Antitubercular activities of Crinum asiaticum bulb extract using aeroso-linduced Mycobacterium smegmatis in mice model

INTRODUCTION

Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis, one major cause of ill health (World Health Organization report, 2020). It was reported in 2019 before the COVID-19 pandemic to be a devastating global health problem and top infectious killer worldwide (Global tuberculosis report, 2019). It is among the top 10 causes of death worldwide and the number one cause of death from a single infectious agent after COVID-19 (World Health Organization report, 2020).

A report in 2017 estimated that out of the 10 million new cases of TB recorded worldwide with 1.8% decline rate, about 1.57 million people died as a result of TB, representing a 3.9% decline compared to the 2016 report. High TB cases were recorded in Africa and Southeast Asia regions (Ghebreyesus, 2019).

In spite of the effort to end TB, most anti-TB drugs have been confronted with the issue of resistance that prolongs TB management and treatment. Several cases of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB have been reported (Ajay et al., 2018). The rate of eliminating TB globally appealed when the annual statistics from TB incidence were compared, including mortalities in recent years (Macneil et al., 2020). This has intensified the efforts by scientists to improve the diagnosis, treatment, and prevention of TB in order to meet the “end TB” target. The insurgence of MDR/XDR-TB is a major problem in the treatment and management of TB (Seung et al., 2015) and has been a challenge in our healthcare system. World Health Organization (WHO) 2020 global TB report estimated 500,000 cases of MDR-TB, of which 186,772 MDR-TB deaths were confirmed (Tiberi et al., 2021). In the past four decades, there has been a surge in multidrug and XDR-TB, while on the other hand, there is a decline in the number of anti TB drugs developed within the same time, leaving options for fewer drugs to treat and manage TB (Espindola et al., 2017; Tiberi et al., 2018). This trend is alarming, and there is a need to discover and develop lead agents that could be used to treat TB and to identify some mechanisms to tackle antimicrobial resistance.

Nature has provided some important sources of new antimicrobials because of their amazing chemical diversity and their validation over the years (Bednarek and Osbourn, 2009; González-Lamothe et al., 2009). Plants, together with some microbes, use their secondary metabolites to fight environmental infections. The antibacterial activities of plants are known based on their ethnomedicinal or folkloric use (Romha et al., 2018; Sisay et al., 2019; Ullah et al., 2020). Crinum asiaticum bulbs are predominant in the southern part of Ghana (Ofori et al., 2021), and the selection of the plant was based on the evidence that it is used traditionally to treat upper respiratory tract infections, skin infections, and severe chest pains (Ofori et al., 2021). The toxicity profile of the chloroformic C. asiaticum bulb extract (CCAE) was recently reported to possess no known toxicity (Ofori et al., 2021). Several scientific validations on the plant have been reported; a few of them are the analgesic and anti-inflammatory effects of C. asiaticum alcoholic leaf extract in animal models reported by Rahman et al. (2013). Antinociceptive and anti-inflammatory effects of C. asiaticum bulb extract were investigated and reported (Ahmed, 2011). Surain reported the anti-Candida potential of C. asiaticum leaf extract against selected oral and vaginal Candida pathogens. There was a study on the anti-inflammatory and antioxidant activity of leaf extract of C. asiaticum (Uddin et al., 2015). Gas chromatography-mass spectrometry analysis conducted on the chloroform extract of C. asiaticum bulb extract reported in the authors’ recent publication revealed the presence of notable compounds (Ofori et al., 2021).

Table 1. Phytochemical compounds identified in the chloroform extract of Crinum asiaticum bulb (Ofori et al., 2021). [Click here to view]

MATERIALS AND METHODS

METHOD

Aerosol infection

The aerosol infection model (Diane and Ian, 2011; Gaonkar et al., 2010; Schwebach et al., 2002) was employed with some modifications using the aerosol chamber (exposure chamber) and fast-growing, nonpathogenic M. smegmatis and a surrogate model of M. tuberculosis. Mycobacterium smegmatis was cultured overnight in MB7H9 broth supplemented with 10% ADC, followed by the addition of 20% Tween 80. Pipette was used to aseptically dispense 1 ml of the Mycobacterium culture into a falcon tube containing 4 ml of sterile water to produce 5 ml of diluted culture suspension (2.0 × 10⁶ colony-forming unit (CFU)/ml) which was used in the inhalation exposure chamber. The standard challenge dose in most TB experiments in mice has CFU of 50–100 and was determined before the aerosol infection (Diane and Ian, 2011; Karaman, 2013). The final suspension (5 ml) was aerosolized in a nebulizer for 30 minutes. Before nebulizing the bacterial suspension, a maximum of 25 mice were placed in the exposure chamber, and the chamber was covered firmly. Glass nebulizer venturi was fixed firmly beside the outer chamber, and the system was run for 30 minutes. The amount of mycobacterial load inhaled during exposure was determined as CFU on a plate culture.

