The anti-tubercular activity of Melia azedarach L. and Lobelia chinensis Lour. and their potential as effective anti-Mycobacterium tuberculosis candidate agents.

OBJECTIVE: To evaluate the anti-mycobacterial activity of Melia azedarach L. (M. azedarach) and Lobelia chinensis Lour. (L. chinensis) extracts against the growth of Mycobacterium tuberculosis (M. tuberculosis).
METHODS: The anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts were evaluated using different indicator methods such as resazurin microtiter assay (REMA) and mycobacteria growth indicator tube (MGIT) 960 system assay. The M. tuberculosis was incubated with various concentrations (50-800 μg/mL) of the extracts for 5 days in the REMA, and for 4 weeks in MGIT 960 system assay.
RESULTS: M. azedarach and L. chinensis extracts showed their anti-M. tuberculosis activity by strongly inhibiting the growth of M. tuberculosis in a concentration-dependent manner in the REMA and the MGIT 960 system assay. Particularly, the methanol extract of M. azedarach and n-hexane extract of L. chinensis consistently exhibited their effects by effectively inhibiting the growth of M. tuberculosis in MGIT 960 system for 4 weeks with a single-treatment, indicating higher anti-M. tuberculosis activity than other extracts, and their minimum inhibitory concentrations were measured as 400 μg/mL and 800 μg/mL, respectively.
CONCLUSIONS: These results demonstrate that M. azedarach and L. chinensis extracts not only have unique anti-M. tuberculosis activity, but also induce the selective anti-M. tuberculosis effects by consistently inhibiting or blocking the growth of M. tuberculosis through a new pharmacological action. Therefore, this study suggests the potential of them as effective candidate agents of next-generation for developing a new anti-tuberculosis drug, as well as the advantage for utilizing traditional medicinal plants as one of effective strategies against tuberculosis.

Introduction

Recently chronic infectious diseases including tuberculosis, hepatitis, filariasis, leishmaniasis, amoebiasis caused by Entamoeba histolytica, as well as acute infectious zoonosis such as malaria, Middle East respiratory syndrome, influenza and Ebola, have been occurred or persisted in various countries, particularly, in less-developed countries globally. Furthermore, the prevalence of tuberculosis has still been causing a serious global challenge of drug-resistant tuberculosis such as extensively drug-resistant tuberculosis (XDR-TB) and multi-drug resistant tuberculosis (MDR-TB) despite various efforts and studies worldwide. Mycobacterium tuberculosis (M. tuberculosis) is one of the major infectious factors causing the highest mortality through the co-infection with HIV/AIDS as well as the most dangerous infectious bacteria causing resistant strain through fast infectivity and the adaptability against environmental variation. Recently, it was estimated that 9.0 million people in the world were confirmed as new tuberculosis cases in 2013, and 1.1 million of them had HIV-positive response. Particularly, tuberculosis cases co-infected with HIV/AIDS showed high-infection rates in the African region compared with other countries, and most of tuberculosis cases in 2013 and 2014 occurred in Asia (56%) and the African region (29%) [1], [2]. In these aspects, various studies for the inhibition and/or the treatment of tuberculosis have been carried out in anti-tuberculosis drug discovery/development field. However, the current anti-tuberculosis drugs not only showed the limit that can't inhibit the increase of the tuberculosis patients, but also caused various side effects. Recently, the global pharmaceutical companies for the development of novel/effective anti-tuberculosis drugs are testing the repurposed compounds such as PA-824, SQ109 and linezolid through chemical remodeling of the existing drugs, as well as newly developed compounds such as Q203, TMC207, pyridomycin, and thiophenes in clinical trials [1], [3], [4], [5], [6], [7]. Nevertheless, the rapid emergence of tuberculosis including MDR-TB and XDR-TB is still inducing serious concerns and problems in the public health field worldwide. Recently, various extracts and/or natural products derived from medicinal plants or traditional oriental medicine were studied or reported for discovering novel anti-M. tuberculosis candidate drugs, which are being progressed for developing anti-tuberculosis drugs of more effective/safe next-generation [8], [9], [10], [11], [12], [13]. Furthermore, various medicinal plants of oriental medicine have been utilized as traditional resources for treating diseases and/or symptoms such as diabetes, arteriosclerosis, hepatitis, parasite, cancer both “in vivo” and “in vitro” [14], [15], [16], [17], [18].

