 328.39 ± 3.10 | 100 | 96.59 |
Positive controls | | MIC in μM | | | | |
Rifampicin | 0.02 | | | | |
Isoniazid | 0.43 | | | | |
Streptomycin | 0.25 | | | | |
TMC207 | 0.02 | | | | |
From the MIC and IC
50 values, we calculated the selectivity index (SI). SI indicates the cytotoxic selectivity or safety of the crude extract or isolated compound against the selected cell lines [
41,
42]. The selectivity index of the derivatives was much higher compared to those of the parent compounds, suggesting the safety of the derivatives is much better compared to that of parent compounds. The derivatives exhibited high selectivity index values, indicating the wider difference between their cytotoxicity and antimicrobial activity.