In vitro antitubercular activity of extract and constituents from the stem bark of Disthemonanthus benthamianus

ABSTRACT

A new C-glycosylflavone, apigenin 7-methyl ether 6-C-[β-xylopyranosyl-(1→3)-β-glucopyranoside] named distemonanthoside was isolated from the stem bark of Distemonanthus benthamianus Baill., Fabaceae, along with six known compounds, sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid, syringic acid, quercetin, 6"-O-acetylvitexin, quercetin 3-O-β-D-glucopyranoside. The structures of those compounds and others were determined through spectral analyses. Compounds distemonanthoside, sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid and quercetin were tested against a clinical isolate strain of Mycobacterium tuberculosis AC 45; they exhibited good to moderate antitubercular activities with MIC values ranged from 31.25 to 125 µg/ml.

Keywords:
Fabaceae; Antitubercular activity; Distemonanthoside; Flavonoids; Phenolic acids

Introduction

Tuberculosis (TB) is a chronic contagious disease caused by several species of Mycobacterium. Due to the fact that there is a doubt nowadays on the efficiency of current antibiotics for the treatment of tuberculosis, micro-organisms developed resistance inducing an increase of the number of patients with the disease in worldwide (WHO, 2016). This increasing of MDR-TB incidence has led to an urgent need for the discovery of new plant natural products that may potentially eradicate TB. Several in vitro growth inhibition of different strains of M. tuberculosis by plant extracts have been reported (Okunade et al., 2004; Copp and Norrie Pearce, 2007; Gautam et al., 2007; Mc Gaw et al., 2008). The Cameroonian medicinal plant Distemonanthus benthamianus Baill., Fabaceae, is a large rainforest tree widely distributed in Africa, especially in equatorial region. This essence is highly appreciated industrially for heavy construction and some countries use to export it as “Movingi”. In Mayombé region (Congo), traditional healers employ the stem bark in the treatment of several diseases as: parasitic, dermatitis, furuncles, aces and chancres. In the Chaillu region (Congo), that plant is used to cure bronchitis affections and children fever (Bouquet, 1969). In previous works carried on D. benthamianus, mainly methoxylated flavonols and flavones were isolated (King et al., 1952; King et al., 1954; Malan and Roux, 1979; Happi and Mpondo, 1994); this paper describes the isolation and structure elucidation of constituents from stem bark of D. benthamianus. The evaluation of antitubercular activities of compounds distemonanthoside (1), sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid (2) and quercetin against resistant strain of M. tuberculosis was also examined.

Material and methods

General procedures

Melting points were uncorrected and were measured on a Mettler Toledo instrument. IR spectra were recorded on an Alpha FT-IR Spectrometer from Bruker, while 1D and 2D NMR spectra were obtained on a Bruker DRX 500 (500 MHz for ¹H and 125 MHz for ¹³C spectra) spectrometer (Bruker, Rheinstetten, Germany) with chemical shifts reported in δ (ppm) using TMS (δ_H) as an internal standard. The HR-ESI-MS was obtained on LTQ-FT instrument (Thermo Scientific). UPLC–MS was measured by a Shimadzu UPLC-MS system. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. Silica gel 60 (230–400 mesh E. Merck, Darmstadt, Germany) was employed for column chromatography, the solvent mixing systems for elution were mainly CH₂Cl₂/MeOH with increasing polarity each.

Plant material

Stem bark of Distemonanthus benthamianus Baill., Fabaceae, were collected at Eséka (Koumoul) near Yaoundé (3º 38' 60.00" N, 10º 46' 0.00" E) in the Centre Region of Cameroon in March 2014 and identified by Victor Nana. A voucher specimen (No. 45488 HCN) was deposited at the National Herbarium in Yaoundé, Cameroon.

