Background
Mycobacterium tuberculosis, the bacillus that causes tuberculosis (TB), is one of the leading causes of illness and death worldwide, accounting for 9.6 million new cases and 1.5 million deaths per year [
1]. Africa carries the most severe TB burden with a prevalence of 281 cases per 100,000 individuals compared to the global average of 133/100, 000 [
2]. Uganda, one of the high TB burden countries, reports approximately 166 new cases per 100,000 people [
3]. The high TB burden is attributed to Human Immune Virus (HIV) co-infection in addition to development of multi- and extensively drug resistant strains (MDR and XDR) of TB [
3]. Worldwide, MDR TB was reported to account for 5% of all new tuberculosis cases [
4] of which 9% was caused by XDR-TB [
5].
Treatment of resistant strains of MDR TB presents a challenge globally because it involves use of more than three drugs for a long period which results in non adherence and development of even more resistant strains [
6]. While new drugs like bedaquiline and delamanid have recently been discovered and approved for use in MDR TB [
7], they are expensive and mostly unavailable in poor resource countries. Furthermore, discovery of these new drugs took approximately 40 years since the last new anti TB drug was introduced. This slow pace in drug development in addition to the rapid emergence and spread of resistant TB calls for continuous search for new agents that are effective against resistant
M. tuberculosis.
Medicinal plants have long been used for treatment of different ailments in local communities [
8]. As such, drugs like chloroquine, artemisinin derivatives and morphine have been derived from different plant sources [
9].
Zanthoxyllum contains about 549 species distributed worldwide; making it the largest genus in
Rutaceae [
10]
Zanthoxyllum leprieurii, a plant species belonging to the same family, is traditionally used in the treatment of HIV/AIDS, malaria, urinary infections, rheumatic pain and is used as an antiseptic in Africa [
11,
12]. In addition, it is used by communities in Uganda for management of TB and cough related infections [
13,
14]. Pharmacological assessment of
Z. leprieurii could potentially lead to the discovery of new anti-TB agents as well as verify its use among local people in Uganda communities. The aims of the study were to; assess the anti mycobacterial activity of
Z. leprieurii, to isolate compounds from
Z. leprieurii and to determine the antimycobacterial activity of the isolated compounds against a pan sensitive, rifampicin resistant and isoniazid resistant strains of
M. tuberculosis.
Discussion
>The search for new anti TB drugs is continuing to be the focus for most research globally due to development of multi drug and extensively drug resistant strains of
M. tuberculosis. Although drugs like bedaquiline and delamanid have recently been discovered and are used in treatment of MDR TB [
7], they are equally expensive and unavailable in poor resource countries. Furthermore reports of totally drug resistant TB in countries like India and South Africa are threatening to make the disease untreatable [
16,
17]. In this study, the anti mycobacterial activity of the crude extracts, fractions and compounds from
Z. leprieurii were evaluated to help demonstrate the plant’s potential as an alternative source for new anti TB agents. The extracts/fractions were considered to be active if the MICs were ≤ 100 μg/ml [
18,
19] while the pure compounds were considered to have antimycobacterial activity if their MIC ≤ 6.25 μg/ml; the maximum tested concentration. In the same way an extract, fraction or compound was considered not to be active if the MIC value was beyond the tested concentration.
Both crude extracts from
Z. leprieurii showed some inhibitory activity with the methanol extract being more active against all the
M. tuberculosis strains as compared to the chloroform extract. This could be due to numerous compounds extracted by methanol which is believed to be an efficient solvent during extraction [
20‐
22]. The methanol extract was fractionated and fractions Za
4 and Za
6 were the most active against all the tested strains; implying that they could possibly contain compounds which could be active on MDR TB warranting further phytopharmacological analysis. However, the activity of the crude extracts was generally lower than that of the pure drugs; isoniazid and rifampicin. The low anti-TB inhibition exhibited by the crude extracts could be due to presence of impurities which may interfere with and reduce the potency of the extracts [
23]. Over all, purification of
Z. leprieurii methanol extract yielded fractions that showed substantial activity than the crude extracts especially against the pan sensitive strain. Among these, fraction Za
4 (MIC = 6.3 μg/ml) was seven times more potent than the methanol crude extract (MIC = 47.5 μg/ml).
Three known acridone alkaloids that is; 2-hydroxy-1, 3-dimethoxy-10-methyl-9-acridone (
1), 1-hydroxy-3-methoxy-10-methyl-9-acridone (
2) and 3-hydroxy-1, 5, 6-trimethoxy-9-acridone (
3) were isolated from fractions Za
4 and Za
7. Their structures were confirmed by analyzing their spectral data and also through comparison with already published work [
24‐
26]. When screened for anti mycobacterial activity, compound
1 was found to have a lower MIC value (MIC = 1.5 μg/ml) than both isoniazid (MIC = 0.2 μg/ml) and rifampin (MIC = 0.4 μg/ml); two first line drugs that form a backbone in TB treatment [
27]. This shows that there is potential for further development of compound
1 into new anti TB agents that could be an alternative to the two most important first line drugs in TB treatment. On the contrary, Compound
2 was inactive on
M. tuberculosis. The variation in activity could not be verified in this study however it is thought that it could be linked to the number and positions of hydroxyl and methoxy groups on rings A and B. Previous studies have shown that the antimycobacterial activity of alkaloids is affected by the number and positions of functional groups [
28].
Previous studies done on Cameroonian spices revealed that
Z. xanthoxyloides and
Z. macrophylla showed weak activity with MICs of 1.024 mg/ml against H37Rv and H37Ra [
29]. However a study by Luo et al., [
30] showed that extracts from
Z. capense had antimycobacterial activity. In addition, decarine, a benzophenanthridine alkaloid was isolated and found to inhibit growth of
M. tuberculosis within macrophages with MIC of 1.6 μg/ml on the pan sensitive strain. Compound
3 has been shown to have cytotoxic effects (IC50 of 86 μM) against liver cancer cell lines by inhibiting glycosyltransferase and aromatase enzymes in the liver [
31]. In addition compound
2 had a moderate activity of 33% mortality against
Anopheles gambiae larvae at a concentration of 1000 ppm [
31]. In a review by Kishore et al., (2009) [
32], different types of alkaloids have been shown to have anti mycobacterial activity however no study has reported activity of acridone alkaloids on resistant strains of TB. Our results add to other plant derived compounds from genus Zanthoxyllum which could be further explored as anti TB agents.
Acknowledgements
We would like to thank all members from Joint Clinical Research Centre, in particular Sam Ogwang for all the advice they gave us while carrying out the bioassays. Also would like to acknowledge support of Colleagues from University of Botswana for all the technical advice they offered during isolation.