Evaluation of anti-Mycobacterium tuberculosis activity of fractions from selected medicinal plants used traditionally for treating cough and respiratory disorders in South West of Nigeria

Tuberculosis is a contagious airborne infection that mostly affects the lungs. The causative agent of tuberculosis in human is Mycobacterium tuberculosis. The emergence and dissemination of M. tuberculosis isolates that are resistant to multiple antimicrobial drugs represent a growing public health threat. Fractions from Alafia barteri, Chasmanthera dependence, Chrysophyllum albidum, Emilia coccinea, Mezoneuron benthamianum, Phyllanthus muellerianus, Secamoni afzeli, Senna alata, Xylopia aethiopica and Acalypha fimbriata were screened for activity against drug susceptible M. tuberculosis H37Rv and the local isolates using proportion and nitrate reduction methods. The organisms used were M. tuberculosis H37Rv strain and the local isolates from TB patients. The standard antitubercular drugs used were isoniazid and rifampicin. No fractions from A. barterii, C. dependens, E. coccinea, S. afzeli, S. alata and X. aethiopica showed sensitivity against the M. tuberculosis strains. The hexane fraction of C. albidum, butanol fraction of M. benthamianum, ethyl acetate fraction of P. muellerianus and ethyl acetate fraction of A. fimbriata showed sensitivity with minimum inhibition concentration of 0.5 mg/ml. The ethylacetate and hexane fractions of M. benthamianum together with hexane fraction of P. muellerianus showed sensitivity with MIC value of 1.25 mg/ml. The highest MIC value of 2.5 mg/ml was obtained from hexane fraction of A. fimbriata. Thus, C. albidum, M. benthamianum, P. muellerianus and A. fimbriata possessed antimycobacterium tuberculosis activity and further research work would be required to assess possible antitubercular agents present in the four medicinal plants.


INTRODUCTION
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis.It is transmitted from an in active tuberculosis patient by exposure to tubercle bacilli air-borne droplets from coughing or sneezing.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Tuberculosis spreads easily in overcrowded settings and in conditions of malnutrition and poverty.In 1993, the World Health Organization (WHO) declared tuberculosis a global emergency because it killed more adults each year than any other infectious disease (Kandel et al., 2008).Tuberculosis still constitutes a major health problem in Nigeria.According to the WHO, the estimated incidence of TB in Nigeria is 322 per 100,000 population with only 15% of the total burden of the disease in the country being notified in 2015 (Onyedum et al., 2017).Over 80 % of TB cases in Nigeria were still undetected while it claimed over 1.5 million lives annually in the country (Olokor, 2017).
Mycobacterium tuberculosis is successful in surviving the presence of toxic compounds because they produce effective permeability barriers comprising the outer membrane and the mycolate-containing cell wall on the surface (Liu et al., 2016).The ability of the organism to remain dormant or persistent within host cells for many years with the potential to be activated allows the bacterium to escape the immune system of the host (Meena and Rajini, 2010).Survival mechanisms of the bacterium include prevention of phago-lysosome fusion (Pieters, 2008), prevention of cell acidification (Queval et al., 2017) and protection against reactive nitrogen intermediates (RNI) (Rousseau et al., 2004).
A person infected with M. tuberculosis incurs 10% risk of developing active TB (WHO, 2007).Major risk factors for TB activation include HIV infection, recent contact with an infectious patient, initiation of an anti-tumor necrosis factor (TNF) treatment, receiving dialysis, receiving an organ or hematologic transplantation, silicosis, being in prison, being an immigrant from high TB burden countries, being a homeless person and being an illicit drug users (WHO, 2018).Alcohol consumption, particularly heavy consumption, is an important risk factor for tuberculosis (Lonroth et al., 2008;Rehm et al., 2009).
Adverse effects of antituberculosis drugs, drug interactions, high cost of drugs, shortage of drugs and a complex long time therapeutic regimen still make TB one of the major health challenges in the world (Arbex et al., 2010;Sotgiu et al., 2015).The emergence of Multi-Drug Resistant Tuberculosis (MDR-TB) and Extended-Drug Resistant Tuberculosis (XDR-TB) strains has threatened the efficacy of many existing antibiotics (Calligaro et al., 2014;Prasad et al., 2017).
Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind.Natural products, either as pure compounds or as standardized plant extracts provide unlimited opportunities for new drug leads because of the unmatched chemical diversity (Mahalingam et al, 2011).Herbal drugs whether extract or decoction used against any pathogen will not cause the problem of drug resistance (Shashidhar et al., 2015).
Pure drugs or synthezised drugs are expensive and sometimes are not available in remote areas (Ammal and Bar, 2013).The search for new plant chemicals as antimicrobial agents becomes paramount because of an increase in antimicrobial resistance by pathogens and the emergence of new drug-resistant pathogens.Among the 11 currently used nature-derived TB drugs, seven of them were either isolated from microbes or semisynthesized from microbial natural products (Liu et al., 2016) for example, streptomycin and kanamycin from Streptomyces griseus and capreomycin isolated from S. capreolus (Copp, 2003;Shu, 1998).
Rifampicin is a semi-synthetic drug that has been derived from Rifamycin, a product of A. mediterranei (Tribuddharat and Fennewald, 1999).Thus, plant kingdom can be looked at as an important source of new drugs for the treatment of TB because of its enormous chemical diversity (Gautam et al., 2007).The new drugs may not necessarily be new antibiotics but rather other drugs that prevent persistence within the host and leave the vegetative cells susceptible to treatment.
Anti-mycobacterium studies of some Nigeria medicinal plants demonstrate that they could be good sources of compounds with anti-mycobacterium activities worth of investigation (Mann et al., 2008;Ibekwe and Ameh, 2014).Some medicinal plants have been reported to possess anti-mycobacterium tuberculosis activity in Nigeria (Adeleye et al., 2008;Faleyimu et al., 2009).The selection of the ten medicinal plants is based on their usage by traditional practitioners in treating tuberculosis, cough or respiratory disorders.
The aim of this research work was to evaluate the antimycobacterium tuberculosis activity of the selected medicinal plants.

