Antituberculosis activity, phytochemical identification of Costus speciosus (J. Koenig) Sm., Cymbopogon citratus (DC. Ex Nees) Stapf., and Tabernaemontana coronaria (L.) Willd. and their effects on the growth kinetics and cellular integrity of Mycobacterium tuberculosis H37Rv.

BACKGROUND: Costus speciosus, Cymbopogon citratus, and Tabernaemontana coronaria are herbal plants traditionally used as remedies for symptoms of tuberculosis (TB) including cough. The aims of the present study were to evaluate the in vitro anti-TB activity of different solvent partitions of these plants, to identify the phytochemical compounds, and to assess the effects of the most active partitions on the growth kinetics and cellular integrity of the tubercle organism. METHODS: The in vitro anti-TB activity of different solvent partitions of the plant materials was determined against M. tuberculosis H37Rv using a tetrazolium colorimetric microdilution assay. The phytochemical compounds in the most active partition of each plant were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The effects of these partitions on the growth kinetics of the mycobacteria were evaluated over 7-day treatment period in a batch culture system. Their effects on the mycobacterial cellular integrity were observed under a scanning electron microscope (SEM). RESULTS: The respective n-hexane partition of C. speciosus, C. citratus, and T. coronaria exhibited the highest anti-TB activity with minimum inhibitory concentrations (MICs) of 100-200 µg/mL and minimum bactericidal concentration (MBC) of 200 µg/mL. GC-MS phytochemical analysis of these active partitions revealed that majority of the identified compounds belonged to lipophilic fatty acid groups. The active partitions of C. speciosus and T. coronaria exhibited high cidal activity in relation to time, killing more than 99% of the cell population. SEM observations showed that these active plant partitions caused multiple structural changes indicating massive cellular damages. CONCLUSIONS: The n-hexane partition of the plant materials exhibited promising in vitro anti-TB activity against M. tuberculosis H37Rv. Their anti-TB activity was supported by their destructive effects on the integrity of the mycobacterial cellular structure.

Analysis of differentially expressed proteins in late-stationary growth phase of Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis is a causative agent of tuberculosis (TB). The ability of M. tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of M. tuberculosis H37Rv over three growth phases, namely mid-log (14-day culture), early stationary (28-day culture), and late stationary (50-day culture), was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry were used to generate proteome maps of M. tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared with the other two phases. These proteins were SucD, TrpD, and Rv2161c, which belong to metabolic pathway proteins; FadE5, AccD5, DesA1, and Rv1139c are proteins involved in cell wall or lipid biosynthesis, whereas TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1, was not detectable in the late-stationary phase, although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection.

Antituberculosis potential of some ethnobotanically selected Malaysian plants.

AIM OF THE STUDY: Many local plants are used in Malaysian traditional medicine to treat respiratory diseases including symptoms of tuberculosis. The aim of the study was to screen 78 plant extracts from 70 Malaysian plant species used in traditional medicine to treat respiratory diseases including symptoms of tuberculosis for activity against Mycobacterium tuberculosis H37Rv using a colorimetric microplate-based assay. MATERIALS AND METHODS: Plant extracts were prepared by maceration in methanol (80%) and antituberculosis screening was carried out using Tetrazolium bromide microplate assay (TEMA) method to determine the minimum inhibitory concentration (MIC). RESULTS: Thirty-eight plant extracts from 36 plant species exhibited antituberculosis activity with MICs in the range of 1600-400 µg/ml. The leaf extract of Angiopteris evecta exhibited the highest activity with MIC of 400 µg/ml. Five other extracts, namely, Costus speciosus (stem and flower), Piper sarmentosum (whole plant), Pluchea indica (leaf), Pluchea indica (flower), and Tabernaemontana coronaria (leaf) exhibited antituberculosis activity, each with MIC of 800 µg/ml. To the best of our knowledge, this is the first report of in vitro high throughput screening of Malaysian medicinal plants for antituberculosis activity. CONCLUSIONS: Antituberculosis activity of extracts of some plants justifies, to a certain extent their ethnomedicinal uses as remedies for symptoms of tuberculosis. These results also support the general view that, selecting the plants based on ethnobotanical criteria would enhance the probability of finding species with antituberculosis activity.

Standardization and in vivo antioxidant activity of ethanol extracts of fruit and leaf of Piper sarmentosum.