CFU = CFU/ml × dilution factor × volume delivered

$\mathrm{where}\mathrm{CFU}\mathrm{/}\mathrm{ml}=\frac{\mathrm{Mean}\mathrm{CFU}}{\mathrm{Dilution}\mathrm{factor}\times \mathrm{Volume}\mathrm{delivered}\mathrm{or}\mathrm{plated}}$

Dilution factor = Dilution factor used to prepare the aerosol suspension,

Volume delivered = Total bacteria suspension in nebulizer before the experiment – Amount left in the nebulizer after the experiment.

To authenticate the aerosol-induced TB work, after 24 hours of postexposure, mice were sacrificed, lungs were homogenized and centrifuged, supernatant-containing bacilli was cultured, and the CFU was determined. This was carried out to show how well the lungs were exposed to the bacterium.

In vivo dose-response studies

After 48 hours of postexposure, the mice were administered by oral gavage with the CCAE and RIF as standard drug once daily for 28 days. Eight groups with seven mice in each group were used for the experiment. The first three groups were orally treated with CCAE with doses of 100, 500, and 1,000 mg/kg, respectively. Groups 4, 5, and 6 were treated orally with 30, 90, and 270 mg/kg of RIF. Group seven was the negative control (the untreated group). Group eight was the naïve group (without TB infection) and was administered with 1 ml/kg of normal saline. Treatment lasted for 28 days after aerosol-induced TB.

The weight of animals was recorded every week and the final weight on the 29th day of treatment. Clinical observations were conducted throughout the treatment days, and mortality was recorded. On the day after the final treatment of the mice, blood samples were collected from each group into ethylenediaminetetraacetic acid-containing tubes for hematological studies. Mice were sacrificed, and lungs were aseptically harvested. The left lobe was placed in 10% formalin for histological analysis, and the right lobe was homogenized and processed for CFU count. The cytokines level for IL-6 and TNF-α was determined since they are crucial in assessing the severity of TB. Assessing the levels of these cytokines is one of the hallmarks of TB management, and it is a contributory factor in TB therapy.

STATISTICAL ANALYSIS

GraphPad Prism 8.0 software was used to carry out statistical analysis using a one-way analysis of variance (ANOVA). Results were quantified as mean ± SEM. Statistical differences between mean values were carried out using Dunnett’s multiple comparison test at p < 0.05.

RESULTS

TNF-ALPHA CONCENTRATION

The CCAE produced a significant dose-dependent decrease in TNF-α concentration within the dose range of 100, 500, and 1,000 mg/kg compared to the negative control group when their levels were assessed in lung tissue homogenate.

HISTOLOGICAL FINDINGS

The histological findings on the lungs of mice after treatment had a strong association with the bacterial load in terms of the CFU count. The histologic examination of the lungs in the negative control group presented with extensive necrotizing lung parenchyma, multinucleated giant cells, scattered lymphocytes, and large areas of collapsing alveoli, and also there was massive nonspecific granulomatous lesion (Fig. 6). These indicators suggest how the lungs were infected by M. smegmatis to a greater extent.

The doses of CCAE treatment groups with the exception of 100 mg/kg produced a significant improvement in relation to the histology of the lungs. Doses (500 and 1,000 mg/kg) produced marked effects on the lung tissues, presented with large areas of viable alveolar spaces, few foci points of necrosis, terminal lymph nodes were normal, and evidence of little blood vessel (BV) congestion (Fig. 6f–h).

RIF as a standard antitubercular agent with doses of 90 and 270 mg/kg produced a high antitubercular effect in regard to lung histology (Fig. 6i–k).

DISCUSSION

Management of TB is threatened with the insurgence of MDR/XDR-TB in medicine and public health becoming one major threat to control around the globe (Angelina et al., 2021). Current anti-TB agents are not functioning effectively due to the emergence of the issue of multidrug and XDR-TB in the healthcare systems (Gygli et al., 2017; Nainu et al., 2021; Prestinaci et al., 2015); therefore, there is a need to discover and develop new compounds with novel mechanisms of action effective in the management of TB.

Natural products such as medicinal plants offer a great platform to meet the needs of new anti TB regimens (Fauziyah et al., 2017; Ramadwa et al., 2019). Several plant species have been investigated for their effects against M. tuberculosis (Kumar et al., 2017). More recently, natural products investigations have reported the antimycobacterial properties of native Indonesian plants that are used traditionally for respiratory diseases (Fauziyah et al., 2017).