For these reasons, various studies for developing the effective/safe anti-tuberculosis drugs with novel mechanisms of action, low cytotoxicity, and the least side-effects are urgently needed to block the tuberculosis. In this aspect, this study was carried out to evaluate anti-M. tuberculosis effect of Melia azedarach L. (M. azedarach) and Lobelia chinensis Lour. (L. chinensis) that are effectively utilized as a medicinal plant in traditional oriental medicine, and to identify the potential of them as candidates for developing novel antitubercular drugs.

Materials and methods

Materials

Various drugs used in this study including rifampicin, isoniazid, resazurin powder and dimethylsulfoxide, were purchased from Sigma-Aldrich Chemical Co. Ltd. (St. Louis, MO, USA), and MGIT™ 960 system indicator 7 mL growth tubes with BACTEC™ MGIT™ 960 supplement kit were purchased from Becton-Dickinson and Company (Sparks, MD, USA). All other chemicals and reagents were purchased from Merck Chemical Co., Ltd. (Darmstadt, Germany) and Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA).

Preparation and extracts of medicinal plants

The dried roots and barks of M. azedarach and dried roots, stem, leaves and flowers of L. chinensis were provided by the Oriental Medical Center, Kyung Hee University (Seoul, Republic of Korea) and Bundang Oriental Hospital, Dongguk University (Seoul, Republic of Korea) for this study. The M. azedarach and L. chinensis were extracted according to solvent extraction system as follows: Five hundred grams of each powdered M. azedarach and L. chinensis were independently extracted with 4 L of n-hexane, chloroform, ethyl acetate, methanol, and distilled water at room temperature for 24 h, respectively. These extracts were filtered using filter paper and a vacuum pump, and then were evaporated under reduced pressure using a rotary evaporator in a vacuum at 45 °C. The extracts were lyophilized after evaporation. All extracts were filtered using a 0.45 μm syringe filter (Roshi Kaisha, Ltd., Tokyo, Japan) and stored at −80 °C until use.

Preparation of anti-M. tuberculosis drugs

The anti-M. tuberculosis first-line drugs, isoniazid, was dissolved in sterile distilled water, and rifampicin was dissolved in dimethylsulfoxide, to a concentration of 50 mg/mL according to the manufacturer's instruction. The anti-M. tuberculosis first-line drugs were used as reference standard drugs. All compounds were filtered using 0.45 μm membrane syringe filter (Roshi Kaisha, Ltd., Tokyo, Japan) before use and stored at −80 °C deep-freezer until use.

Preparation and growth conditions of M. tuberculosis

M. tuberculosis H37Rv (ATCC 27294) and H37Ra (ATCC 25177) used in this study were purchased from American Type Culture Collection (Manassas, VA, USA). M. tuberculosis H37Rv and H37Ra were grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA) supplemented with 10% (v/v) oleic acid/albumin/dextrose/catalase enrichment (Becton–Dickinson and Company, Sparks, MD, USA) and 0.05% (v/v) Tween 80 (Sigma–Aldrich Chemical, St. Louis, MO, USA) to the log phase at 37 °C for 4–5 weeks on shaking incubation of 120 r/min.

