Background
Mycobacterium tuberculosis, an obligate aerobe belonging to the
M. tuberculosis complex (also include
M. bovis, M. africanum and M. microti), is the most important cause of TB in humans. In addition to
M. tuberculosis complex,
Mycobacterium avium complex (include
M. avium, M. intracellularae, and M. kansasii) can also cause closely related mycobacterium diseases in AIDS patients and is difficult to distinguish as a group clinically [
1].
In 1993, during the world TB day, WHO declared TB as a ‘global emergency,’ which requires emergency action and launched several programs to combat the disease, including the search for newer remedies and/or anti-TB agents to complement currently used agents [
2]. In the year 2014, TB killed 1.5 million people (1.1 million HIV-negative and 0.4 million HIV-positive) and 9.6 million new cases of active tuberculosis globally caused by TB [
3].
Mycobacteria are slow growing organisms that require administration of a combination of drugs for extended periods to achieve effective therapy and to prevent the emergence of resistance. The risk of adverse reactions therefore must be a major consideration in drug selection. Apart from significant toxicity, lengthy therapy also creates poor patient compliance. Non-compliance is a frequent cause of a deadly multidrug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB). Drug toxicity coupled with the problem of mycobacterial persistence highlights the need to develop novel TB drugs that are active against drug resistant bacteria and kill persistent bacteria as well as shorten the length of TB treatment [
4].
Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity [
5]. Scientific interest in medicinal plant has grown in recent times because natural products are evolutionary shaped drugs or drug‐like molecules. Nature’s biosynthetic machinery produces innumerate natural products with distinct biological properties that make them valuable as inhibitors or promoters of biological action [
4,
6]. Moreover, higher plant extracts have been considered as promising sources of novel anti-TB leads [
7]. This has prompted us to investigate selected Ethiopian medicinal plants, including
Carissa edulis Vahl (Apocynaceae),
Otostegia integrifolia Benth (Lamiaceae (Labiatae)),
Persea Americana Mill (Lauraceae),
Pterolobium stellatum (Forsk.) Brenan. (Fabaceae), and
Vernonia amygdalina Del. (Asteraceae) for possible anti-TB activity. The plants have been traditionally used to treat respiratory or lung-related diseases, including TB for a very long time [
8‐
11]. The present study attempted to screen the antmycobacterial activity of different solvent extract of these five Ethiopian medicinal plants, and
Pterolobium stellatum was found to be the most promising plant for further investigation.
Discussion
Current TB therapy consists of treatment with a combination of drugs. This combination therapy causes hepatotoxicity as the major side effect as well as development of drug resistance. To avert toxicity and reduce ineffectiveness of current anti-TB drugs, medicinal plants are considered as potential anti-tuberculosis agents that can be used in combination with the standard anti-tuberculosis drugs or alone [
20]. In this study, antimycobacterial activity of crude extract of
O. integrifolia,
V. amygdalina,
C. edulis, P. americana,
P. stellatum and as well as fractions of the most active crude extract was investigated.
From the aerial parts of
O. integrifolia otostegin A, otostegin B, 15-
epi-otostegin B, preleoheterin, leoheterin, and related compounds, including leopersin C, 15-
epi-leopersin C, ballonigrin, vulgarol, and 8-O-acetylharpagide were isolated and reported. In addition, the essential oil and chloroform extract of air-dried leaves of
O. integrifolia constitute monoterpenes, sesquiterpenes, diterpenes and their derivatives were identified [
21]. Phytochemical screening study report indicated that saponins, glycosides and tannins, which are known to be bioactive purgative principles were present in
V. amygdalina extract. Flavonoids are also present in the plant that possess antioxidant activity and may play a beneficial role in cancer prevention and offer some protection against diabetes and atherosclerosis [
22].
The chemical compositions of
C. edulis have extensively been reported. Roots contain an active ingredient, carissin that may prove useful in the treatment of cancer. The twigs contain quebrachytol and cardioglycosides that are useful as an anthelmintic against tapeworm [
23] and the roots contain lupeol (has antiviral activity), oleuropein, carissol and β-amyrin [
24]. Major chemical constituents of
P. americana include the following: the leaf contains volatile oil, flavonoids and coumarins; the fruit contains sesquiterpenes and carbohydrates; the seed contains fixed oil consisting of vitamins A, D
3, alpha tocopherol and cholesterol. The fruit is a significant source of protein, monounsaturated fatty acids, vitamin A, thiamin, riboflavin, niacin, vitamin B6, vitamin C, vitamin E, folate, vitamin K, pantothenic acid, magnesium, manganese, phosphorus and the amino acids tryptophan, valine, tyrosine, threonine, phenylalanine and methionine [
25]. Chemical classes present in
P. stellatum 80% root extract are terpenoids, saponins and tannins and had antibacterial activity as reported by previous study.