The present study aimed to investigate standardized ethanol extracts of fruit and leaves of Piper sarmentosum for their in vivo antioxidant activity in rats using a CCl (4)-induced oxidative stress model. The standardization was based on the quantification of the markers pellitorine, sarmentine and sarmentosine by high performance liquid chromatography (HPLC), and determination of total primary and secondary metabolites. The rats, divided into 7 groups each (n = 6), were used as follows: group 1 (CCl (4), negative control), group 2 (untreated, control), groups 3 and 4 (fruit extract 250 and 500 mg/kg, respectively), groups 5 and 6 (leaf extract 250 and 500 mg/kg, respectively) and group 7 (vitamin-E 100 mg/kg, positive control). The doses were administered orally for 14 days; 4 h following the last dose, a single dose of CCl (4) (1.5 mg/kg) was given orally to all the groups except group 2, and after 24 h, blood and liver of each animal were obtained. Analysis of plasma and liver homogenate exhibited significant preservation of markers of antioxidant activity, total plasma antioxidant activity (TPAA), total protein (TP), superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid reactive species (TBARS), in the pretreated groups as compared to the CCl (4) group (p < 0.05). Histology of the liver also evidenced the protection of hepatocytes against CCl (4) metabolites in the pretreated groups. The results of this study indicate the IN VIVO antioxidant activity of both extracts of the plant, which may be valuable to combat diseases involving free radicals.

(E)-N'-(2,4,6-Trihydroxy-benzyl-idene)isonicotinohydrazide sesquihydrate.

In the title compound, C(13)H(11)N(3)O(4)·1.5H(2)O, the pyridine ring forms a dihedral angle of 1.50 (6)° with the benzene ring. An intra-molecular O-H⋯N hydrogen bond forms a six-membered ring with an S(6) ring motif. In the crystal structure, one water mol-ecule is disordered over two positions around an inversion centre with site-occupancy factors of 0.5. Inter-molecular O-H⋯N, O-H⋯O, N-H⋯O and C-H⋯O hydrogen bonds consolidate the structure into a three dimensional network. A π-π stacking inter-action with a centroid-centroid distance of 3.5949 (7) Šis also present.

(E)-N'-(2,3,4-Trimethoxy-benzyl-idene)isonicotinohydrazide.

In the title compound, C(16)H(17)N(3)O(4), the mol-ecule exists in an E configuration with respect to the C=N double bond. The mol-ecule is not planar, the dihedral angle between the pyridine and benzene rings being 71.67 (8)°. In the crystal structure, mol-ecules are linked into chains along the b axis by bifurcated N-H⋯O and C-H⋯O hydrogen bonds. These chains are linked into a three-dimensional network by C-H⋯O and C-H⋯π inter-actions.

(E)-N'-(2,4,5-Trimethoxy-benzyl-idene)isonicotinohydrazide dihydrate.

The asymmetric unit of the title compound, C(16)H(17)N(3)O(4)·2H(2)O, contains one Schiff base mol-ecule and two water mol-ecules. The Schiff base mol-ecule exists in an E configuration with respect to the C=N double bond and is essentially planar, the dihedral angle between the benzene and pyridine rings being 5.48 (8)°. The three meth-oxy groups are also coplanar with the benzene ring [C-O-C-C torsion angles = 3.9 (2), 178.51 (15) and 0.8 (2) Å]. In the crystal structure, the water mol-ecules link the mol-ecules into a three-dimensional network via inter-molecular N-H⋯O, O-H⋯O, O-H⋯N and C-H⋯O hydrogen bonds.

(E)-N'-(2-Benzyl-oxybenzyl-idene)isonicotinohydrazide methanol solvate monohydrate.

The title compound, C(20)H(17)N(3)O(2)·CH(4)O·H(2)O, was synthesized by the condensation reaction of 2-benzyl-oxybenzaldehyde with isoniazid (isonicotinic acid hydrazide). The tricyclic compound displays a trans configuration with respect to the C=N double bond. The central benzene ring makes dihedral angles of 8.83 (7) and 70.39 (8)° with the pyridine ring and the terminal benzene ring, respectively. The dihedral angle between the pyridine ring and the terminal benzene ring is 73.11 (8)°. In the crystal structure, mol-ecules are connected by inter-molecular N-H⋯O, O-H⋯O, O-H⋯(N,N) and C-H⋯O hydrogen bonds, forming a two-dimensional network perpendicular to the a axis.

