Antimycobacterial potential of <i>Trachyspermum ammi</i> seed essential oil via fume contact and determination of major compounds

Trachyspermum ammi (L.), commonly known as carrom seeds or Ajwain, has been extensively studied for its medicinal properties. In this study, anti-mycobacterial effect of AEO in liquid and fume form was investigated against Mycobacterium smegmatis and Mycobacterium tuberculosis (M. tb). Results showed that AEO inhibits the growth of M. smegmatis at 0.03 mg/mL and becomes bactericidal at 0.125 mg/mL. MICs were observed at 0.03, 0.125 and 0.06 mg/mL against M. tb (H37Rv), isoniazid- and rifampicin-resistant (RIF-R) strains. Inverted disc-fume assay revealed AEO and Thymol efficiently inhibit the growth of M. smegmatis and M. tb. Similarly, in fume contact AEO and Thymol demonstrated antibiofilm activity at a dose of 1.25 mg/mL air and 40 mg/mL air against M.smegmatis effectively. GC-MS analysis showed that Thymol was the dominant compound. These findings suggest that the use of AEO in fume form may serve as a promising strategy as an anti-mycobacterial activity against M. tb</p

Mean fluorescent intensity and percent positive cells in FACS analysis (Figure 2B).

<p>Data represent Mean ± SD from three preparations.</p

Effect of AE and AC- treatments on parasitemia in mice infected with <i>P.berghei</i> for 72 hr.

<p>(A) Changes in parasite load in blood at different time periods through real-time PCR analysis of parasite 18S rRNA after drug treatment. UT, untreated; C, curcumin, AE, ART alone; AC, ART+CUR. D, died; N, not detectable. Data provided represent Mean ± S.D. from three animals. The whole series was repeated thrice (total of 9 animals in each group) and number of animals surviving on day 30 in each group were as follows: UT, 0; C, 0; AE, 1; AC, 9. (B) Semiquantitative RT-PCR analysis of parasite 18S rRNA in blood, liver and spleen on day 10 and 15 after infection. GAPDH was used as a control. *, positive control. (C) Effect of injection of blood from recrudescing animals after AE and AC-treatments into naïve animals. Five animals were used in each group.</p

Effect of AE and AC-treatments on changes in serum cytokine levels and survival response of TLR2^−/− and IL-10^−/− mice to <i>P.berghei</i>-infection.

<p>(A) Changes in the serum levels of INFγ, IL-10 and IL-12. The data represent Mean + S.D. from three sera preparations and collected during day 6 (D6) to day 23 (D23) at intervals. The day 0 values correspond to those of uninfected animals. The values (pg/ml) corresponding to infected animals, which all died on day 6, are as follows: INFγ, 274±22; IL-10, 422±29; IL-12, 510±39. The values corresponding to infected animals treated with curcumin alone and which all died between, 7–8 days are as follows: INFγ, 243±22; IL-10, 310±33; IL-12, 377±39. (B) Effect of IL-10 injection in AE-treated animals. IL-10 was injected on alternate days from day 10 in five doses ranging from 10 ng to 200 ng. Alternate i.p. and i.v. routes were used. (C) Survival response of <i>P.berghei</i>infected C57BL/6 mice to AE and AC-treatments. The data are from two experiments with five animals in each group. (D) Survival response of TLR^−/− animals with and without IL-10 injection (100 ng per animal for 5 days). (E) Survival response of IL-10^−/− animals. Four animals were used in each group for the experiments in (B), (D) and (E). The knock-out animals were in C57BL/6 background. While, the animals were injected with around 10⁴ parasites in general, the IL 10^−/− animals received around 10³ parasites. UI, unifected; I, infected; UT, untreated; C, curcumin; AE, ART alone; AC, ART+CUR.</p

Effect of AE and AC-treatments on changes in spleen mass and mRNA levels of cytokines, TLRs and IgG-sub-class antibodies in <i>P.berghei</i>-infected mice.