Drug susceptibility testing of M. tuberculosis

The in vitro anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts was confirmed by resazurin microtiter assay (REMA) using a 96-well micro-plate. Briefly, M. tuberculosis was grown in fresh Middlebrook 7H9 broth supplemented with 10% (v/v) oleic acid/albumin/dextrose/catalase enrichment and 0.05% (v/v) Tween 80 until the culture reached a turbidity equal to that of 1.0 McFarland standard (3.0 × 108 CFU/mL) at 37 °C. The bacteria were adjusted to a density of 2 × 106 CFU/mL in fresh culture broth. Finally, the bacterial suspensions were inoculated into all wells of a 96-well microtiter plate containing final concentrations (50–200 μg/mL) of M. azedarach and L. chinensis extracts and anti-tuberculosis first-line drugs (1.25–5.00 μg/mL). M. tuberculosis growth controls containing no anti-M. tuberculosis first-line drugs and blank controls without inoculation were also included. The 96-well plates, covered with lids, placed in a plastic bag, were incubated at 37 °C for 5 days. After incubation, 20 μL of freshly prepared 0.05% (w/v) resazurin solution was added to all wells, and the plates were re-incubated at 37 °C for 36 h. A change in color from blue to pink indicating bacterial growth was observed after 36 h of incubation. The minimum inhibitory concentration (MIC) was expressed as the lowest concentration of the drug that inhibited M. tuberculosis growth or prevented change in color of the resazurin solution from blue to pink based on a REMA.

Evaluation of drug susceptibility of M. tuberculosis by MGIT 960 system assay

For evaluate anti-M. tuberculosis effects of M. azedarach and L. chinensis extracts against the growth of M. tuberculosis, the drug susceptibility testing of the strain was further performed using the BACTEC™ MGIT 960 system (Becton, Dickinson and Company, Sparks, MD, USA). In brief, 100 μL of a suspension of M. tuberculosis culture, adjusted to 9.6 × 106 CFU/mL, was inoculated in an MGIT growth media tube with BACTEC™ MGIT 960 growth supplement (Becton, Dickinson and Company), which were incubated with different concentrations (25–800 μg/mL) of the extracts and anti-M. tuberculosis first-line drugs (10 μg/mL), and M. tuberculosis growth controls containing no anti-M. tuberculosis first-line drugs were also included. They were incubated into the BACTEC™ MGIT 960 system device for 4 weeks for the accurate determination of M. tuberculosis drug-susceptibility.

Statistical analysis

All results were expressed as mean ± SD of three independent experiments. Statistical analysis of the data was performed using the Student's t-test and One-way ANOVA. P < 0.05 was considered to be statistically significant.

Results

Evaluation of anti-M. tuberculosis activity of the extracts using REMA

The anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts were evaluated using the REMA. The M. azedarach and L. chinensis were independently extracted using different solvents at room temperature for 24 h, respectively (Figure 1 ). After the bacteria were incubated with various concentrations (50–200 μg/mL) of the extracts and anti-M. tuberculosis first-line drugs (rifampicin and isoniazid, 1.25–5.00 μg/mL) for 5 days, the growth of M. tuberculosis were markedly inhibited in a concentration-dependent manner, and then their viabilities were 0% at a concentration of 100 μg/mL of all extracts (Table 1 ). The minimal inhibitory concentration (MIC) values of all extracts against the viability of M. tuberculosis were measured as 100 μg/mL (Table 2 ). Furthermore, the extracts obviously demonstrated their M. tuberculosis-inhibitory effect through the REMA. Although the superiority and specificity between the extracts regarding anti-M. tuberculosis effect were not confirmed in the REMA, the extracts showed similar anti-M. tuberculosis activity in both M. tuberculosis H37Ra and M. tuberculosis H37Rv. These results indicate that M. azedarach and L. chinensis extracts have not only anti-M. tuberculosis effects by strongly inhibiting the growth of M. tuberculosis but also the potential that can be utilized or developed as promising anti-M. tuberculosis candidate agents through their new pharmacological activity.

Figure 1

Extraction process of M. azedarach (A) and L. chinensis (B).

Table 1

The inhibitory effects of M. azedarach and L. chinensis extracts against the growth of M. tuberculosis (H37Rv and H37Ra) determined by the REMA.

