Tuberculosis (TB) is a communicable disease that has caused ill health to humanity for many years. TB was the leading cause of death worldwide from a single infectious agent until 2020, when the coronavirus (COVID-19) pandemic emerged. According to the World Health Organization (WHO), approximately 10.6 million people fell ill with TB in 2021, which represents a 4.5% increase from 10.1 million in 2020. The disease also claimed 1.6 million lives globally, with the burden mainly confined within 30 countries, which account for over 86% of all cases [1] .

Although TB treatment is effective, its duration is long, and anti-TB drugs can cause serious side effects. These factors, among others, confer poor adherence of patients to chemotherapy, and patients frequently abandon treatment. This, consequently, may result in secondary resistance to anti-TB drugs and eventual spread of drug-resistant bacilli. Ultimately, this leads to primary resistance (presence of resistant Mycobacterium tuberculosis in patients with no prior history of treatment) [2] .

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB continues to be a public health threat. In 2021, the World Health Organization estimated that the proportion of people with MDR-TB to be 3.6% and 18% among new and previously treated cases respectively. In the same year, 25,038 new cases of XDR were detected globally [1] . The standard short-course treatment of TB requires a combination of anti-TB drugs with a duration of six months. This regimen has been found to give an 85% - 90% success rate in 100% complaint TB patients. Patients with MDR-TB are treated with a regimen of four to five anti-TB drugs for a period of greater than 15 months after a negative culture is obtained [3] . This long duration, accompanied by frequent serious adverse effects, leads to poor compliance and cessation of treatment [4] [5] .

Medicinal plants have for centuries been used as a source of medicine and in Uganda, traditional medicine use is found to be between 60% - 80% [6] [7] . Many plant species have broadly been studied for their activity against Mycobacterium tuberculosis. Recently, a number of studies have reported that native Ugandan plants used traditionally for respiratory diseases and other ailments have antimycobacterial activity [8] - [12] . It has been reported that quite a number of people in Africa use both medicinal plants and conventional medications concurrently without any knowledge of possible interactions [13] . The deficiency of knowledge of interactions between medicinal plants and conventional drugs and as well as the lack of reporting of traditional medicine use to healthcare practitioners can pose a serious risk to patient safety and treatment outcome [14] [15] . It has also been noted that even in some of the world-class hospitals in Africa, traditional medicine is found to be used concurrently with conventional therapies [16] .

Bunalema and colleagues reported that medicinal plants, Zanthoxylum leprieurii Guill. & Perr. and Rubia cordifolia L. have been extensively used by traditional medicine practitioners (TMPs) for the treatment of tuberculosis either singly or in combination with standard anti-tuberculosis drugs [8] . This study therefore aimed to investigate the in vitro activity of extracts of Zanthoxylum leprieurii and Rubia cordifolia in combination with standard anti-TB drugs against two MTB strains (pansensitive H37Rv and MDR-TB strain).

The study was performed as an antimycobacterial assay. Data collected were

MTB colony counts on 7H11 media for 8 weeks. The collected data were then processed using the proportion method, which resulted in a percentage inhibition as shown in Table 2 for the preliminary study and Table 3 for the extract-drug combination assay.

Starting with the preliminary study (individual extract antimycobacterial activity), the methanol extract of Zanthoxylum leprieurii exhibited 100% growth inhibition against both H37Rv and MDR Mycobacterium tuberculosis strains at concentration of 100 µg/mL and above. Both extracts showed preferentially high inhibition rates against MDR strain. A concentration of 500 µg/mL was used as the highest concentration for both Z. leprieurii and R. cordifolia extracts for the combination study because it gave 100% growth inhibition for both MTB strains. The results indicated a dose‒response relationship, thus justifying the lowering of extract concentrations to 50 µg/mL in the combination assay.

The results indicated an additive effect when rifampicin was combined with both plant extracts against the H37Rv strain. Specifically, both plant extracts exhibited complete (100%) inhibition at a lower concentration of 50 µg/mL against the pansensitive MTB strain when combined with rifampicin. An increased potency for the combination of ethambutol with the extracts against the MDR-TB strain was observed. In contrast, the results indicated a decrease in potency for the combination of rifampicin and isoniazid against MDR-TB isolates. Noticeably, Z. leprieurii extract alone exhibited 100% growth inhibition at 50 µg/mL, while its combination with isoniazid at a concentration of 100 µg/mL yielded 94% and 95% inhibition against H37Rv and MDR-TB, respectively. There was a remarkable increase in the percentage inhibition of extract-drug combinations against MDR-TB compared to individual single-standard anti-tuberculosis

*Colony Forming Unit.

drugs. Individual standard anti-TB drugs showed a growth inhibition ranging from as low as 5% to 52% against MDR-MTB strain, whereas on combination with both extracts, ethambutol and streptomycin exhibited complete inhibition (100%) against the same strain.