Plant materials
The medicinal plants were obtained from Olokemeji Forest Reserve in Oyo state, Iberekodo market in Ogun state and Mushin market in Lagos state.The plants were identified by Mr T. K. Odewo of the Forestry Research Institute of Nigeria, (FRIN), Ibadan.

Preparation of extracts
80% ethanol solutions were added to the dried powdered samples of the plants.The mixtures were kept at room temperature for 72 h with gentle and intermittent shaking and thereafter filtered.Filtrates were dried at 42.5°C.Sequential extraction with hexane, ethyl acetate and butanol solvents were carried out.

Preparation of samples of roasted seeds of C. albidum
In line with the folklore usage of the seeds of C. albidum in treating tuberculosis infection, 100 sun dried seeds of the plant were put in a closed crucible and heated for 30 min at 50, 100 and 120°C separately.The seeds were removed from their shells after heating and were ground to powder for hexane extraction at room temperature.The yields were 0.13, 0.15 and 0.16 g of hexane extracts respectively.Various concentrations of the 50°C and the 120°C were subjected to anti-M.tuberculosis test as described above.

The test organisms
The reference M. tuberculosis strain H37RV labelled PT12 and the local isolates labelled PT10 were used.The local isolates were isolated from TB patients using standard methods.The organisms were sub-cultured in Middle Brook 7H9 broth supplemented with OADC at 37°C for 21-28 days and were confirmed acid fast gram positive bacillus using Ziehl Nelson stain.

Anti-M. tuberculosis test
The anti-M.tuberculosis test was done using proportion method.5 ml of the filtered extract solutions (DMSO as solvent) was added to 15 ml of the homogenized egg LJ media to arrive at various concentrations ranging from 0.5 to 0.5 mg/ml.Each 20 ml medium was divided into 10 ml in universal containers.Standard drugs, isoniazid and rifampicin, at 0.2 and 0.4 µg/ml respectively, were added to LJ media accordingly.The media were slanted to form slopes.The LJ slopes without extracts and drugs were used as control.The slopes were insipissated (the slopes were thickened) at 85° C for 45 min, cooled and stored in a refrigerator at 4°C.Sterility and viability check were carried out before inoculation.

Inoculation of slopes with the bacteria
Bacterial dilutions 10 -5 and 10 -3 mg/ml were prepared for inoculation.0.1 ml of the chosen bacterial dilutions was inoculated into all the labelled LJ slopes (Adeleye et al., 2008).The universal containers were loosely closed with caps to allow evaporation and were incubated at 37°C.The specimens were checked on the 7 th , 14 th , and 21 st days to ensure no contaminations.Readings were done on the 28 th day.

Nitrate reduction test
Nitrate reduction test was performed on all the slopes after 28 days.This involved addition of 2 ml Nitrate Substrate Broth, incubation at 37°C for 2 h, addition of 1 drop of 50% hydrochloric acid, 2 drops of AFB Nitrate Reagent A (sulfanilamide 0.2%), 2 drops of AFB PT12, The standard strain of H37Rv; PT10, the local isolate strain, R, resistance (no inhibition of bacteria growth), S, sensitive (bacteria growth was inhibited).
Nitrate Reagent B (naphthylethylenediamine dihydrochloride, 0.1 %) and a pinch of Nitrate Reagent C (Zinc dust).Colour change was examined for resistance while no colour changes were examined for sensitive.