Extraction and isolation

Dried and powdered stem bark of D. benthamianus (254 g) were extracted for 48 h with MeOH (3 × 1l) at room temperature. After filtration and evaporation of solvent, the crude MeOH extract (16 g) was subjected to CC (150 × 3 cm) [(SiO₂), eluting with a gradient solvent system (CH₂Cl₂/MeOH)] giving four main fractions: I (1.9 g), II (3.8 g), III (3.6 g) and IV (6.7 g). Fractions (100 ml) were collected and grouped on the basis of TLC analysis. Fraction II (3.8 g) was submitted to CC (SiO₂, 100 × 1 cm) using solvent system CH₂Cl₂/MeOH (50/1) to give sitosterol 3-O-β-D-glucopyranoside (65 mg). Fraction III (3.6 g) was submitted to CC (SiO₂, 100 × 1 cm) using solvent system CH₂Cl₂/MeOH (60/1 to 5/1) to give four sub-fractions (IIIa, IIIb, IIIc and IIId). Sub-fraction IIIc (1 g) was chromatographed (SiO₂, 50 × 1 cm) using CH₂Cl₂/MeOH (40/1–15/1) to afford compound 2 (480 mg) and compound 3 (3 mg). Sub-fraction IIId (0.65 g) was subjected to a silica gel column in gradient elution mixture solvent composed of CH₂Cl₂/MeOH (25/1–5/1) to afford quercetin (8 mg) and quercetin 3-O-β-D-glucopyranoside (11 mg). Using the same process, fraction IV (6.7 g) gave three sub-fractions (IVa, IVb and Vc). Sub-fraction IVa (0.98 g) was further chromatographed on a silica gel column (100 × 1 cm) using CH₂Cl₂/MeOH (10/1) to afford compound 4 (4 mg). Sub-fraction IVb (2.8 g) was purified by repeated CC on silica gel (100 × 1 cm) with the solvent system CH₂Cl₂/MeOH (10/1–1/1) to provide compound 1 (28 mg).

Structural characterization of distemonanthoside (1)

Yellow solid; [α]_D²⁵= −54º (c 0.05, MeOH); m.p. 285–287 ºC; IR_υmax^KBr cm⁻¹: 3267, 2923, 2853, 1595, 1512, 1226, 1159; TLC, R_f: 0.28 (CH₂Cl₂/MeOH: 90/10); ESI-MS m/z: ESI-MS: 577.4 [M−H]^−•, LC–MS: m/z 579 [M+H]⁺ and ESI-MS: m/z 601.5 [M+Na] + (Calcd for C₂₇H₃₀O₁₄Na = 601.5); ¹H NMR (500 MHz, DMSO-d₆), δ_H: 8.09 (2H, d, J = 8.8 Hz, H-2' and H-6'), 6.96 (2H, d, J = 8.8 Hz, H-3' and 5'), 6.82 (1H, s, H-3), 6.44 (1H, s, H-8), 4.81 (1H, d, J = 10.0 Hz, H-1"), 4.01^b (1H, H-3"), 3.86 (3H, s, –OCH₃), 3.82 (1H, d, J = 7.0 Hz, H-1'''), 3.72 (1H, dd, J = 11.3; 2.4 Hz, H-6"), 3.42^b (1H, H-6"), 3.39^b (1H, H-2"), 3.36^b (1H, H-4"), 3.21^b (1H, H-5"), 2.83 (1H, dd, J = 11.5; 4.0 Hz, H-5'''), 2.81^b (1H, H-3'''), 2.78^b (1H, H-2'''), 2.39^b (1H, H-5'''); ¹³C NMR (125 MHz, DMSO-d₆): δ_C: 182.1 (C-4), 164.1 (C-2), 163.4 (C-7), 161.4 (C-9), 161.2 (C-4'), 155.5 (C-5), 128.9 (C-2' and C-6') 121.3 (C-1'), 115.8 (C-3' and C-5'), 105.3 (C-1'''), 104.7 (C-6), 104.1 (C-10), 102.3 (C-3), 94.8 (C-8), 81.7 (C-5"), 80.4 (C-3"), 78.1 (C-2"), 76.4 (C-3'''), 75.6 (C-2'''), 73.5 (C-1"), 71.3 (C-4"), 69.2 (C-4'''), 65.3 (C-5'''), 60.8 (C-6"), 56.5 (–OCH₃).

^bSignal patterns are unclear due to overlap.