Medicinal plant	Extracts	Concentrations (μg/mL)
		H37Rv			H37Ra
		200	100	50	200	100	50
M. azedarach	n-Hexane extract	+	+	−	+	+	−
	Chloroform extract	+	+	+/−	+	+	+/−
	Ethyl acetate	+	+	+/−	+	+	+/−
	Methanol extract	+	+	+/−	+	+	+/−
	H2O extract	+	+	+/−	+	+	+/−
L. chinensis	n-Hexane extract	+	+	+/−	+	+	+/−
	Chloroform extract	+	+	−	+	+	−
	Ethyl acetate	+	+	+/−	+	+	+/−
	Methanol extract	+	+	+/−	+	+	+/−
	H2O extract	+	+	+/−	+	+	+/−

The M. tuberculosis was incubated with different concentrations (50–200 μg/mL) of the extracts at 37 °C for 5 days. The “+” and “−” sign indicates anti-M. tuberculosis activity and no activity respectively, and “+/−” sign shows 55%–65% inhibitory rates of the extracts against the growth of M. tuberculosis.

Table 2

MICs of M. azedarach and L. chinensis extracts against the growth of M. tuberculosis (H37Rv and H37Ra) determined by different anti-M. tuberculosis indicator assays.

Medicinal plants	Extracts	MIC (μg/mL)
		REMA	MGIT 960 system
M. azedarach	n-Hexane extract	100	> 800
	Chloroform extract	100	> 800
	Ethyl acetate	100	> 800
	Methanol extract	100	400
	H2O extract	100	> 800
L. chinensis	n-Hexane extract	100	800
	Chloroform extract	100	> 800
	Ethyl acetate	100	> 800
	Methanol extract	100	> 800
	H2O extract	100	> 800

M. tuberculosis (H37Rv and H37Ra) were incubated with different concentrations (25–800 μg/mL) of the extracts at 37 °C, and their susceptibility was evaluated by different anti-M. tuberculosis-indicator assays. The results of the REMA and the MGIT 960 system assay were determined after 5 days and 28 days of incubation, respectively.

Anti-M. tuberculosis effects of the extracts using the MGIT 960 system assay

The anti-M. tuberculosis effects of the extracts were further evaluated using the MGIT 960 system assay. The bacteria were incubated with various concentrations (25–800 μg/mL) of the extracts and anti-M. tuberculosis first-line drugs (10 μg/mL) in an MGIT growth media tube of the BACTEC™ MGIT 960 system device for drug susceptibility testing for 4 weeks, and their growth units were markedly inhibited in a concentration-dependent manner (Figure 2 ). Particularly, the methanol extract of M. azedarach and n-hexane extract of L. chinensis strongly inhibited the growth of M. tuberculosis compared with other extracts. When M. tuberculosis was incubated with 200 μg/mL of the methanol extract of M. azedarach, their growth units were detected at about 8.7 days, whereas 400 μg/mL of the methanol extract strongly inhibited the growth of M. tuberculosis compared with other extracts, and the growth unit of M. tuberculosis was not detected until 28th day. Furthermore, when M. tuberculosis was incubated with 400 μg/mL of the n-hexane extract of L. chinensis, their growth units were detected at 20.6 days, whereas 800 μg/mL of the n-hexane extract strongly inhibited the growth of M. tuberculosis, and the growth unit of M. tuberculosis was not detected until 28th day. In this MGIT 960 system assay, the methanol extract of M. azedarach and the n-hexane extract of L. chinensis consistently inhibited the growth of M. tuberculosis for 4 weeks with a single treatment (400 μg/mL and 800 μg/mL) respectively, and they showed more effectively the selective anti-M. tuberculosis activity as well as anti-M. tuberculosis action compared with other extracts. In addition, the extracts indicated higher activity against M. tuberculosis H37Ra than M. tuberculosis H37Rv in the MGIT 960 system assay (Figure 2). Particularly, the differences in anti-M. tuberculosis activity of them were obviously confirmed in the MGIT 960 system assay compared with the REMA assay (Table 2). These results demonstrate that the extracts not only induce anti-M. tuberculosis activity causing the inactivation of M. tuberculosis by effectively or consistently inhibiting the growth of M. tuberculosis, but also have unique anti-M. tuberculosis properties that inhibit or block the growth of M. tuberculosis in a concentration-dependent manner.

