Anti-mycobacterium tuberculosis activity of polyherbal medicines used for the treatment of tuberculosis in Eastern Cape, South Africa

Background The emergence of drug-resistant strains of Mycobacterium tuberculosis has become a global public health problem. Polyherbal medicines offer great hope for developing alternative drugs for the treatment of tuberculosis. Objective To evaluate the anti-tubercular activity of polyherbal medicines used for the treatment of tuberculosis. Methods The remedies were screened against Mycobacterium tuberculosis H37Rv using Middlebrook 7H9 media and MGIT BACTEC 960 system. They were liquid preparations from King Williams Town site A (KWTa), King Williams Town site B (KWTb), King Williams Town site C (KWTc), Hogsback first site (HBfs), Hogsback second site (HBss), Hogsback third site (HBts), East London (EL), Alice (AL) and Fort Beaufort (FB). Results The susceptibility testing revealed that all the remedies contain anti-tubercular activity with KWTa, KWTb, KWTc, HBfs, HBts, AL and FB exhibiting more activity at a concentration below 25 µl/ml. Furthermore, MIC values exhibited inhibitory activity with the most active remedies from KWTa, HBfs and HBts at 1.562 µg/ml. However, isoniazid showed more inhibitory activity against M. tuberculosis at 0.05 µg/ml when compare to the polyherbal remedies. Conclusion This study has indicated that these remedies could be potential sources of new anti-mycobacterial agents against M. tuberculosis. However, the activity of these preparations and their active principles still require in vivo study in order to assess their future as new anti-tuberculosis agents.


Introduction
Mycobacterium tuberculosis, the leading causative agent of tuberculosis (TB) is responsible for the morbidity and mortality of a large population worldwide 1 . TB has a long co-evolutionary history with humans. It does not exhibit any symptom of disease except when impairment of immunity arises due to malnutrition, diabetes, malignancy and AIDS 2 ; however, about 10% of healthy individuals may develop active TB in their life time due to genetic factors. The ability of TB to resist drugs and the influence of HIV epidemic has made the disease remain a devastating global public health problem 3 . According to WHO 4 , one-third of the world's population have been infected with Mycobacterium tuberculosis (MTB). In 2014, an estimated number of 9.6 million new TB infections were reported, of which 5.4 million were men; 3.2 million were women and 1.0 million children 3 . This disease is responsible for approximately two million deaths annually 5 . Some of the main obstacles to the global control of the disease are the HIV epidemic that has dramatically increased the risk of developing active TB, increasing emergence of multidrug resistant-TB (MDR-TB: resistance to isoniazid and rifampin) and refractory nature of latent TB treatment to conventional anti-TB drugs 6,7, 8,9 . The situation is further exacerbated by the increasing development of extensively drug-resistant (resistant to MDR-TB, all fluoroquinolones and at least one of the second-line anti-TB injectable drugs including amikacin, kanamycin and/or capreomycin) 10,11 . According to the modes of action of these drugs, they can be grouped as cell wall inhibitors (isoniazid, ethambutol, ethionamide, cycloserine), nucleic acid synthesis inhibitors (rifampicin and quinolones), protein synthesis inhibitors (streptomycin, kanamycin) and inhibitors of membrane energy metabolism (pyrazinamide) 12,13,14 . For instance, Isoniazid (INH )is the most widely used treatment for TB and its latent infections 15 . This drug enters the cell as a pro-drug, which is activated by MTB catalase-peroxidase enzyme (KatG). The enzyme activates INH and facilitates its interaction with various toxic reactive species (oxides, hydroxyl radicals and organic moieties) in the bacterial cell 16 , thereby, weakening the components of the cell wall and finally, the death of the bacteria 17 . INH targets inhA enzyme (enoylacyl carrier protein reductase), which is involved in the elongation of fatty acids in mycolic acid synthesis 18 . The replacement of an amino acid in the NADH binding site of inhA results into INH resistance, preventing the inhibition of mycolic acid biosynthesis 19 . INH-resistant strains often lose catalase and peroxidase activities due to KatG Ser315Thr mutation 20 . Resistance to INH can also occur through mutations in the promoter region of inhA, leading to over expression of inhA, or by mutations at the inhA active site, thereby lowering inhA affinity for INH 21 .