Antitubercular activity

MIC values were determined for the extract against M. tuberculosis strain AC 45 (clinical isolate obtained from Sangmelima district's Hospital in South Region of Cameroon) employing the microplate Alamar Blue assay, using Rifampicin as reference. The 96 wells plate received 100 µl of Middlebrook 7H9 medium supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) 2% glycerol and 0.05% v/v of tween 80. Broth and serial dilution of compounds were made directly on the plate with drug concentrations of 0.244 to 250 µg/ml. Plates were covered and sealed with parafilm and incubated at 37 ºC for 14 days. Then, 40 µl Alamar Blue solution was added to the plate and incubated for 24 h. A blue colour in the well was interpreted as no bacterial growth and pink colour was scored as growth. The MIC was defined as the lowest drug concentration, which prevented colour change from blue to pink. The result of antitubercular activity depicted in Table 1. The MIC and MBC were determined according to the guidelines of CLSI (2011). Each experiment was performed at least twice according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2011).

Table 1
MIC and MBC values of the methanol extract and the isolated compounds against clinical isolate strain of Mycobacterium tuberculosis (AC 45).

Plant species/compounds	MICa (µg/ml)	MICa (µM)	MBCb (µg/ml)	MBC/MIC
D. benthamianus	1250	ndc	2500	2
1	125	216.3	125	1
Sitosterol 3-O-β-d-glucopyranoside	62.5	108.5	125	2
2	31.25	169.8	125	4
Quercetin	62.5	207.0	125	2
RMP	0.976	ndc	7.8125	8

• RMP, Rifampicin.
• ^a
  MIC, Minimum Inhibitory Concentration.
• ^b
  MBC, Minimum Bactericidal Concentration.
• ^c
  nd, Not determined.

Acid hydrolysis of 1

Compound 1 (8 mg) was dissolved in7% H₂SO₄ (0.5 ml) and heated on an aqueous bath at 100 ºC for 4 h. The reaction mixture was diluted with H₂O and extracted with CH₂Cl₂. The CH₂Cl₂ layer was evaporated to dryness and purified by preparative TLC over silica gel with CH₂Cl₂–MeOH (5/1) as eluent. Apigenin-7-methyl ether 6-C-glucoside (3 mg) was isolated and identified through direct comparison with authentic samples (TLC, MP, and IR). The neutralized and lyophilized aqueuous hydrosylates of the aqueous solution gave only xylose. GC-MS (Column: 5% phenyl and 95% methyl silicone on ultra 2, 0.2 × 46 m, column temp.: 250 ºC, carrier gas: He 0.8 ml/min, sample: trimethylsilyl derivatives: t_R (min) xylose (19.29 for 1).

Results and discussion

The detailed investigation of methanol extract of the stem bark of D. benthamianus led to the isolation of seven compounds. Six of them were identified as the known sitosterol 3-O-β-D-glucopyranoside (Ngono Bikobo et al., 2014), 4-methoxygallic acid (2) (Ouyang et al., 2007), syringic acid (3) (Bayiha Ba Njock et al., 2011), quercetin (Güvenalp and Demirezer, 2005), 6"-O-acetylvitexin (4) (Bayiha Ba Njock et al., 2011), quercetin 3-O-β-D-glucopyranoside (Murai et al., 2014). The structures of these compounds were elucidated by NMR spectroscopy analysis, including 1D and 2D techniques and also by comparing experimental data with respective literature data

