Evaluation of in vivo antimycobacterial activity of some folklore medicinal plants and enumeration of colony forming unit in murine model.

OBJECTIVES: The present study was carried out to investigate the in vivo antimycobacterial activity of methanol extract of Alstonia scholaris and Mucuna imbricata in murine model.
MATERIALS AND METHODS: Female BALB/c mice were infected with the Mycobacterium tuberculosis H37Rv suspension. Extracts were administered orally for 2 weeks from 7th day postinfection at a dose of 200 mg/kg and rifampicin at 20 mg/kg as standard. The synergistic groups were 10 and 100 mg/kg for rifampicin and extract, respectively.
RESULTS: The final body weight of mycobacteria-infected group was significantly reduced (15.41 ± 0.42, P < 0.01), but following treatment with the plant extract plus rifampicin could elevate the body weight. Colony forming unit (CFU) count of lung (8.71 ± 0.01) and spleen (8.59 ± 0.01) was significantly higher in infected and untreated group (P < 0.01). It was observed that activity of the synergistic group displayed powerful and maximum response against tuberculosis (TB) infection with lower CFU counts. Histopathology study showed cells such as lymphocytes, epithelioid, Langhans giant cell, and fibrous tissue proliferation in lungs; depletion of lymphocytes in the spleen.
CONCLUSIONS: The data indicate that methanol extract of A. scholaris has potential antimycobacterial activity, and the synergistic group consisting of rifampicin and A. scholaris could be a rational choice for the treatment of TB.

One of the most challenging aspects of developing antituberculosis (TB) drug of efficacy to reduce or prevent the disease is drug resistance toward modern medicines.[12] Chemotherapy of drug-susceptible TB consists of three or four drugs regimen, administered for a period of 6 months. A major problem of TB control is its lengthy drug regimens which lead to noncompliance with therapy, relapse, and development of multidrug resistance.[23] Development and evaluation of new chemical entities against TB are a challenging task. There is no doubt that the discovery of effective new agents is needed to deal with the current situation. The global threat of TB demands for the search for alternative antimycobacterial drugs.

Plant-derived drugs have long been used worldwide in traditional medicine for the treatment of various infectious diseases and have received considerable attention as potential anti-TB agents.[4567] Alstonia scholaris (Assamese name: Satiana) and Mucuna imbricata (Assamese name: Ghila) invite the attention of the researchers worldwide for its multifarious pharmacological activities ranging from antimalarial to anti-TB.[8] A. scholaris is a large evergreen tree commonly found in the subtropical regions of South Asia and Africa. Almost all parts of the plants are used in medicine. Alstonia spp. is used as an anthelmintic, astringent, antiperiodic, and also used to treat chronic diarrhea, dysentery, beriberi, congestion of liver, dropsy, and ulcers. Methanolic extracts of A. scholaris (MEAS) have exhibited potent antimicrobial activity.[9] M. imbricata belongs to family Fabaceae is found in both temperate and tropical countries including India. Mucuna spp. is used traditionally in diarrhea, cough, asthma, muscular pain, gout, diabetes, uterine stimulant, Parkinson's disease, and cancer.[10] To date, a very few information on antimycobacterial activity of A. scholaris (MEAS) and M. imbricata (MEMI) is available.[891011]

Therefore, the present study was carried out to authenticate the folklore claim of A. scholaris and M. imbricata against mycobacteria. The objective of the study was designed to evaluate in vivo antimycobacterial activity of methanol extract of leaves and seeds of these plants in murine model along with synergistic effect with rifampicin.

Materials and Methods

Organism

M. tuberculosis H37Rv was procured from Indian Veterinary Research Institute, Izatnagar, India. The Mycobacterium strains were grown in Middlebrook 7H9 medium (HiMedia, India) supplemented with 10% OADC (HiMedia, India). Log phase cultures were centrifuged, washed with sterile saline, and adjusted to McFarland standard corresponding to 1 × 106 colony forming unit/ml (CFU). The size of inoculums was confirmed by plating serial dilutions on Middlebrook 7H11 media plates supplemented with 10% OADC. The plates were incubated for 4 weeks before CFU enumeration.