Rifampicin (RIF) have been used as first-line drug in combination with other therapies for the treatment of TB infections. RIF is believed to inhibit bacterial DNA-dependent RNA polymerase 9 . This drug interferes with RNA synthesis by binding to the β subunit of mycobacterial RNA polymerase, which is encoded by rpoB, thereby killing the organism. Resistance to RIF arises due to missense mutations in the gene. Mtb resistance to RIF occurs at a frequency of 10−7 to 10−8 as a result of mutations in rpoB 22 . About 96% of all mutations are found in the 81-bp core region of the gene between codons 507 and 533, with the most common changes occurring in codons Ser531Leu,His526Tyr and Asp516Val 23 .
Pyrazinamide (PZA) is another vital first-line drug used for the treatment of TB. It plays an important role in reducing the duration of TB treatment 24 . PZA is a pro-drug that requires conversion to its active form, pyrazinoic acid (POA) by the mycobacterial enzyme pyrazinamidase/nicotinamidase. The efflux system of the mycobacterial cell enables massive accumulation of POA in the bacterial cytoplasm, leading to disruption of the bacterial membrane potential 25,26 . The exact mechanism of PZA resistance remains unknown 9 . However, PZA resistance has been associated with defective pyrazinamidase/nicotinamidase activity which results from mutations that might occur at different regions (3-17, 61-85 and 132-142) of pyrazinamidase/ nicotinamidase 27 .
Ethambutol (EMB) is a first-line drug used in combination with INH, RIF and PZA preventing the emergence of drug resistance mycobacterium. This drug interferes with the cell wall of MTB through a synthetic mechanism thereby inhibiting arabinosyl-transferase (embB), an enzyme involved in cell wall biosynthesis28. The enzyme has been proposed as the target of EMB in Mtb11. Mutation is the cause of EMB resistance and it occurs at a rate of approximately 1 in 107 organisms. It increases the production of arabinosyl-transferase, which overwhelms the inhibitory effects of EMB. Studies have revealed five mutations in codon 306 accounting 70-90% of all EMB resistant strains 29 . The resistance of Mtb to TB-drugs is mostly due to mutation which is a cause for concern. Therefore, it is important to search for new anti-tuberculosis agents, preferably those that can be readily and simply produced from medicinal plants.
It has been estimated that about 80% of South African population is infected with tuberculosis, with 88% highest prevalence of latent TB among the age group of 30-39 years old living in the rural settlements 30 . However, the strains of drug resistant tuberculosis have been on increase yearly in the country 31 . Polyherbal remedies have been used extensively for the treatment of various diseases for many centuries. They are mixtures of various herbs which contain multiple active constituents and act synergistically against infections 32 . Natural products and/or their semi-synthetic derivatives are important sources of new chemical compounds that might play an important role in the chemotherapy of tuberculosis 33 . Several studies on the use of polyherbal medicines have revealed that these therapies possess pharmacological functions. For instance, Rajanyamalakadi, a polyherbal preparation which contains three herbal ingredients has been proven to show significant anti-diabetic, hypolipidemic and anti-oxidant properties 34 . Also, Polyherbal health tonic tea used for the treatment of an array of diseases affecting humans and Sanjivani Vati used for the treatment of cough and cold have been shown to possess significant pharmacological activities 35,36 . Other Polyherbal remedies such as Livina, Rhumapar tablet, Diakyur and Sugar Remedy have been proven to contain pharmacological activities 37,38,39,40 .
Many researchers have reported on the inhibitory properties of medicinal plants against Mycobacterium tuberculosis both in South Africa and in other countries 33,41,42 but there is a dearth of information on the inhibitory properties of polyherbal medicines against this organism. The aim of the present study therefore was to evaluate polyherbal remedies used for the treatment of TB for anti-Mycobacterium tuberculosis activities. The small number of remedies obtained in this study was due to the fact that only a few traditional healers treat and sell remedies for TB. They claim to have acquired the knowledge from their ancestors; and this knowledge is been transferred from one generation to another. The herbal ingredients present in each of the remedies are shown in Table 1