Figure 2

The anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts against the growth of M. tuberculosis H37Rv (A) and H37Ra (B).

Control: Untreated growth-M. tuberculosis; R: Rifampicin 10 μg/mL; I: Isoniazid 10 μg/mL; A: n-Hexane extract; B: CHCl3 extract; C: Ethyl acetate extract; D: Methanol extract; E: Distilled water extract. The M. tuberculosis (H37Rv and H37Ra) were incubated with different concentrations (25–800 μg/mL) of the extracts in the MGIT 960 system device at 37 °C for 4 weeks, respectively. ∗: P < 0.05 was considered to be statistically significant compared with control.

Discussion

The recent zoonotic diseases such as Middle East respiratory syndrome, avian influenza and Ebola have been occurred in various countries worldwide through diverse infectious pathways, particularly, in Asia, Middle East and Africa. Furthermore, the serious infectious pathogens such as influenza, M. tuberculosis, and methicillin-resistant Staphylococcus aureus have strongly enhanced their infectivity and viability through resistance or adaptability to drugs as well as environmental variation for the past decades, which induces serious difficulty to treatment and prevention of the disease as well as the development of the effective drug. In particular, these diseases have caused high risk symptoms to young children whose immune function is low and the elderly who have other underlying diseases or complications compared with normal adults. In this aspect, tuberculosis is a disease to cause infection through droplets, which has been causing high mortality and prevalence rate compared with other contagious diseases worldwide. Tuberculosis was one of the leading causes of death among various infectious diseases in 2013 and 2014 globally, particularly, in Africa [1], [2]. It is the most dangerous infectious disease causing complications such as bronchiectasis, emphysema, and pneumothorax through chronic symptom. In addition, the rapid increase and appearance of XDR-TB/MDR-TB imply an urgent need to develop anti-tuberculosis drugs of effective/safe next-generation for treating tuberculosis. Until recently, despite various efforts to discover and to develop new anti-tuberculosis drugs, the development of effective/safe anti-tuberculosis drugs is still facing with direct or indirect-difficulty including the cost or time as well as the economic aspect and the profitability of pharmaceuticals. Furthermore, the overuse of antibiotics and antibacterial drugs has rapidly increased the prevalence and incidence rate of resistant-tuberculosis such as XDR-TB and MDR-TB, which has caused more difficulty to treat tuberculosis-patients compared with the past. In addition, the current anti-tuberculosis drugs induce various/serious side effects including hepatotoxicity, ototoxicity, and nephrotoxicity. In this aspect, as one of feasible alternatives for overcoming limitations of the drug's side effects, particularly, including rifampicin, isoniazid, ethambutol and moxifloxacin, various studies for developing anti-tuberculosis drugs were reported from active substances derived from traditional diverse medicinal plants and biological resources [19], [20], [21], [22], [23]. However, anti-tuberculosis agents of effective/safe next-generation that can be utilized or used as potential/novel first-line anti-tuberculosis drugs, particularly, the extracts, natural products, and/or semi-synthetic compounds, have not yet been reported in the global pharmaceutical market.

