Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori

Abstract

Background and aim: Tuberculosis (TBC) is a deadly disease and major health issue in the world. Emergence of drug resistant strains further worsens the efficiency of available anti-TBC drugs. Natural compounds and particularly traditional medicines such as Unani drugs are one of the promising alternatives that have been widely used nowadays. This study aims to evaluate the efficacy of unani drug Qurs-e-Sartan Kafoori (QSK) on Mycobacterium tuberculosis (MTB).

Experimental procedures: Drug susceptibilities were estimated by broth microdilution assay. Cell surface integrity was assessed by ZN staining, colony morphology and nitrocefin hydrolysis. Biofilms were visualized by crystal violet staining and measurement of metabolic activity and biomass. Lipidomics analysis was performed using mass spectrometry. Host pathogen interaction studies were accomplished using THP-1 cell lines to estimate cytokines by ELISA kit, apoptosis and ROS by flow cytometry.

Results: QSK enhanced the susceptibilities of isoniazid and rifampicin and impaired membrane homeostasis as depicted by altered cell surface properties and enhanced membrane permeability. In addition, virulence factor, biofilm formation was considerably reduced in presence of QSK. Lipidomic analysis revealed extensive lipid remodeling. Furthermore, we used a THP-1 cell line model, and investigated the immunomodulatory effect by estimating cytokine profile and found change in expressions of TNF-α, IL-6 and IL-10. Additionally, we uncover reduced THP-1 apoptosis and enhanced ROS production in presence of QSK.

Conclusion: Together, this study validates the potential of unani formulation (QSK) with its mechanism of action and attempts to highlight its significance in MDR reversal.

Keywords: Biofilm; Cell membrane; Ethambutol, (EMB); Isoniazid, (INH); Lipidomics; Mycobacterium; Qurs sartan kafoori, (QSK); ROS; Reactive oxygen species, (ROS); Rifampicin, (RIF); Streptomycin, (STP); fatty acid, (FA); glycerolipids, (GL); glycerophospholipids, (GPL); unani drug.

Graphical abstract

Fig. 1

Effect of QSK on anti-TB drugs susceptibility. The upper panel display MIC of anti-TB drugs (RIF, INH, EMB and STP) and combination with QSK by REMA plate method. The visible color change from blue (resazurin) to pink (resorufin) shows reductive activities of the cells, indicating cell growth. The lower panel represents analogous depiction of broth micro dilution assay of anti-TB drugs concentration and combination with QSK represented by color bars.

Fig. 2

Effect of QSK on cell surface integrity (A) ZN staining images of control and cells treated with QSK. Scale bar depicts 20 μm. (B) Colony morphology of control and cells treated with QSK observed on 7H10-based medium at 10x magnification. Scale bar depicts 20 μm. (C) Cell sedimentation. Left panel shows O.D₆₀₀ of control cells and treated with QSK depicted on y axis with respect to time (hours) on x-axis. (D) Nitrocefin hydrolysis of control and cells treated with QSK. Mean of O.D₄₈₅ ± SD of three independent sets are depicted on y axis.

Fig. 3

Effect of QSK on biofilm formation. (A). The upper panel show CV staining depicting biofilm formation in control and in presence of QSK. Scale bar depicts 50 μm. Lower panel depicts bioflim formation on polystyrene surface in presence of QSK. (B) Biofilm metabolic activity (expressed as O.D₄₅₀) depicted as a bar graph. Data are expressed as mean ± SD of three independent sets of experiments and ∗ depicts P value < 0.5. (C) Biofilm biomass (dry weight) of control and QSK treated cells. Mean of dry weight ±SD of three independent experiments are depicted on the Y-axis ∗ depicts P value < 0.5.

Fig. 4

Changes in FA composition in response to QSK. (A). Web graph depicts number of FA subclasses identified from m/z values at WR 0.5 from total lipid through MS-LAMP. (B) Graph represents number of mycocerosic acid and species level changes. (C). Graph represents number of phthioceranic acid and species level changes.

Fig. 5

Changes in GL composition in response to QSK. (A). Bar graph depicts number of GL subclasses (MG, DG and Tg) identified from m/z values at WR 0.5 from total lipid through MS-LAMP. (B) Graph represents number of MG and species level changes. (C). Graph represents number of DG and species level changes. (D). Graph represents number of TG and species level changes.

Fig. 6

Changes in GPL composition in response to QSK. (A). Circular graph depicts number of GPL subclasses identified from m/z values at WR 0.5 from total lipid through MS-LAMP. Outer circle and inner circle represents control and QSK respectively. (B) Graph represents number of PE and species level changes. (C). Graph represents number of lyso-PE and species level changes. (D). Graph represents number of PG and species level changes. (E). Graph represents number of lyso-PG and species level changes.

Fig. 7

Intracellular growth of MTB and Cytokine profiling in presence of QSK. (A) Bar graph represents CFU/ml on y-axis with respect to number of days on x-axis. THP-1 cells were infected with MTB at MOI of 10 for 0, 2, 4, 6 days. After each incubation time, the infected cells were lysed and colonies were counted after 30 days of plating. Three such independent experiments were carried out and the data points represent mean of all three experiments. (B–D) Bar graph represents cytokine levels measured in pg/ml on y-axis with respect to time on x-axis. Cells were infected with MTB at a MOI of 10, and supernatants were collected at different time points after infection and analyzed with specific ELISA for proinflammatory cytokines (B) TNF-α (C) IL-6 (D) IL-10. The results represent the means of three separate experiments, ∗ depicts P value < 0.5.

Fig. 8

Effect of QSK on apoptosis and ROS production. (A). Flow-cytometry analysis of Annexin V–positive cells, uninfected THP-1 cell and MTB infected THP-1 in the presence and absence of QSK. (B) The bar graph represents percentage of Annexin V or propidium iodide–positive cells for early and late apoptosis respectively. (C) Effect of QSK on intracellular ROS generation with DCFDA observed by using flowcytometer. Annexin V-FITC staining Mtb-infected (blue overlay) THP-1 cells and QSK treated showed green overlay.(D) Bar graph represents mean fluorescence intensity on y-axis with respect to control, QSK and hydrogen peroxide (positive control).