Plant Materials and Extraction

Leaves of A. scholaris were collected from Medicinal Garden of College of Veterinary Science, Guwahati, and seeds of M. imbricata were collected from Karbi Anglong District in the month of March-April of 2015. Plants were identified and authenticated by Botanical Survey of India, Shillong, Meghalaya, India. The voucher specimen number of A. scholaris and M. imbricata was 4732 and 4757, respectively. Leaf of MEAS and seed of MEMI were prepared as per the method of Mann et al.[8]

Animals

Female BALB/c mice of 19–21 g body weight were obtained from the Department of Pharmacology and Toxicology and maintained in 12 h light/dark cycle. The animals were provided food and water ad libitum. All studies were performed as per guidelines approved by the Institutional Animal Ethics Committee (CPCSEA/770/ac/IAEC/22).

Mice were divided into seven groups of 10 animals each, namely, Group I: Control (uninfected and untreated), Group II: Infected and placebo treated, Group III: Standard drug rifampicin (RIF) treated, Group IV: MEAS treated, Group V: MEMI treated, Group VI: Rifampicin + MEMI treated, and Group VII: Rifampicin + MEAS treated. Mice were weighed and data were recorded at the initiation of the experiment. BALB/c mice were infected via tail vein method with 0.2 ml of M. tuberculosis H37Rv suspension in phosphate-buffered saline (PBS) supplemented with 0.05% Tween 80. Reproducibility of the challenging dose was censured by standardizing its optical density to obtain the desired CFU in Middlebrook 7H9 broth. On day 7, following infection, six of the infected mice were sacrificed, spleens and lungs were recovered and homogenized. Five-fold serial dilutions of organ homogenates in PBS with 0.05% Tween 80 were placed on Middlebrook 7H11 agar to determine CFU counts in organs.[12] Drugs were administered for 2 weeks from 7th day postinfection at an oral dose of 20 mg/kg for rifampicin and 200 mg/kg for extracts in individual groups. The doses for synergistic groups were 10 and 100 mg/kg for rifampicin and extract, respectively.

Histopathology

Three mice per infected group were sacrificed under ether anesthesia on day 0, 7, and 20. The right lobe of lungs was fixed with ethanol then embedded in paraffin. The left lobe of lung and spleen was rapidly frozen and kept at −80°C for microbiological studies.[1314]

Colony Forming Unit Enumeration

Organs were homogenized with a Polytron homogenizer in sterile tubes containing 1 ml of PBS with 0.05% Tween 80. Ten microliters of the original concentration and five-fold dilutions of each homogenate were spread onto Bacto Middlebrook 7H11 agar (HiMedia) medium supplemented with 10% OADC to enumerate the total CFU of M. tuberculosis per organ per mouse.

Statistical Analysis

CFU counts were converted to log10 values and compared using Student's t-test. Correlation was calculated between CFU and bodyweight.

Results

The TB challenged model was generally assessed by recording weight gain or loss as an indicator of time of onset of disease and drug activity. By 20 days, all the untreated mice lost about 35–40% of their body weight [Figure 1]. Mice treated with rifampicin or rifampicin + MEAS did not lose weight; they even continued to gain weight. After 20 days, the body weights of 2–3 mice of the infected but untreated group had dropped to a weight of 14.5 g, whereas body weights of the drug-treated mice attained weight near to the pretreatment level. The difference between body weight of untreated mice and the other group was highly significant [P < 0.01, Table 1].

Figure 1

Differences in mean body weights at day 1, 7, and 20 of different group of mice

Table 1

Mean±standard error of body weight, spleen colony forming unit, and lung colony forming unit of different treatment groups

Histopathology showed edema with lymphocytic infiltration, fibrous tissue proliferation, giant cell formation, and TB granuloma in lung tissues of infected but nontreated group. This confirmed the TB infection in tissues [Figures 2 and 3]. Extensive edema was observed in lung tissues of mice of infected and untreated group on day 20. Figure 4 shows the effect of synergistic treatment of rifampicin + MEAS; the tissues were devoid of giant cell, tissue proliferation, and other symptoms of TB. The response of rifampicin + MEAS was almost equivalent to the single treatment of rifampicin. Development of secondary follicles of spleen in isolated areas commonly observed. Very less or no depletion of lymphocytes in spleen indicating curing stage of day 20 sample of Group VII (rifampicin + MEAS). In our study, most of the mice treated with effective doses of rifampicin, MEAS, and MEMI were alive and maintained body weights longer than 20 days postinfection, whereas 75% of infected, nontreated mice died during the experiment.

