In 2018, an estimated 10 million new cases of TB and 1.5 million deaths due to TB worldwide were reported. Drug-resistant TB is major concern today globally. In 2018, WHO estimated that there were 4,84000 new cases with drug resistance to rifampicin (WHO TB report 2019). Today there is an urgent need to search for a new anti-tubercular drug, preferably those that can be quickly and easily produced from derivatives of herbal plants. Being a natural product, it is thought that these may possess lower chances of resistance and hepatotoxicity that might play an important role in the chemotherapy of tuberculosis (Arellanes J. et al. 2016). Cressa cretica (Rudravanti) traditionally used as anti-tuberculosis agent, widely accepted for the treatment of respiratory diseases due to its bronchodilatory, antitussive, antibacterial, antipyretic, and analgesic effects (Jaafar Noor S. et. al. 2021). It acts both as a bacteriostatic as well as a bactericidal agent. It has been shown to be effective for reducing lung lesions due to tuberculosis and helps to gain weight (Jaafar Noor S. et. al. 2021). P. longum is reported as a good herbal remedy for treating respiratory tract infections and also found to increase the circulating antibody titre and antibody-forming cells indicating its stimulatory effect on the humoral immunity (Sunila ES. 2004). The immunomodulatory activity of P. longum may be due to the combined action of humoral and cell-mediated immune responses (Sunila ES. 2004). Calotropis gigantea used in the traditionally medicinal system for the treatment of various infectious diseases such as leprosy and TB. It is reported to have antimicrobial, anticancer, anti-inflammatory, antidiabetic, hemolytic, antioxidant and larvicidal properties (Kadiyala M. et. al. 2013). Many researchers across the world including in India have reported on the inhibitory properties of these medicinal plants against Mtb. (WHO 2006; Askun T. et. al. 2012). In this study, we present the in vitro antimycobacterial activity exhibited by methanolic extracts of these medicinal plant and their therapeutic potential against Mtb.
Preparation of Plant material:
Plants were collected from the herbal garden, Jawaharlal Nehru Agricultural University, Jabalpur. The collected plant materials were washed with tap water and then distilled water 2-3 times. The washed plant material was shade dried and crushed in a mechanical grinder to make fine powder and stored at room temperature. Five grams of powdered material was used for extraction with 250 mL of 100% methanol as solvent in soxhlet apparatus at low temperature (15-25°C) continuously for 24-48 hours until the solvent appeared colourless. This concentrated extract was stored at 4oC in a desiccator for all further experiments. Stock solutions of crude extracts were prepared using Dimethylsulfoxide (DMSO) to a concentration of 70mg/mL in accordance with MGIT protocol and sterilized through a syringe filter of 0.2 µm pore size (Yadav R. 2011).
Mtb strain and culture medium:
H37Rv (ATCC 25618) was used as reference strain for the anti-mycobacterial assay, Mtb culture with DMSO (1.2%), isoniazid (INH) at MIC99 (0.05μg/mL) and Rifampicin at MIC99 (0.12 μg/mL) were used as controls.
Inoculum and extract preparation
The inoculum was prepared from solid culture (L–J slants), as described by (Askun T et al. 2012). 500 µl of 1:5 dilution of 0.5 McFarland bacterial suspension of Mtb-H37Rv was inoculated in MGIT (BD, USA), tubes containing test compounds and controls (Askun T et al. 2012). The bacterial growth was monitored as per the manufacturer’s instructions. 100μl of extract was added individually in MGIT tubes, containing Mtb and allowed for incubation at 37°C in the BACTEC system. The growth units (GU-400) were monitored for six days. For the minimum inhibitory concentration (MIC) evaluations, a 1% bacterial suspension of 0.5 McFarland was prepared and cultured as a control without any extract in MGIT tube. All the extract preparations were tested at two-fold decreasing concentration from 1mg/mL to 0.12mg/mL. The MIC of the extract determined in comparative to the growth units of the control (GU–400) as the lowest extract concentration that equals or lower than GU–400. Growth or inhibition of H37Rv was observed in extract containing and extract free control after 42 days of incubation at 37°C were recorded.
The preliminary in-vitro screening revealed that the methanolic extract of Piper longum and Cressa cretica inhibit the H37Rv with a MIC value of 125μg/mL individually. C.gigantea inhibited at 250μg/mL but to a lesser extent than. P longum and C. cretica at all concentrations tested. All 3 methanolic extracts showed growth inhibition against Mtb- H37Rv at MIC between 125μg/mL to 250μg/mL. On the other hand, both isoniazid and rifampicin showed relatively higher inhibitory activity against Mtb-H37Rv even at 0.05μg/mL and 0.12μg/mL as compared to the crude extract (Table 1).
In many developing countries rural and tribal population largely rely on conventional medicines obtained from medicinal plants (Askun T. et. al. 2012). According to the United Nations conference on trade and development, more than 33% of medicines are produced by industries that are plant-derived. More than 20,000 types of medicinal plants are named by WHO for their medicinal properties (WHO 2006). In general, medicinal plants do not confer resistance while exerting their biological activity (Yadav R. 2011). Previously reported medicinal plants for the TB treatment are Acalyphaindica, Adhatodavasica, Allium cepa, Allium sativum, A. Indica and Aloe vera etc. (Gupta R et. al. 2010). Based on ethnomedicinal values and uses, the present study was carried out to identify the in-vitro antimycobacterial potential of P. longum, C. cretica and C. gigantiea. There are several reports available using MGIT for the determination of MIC value of available anti TB drugs in contrast this study uses MGIT for plant extract.
P. longum has been indicated to an effective remedy for the treatment of respiratory tract infections and in the treatment of TB. C. Cretica possesses a variety of biological properties such as antipyretic, anti-microbial, antioxidant, immunomodulatory, anti-inflammatory, hypoglycaemic and anti-cardiovascular properties and also reported as an anti-tubercular activity for its bronchodilatory properties (Ali AS 2016). C. gigantiea has been used for syphilis, leprosy, tuberculosis and lupus bacterial infections (Askun T et. al. 2012). In the present studies, the MIC value of P. longum and C. Cretica was found to be 125μg/mL against Mtb while it was 250μg/mL for C. gigantiea. To the best of our knowledge the MIC values of these compounds are reported for the first time. The methanolic extract of P. longum, C. cretica and C. gigantiea showed appreciable anti-mycobacterial activity. The active compound in these medicinal plant and their combine action could be the reason for enhanced anti-mycobacterial activity. This study shows that these plants have potential for antimycobacterial activity. The benefits of plant derivative compounds using as anti-mycobacterial agent includes fewer side effects, better patient acceptance due to a long history of use, reduced costs and cultivability rendering them renewable in nature (Gupta P et. al. 2014).
The limitation of the study is that it was done on crude extract and hence the MIC is very high compared to the in use anti TB drugs.
This study has revealed that of P. longum, C. cretica and C. gigantiea have the potential to be an effective anti TB drug. These plants have potential of anti-tubercular agents as all 3 showed activity against Mtb. However, these crude extracts and their active component needs to be purified and their active principles characterized both in vitro as well as in vivo study for identifying the clinical potential.