Sample preparation
The already prepared water remedies were put in 2-liter containers. Each remedy was filtered with a Buchner funnel and Whatman No. 1 filter paper. The filtrate obtained was frozen at -40°C and freeze dried for 48h using a freeze dryer (Vir-Tis benchtop K, Vir-Tis Co., Gardiner, NY). The resulting sample was dissolved in 100% dimethylsulfoxide (DMSO) to a concentration of 50 mg/ ml to make a stock solution 45 .

Microbial strain and medium used for the assays
Reference MTB strain H37Rv (ATCC 25618) was used for the anti-Mycobacterium tuberculosis assay. It was obtained from American Type, MD, USA Culture Collection. Bacterial culture with DMSO (1.2%), isoniazid (INH) at MIC99 (0.05 µg/ml) and bacterial culture only were used as controls 46 .

Bacterial culture and drug preparation
Suspensions of Mycobacterium tuberculosis H37Rv were grown using mycobacterial growth indicator tubes (MGIT). The inocula were prepared from Lowenstein-Jensen slants. To prepare an inoculum that was less than 15 days old from a culture grown on Lowenstein-Jensen medium, a suspension was prepared in saline and adjusted to a 1.0 McFarland standard. The suspension was vortexed for several minutes and was allowed to stand for 20 min for the initial settling of larger particles. The supernatant was transferred to an empty sterile tube and was allowed to stand for an additional 15 min. After being transferred to a new sterile tube, it was then adjusted to a 0.5 McFarland turbidity standard by visual comparison. A 1:5 dilution of the bacterial suspension was prepared, and 0.5 ml was inoculated into MGIT 7H12® (MGIT 960 system, Becton Dickinson, Sparks, USA) tubes containing test and control compounds 46 . The growth of the organism was monitored through fluorescent changes due to oxygen consumption in the medium during active growth. Aliquots (100 µl) of each herbal medicine was added to the MGIT tubes containing bacteria in Middlebrook 7H12® media, with the final DMSO concentration not exceeding 1.2%. The tubes were incubated at 37°C in MGIT system, and growth units (GU) were monitored for six days. All the remedies were tested at concentrations of 50 and 25 ug/ml 46 . For MIC99 evaluations, a 1% bacterial control culture was prepared in a drug-free MGIT tube and the MIC 99 of the compound determined relative to the growth units of the control (GU-400). The MIC was determined as the lowest drug concentration that equals or lower than GU of the 1% bacterial culture. Controls that were also included are bacterial culture with DMSO (1.2%), isoniazid (INH) and bacterial culture only. All the herbal preparations were tested at two-fold decreasing concentration 46 .

Results
In the present study, the susceptibility and minimum inhibitory concentration (MIC) of nine polyherbal medicines were determined against M. tuberculosis H37Rv, in vitro. The susceptibility testing revealed that all the remedies have anti-tubercular activity against M. tuberculosis H37Rv at concentrations below 50 ug/ml. Seven of these polyherbal preparations, namely; KWTa, KWTb, KWTc, HBfs, HBts, AL and FB showed activity at concentrations below 25 ug/ml, with the remaining remedies showing activity at concentrations between 25 and 50 ug/ml ( Table 2). All the remedies exhibited inhibitory activity against M. tuberculosis H37Rv with KWTa, HBfs and HBts as the most active remedies at 1.562 µg/ml, followed by AL remedy which showed growth inhibition at 3.125 µg/ml. The remaining preparations from KWTb, KWTc, HBss, EL and FB showed growth inhibition against M. tuberculosis at 25 µg/ml. However, isoniazid showed more inhibitory activity against M. tuberculosis H37Rv at 0.05 µg/ml when compared to the polyherbal remedies (Table 2).  51,15,52 . Allium sativum is a plant that has been reported as an established remedy for the treatment of tuberculosis 53 . It possesses variety of biological properties such as anti-cancer, anti-microbial, antioxidant, immunomodulatory, anti-inflammatory, hypoglycaemic and anti-cardiovascular properties 54 . Several studies conducted on the in vitro activity of Allium sativum against Mycobacterium tuberculosis revealed that this plant possesses anti-tubercular properties 41,42,53,53 . The presence of sulphur compounds such as allicin, ajoene, allylmethyltrisulfide, diallyltrisulfide, diallyldisulphide has been associated with the anti-tubercular activity of this Allium sativum 55 .
Information on the use of Strychnos decussate as an anti-tubercular agent has not been reported. This study is the first to report the use of this plant as a remedy for the treatment of TB. However, it has been reported to possess anti-fungal activity 56 . Daucus carota is a root vegetable. There are only a few reports on the anti-tubercular activity of this plant 57,58 . However, it has been reported to be used as an anti-bacterial 59 , anti-fertility 60 , anti-oxidant 61 , ophthalmic and stimulant 62 , anti-septic, diuretic, hepatoprotective, anti-inflammatory 63,64 , anti-helmintic, carminative 65 , deobstruent, diuretic and galactogogue. According to the reports, phenolics, polyacetylenes, carotenoids, ascorbic acid and tocopherol are the most abundant phytonutrients present in this plant 66 . Hypoxis argentea has also been reported to be used as a remedy for the treatment of TB 58 . Species of the genus Hypoxis have been used as anti-bacterial, anti-fungal, anti-viral, anti-oxidant, anti-inflammatory, anti-diabetic, cardiovascular, anti-convulsant and anti-cancer 67,68,69,70,71 . The presence of several compounds, especially glucosides, sterols and sterolins could be responsible for the different activities found in Hypoxis 72 . Rapanea melanophloeo has been screened for activity and found active against drug-resistant and drug-sensitive strains of Mycobacterium tuberculosis 73,74 . This plant has been reported to contain bioactive compounds such as benzoquinones, saponins and tannins which could probably contribute to its activity 73 . The high activity of these polyherbal remedies against M. tuberculosis could be attributed to the presence of multiple active constituents which may act in synergy and produce greater anti-mycobacterial activity. This is an indication that many natural products are potential source of antimycobacterial agents 42 .

Conclusion
This study has revealed that polyherbal remedies have the potential to cure tuberculosis. This is the first research work on the anti-tuberculosis activity of polyherbal medicines used for the treatment of tuberculosis in South Africa. The remedies might be potential sources of new anti-mycobacterial agents as they all showed activity against M. tuberculosis. However, the activity of these remedies and their active principles still require in vivo study in order to validate their potential as anti-tuberculosis agents.