Figure 2

Histopathological changes showing (a) edema with lymphocytic infiltration, fibrous tissue proliferation (b) edema, macrophage giant cell, epithelioid cell, lymphocytic proliferation of lung of infected group at day 7 sample (H and E, ×100)

Figure 3

Histopathological changes showing (a) extensive edema and infiltration of lymphocytes in lung (H and E, ×100) (b) depletion of lymphocyte in spleen (H and E, ×400) of day 20 sample of infected group

Figure 4

Histopathological changes showing (a) clear curing stage of lung (H and E, ×100) (b) curing stage of spleen (H and E, ×400) of day 20th sample of Group V (rifampicin + Methanolic extracts of A. scholaris)

Mice were inoculated with 1 × 106 CFU of M. tuberculosis H37Rv to develop a rapid and progressive TB disease. Chemotherapy was initiated 7 days after inoculation, when bacteria in spleens and lungs achieved significant levels (6.11 ± 0.01 and 6.26 ± 0.01 mean log10 CFU, respectively, at P < 0.01). Mean log10 CFU in organs of infected and untreated mice continued to increase and reached 8.59 ± 0.01 in spleen and 8.71 ± 0.01 in lungs by 20 days (P < 0.01). Mean log10 CFU in organs of MEAS treated group (spleen 5.26 ± 0.08; lung 5.97 ± 0.02) did not significantly differ (P < 0.01) from rifampicin-treated group (spleen 4.22 ± 0.12; lung 4.29 ± 0.12). Untreated infected mice which were inoculated with 1 × 106 CFU of H37RV per mouse, started dying from day 10, only 25% of the untreated control survived after 20 days. Ninety percent survival rate was observed in mice treated with standard drug rifampicin at 20 mg/kg; on the contrary, 85% survival rate was observed in the synergistic groups, i.e. the mice treated daily with rifampicin + MEAS or rifampicin + MEMI. The survival rates for mice treated daily only with MEAS or MEMI at 200 mg/kg were 62.5% and 50%, respectively, so the survival rate for the mice treated with MEAS was higher than group treated with MEMI, significantly lower than those for the above-mentioned synergistic and rifampicin-treated groups (P < 0.01).

The drug efficacy and weight loss in mice at the end of the experiment were inversely correlated in this study. The drugs that reduced bacterial counts in lung and spleen (from 1 × 106 to 1 × 103 CFU) showed body weights near to the level of uninfected mice. The mice lost body weight if their lung CFU was >1 × 106. Without treatment, the infection became severe day by day with the gradual loss of body weight. The corresponding CFU in lung and spleen of the mouse was determined after sacrifice for all the mice separately, and the correlation of body weight with CFU was assessed. A high correlation (r = 0.972) was noticed between lung and spleen CFU calculated throughout the experiment. The correlation between CFU in lung and mouse body weight was − 0.937 (P = 0.0058) and that of spleen and body weight was − 0.975 [P = 0.0009, Figure 5], indicating higher CFU in lungs and spleen had a negative impact on body weight.

Figure 5

Correlation of colony forming unit of Mycobacterium tuberculosis H37Rv in spleen and lung with final body weight of mice on 20 days of postinfection. (Roman numbers indicate different treatment groups)

Discussion

In this murine model, we studied antimycobacterial activity of MEAS and MEMI along with synergistic effect with rifampicin. The comparison was done among MEAS, MEMI, rifampicin, and synergistic activity on the basis of CFU in lung, spleen, mortality rate as well as body weight changes including histopathological changes. Infected but untreated mice began to lose weight after few days of inoculation. This might be due to increased bacterial load which adversely affects growth and development. These data correlated with the results published by Lavebratt et al.[15] and Nikonenko et al.[16]