Compound 1 was obtained as yellow amorphous powder, [α]D25=−54º (c = 0.05, MeOH). Its molecular formula, C₂₇H₃₀O₁₄ was established by negative-ion HR-ESI-MS (Fig. S4). The spectrum displayed the deprotonated molecule peak [M−H]⁻ at m/z = 577.4 in agreement with the above formula (calcd, 577.44). The IR spectrum of 1 showed absorption bands characteristic of hydroxyl groups (3219 cm⁻¹), conjugated carbonyl groups (1652 cm⁻¹) and aromatic rings (1603 and 1572 cm⁻¹). UV spectral properties of 1 showed absorption maxima at λ_max 340 nm and 268 nm in MeOH, characteristic for a substituted flavone (Mabry et al., 1970). In addition, acid hydrolysis of 1 gave apigenin 7-methyl ether 6-C-glucoside and β-xylose which were identified by TLC analysis and comparison with authentic samples (GC; t_R 19.29 min). In the ¹H NMR spectrum (Table 2) the set of ortho-coupled AA'BB' type protons at δ_H 8.04 (2H, d, J = 8.8 Hz) and 6.96 (2H, d, J = 8.8 Hz), was respectively assigned to H-2'/6' and H-3'/5' protons of the B-ring of the molecule, while an isolated aromatic proton appeared at δ_H 6.44 (s, H-8) from A ring. The spectrum also revealed the presence of a methoxyl group at δ_H 3.86 and two signals assignable to anomeric sugar protons, which were identified to be an inner β-glucopyranose and a terminal β-pyranose structure of xylose. This was strengthened by the observation in ¹³C NMR and DEPT spectra of eleven carbon signals (Table 2) among which two are anomeric carbon signals at δ_C 73.5 and 105.3, seven methine carbon signals, two oxymethylene carbons at δ_C 60.8 and 65.3. Since the anomeric protons of glucose and xylose at δ_H 4.81 and 3.82 exhibited large coupling constants (J = 10.0 and 7.0 Hz), the sugars were considered of the β-pyranose type. The HMBC spectrum of compound 1 revealed correlations of the anomeric proton at δ_H 4.81 (H-1") and carbons at δ_C 161.3 (C-7), 155.6 (C-5) 104.7 (C-6) and 81.7 (C-5") (Fig. 1), indicating the C-C bond between the inner β-glucopyranosyl moiety and the aglycone at 6-position. In addition, H-1''' at δ_H 3.82 correlates to both C-3" (δ_C 80.4) and C-5''' (δ_C 65.3) revealing that the β-xylopyranosyl moiety was linked to C-3" at δ_C 80.4, showing that glucose and xylose are linked through a 1→3 type. This was strengthened by the NOESY crosspeaks of the protons H-3" (δ_H 4.01) with H-1''' (δ_H 3.82) confirming the aforementioned bonding. The attachment of a methoxyl group to the 7-position was shown by the observation of the crosspeaks at δ_H 3.86 (3H, s, OMe) and δ_C 164.1 (C-7) in the long-range HMBC spectrum. this position through the correlation between H-8 (δ_H 6.44) and the methoxyl proton signals at δ_H 3.86. The complete assignment of all proton and carbon resonances was achieved after careful analysis of COSY, HSQC and HMBC techniques.

Table 2
¹H and ¹³C NMR spectroscopic data of compound 1 (500 and 125 MHz in DMSO-d₆ ) δ in ppm.

Position	δ C	DEPT	δH (J in Hz)	HMBC (C→H)
Apignenin
2	164.1	C		H-C(3); H-C(2')
3	102.3	CH	6.82 (s)
4	182.1	C		H-C(3)
5	155.5	C		H-C(1")
6	104.7	C		H-C(1")
7	163.4	C		H-C(1");CH3O-
8	94.8	CH	6.44 (s)
9	161.4	C		H-C(8)
10	104.1	C		H-C(3)
1'	121.3	C		H-C(3); H-C(2', 6')
2'	128.9	CH	8.04 (d, 8.8)	H-C(3', 5')
3'	115.8	CH	6.96 (d, 8.8)	H-C(2', 6')
4'	161.2	C		H-C(2'); H-C(5')
5'	115.8	CH	6.96 (d, 8.6)	H-C(2', 6')
6'	128.9	CH	8.04 (d, 8.6)	H-C(3', 5')
7-OCH3	56.5	CH3	3.86 (s)
Inner glucose
1"	73.5	CH	4.81 (d, 10)	H-C(2"); H-C(5")
2"	78.1	CH	3.39a	H-C(1")
3"	80.4	CH	4.01a	H-C(1")
4"	71.3	CH	3.36a	H-C(2"); H-C(5")
5"	81.7	CH	3.21a	H-C(1"); H-C(6")
6"	60.8	CH2	3.72 (dd, 11.3; 2.4) 3.42a	H-C(5")
Terminal xylose
1‴	105.3	CH	3.82 (d, 7.0)	H-C(2‴); H-C(5‴)
2‴	75.6	CH	2.78a	H-C(1‴)
3‴	76.4	CH	2.81a	H-C(1‴); H-C(5‴)
4‴	69.2	CH	2.89a	H-C(5‴)
5‴	65.3	CH2	2.83 (dd, 11.5; 4.0) 2.39a	H-C(1‴); H-C(4‴)

• ^a
  Signal patterns are unclear due to overlap.