In the disease progression of TB, there is suppression of appetite due to decreased plasma leptin concentrations and therefore the need for nutritional support (Kim et al., 2010). It has been reported that the serum leptin level expressed in pulmonary TB may be implicated independently by inflammation and weight loss (Ye and Bian, 2018). In clinical research, it has been found that serum leptin level becomes very low in pulmonary TB due to the loss of body weight and that confirms that prolonged production of inflammatory cytokines may further suppress leptin production (Herlina et al., 2011; Kim et al., 2010).

In this study, the albino mice body weight was determined, and there were gradual increases in the weight of mice throughout the experiment for the CCAE treatment groups, the RIF treatment groups, and the naïve control group (Fig. 1) compared to the negative control group showing a decrease in the mean body weight. This suggested that, in the CCAE administration, the disease did not get to a stage that would suppress appetite and that could have caused loss of weight in the various treatment groups compared with the negative control. This confirms an investigation by (Kim et al., 2010) which reported that there is production of proinflammatory cytokines in TB that suppress plasma leptin level and that would obviously lead to wasting (Buyukoglan et al., 2007; D’Attilio et al., 2018; Kim et al., 2010; Wieland et al., 2005).

Abnormalities in hematological parameters are common in pulmonary tuberculosis patients, which is one of the major public health problems globally (Abay et al., 2018). TB effect on hematological parameters has been reported to cause a decrease in HGB (Iqbal et al., 2015), RBC count, and altered WBC count (Kulkarni and Jaju, 2017). This spells out one major reason why most TB patients are diagnosed with iron deficiency anemia (Chu et al., 2019; Gunda et al., 2016; Isanaka et al., 2012), and it is as a result of hematopoietic cells destruction (Iqbal et al., 2015). The WBC count elevation in (Fig. 2) was due to the existing infection. The negative control group showed a decrease in HGB and RBC (Fig. 2), but there was an increase in the WBC (Fig. 2), while on the other hand, the CCAE treated groups showed a dose-dependent increase in RBC and HGB levels and a decrease in WBC compared with the negative control (Fig. 2). The effect of CCAE on hematological parameters showed how effective the extract was in elevating the levels of parameters such as RBC, HGB, and WBC during TB infection (Fig. 2). In TB, some cardinal signs are investigated as evidence of existing TB in an individual (WHO Report, 2013), which include the presence of viable M. tuberculosis and pathological markers such as granulomatous lesion, caseous necrosis, and multinucleated giant cells (Kumar et al., 2013; Shah et al., 2017).

In this study, the number of viable M. smegmatis on the lungs was determined as their CFU/ml. The doses of CCAE significantly (p ? 0.005) decreased the CFU count on the lungs in the infected mice, suggesting how well the CCAE was able to inhibit the mycobacterial (Fig. 3). Mycobacterium smegmatis presence within the alveolar spaces induces the activation of macrophages which modulate the immune response by upregulating the presence of inflammatory cytokines such as IL-1β, IL-6, and TNF-α (Arora et al., 2020; Li et al., 2014; Yamawaki et al., 2016).

Figure 1. Effect of CCAE on body weight of mice (CCAE: chloroformic C. asiaticum bulb extract; RIF: rifampicin). [Click here to view]

Figure 2. Assessment of hematological parameters during TB management with CCAE (CCAE: chloroformic C. asiaticum bulb extract, RIF: rifampicin, WBC: white blood cells, RBC: red blood cells, and HGB: hemoglobin). [Click here to view]

Figure 3. Effect of CCAE on M. smegmatis CFU/ml count. One-way ANOVA. ****p-value ? 0.0001 of treatments versus negative control (CCAE: chloroformic C. asiaticum bulb extract; RIF: rifampicin). [Click here to view]

Figure 4. Effect of CAE, negative control, naïve, and RIF on IL-6 expression. Data are expressed as mean ± SEM. n = 5, one-way ANOVA followed by Dunnett’s multiple comparison test, ****p < 0.0001 of negative control versus 500 and 1,000 mg/kg CCAE (CCAE: chloroformic C. asiaticum bulb extract; RIF: rifampicin). [Click here to view]

Figure 5. Effect of CAE, negative control, naïve, and RIF on TNF-α expression. Data are expressed as mean ± SEM. n = 5, one-way ANOVA followed by Dunnett’s multiple comparison test, ****p < 0.0001 of negative control versus 100, 500, and 1,000 mg/kg CCAE. [Click here to view]

Assessing the levels of cytokines such as TNF-α and IL-6 is one of the hallmarks in TB management (Basaraba, 2008), and it is a contributory factor in TB therapy. Low levels of TNF-α and IL-6 combined with other markers depict the effectiveness of antitubercular agents (Joshi et al., 2015; Mesquita et al., 2016). It was reported that the levels of cytokines such as TNF-α and IL-6 levels are elevated in TB -infected mice (Domingo-gonzalez et al., 2017; Kramnik et al., 2016). The elevation is attributed to the aggregation of macrophages and other immune cells that are produced when there is infection. CCAE markedly decreased the level of IL-6 (Fig. 4) and TNF-α (Fig. 5), and that suggested how the extract was to control the infection and how effective the extract was in managing TB.