The result of the study revealed that three of the experimental plants had significant anti-tuberculosis activity. The activity was seen with chloroform and methanol extracts of
P. stellatum and
O. integrifolia as well as with methanol and acetone extracts of
P. americana. In addition, fractions from the most active plant,
P. stellatum had demonstrated promising antimycobacterial activity [
8].
MICs of all experimental plant extracts and solvent fractions of the most active plant extract were determined by CFU method and microplate resazurin assay method. As evidenced from Tables
2 and
3,
P. stellatum chloroform extract was endowed with lower MIC by CFU method than by microplate resazurin assay method, although test organisms were different. It has been reported that the MIC exhibited by a compound/extract depends on the technique used for determination. MICs obtained in a liquid medium are lower than that obtained from a solid medium, as the drug has to diffuse through the matrix in the solid medium in order to exert activity [
20].
MIC of
P. stellatum chloroform extract was evaluated using CFU and Resazurin indicator method. The finding in the later method revealed that the MICs were 0.078, 0.156 and 0.156 mg/ml against AOZ8W-4, AOA8W-4 and SO38SW-4 MDR-TB clinical isolates, respectively. However, the extract had lower MIC (0.039 mg/ml) against the standard strain. The difference in MICs might be ascribed to the difference in susceptibility of the standard strain and MDR-TB clinical isolates towards the extract. Furthermore, this apparent difference might be attributed to more favorable growth conditions provided by agar culture medium to extract-treated bacilli in colony count method or the drug susceptible standard strain and the drug resistant strain might have different growth conditions. Another cause for the different MICs of
P. stellatum chloroform extract could be the slight difference in sensitivities of colony count method and resazurin indicator method as reported by Taneja and Tyagi [
26]. According to this study, Resazurin indicator assay was noted to be superior to the colony count assay in that it distinguished between metabolically active dormant bacteria and non-viable organisms, unlike the colony count assay that could not differentiate between these two populations.
In this study,
P. stellatum chloroform extract showed promising activity against
M. tuberculosis H37Rv and AOZ8W-4, with complete inhibition at 0.039 mg/ml and 0.078 mg/ml, respectively. These values are within the range stated by the Clinical and Laboratory Standards Institute [
27]. In addition, the MDR-TB clinical isolates were resistant to isoniazid, while this plant was active against the clinical isolates of MDR-TB, possibly suggesting that the plant could have an obvious advantage over the standard drug. Moreover, the activity displayed by this extract makes the plant to be a promising plant according to Tosun et al. [
28], which states that compounds with an MIC of less than 10 μg/ml, and ideally less than 2 μg/ml could have a potential for further investigation.
There is sparse data in the literature about the antimycobacterial activity of
P. stellatum, making comparison a bit difficult. A study reported that 80% ethanol leaf extract of the plant had shown activity (MIC = 250 mg/ml) on
M. tuberculosis H37Rv [
9]. This value is much higher than the value obtained in this study, possibly indicating that non-polar constituents might be more responsible for the observed antitubercular activity. Efforts made to compare activity of the chloroform extract of
P. stellatum with other plants showed either better or comparable activity. Activity was better compared to stem bark extract of
Anogeissus leiocarpus [
13] and comparable to that of the chloroform leaf extract of
Byrsonima fagifolia [
29].
Although 80% methanol extract of
P. stellatum (MIC = 0.312 mg/ml),
P. americana (MIC = 2.5 mg/ml) and
O. integrifolia (MIC = 0.312 mg/ml) as well as chloroform extract of
O. integrifolia (MIC = 0.312 mg/ml) and acetone extract of
P. americana (MIC = 1.25 mg/ml) had significantly higher activity than vehicle-treated controls, they failed to exhibit promising activity according to Tosun [
30] or Sánchez [
31]. The MICs in this finding are lower/equal than a study done on methanol extract of
Pelargonium sidoides (MIC = 5000 μg/ml) as well as methanol extract of
Capparis brassi,
Entada africana and
Combretum species (MIC = 1250 μg/ml each) against
M. tuberculosis H37Rv [
12]. However, the MICs were higher than that of chloroform extract of
Byrsonima fagifolia (MIC = 62.5 μg/ml) [
29]. This difference might be imputed to the fact that different plant species may contain different active constituents at varying amount and/or different in vitro methods were used.
Previous studies reported several plants with promising anti-tubercular activity [
13,
29‐
33]. Studies had mostly reported activity in the plant families of Asteraceae, Lamiaceae, Fabaceae and Apiaceae, among others [
34]. It is noticeable from the present study that plants exhibiting activity belong to Fabaceae, Lamiaceae and Lauraceae families, which is in agreement with the plant families previously reported [
34].
Antimycobacterial activity evaluation revealed that P. stellatum chloroform root extract to have a better activity and further fractionation was pursued and activity of the fractions was evaluated. Accordingly, the ethyl acetate fraction was found to be the most active fraction, based on MIC values (0.195 μg/ml), which indicated that antimycobacterial constituents were contained in this fraction. Further work is underway to isolate and characterize active principles from this fraction.