Tuberculosis treatment is intrinsically associated with major challenges, such as complex and lengthy treatment. These have a direct impact on patient compliance and treatment outcome. Recently, there has been another concern regarding the increasing incidence of MDR and XDR tuberculosis [21] . As a result of the growing burden of drug-resistant genotypes of Mycobacterium tuberculosis coupled with TB-HIV coinfection, the burden of TB is now quite difficult to manage. Therefore, there is a global imperative to discover and develop new and efficacious antituberculosis agents, which should be an urgent biomedical priority [23] [24] . The plants selected in the current study are commonly used in Ugandan traditional medicine to treat tuberculosis and related ailments and have also been studied for their antimycobacterial activity against different strains of Mycobacterium tuberculosis [4] [12] [25] . However, little is known about the effects of combination of medicinal plants and conventional antituberculosis drugs.

This study is the first to report on the combination effect of Zanthoxylum leprieurii and Rubia cordifolia with standard antituberculosis drugs in Uganda. Previous studies have only focused on the antimycobacterial activities of these plants. It has been demonstrated that when used in combination, drugs with significantly low efficacy have therapeutic effects, even against drug-resistant strains [3] . Drug combination treatment has long been employed as a standard treatment strategy for many chronic diseases, including tuberculosis. With an objective to elucidate any potential synergistic interactions of the two commonly used medicinal plant extracts with standard first-line anti-TB drugs, we evaluated the antimycobacterial activity (percentage inhibition) of Z. leprieurii and R. cordifolia methanol extracts in combination with rifampicin, isoniazid, ethambutol and streptomycin against M. tuberculosis (H37Rv and MDR) strains.

Rifampicin alone exhibited 100% and 29% growth inhibition at 1 µg/mL against pansensitive H37Rv and MDR strains, respectively. On combination with Z. leprieurii and R. cordifolia extracts at a concentration of 50 µg/mL, an increase in growth inhibition was observed at 86% and 63% respectively against MDR-MTB. When each of the extracts at a concentration of 50% µg/mL was combined with isoniazid at 0.2 µg/mL, an additive effect was observed against the MDR-strain, resulting in 91% and 62% inhibition by Z. leprieurii and R. cordifolia against the MDR strain, respectively. The mechanism contributing to this synergistic effect against MDR strains is not yet known; however, there is a likelihood that since isoniazid inhibits mycolic acid synthesis, these plant extracts inhibit cell wall synthesis [3] [26] .

All anti-TB drugs used in this study, rifampicin at 1 µg/mL, isoniazid at 0.2 µg/mL, ethambutol at 7.5 µg/mL and streptomycin at 2 µg/mL, exhibited a growth inhibition of not more than 52% against the MDR strain. However, a synergistic effect was observed when the standard drugs were combined with both extracts at even lower concentrations. They exhibited mycobacterial growth inhibition of between 62% and 100% against the MDR strain. This finding was consistent with other standard anti-TB drugs studied against MDR strains. This is in tandem with other previous studies that indicated that extracts of Z. leprieurii and R. cordifolia inhibited the growth of the MDR strain of Mycobacterium tuberculosis [4] [25] [27] .

This study demonstrated that extracts of Z. leprieurii and R. cordifolia possess significant in vitro antimycobacterial activities against both pansensitive (H37Rv) and multidrug-resistant (MDR) MTB strains. Additionally, the extracts exhibited additive and/or synergistic interactions when combined with standard anti-tuberculosis drugs, more remarkably against MDR-MTB. This implies that pure compounds of these plants have the capability of reducing the doses and duration of treatment when combined with conventional anti-TB drugs. This may result in a multitude of advantages, ranging from preventing the emergence of resistance, increased compliancy and general reduction in global tuberculosis burden.

This study demonstrated that Zanthoxylum leprieurii Guill. & Perr. and Rubia cordifolia L. extracts increase inhibitory activity of standard anti-tuberculosis drugs, especially against MDR-TB strain. This study verified local use of these plants in the traditional treatment of tuberculosis in Uganda. However, the use of these medicinal plants as traditional anti-tuberculosis remedies in combination with standard anti-TB drugs should be done cautiously, especially on individuals with susceptible tuberculosis, since not all extract-drug combinations showed good in vitro combination effects. These plants’ cytotoxicity and intracellular activities should be determined to further justify their traditional use either singly or in combination with conventional anti-tuberculosis drugs.

The authors express gratitude to PHARMBIOTRAC-African Center of Excellence (ACE II) project of Mbarara University of Science and Technology for funding this research. We are also pleased with the team from Mycobacteriology BSL-3 Laboratory, Department of Microbiology, College of Health Sciences, Makerere University for allowing this study to be carried in their facility.

MM developed the concept and proposal; MM and KK collected, sorted the data; MM analyzed the data and wrote the manuscript; JB, DS, PO supervised the study and reviewed the final manuscript. All authors reviewed and approved the final manuscript.

Approval was sought from Institutional Review Board of Mbarara University of Science and Technology. Permission to collect plant parts from Mabira forest was obtained from National Forest Authority (NFA), Kampala, Uganda.