Fig. 1
Selected HMBC (1.) correlations of compound ) and NOESY (

Some significant HMBC correlations are shown in Fig. 1 and in Table 2. Compound 1 is closely related to the previously reported swertisin 2"-O-arabinoside from the tall bearded iris (Takayuki et al., 2012); meanwhile, differences occur in the sequence of sugar moieties and this is exemplified by the values of the retention times of xylose [which is close to reported data (Liu et al., 2009)]. This assertion is also strengthened by the upper chemical shift values of protons of the xylose moieties compared to those of arabinose (Gu et al., 2011).

Accordingly, 1 was defined as apigenin 7-methyl ether 6-C-[β-xylopyranosyl-(1→3)-β-glucopyranoside] named distemonanthoside. To the best of our knowledge, this is the first report of the isolation of this compound and others (phenolic acids and sterols) from D. benthamianus.

According to Cantrell et al. (2001) isolated compounds that exhibit a MIC of 64 µg/ml or lower are considered promising. For crude extracts, the MIC should be equal to or lower than 125 µg/ml (Gu et al., 2004). Thus, the values of 125, 62.5, 31.25 and 62.5 µg/ml for 1, sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid and quercetin, respectively obtained here, are as good as a promising isolated compounds except for compound 1 (Table 1). According to Gu et al. the methanol extract of D. benthamianus showed poor inhibitory activity against M. tuberculosis, exhibiting a MIC and MBC of 1250 and 2500 µg/ml respectively, suggesting the low lipophilicity of its constituents (more polar compounds) when they act mutually in synergy. According to Peterson and Shanholtzer (1992) bacteriostatic activity has been defined as a ratio of MBC to MIC of >4. Thus, all tested compounds exhibited bactericidal activity. The results of the present study are in accordance with previous report regarding the values of MIC of isolated compounds (Gu et al., 2004; Jiménez-Alleranes et al., 2007).

Conclusion

The species D. benthamianus, is known as abundant sources of flavonoids. Compounds 1, sitosterol 3-O-β-D-glucopyranoside and 4-methoxygallic acid were isolated for the first time from this species. The bioactivity study of the isolated compounds indicated that three compounds (sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid and quercetin) exhibited interesting antitubercular activity.

Acknowledgements

The authors gratefully acknowledge financial support from the Swiss National Science Foundation (SNSF) (No: IZK0Z2-157272) for research fellowships in Switzerland to D. S. Ngono Bikobo. We thank Mr V. Nana for the collection and identification of plant material. We thank Mr Felix Fehr of Department of Chemistry of University of Fribourg and Koert's team, particularly Mr Oliver Born of Philipps-Universität Marburg for spectral analysis.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2017.09.006.

