In vitro Anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis Strains

Background Tuberculosis (TB) is a global burden with one –third of the world’s population infected with the pathogen Mycobacterium tuberculosis complex and annually 1.4 million deaths occur due to the disease. This high incidence of infection and the increased rate of multi-drug resistant and extensively-drug resistant strains of the organism further complicated the problem of TB control and have called for an urgent need to develop new anti-TB drugs from plants. In this study, the in vitro activity of root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis were evaluated against M. tuberculosis and M. bovis strains. Methods Five Ethiopian medicinal plants, root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis used locally for the management of TB. They were investigated for in vitro antimycobacterial activity against M. tuberculosis and M. bovis strains. 80% methanolic extracts of the plant materials were obtained by maceration. The antimycobacterial activity was determined using 96 wells of microplate with the help of visual Resazurin Microtiter Assay. Results The crude 80% methanolic extracts of the root of C. aurea, seeds of O. basilicum, and leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis had anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 6.25–100 μg/mL. The MIC of 80% methanol extracts in the order mentioned above ranged 25-100 μg/ml and 12.5-75 μg/mL, 25–100 μg/mL and 25–50 μg/mL, 6.25-50 μg/mL and 12.5-50 μg/mL, 12.5-100 μg/mL and 18.25-50 μg/mL and 6.25-50 μg/mL and 12.5-50 μg/mL, respectively for M. tuberculosis and M. bovis strains. Conclusions The results support the local use of these plants in the treatment of TB and it is suggested that these plants may have therapeutic value in the treatment of TB. However, further investigations are needed on isolating chemical constituents responsible for eliciting the observed activity in these plants.


Background
Tuberculosis (TB) is caused by a set of closely related mycobacterial strains such as Mycobacterium tuberculosis, M. bovis, M. africanum and others, known collectively as the M. tuberculosis complex (MTC) [1]. Currently about one -third of the world's population is assumed to be infected with the tubercle bacillus [2,3]. It is responsible for 1.4 million deaths per a year [4]. Moreover, up to 50 million people are said to be infected with drug-resistant forms of TB from which about 500,000 cases of multidrug resistant (MDR) TB worldwide per a year [5].
Management of TB/MDR-TB patient requires intense multi-chemotherapy for at least six months to two years. It is very hurtful to a patient's health due to high levels of drug toxicity and its adverse effects [6][7][8]. The emergence of MDR TB and extensively-drug resistant (XDR) TB to the medicines now in use makes urgent search for new anti-TB agents worldwide [9,10]. Medicinal plants offer a great hope to overcome these needs because of their chemical diversity and their significant role in the drug sighting and development [11]. They are also recognized as a useful source of highly active antimycobacterial metabolites [12].
In Ethiopia there are many plant species reported to be used traditionally to treat TB and respiratory tract infections. For instances, leaves and fruits of Allium ursinum were locally used for the treatment of bronchitis and TB respectively in Central Zone of Tigray [13]. It was also reported that locally available plant species such as Croton macrostachyus and Oenanthe procumbens in Farta wereda of Amhara region [14] and Arisaema schimperianum around Bale mountains national park [15] are to used to treat TB respectively. Likewise, roots of Calpurnia aurea are used for lung TB [16]. Additionally it was reported that fruits/seeds of Ocimum basilicum and Ocimum americanum, leaves of Eucalyptus camaldulensis, Artemisia afra and Artemisia abyssinica are used for cough and TB [17]. Leaves of Aloe pubescens, root of Indigofera amorphoides and Psydrax schimperiana are reported to be some of the medicinal plants locally used to treat TB and other respiratory illness and other diseases [18]. Moreover, in Chifra District, Afar region, it was reported that Kanahia laniflora and Aloe species were locally used for treatment of TB [19]. However, their efficacy remains unknown. Thus, the aim of this study was to evaluate the in vitro anti-mycobacterial activity of root of Calpurnia aurea, seeds Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis against M. tuberculosis and M. bovis strains.

Study design
This study was conducted at Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University (AAU) using an experimental study design. Five plant species, namely C. aurea, O. basilicum, A. abyssinica, C. macrostachyus, and E. camaldulensis were selected for their in vitro activity test against M. tuberculosis and M. bovis strains. Susceptibility tests and MIC of the extracts were determined using Resazurin Microtiter Assay (REMA). Rifampicin was used as positive control while drug and extract free medium were used as negative controls.

Collection of plants materials
The leaves of C. macrostachyus, roots of C. aurea, and seeds of O. basilicum were collected from Addis Ababa. Leaves of E. camaldulensis, and A. abyssinica were collected from Harar (525 km East of Addis Ababa) and Muka Turi (Selale/North Shoa Zone) 80 km north of Addis Ababa, respectively ( Table 1). The identities of each plant specimen were identified by Dr. Mirutse Giday (a botanist) at the Endod and Other Medicinal Plants Research Unit, ALIPB. A voucher specimen of each plant was deposited at the Endod and Other Medicinal Plants Research Unit, with voucher numbers AG-01 (O. basilicum), AG-02 (C. macrostachyus), AG-03 (C. aurea), AG-04 (E. camal dulensis), and AG-05 (A. abyssinica). All parts of the plant materials were made to become dry in an open air and protected from direct exposure to sunlight. The dried plant materials were separately powdered to suitable size by using mortar and pestle, and then made ready for extraction.