The histological examination on the lungs of mice helps to investigate how extensive the disease destroys the cells of the lungs (Fukushi et al., 2011; Ravimohan et al., 2018). In TB, there are inflammatory cells infiltrations and macrophages adherence, which affect the lung parenchyma, and therefore the formation of granulomatous lesions and necrosis, which may cause alveolar collapsed (Guirado and Schlesinger, 2013; Kradin and Mark, 2018; Rosen, 2020). The histology of the lungs of infected mice showed dose-dependent improvement in lung function following the administration of CCAE (Fig. 6). In the negative control group (Fig. 6b and c), there were a large focal area of caseous necrosis, several alveolar spaces collapsed, bronchi infiltrations with inflammatory cells, and many congested BV compared to the naïve control group (Fig. 6a), where the lung architecture was maintained devoid of any inflammatory cells infiltrations. The treatment groups (Fig. 6d, e, f g, and h) showed dose-dependent improved lung functions, and the biggest improvement was seen in the 1,000 mg/kg CCAE (Fig. 6h). Comparatively, the level of lung tissues maintained in the CCAE treatment groups (Fig. 6d, e, f, g, and h) was similar to the RIF treatment groups (Fig. 6i, j, and k). The histological improvement of the lungs of mice after TB induction using M. smegmatis as a surrogate model for screening drugs against M. tuberculosis (Gupta and Bhakta, 2012; Namouchi et al., 2017) suggested the anti-Mycobacterium activity of CCAE.

CONCLUSION

This study demonstrated that C. asiaticum bulbs extract has an anti TB effect, was achieved in the inhibition of M. smegmatis in vivo, and improved weight loss and hematological parameters as well as reducing some notable inflammatory cytokines and the maintenance of lung architecture in the histological analysis. This would serve as a lead in the drug discovery process in developing novel compounds that could exhibit diverse mechanisms to treat TB and tackle associated drug-resistant TB.

Figure 6. Photomicrograph of the histology of mice lungs after TB treatment with CCAE in H and E stains. (a) Magnification ×10 is naïve control presented with intact terminal bronchiole (B), BV not congested, and well-defined alveolar spaces (A). (b and c) are the negative control groups (×4 and ×10, respectively) and showed extensive caseous necrosis (C), giant cells, granulomatous lesion, extensive collapse of alveoli (A), and numerous BV congestion. (d and e) represented 100 mg/kg CCAE (×4 and ×10, respectively) and showed collapsed alveolar spaces (A), a large field of caseous necrosis (C), and granulomatous lesion and numerous BV congestion. (f and g) (×4 and ×10, respectively) were 500 mg/kg CCAE-treated groups presented with few collapsed alveolar spaces (A), small foci of necrosis, little BV congestion, and little terminal bronchiole congestion (B). Micrograph (h) is 1,000 mg/kg CCAE (×10) which showed small foci of caseous necrosis, normal terminal bronchioles (B), and a large area of normal alveolar space (A). (i) represents 30 mg/kg of RIF (×10) and showed a large area of collapsed alveolar spaces (A), wider areas of granulomatous lesion and necrosis, and presence of residual alveolar (A). (j) was 90 mg/kg of RIF (×10) and showed few collapsed alveolar spaces (A), little BV congestion, and little terminal bronchiole congestion (B), and (k) represents 270 mg/kg of RIF (×10) with small foci of necrosis and granulomatous lesion, and terminal bronchiole bronchus appeared normal (B). [Click here to view]

ACKNOWLEDGMENTS

The authors would like to thank the technical staff at the animal house and anti-infective drug discovery laboratory in the Department of Pharmacology, KNUST, for their immense contribution towards this work.

LIST OF ABBREVIATIONS

CCAE: chloroformic Crinum asiaticum bulb extract; CFU: colony-forming unit; HGB: hemoglobin; MDR: multiple drug resistance; RBC: red blood cell; WBC: white blood cells; XDR: extensively drug-resistant.

AUTHORS’ CONTRIBUTIONS

MO and CAD conceived and designed the study and were involved with data analysis and interpretation. MO and INN carried out the experiments, generated the data, and wrote the first draft of the manuscript. TN, PPSO, and PD contributed equally by providing the plant material, processing, and extraction. RG conducted the biochemical aspect of this work. All authors contributed to revising the final manuscript

FUNDING

There is no funding for this work.

CONFLICT OF INTERESTS

This is to certify that the authors have no conflict of interest.

ETHICAL APPROVAL

Ethical clearance from the animal ethical committee, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, was issued with ethical code FFPS-AEC/CA05/18, dated May, 2018.

DATA AVAILABILITY

All data generated and analyzed are included within this research article.

PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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