<p>(A) Changes in spleen weight through out the course of infection and drug treatments. Data provided represent Mean ± S.D. from five animals. *, infected animals died 5/6 day (B) Semi-quantitative RT-PCR analysis of spleen RNA on day 14. UI, unifected; I, infected; AE, ART alone; AC, ART+CUR.</p

Effect of AE and AC-treatments on hemozoin content and ROS generation in <i>P.berghei</i>infected mice.

<p>(A). Hemozoin content in parasite 8 hr and 12 hr after a single injection of ART and one oral dose of curcumin. The data represent Mean ± S.D. from three animals. (B) FACS analysis of <i>P.berghi</i>–infected red blood cells for ROS measurement 12 hr after drug treatment as per (A).</p

Effect of AE and AC-treatments on changes in serum total IgG and IgG-subclass antibody levels and Western analysis of parasite proteins with such sera in <i>P.berghei</i>-infected mice.

<p>Antibody levels were quantified in sera by ELISA using microtitre plates coated with parasite lysate from day 6 (D6) onwards. (A) Changes in the serum levels of total IgG and IgG-subclass antibodies against parasite lysate. The data represent Mean ± S.D. from three sera preparations (pooled from two animals each) and collected during day 6 (D6) to day 23 (D23) at intervals. The day 0 values correspond to those of uninfected animals. The values (A₄₀₅) corresponding to infected animals, which all died on day 6, are as follows: IgG, 0.07±0.002; IgG1, 0.03±0.005; IgG2a, not detectable; IgG2b, not detectable; IgG3, not detectable; IgM, 0.22±0.007. (B) Anamnestic response of AC-treated animals. The animals were injected with fresh parasitized blood 10 days before the day mentioned in the Figure. The antibody titre (IgG) was measured before and after challenge. The data represent an average from two sera preparations. (C) Western blot analysis of parasite proteins with the different sera preparations. 1, uninfected; 2, day 5- infected; 3, AE -day 22; 4, AC day-22; 5 and 6, AC - day 75, before and after challenge; 7 and 8; AC - day 180, before and after challenge.</p

Activation of infected macrophages and inter-compartmental localization of mycobacteria within A.

<p>The plots depict reduced activation of H37RV/ARPC4 infected macrophages. <b>a)</b> Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. <b>b)</b> Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. <b>c)</b> Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. <b>B.</b> Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.</p

Effect of ARPC4 on the growth of <i>M. tuberculosis</i>.

<p>A. An individual colony each from mycobacteria harbouring either ARPC4pVV16 (H37Rv/ARPC4) or pVV16 empty vector (H37Rv/pVV16) was picked for growth curve analysis. Optical density of both cultures was measured at 600 nm. Mean±s.d values are plotted against time (in Days). A similar growth curve pattern was observed each time the experiment was repeated. Triangular data points represent OD₆₀₀ values of H37Rv/pVV16 samples (control); circles represent OD₆₀₀ values of H37Rv/ARPC4 samples. <b>B.</b> Cultures were grown in 7H9 medium at 37°C with shaking under axenic conditions. The figure shows result of CFU counts of H37Rv/ARPC4 and H37Rv/pVV16 at days 0, 7 and 14. A significant decline in the survival of mycobacteria carrying ARPC4pVV16 was observed in comparison to the vector control. <b>C.</b> Effect of ARPC4 expression on cell morphology growth of <i>M. tuberculosis</i>. Transmission electron micrographs of H37Rv (panel a) and mycobacterial cells harboring ARPC4pVV16 (H37Rv/ARPC4) (panels b and c). <b>D.</b> Relative fold change in the transcript levels of <i>rv1626</i> and <i>bfrB</i> genes. The <i>rv1626</i> gene (dark grey) was found to be significantly down-regulated (6-fold) in H37Rv/ARPC4 cells when compared with the H37Rv mycobacterial cells, whereas the unrelated <i>bfrB</i> gene (light grey) expression was not much perturbed. The 16S rRNA gene was used as the normalization internal control. Experiment was performed in triplicates.</p

Sequences of the DNA primers used for PCR amplification of various genes described in the present study.

<p>Sequences of the DNA primers used for PCR amplification of various genes described in the present study.</p