The histopathology results indicated that the treatment effectively prevented further development of splenomegaly due to infection; synergistic treatments were, however, superior than that of single drug. Presence of lymphocytes, epithelioid, Langhans giant cell, and fibrous tissue proliferation in lung; depletion of lymphocytes in the spleen showed the severity of TB infection in mice. Our histopathological results corroborate with the reports of previous studies on histopathological changes due to mycobacterial infection.[17]

Treatment of mice with any drug at its known effective concentration eliminated the increase in CFU in organs and reduced bacterial concentrations to levels below the CFU observed at the start of drug treatment. The mean number of CFU in placebo group significantly increased on day 20 in comparison with that of day 0 (P < 0.01). On day 20, the mean numbers of CFU for all the treated groups were significantly lower than those for the corresponding placebo group (P < 0.01), indicating that all the treatments displayed various degrees of antimycobacterial activity. Byrne et al.[18] recorded 5.66 ± 0.26 log10 CFU in lung and 3.23 ± 0.65 log10 CFU in spleen of BALB/c mice and in another study by Boris et al.[19] recorded 1.2 × 105 ± 0.5 × 105 CFU in spleen and 8.0 × 105 ± 2.5 × 105 CFU in lung with rifampicin-treated group.

The mean numbers of CFU for mice treated daily with MEAS and MEMI were virtually the same; both of them were significantly greater than pretreatment value. The anti-TB activity of MEAS was so close to MEMI that the growth curves of M. tuberculosis for both groups overlapped.

Studies in this murine TB model indicated that the daily treatment of rifampicin + MEAS had a greater activity than the individual treatment of MEAS or MEMI. It was very encouraging to note that daily treatment of mice with MEAS at 200 mg/kg resulted in a very powerful bactericidal action against M. tuberculosis which was almost similar with the effect of rifampicin. The synergistic groups responded better compared to individual plant extract groups in case of all the parameters evaluated in this experiment. A tendency for the same order was also observed for survival rate, body weight, spleen weights, and lung lesions although the differences were less than those of the CFU enumeration. Above results suggested that the body weight of mouse was good evidence of CFU levels in organs. Therefore, the CFU in organs such as lung or spleen equally reflected the specificity and sensitivity toward the drug treatment.

A drug which prevents body weight loss could be considered as a very protective agent against TB. The data suggested that the rapid screening assay could easily distinguish effective drugs from drugs with lower efficacy. In this study, the protective effect of synergistic treatment of MEAS or MEMI with rifampicin was superior to the single treatment of MEAS or MEMI. The rapid screening model of this study differs in intent from the acute and chronic TB models that are usually used for a detailed study of drug activity. To date, not much information about the in vitro and/or in vivo action of A. scholaris and M. imbricata against M. tuberculosis is available but need much more specific study.[20] The phytochemical study of the extracts of A. scholaris and M. imbricata led to the isolation of compounds with different scaffolds, most of them alkaloids, flavonoids, and glycoside that might have potent antimycobacterial activity.[212223]

Treatment of established TB infection in animal models, as well as in patients, requires long-duration therapy. This study showed that the additive effect of MEAS and/or MEMI to the standard regimen of rifampicin resulted in increased killing of bacilli in the 1st month of treatment. For further studies, one can use a high-dose challenge or a chronic model of TB on these selected plants extracts for effective doses and mechanism(s) of action.

Conclusions

Long period of TB treatment makes the case more complicated, thus this protocol could be a very essential and useful tool to investigate and develop a new drug against TB. Although we have enumerated CFU in lung and spleen, bacteria may harbor in other organs such as lymph nodes and liver. The research has been performed with crude extracts only, but isolation of pure and active compound from the plants may show a better result. The present study provided the scientific basis for the use of A. scholaris and M. imbricata against TB, which have been traditionally used against TB/respiratory diseases in Northeast India. Synergistic treatment with rifampicin and A. scholaris against TB could be a powerful and effective regimen.

Financial Support and Sponsorship

This work was financially supported by ICAR, New Delhi as project grant of “Outreach Programme on Zoonotic Diseases” (F. No. AS/14/1/2013/ASR-IV).

Conflicts of Interest

There are no conflicts of interest.