Preparation of the extract
The powdered plant materials were weighed using analytical balance and prepared for solvent extraction. The crude 80% methanolic extracts of the plant materials were obtained by maceration. The total crude 80% methanolic extracts were obtained by soaking powdered plant material (about 50 g) in 400 mL methanol for 48-72 h on an orbital shaker [20]. Extracts were filtered using Whatman No.1 filter paper and concentrated using a rotary evaporator (Laborota 4000, SN 090816862, Germany) in a water bath set at 40°C. The dried methanolic extracts obtained from each plant were air-dried then packed in glass bottles with proper labeling for future reference. The extracts were kept refrigerated and far away from light. Stock solutions were prepared in dimethyl sulphoxide (DMSO) at a concentration of 20 mg/mL and stored at −20°C until use [20,21]. Finally, all the crude 80% methanolic extracts of each plant species were tested for antimycobacterial activity.

Preparation of Inoculum
The isolates grown on Lowenstein Jensen medium (LJ) were sub-cultured in Middle Brook 7H9 broth supplemented with OADC at 37°C for 14 -21 days. The antimycobacterial activities of the extracts were evaluated against ten M. tuberculosis strains and three M. bovis strains by maintaining on Middle Brook 7H9 medium for about 14-21 days. The bacterial suspension was homogenized by vortex shakeup and the turbidity was adjusted in agreement with tube which is the scales of McFarland no.1 (3.2 × 10 6 cfu/mL). The inoculum was prepared diluting the bacterial suspension in the proportion of 1:20 in Middle Brook 7H9 broth medium. This diluted suspension (100 μL) was used to inoculate each well of the plate [22].

Anti-mycobacterial activity tests
The antimycobacterial activity of 80% methanolic crude extracts of O. basilicum seeds, C. aurea root, C. macro stachyus, A. abyssinica and E. camaldulensis leaves were tested using the REMA. The susceptibility test was accomplished in 96 microplates (wells) using the resazurin as an indicator of cellular viability or growth inhibition. Working solutions of the tested extracts were diluted in Middle Brook 7H9 broth supplemented with OADC to obtain final sample concentrations that ranges from 0.78 μg/mL to 100 μg/mL. Rifampicin was dissolved in DMSO and used as positive control drug and extracts/ drug free medium with strain suspensions were used as negative control. One hundred micro liters of Middle Brook 7H9 broth and the test inoculum were added to all testing wells, as well as to the drug/extract-free control wells. Then, one hundred micro liters working extract solutions were poured into the first well of each row from which two-fold dilution series were made through the micro plate column. Each extract concentration was assayed in duplicate. Each micro plate was then sealed with parafilm and incubated for 5-7 days at 37°C in normal atmosphere. After the incubation period, 25 μL of resazurin 0.02% w/v was added to each wells and reincubated at 37°C for 24 h for color development. The visual MIC was defined as the lowest drug/extract concentration that prevented the color change of resazurin reagent from blue to pink. Blue color in the well was interpreted as there is no mycobacterial growth and pink color was scored as growth occurrence [23,24].

Determination of the Minimum Inhibitory Concentration (MIC)
MIC was determined by using the REMA in 96 well micro titer plates. One hundred micro liters of Middle Brook 7H9 broth and M. tuberculosis and M. bovis strains were dispensed into all wells of a sterile 96-well microtitre plate.
In the first column (no.1 well of all plate), one hundred micro liters of extracts were added to each first wells using a unique pipette for each extracts. The extracts were mixed thoroughly and fifty micro liters of extracts were transferred to well 2 to well 8 from which fifty micro liters were discarded. Well 9 up to well 12 were served as sterility and negative controls. The working solution of extracts (100 μg/mL) were diluted out across a 96well in a two-fold serial dilution to give final testing concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 μg/mL. The same procedure was followed for rifampicin with the initial concentration of 32 μg/mL with subsequent dilution to the final testing concentrations 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.06 μg/mL. The plates were then incubated for 5-7 days at 37°C. After 7 th day, 25 μL of resazurin was added to all wells and re-incubated overnight for development. The MIC was defined as the lowest concentration of the extracts/drugs that prevented a colour of resazurin to be changed from blue to pink (visual determination) [24][25][26][27]. According to Ramos [22], extracts were considered as active if they inhibited growth of mycobacterium at MIC ≤ 100 μg/mL. Each extract was tested in duplicate against each strain.