In this perspective, the new pharmacological activity of medicinal plants used in oriental medicine or traditional medicine can provide the advantages of both the safety and efficacy compared with newly developed drugs such as semi-synthetic compounds or biomedicine, which may be used or utilized as one of effective/feasible strategies for developing anti-tuberculosis drugs of effective/safe next-generation. Moreover, the plants selected from the existing medicinal plants through new pharmacological action may increase the potential which can be utilized as a useful/feasible resource for developing new substances in a medical field. For these reasons, this study has been focused on major key points for the discovery of new candidate substances as one of different strategies for developing anti-tuberculosis drugs, which has included crucial factors such as the minimization of side effects and the safety of drugs, as well as the finding of novel pharmacological activity and function of the medicinal plants used in Korean traditional medicine. In these aspects, M. azedarach and L. chinensis are medicinal plants that are used as biological resources of traditional oriental medicine in Korea, Japan and China as well as various countries and regions globally, which include various bioactive substances. Until recently, pharmacological activity and function of them have been variously reported as follows: (1) the aqueous extracts from M. azedarach induce the effect of wound healing by activating the growth of keratinocyte [24]; (2) the pediculicidal activity of ethanol extracts of M. azedarach [25]; (3) the leaves and bark extracts of M. azedarach through cyclooxygenase-2 and the inducible NO synthase inhibition cause anticancer activity and anti-inflammatory effects [26], [27], [28]; (4) the anti-diabetic activity of ethanol extract of M. azedarach [29]; (5) the anti-parasitic effect of hexane extract of M. azedarach against gastrointestinal nematodes [30]; (6) the antibacterial effect and antifungal activity of the extracts of M. azedarach against pathogenic bacterial and fungus strains [31], [32]; (7) the methanol extracts and the compounds isolated from L. chinensis induce anti-oxidant activity and anti-inflammatory effects through the nuclear factor-κB pathways [33], [34]; (8) the anti-viral effects of L. chinensis extracts in mouse model infected by herpes simplex virus type 1 [35]; (9) the anticancer effects of the compounds isolated from L. chinensis against human lung cancer cells [36], [37].

Taken together, these studies show substantial evidence that M. azedarach and L. chinensis can be used or utilized as therapeutic agents through their unique pharmacological activity and function in both “in vitro” and “in vivo”. However, despite the pharmacological activity or actions of them identified through these studies, their anti-tubercular activity have not yet been reported in both “in vitro” and “in vivo”. For this reason, this study has been begun from hypothesis that M. azedarach and L. chinensis may strongly inhibit or block and effectively modulate the growth of M. tuberculosis. As mentioned above, the results of this study showed novel anti-tubercular activity of M. azedarach and L. chinensis extracts by effectively inhibiting the growth of M. tuberculosis through their novel pharmacological activity and properties. Particularly, anti-M. tuberculosis activity and the ability of M. azedarach and L. chinensis extracts were obviously demonstrated through different M. tuberculosis susceptibility-indicator assays such as the REMA and MGIT 960 system assay, and they consistently showed their anti-M. tuberculosis activity by strongly inhibiting the growth of M. tuberculosis for 4 weeks with a single treatment (400 μg/mL and 800 μg/mL) in MGIT 960 system assay, respectively. In addition, the specificity and differences in anti-M. tuberculosis activity of them were obviously confirmed in the MGIT 960 system assay compared with the REMA assay, and the methanol extract of M. azedarach more effectively inhibited the growth of M. tuberculosis compared with those of L. chinensis extracts. These results imply that the intracellular signaling-pathways for replication as well as key-proteins for regulating cell cycle in cytoplasm that accelerate the growth of M. tuberculosis and/or functions of the cell wall of M. tuberculosis are strongly inhibited or deactivated by the binding of the extracts. Furthermore, the extracts showed higher anti-M. tuberculosis activity in M. tuberculosis H37Ra than M. tuberculosis H37Rv in the MGIT 960 system assay, and it suggests that these results can be associated with differences in pathogenesis and virulence of the two strains or different pathogenic phenotypes between H37Rv and H37Ra.

In conclusion, M. azedarach and L. chinensis extracts effectively inhibited the growth of M. tuberculosis that can cause active tuberculosis in those with weakened immune systems through their novel pharmacological activity or function, and their anti-M. tuberculosis activity were obviously demonstrated through different anti-M. tuberculosis indicator assays such as the MGIT 960 system and the REMA. These results showed that the effective use of M. azedarach and L. chinensis extracts can be utilized as potential anti-M. tuberculosis agents for consistently inhibiting or blocking tuberculosis causing various complications in clinical fields. Therefore, this study provides significant evidence and the potential that M. azedarach and L. chinensis extracts can be used or utilized as promising candidate substances for developing novel anti-tubercular drugs of the effective next-generation in the near future through their new pharmacological activity concerning anti-M. tuberculosis effect.

Conflict of interest statement

We declare that we have no conflict of interest.