Determination of minimum inhibition concentration (MIC)
Minimum inhibition concentration of the eight anti-M.tuberculosis fractions from the four medicinal plants was determined.The stock solution for each fraction contained 0.2 g of sample in 20 ml of DMSO.Various dilutions were made to arrive at 2.5, 1.25, 0.75, 0.5 and 0.25 mg/ml.The LJ media were duplicated to serve both the standard Mtb strain, PT12 and the local strain, PT10 to obtain 80 LJ slopes of samples.The anti-mycobacterium tuberculosis test procedure described above was used.

RESULTS AND DISCUSSION
Fractions from ethanolic extracts of the ten medicinal plants were evaluated for anti-mycobacterium tuberculosis activities.Table 3 shows the results of the anti-mycobacterium tuberculosis test.
Butanol, ethylacetate and hexane fractions from M. benthamianum, The negative result obtained for X. aethiopica as shown in Table 3 is consistent with the earlier report by Adeleye et al. (2008) and Ogu (2011), of the ineffectiveness of the plant in inhibiting the growth of the M. tuberculosis.X. aethiopica had been reported to be antihypertensive (Gbadamosi and Kalejaye, 2017) and the association between hypertension and tuberculosis had been reported (Seegert et al., 2017).Thus, the use of X. aethiopica in combination therapy with other antitubercular medicinal plants by traditional practitioners of Southwestern Nigeria could have the advantage of limiting hypertension of the TB patients during the treatment period.
Figure 1 shows the results of the MIC.Hexane fraction of C. albidum, butanol fraction of M. benthamianum, ethylacetate fractions of both P. muellerianus and A. fimbriata had MIC value of 0.5 mg/ml.The ethylacetate and hexane fractions of M. benthamianum together with the hexane fraction of P. muellerianus showed minimum inhibition concentration of 1.25 mg/ml.The highest MIC value obtained was 2.5 mg/ml for hexane fraction of A. fimbriata.Extracts of A. fimbriata are used in the treatment of asthma and respiratory tract inflammation (Essiett and Okoko 2013).The anti-tuberculosis activity of an Acalypha specie, Acalypha indica, against multidrug resistant M. tuberculosis isolates had been reported (Gupta et al., 2010).
The ethylacetate and hexane fractions from P. muellerianus showed inhibition of the bacteria.Its butanol fraction was not sensitive as shown in Table 3.The leaves extract of P. muellerianus was reported to inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa (Doughari and Sunday, 2008).The leaves extract of P. muellerianus possessed anti-inflammatory property and the main constituent isolated from the plant, geraniin, had been shown to be anticarcinogenic, antihyperglycemic and antihypertensive (Boakye et al., 2016, Elendran et al., 2015).Only the hexane fraction of C. albidum inhibited the growth of M. tuberculosis.The traditional practitioners in Abeokuta, Ogun state part of There was no bacteria growth on the agar without inoculation (negative control) while there was growth on the agar inoculated (positive control).Both the hexane extracts of the 50 and 120°C roasted seeds showed sensitivity at 0.4 mg/ml.The fruit and the leaves extracts of C. albidum had been reported to possess high antimicrobial activity (George et al., 2018;Olasehinde et al., 2015).

Conclusion
There were no fractions from A. barterii, C.dependens, E. coccinea, S. afzeli, S. alata and X. aethiopica that showed sensitivity against the drug susceptible M. tuberculosis strains.Hexane fraction from C. albidum, butanol, ethylacetate and hexane fractions from M. benthamianum, ethylacetate and hexane fractions from P. muellerianus and A. fimbriata showed sensitivity against M. tuberculosis H 37 RV and the local isolate from TB patients.The hexane extracts of the roasted seeds of C. albidum were also sensitive to the M. tuberculosis strains.The active fractions would be investigated for the presence of anti-mycobacterium tuberculosis agents.

Table 1 .
Table 1 shows the ten plants evaluated for anti-mycobacterium tuberculosis.Only A. fimbriata and P. muellerianus are of the same family, Euphorbiaceae, while others belong to different families.The Table 1 also shows the part of the plants used in the research work.The local name of the plant represents the name in Yoruba language.Table 2 shows the yield in both the 80% ethanol extraction and in the fractionation.No hexane fraction was obtained for A. barteri, C. dependens, E. coccinea, S. afzelii, S. alata and X. aethiopica. A. fimbriata was restricted to only hexane and ethylacetate partitioning.Plants for the anti-mycobacterium tuberculosis evaluation.

Table 2 .
Weight of extracts and fractions from the medicinal plants.

Table 3 .
Results of anti-mycobacterium tuberculosis test.

Table 4 .
Results of anti-mycobacterium tuberculosis test of the extracts of roasted seeds of Chrysophyllum albidum.Nigeria, put roasted and powdered cotyledon of the seeds of C. albidum in honey for TB patients to lick for a period of one month.Table4shows the results of anti-M.tuberculosis test of the roasted seeds of C. albidum.