Data analysis
The experimental results were computerized using Epi-Data version 3.1 and STATA version 11 statistical software for data entry and analysis purposes respectively. The MIC results were presented as mean value. The t-test was employed to test significance for the difference between 80% methanolic extract results of each plant and rifampicin against M. tuberculosis and M. bovis strains. All the data were analyzed at 95% confidence interval and considered as statistically significant.

Ethical consideration
The study was conducted after its approval by the Institutional Review Board of ALIPB. The test was done in the TB laboratory where the necessary protective wears such as respirators and gloves as well as safety cabinets were used, to minimize the risk of exposure to M. tuberculosis and M. bovis isolates.

Results and discussion
Anti-mycobacterial activity of the crude extracts  (Table 2).

Minimum inhibitory concentration
The MIC of the extracts was determined for their antimycobacterial activity using resazurin as an indicator for M. tuberculosis and M. bovis strains viability in 96-well microplates. The MIC of 80% methanolic extracts of root of C. aurea, seeds of O. basilicum, leaves of A. abyssinica, C. macrostachyus and E. camaldulensis ranged from 25-100 μg/mL and 12.5-75 μg/mL, 25-100 μg/mL and 25-50 μg/mL, 6.25-50 μg/mL and 12.5-50 μg/mL, 12.5-100 μg/mL and 18.25-50 μg/mL and 6.25-50 μg/mL and 12.5-50 μg/mL, respectively for M. tuberculosis and M. bovis strains. The investigation showed that leaves of E. camaldulensis extract was the most active against both M. tuberculosis and M. bovis strains (MIC 6.25-50 μg/mL). The mean MIC results of 80% methanolic crude extracts of each plant showed significantly lower antimycobacterial activities in comparison to rifampicin for all strains (Table 2). Although all extracts were found less active than rifampicin, extracts of leaves of A. abyssinica and E. camaldulensis showed interesting antimycobacterial activity. However, reference strain of M. tuberculosis H37Rv, SIT73, SIT149, SIT149, SB1176 and SB1953 strains were comparatively the most susceptible strains to the extracts of the study plants with MIC ≤ 50 μg/mL. In contrast, SIT37 and SIT336 strains were the most resistant to extracts as compared to other strains. The comparative mean MIC values of plant extracts and rifampicin against clinical isolates and reference strain of M. tuberculosis H37Rv and M. bovis strains is shown in (Table 2). Despite intense hard work to control TB disease, it remains one of the global health problems claiming 1.4 million human lives per annum. Furthermore, in the worldwide up to 50 million people are said to be infected with drug-resistant forms of TB with about 500,000 cases of multi-drug resistant MDR-TB per a year [5). Thus, with the emergence of the drug-resistant strains of MTC, lengthy of therapy, which is very hurtful to a patient's health due to high levels of drug toxicity and its various adverse effects, the need to search for new effective anti-TB agents has become very necessary. Using medicinal plants for the treatment of TB offers a great hope to fulfill these needs because of their chemical diversity and they have been used for curing diseases for many centuries [11]. In addition, natural herbs continue to play a great significant role in the drug discovery and development of highly active antimycobacterial metabolites and they can be used as pure compounds or as crude materials [12].
In Ethiopia, many medicinal plants have been found which are used in the treatment of microbial infections including TB. It is our interest to report the anti-TB activity of these plants since these plants are reported from communities to treat TB and other respiratory infections. During ethnobotanical study these plants had been reported to possess antimicrobial activity. In the present study, the crude 80% methanol extracts of seeds of O. basilicum, root of C. aurea, and leaves of C. macrostachyus, A. abyssinica and E. camaldulensis showed promising antimycobacterial activity against M. tuberculosis and M. bovis strains. This may be due to the bioactive constituents, such as alkaloids, tannins, saponins, phenols, flavonoids and others that are present in the extracts [28,29]. Although the antibacterial activity of O. basilicum, C. aurea, C. macrostachyus, and A. abyssinica had been reported against other various pathogenic bacteria [30,31], no report was found during a literature search against mycobacterium strains apart from ethnobotanical reports on these plants. Therefore, this investigation would be the first report on their anti-mycobacterial activities. The crude 80% methanolic extracts of leaves of E. camaldulensis exhibited the most antimycobacterial activity against both M. tuberculosis and M. bovis strains. This could be due the bioactive tannins and saponins present in the extracts. This report agrees with the previous reports of hexane, chloroform, methanol extracts of E. camaldulensis [32,33]. In Ethiopia, the present work demonstrates antimycobacterial properties of these plants having therapeutic value in the treatment of TB. However, it is generally recognized that the more antimycobacterial activity of extracts depends on their lipophilic nature and showed better activity [20,34].

Conclusions
Even though this investigation supports the traditional medicinal usage in the Ethiopian community and the medicinal uses of these plants in different regions of Ethiopia; to obtain the most promising antimycobacterial activity and to conclude their anti-TB activity, these plants should be fractionated with less polar solvents. In addition, further investigations should focus on isolating chemical constituents responsible for pharmacological activities and identifying the compounds eliciting the activity observed in these plants.