(E)-N'-[(E)-2-Methyl-pent-2-enyl-idene]isonicotinohydrazide.

The asymmetric unit of the title Schiff base compound, C(12)H(15)N(3)O, contains two crystallographically independent mol-ecules, with both existing in an E configuration with respect to the C=N double bonds. In the crystal structure, inter-molecular N-H⋯N and C-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network.

(E)-N'-(2,4,6-Trimethyl-benzyl-idene)isonicotinohydrazide.

The title isoniazid derivative, C(16)H(17)N(3)O, exists in an E configuration with respect to the Schiff base C=N bond. The pyridine ring is essentially planar [maximum deviation = 0.009 (3) Å]. The mean plane through the hydrazide unit forms dihedral angles of 38.38 (16) and 39.42 (16)°, respectively, with the pyridine and benzene rings. In the crystal structure, symmetry-related mol-ecules are linked via inter-molecular N-H⋯O hydrogen bonds into chains along [100]. The crystal structure is further stabilized by weak inter-molecular C-H⋯π inter-actions.

(E)-N'-[(E)-3-(4-Hydr-oxy-3-methoxy-phen-yl)allyl-idene]isonicotinohydrazide.

In the title compound, C(16)H(15)N(3)O(3), the dihedral angle between the pyridine and benzene rings is 7.66 (5)°. The crystal packing is consolidated by inter-molecular C-H⋯O and O-H⋯N inter-actions, which link the mol-ecules into zigzag chains propagating along [010]. The chains are further linked into a three-dimensional network by N-H⋯O, C-H⋯N, C-H⋯O and C-H⋯π inter-actions.

(E)-N'-(2,4,5-Trifluorobenzyl-idene)isonicotinohydrazide monohydrate.

In the Schiff base mol-ecule of the title compound, C(13)H(8)F(3)N(3)O·H(2)O, the benzene ring and the pyridine ring are nearly coplanar, making a dihedral angle of 6.64 (7)°. The mol-ecule exists in an E configuration with respect to the C=N double bond. In the crystal structure, mol-ecules are linked via the water mol-ecules into two-dimensional planes parallel to the ab plane through inter-molecular N-H⋯O, O-H⋯O O-H⋯N and C-H⋯O hydrogen bonds.

Bis{(E)-N'-[2,4-bis(trifluoro-meth-yl)benzyl-idene]isonicotinohydrazide} monohydrate.

The asymmetric unit of the title compound, 2C(15)H(9)F(6)N(3)O·H(2)O, contains two independent Schiff base mol-ecules and one water mol-ecule. Both Schiff base mol-ecules exist in an E configuration with respect to the C=N double bonds and the dihedral angles between the benzene and the pyridine rings in the two mol-ecules are 17.53 (12) and 20.62 (12)°. In the crystal structure, mol-ecules are linked by inter-molecular N-H⋯O and C-H⋯O hydrogen bonds into infinite one-dimensional chains along the a axis. In addition, inter-molecular O-H⋯N, O-H⋯F, C-H⋯F and C-H⋯O hydrogen bonds further link these chains into a three-dimensional network. Weak π-π inter-actions with centroid-centroid distances in the range 3.6495 (17)-3.7092 (16) Šare also observed.

(E)-N'-(2,3,4-Trihy-droxy-benzyl-idene)-isonicotinohydrazide dihydrate.

In the title isoniazid derivative, C(13)H(11)N(3)O(4)·2H(2)O, the Schiff base mol-ecule exists in an E configuration with respect to the acyclic C=N bond. An intra-molecular O-H⋯N hydrogen bond forms a six-membered ring, producing an S(6) ring motif. The essentially planar pyridine ring [maximum deviation = 0.0119 (8) Å] is inclined at a dihedral angle of 7.30 (4)° with respect to the benzene ring. In the crystal, inter-molecular O-H⋯N, O-H⋯O, N-H⋯O and C-H⋯O hydrogen bonds link the mol-ecules into two-dimensional arrays lying parallel to the (10) plane. These arrays are further inter-connected into a three-dimensional extended network via O-H⋯O and C-H⋯O hydrogen bonds. A weak inter-molecular π-π inter-action [centroid-to-centroid distance = 3.5627 (5) Å] is also observed.