References

• Bayiha Ba Njock, G., Bartholomeusz, T.A., Foroozandeh, M., Pegnyemb, D.E., Christens, P., Jeannerat, D., 2011. NASCA-HMBC, a new NMR methodology for the resolution of severely overlapping signals: application to the study of agathisflavone. Phytochem. Anal. 23, 126-130.
• Bouquet, A., 1969. Fetish and traditional medicine of Congo (Brazzaville). O.R.S.T.O.M. Paris 36, 177-178.
• Cantrell, C.L., Franzblau, S.G., Fischer, N.H., 2001. Antimycobacterial plant terpenoids. Planta Med. 67, 685-694.
• CLSI, 2011. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes, 2nd ed. Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, USA, Approved Standard M24-A2.
• Copp, B.R., Norrie Pearce, A., 2007. Natural product growth inhibitors of Mycobacterium tuberculosis Nat. Prod. Rep. 24, 278-297.
• Gautam, R., saklani, A., Jachak, S.M., 2007. Indian medicinal plants as a source of antimycobacterial agents. J. Ethnopharmacol. 110, 200-234.
• Gu, J.Q., Wang, Y., Franzblau, S.G., Montenegro, G., Yang, D., Timmermann, B.N., 2004. Antitubercular constituents of Valeriana laxiflora Planta Med. 70, 509-514.
• Gu, X., Lee, S.G., Bar-Peled, M., 2011. Biosynthesis of UDP-xylose and UDP-arabinose in Sinorhizobium meliloti 1021: first characterization of a bacterial UDP-xylose synthase, and UDP-xylose 4-epimerase. Microbiology 157, 260-269.
• Güvenalp, Z., Demirezer, O., 2005. Flavonol glycosides from Asperula arvensis L.. Turk. J. Chem. 29, 163-169.
• Happi, E.N., Mpondo, T.N., 1994. Two polymethoxylated flavones from Distemonanthus benthamianus J. Nat. Prod. 57, 291-293.
• Jiménez-Alleranes, A., Meckes, M., Torres, J., Herrera-Luna, J., 2007. Antimycobacterial triterpenoids from Lantana hispida (verbenaceae). J. Ethnopharmacol. 111, 202-205.
• King, F.E., King, T.J., Sellars, K., 1952. The chemistry of extractives from hardwoods. Part V. The isolation of 3,7,4'-trimethylquercetin (ayanin) from the heartwood of Distemonanthus benthamianus J. Chem. Soc. 0, 92-95.
• King, F.E., King, T.J., Stokes, P.J., 1954. The chemistry of extractives from hardwoods. Parts XIX. The structures of further new flavones occurring in ayan (Distemonanthus benthamianus). J. Chem. Soc. 0, 4587-4594.
• Liu, R., Ma, S., YU, S., Pei, Y., Zhang, S., Chen, X., Zhang, J., 2009. Cytotoxic oleanane triterpene saponins from Albizia chinensis J. Nat. Prod. 72, 632-639.
• Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer, 354 p.
• Malan, E., Roux, D.G., 1979. Flavonoids from Distemonanthus benthamianus Baillon. Methoxylated flavones and inter-relationships of benthamianin, a [2]-benzopyrano-[4,3-b] [I]-benzopyran. J. Chem. Soc. 1, 2696-2703.
• Mc Gaw, L.J., Lall, N., Meyer, J.J.M., Eloff, J.N., 2008. The potential of South African plants against Mycobacterium infections. J. Ethnopharmacol. 119, 482-500.
• Murai, Y., Kitajima, J., Iwashina, T., 2014. Flavonoids from two alpine Campanula species in Japan. Bull. Natl. Mus. Nat. Sci. 40, 113-118.
• Ngono Bikobo, D.S., Mosset, P., Abouem, A., Zintchem, A., Atchadé, A.T., Balemaken Missi, M., Mbabi Nyemeck, N., Pegnyemb, D.E., 2014. Campylospermine, an N-hydroxy alkaloid from the leaves of Campylospermum densiflorum (Ochnaceae). Int. J. Pharm. Phytopharmacol. Res. 6, 719-728.
• Okunade, L., Elvin-Lewis, M.P., Lewis, W.H., 2004. Natural antimycobacterial metabolites: current status. Phytochemistry 65, 1017-1032.
• Ouyang, M.A., Wein, Y.S., Su, R.K., Kuo, Y.H., 2007. Rhusemialins A-C, new cyclolignan esters from the roots of Rhus javanica var. roxburghiana Chem. Pharm. Bull. 55, 804-807.
• Peterson, L.R., Shanholtzer, C.J., 1992. Tests for bactericidal effects of antibacterial agents: technical performance and clinical relevance. Clin. Microbiol. Rev. 5, 420-432.
• Takayuki, M., Tsutomu, Y., Nobuhiro, S., Tsukasa, I., 2012. Phenolic compounds, including novel C-glycosylflavone, from the flowers of the tall bearded Iris cultivar ‘Victoria Falls’. Nat. Prod. Commun. 7, 1591-1594.
• WHO, 2016. Global Tuberculosis Report. Global and Investments Fall Far Short of Those Needed to End the Global TB Epidemic. World Health Organisation, Geneva, Switzerland, http://www.who.int/tb/publications/globalreport/gtbr2016maintext.pdf [accessed 03.05.17].
  » http://www.who.int/tb/publications/global_report/gtbr2016_main_text.pdf

Publication Dates

• Publication in this collection
  Nov-Dec 2017

History

• Received
  10 May 2017
• Accepted
  12 Sept 2017