N'-[(3-Methyl-2-thien-yl)carbon-yl]isonicotinohydrazide.

In the title compound, C(12)H(11)N(3)O(2)S, the pyridine ring is inclined to the thio-phene ring, forming a dihedral angle of 34.96 (7)°. The mean plane through the hydrazide unit forms dihedral angles of 21.57 (8) and 53.08 (8)°, respectively, with the pyridine and thio-phene rings. The two O atoms are twisted away from each other, as indicated by the C-N-N-C torsion angle of -81.27 (15)°. In the crystal structure, mol-ecules are linked into an extended three-dimensional network by inter-molecular N-H⋯N, N-H⋯O and C-H⋯O hydrogen bonds. The crystal structure also features a short S⋯O [3.2686 (10) Å] inter-action and a weak inter-molecular C-H⋯π inter-action.

(E)-N'-(3-Benz-yloxy-4-methoxy-benzyl-idene)isonicotinohydrazide.

In the title compound, C(21)H(19)N(3)O(3), the pyridine ring forms a dihedral angle of 15.25 (6)° with the benzene ring. The dihedral angle between the two benzene rings is 83.66 (7)°. The meth-oxy group is slightly twisted away from the attached ring [C-O-C-C = 7.5 (2)°]. In the crystal structure, mol-ecules are linked into a three-dimensional network by inter-molecular N-H⋯N and C-H⋯O hydrogen bonds. The structure is further stabilized by C-H⋯π inter-actions.

N'-(Cyclo-hexyl-carbon-yl)isonicotino-hydrazide.

In the title compound, C(13)H(17)N(3)O(2), the mean plane of the cyclo-hexane ring forms a dihedral angle of 33.12 (5)° with the pyridine ring. The two O atoms are twisted away from each other, as indicated by the C-N-N-C torsion angle of -74.97 (9)°. In the crystal structure, mol-ecules are linked into a three-dimensional network by inter-molecular N-H⋯N, N-H⋯O and C-H⋯O hydrogen bonds. The structure is also stabilized by C-H⋯π inter-actions.

Elucidating isoniazid resistance using molecular modeling.

The continuing rise in tuberculosis incidence and the problem of drug resistance strains have prompted the research on new drug candidates and the mechanism of drug resistance. Molecular docking and molecular dynamics simulation (MD) were performed to study the binding of isoniazid onto the active site of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) in an attempt to address the mycobacterial resistance against isoniazid. Results show that isonicotinic acyl-NADH (INADH) has an extremely high binding affinity toward the wild type InhA by forming stronger interactions compared to the parent drug (isoniazid) (INH). Due to the increase of hydrophobicity and reduction in the side chain's volume of A94 of mutant type InhA, both INADH and the mutated protein become more mobile. Due to this reason, the molecular interactions of INADH with mutant type are weaker than that observed with the wild type. However, the reduced interaction caused by the fluctuation of INADH and the mutant protein only inflected minor resistance in the mutant strain as inferred from free energy calculation. MD results also showed there exists a water-mediated hydrogen bond between INADH and InhA. However, the bridged water molecule is only present in the INADH-wild type complex, reflecting the putative role of the water molecule in the binding of INADH to the wild type protein. The results support the assumption that the conversion of prodrug isoniazid into its active form INADH is mediated by KatG as a necessary step prior to target binding on InhA. Our findings also contribute to a better understanding of INH resistance in mutant type.

Effects of isoniazid on viability, cell morphologies and acid fastness properties of Mycobacterium avium NCTC 8559 during the growth cycle.

Our previous study demonstrated that the effects of isoniazid (INH) on Mycobacterium tuberculosis at the cellular level varied according to the growth phases. In this study, the variations in the INH action on M. avium strain NCTC 8559 are reported. M. avium cells grown on Middlebrook 7H10 agar were harvested at different stages of their growth cycle, exposed to the minimum inhibitory concentration of INH, stained with acid-fast staining for morphological changes and acid fastness properties, and the number of colonies were evaluated for viability studies. The study demonstrated that M. avium NCTC 8559 cells at the initial and fragmentation stages of the growth cycle were most susceptible to INH.