GC-MS profiling of Vitex pinnata bark lipophilic extract and screening of its anti-TB and cytotoxic activities

Abstract Tuberculosis is a highly infectious ailment worldwide. The emergence of multi-drug resistance and serious adverse effects of anti-TB drugs have led to the continuous search of natural candidates. This study aimed to analyse the chemical profile of Vitex pinnata (VP) bark lipophilic extract using GC-MS also evaluating its anti-TB and cytotoxic activities. GC-MS revealed a total of 81 compounds which representing 86% identified compounds. In vitro anti-TB of VP lipophilic extract was evaluated using the Microplate Alamar Blue Assay which exhibited MIC value of 62.5 µg/mL. In vitro cytotoxicity was evaluated using Water Soluble formazan assay recording IC50 > 100 and 200 µg/mL using Vero and A-549 cell lines, respectively. In silico docking study was performed on the major identified compounds, n-nonane showed the most favourable binding affinity (ΔG) equals to −33.34 Kcal/mol. The results obtained herein unravelled the potential use of VP n-hexane extract as a natural anti-TB. Graphical Abstract


Introduction
Tuberculosis (TB) is a global infectious ailment caused by Mycobacterium tuberculosis (Mtb) (Lakshmanan et al. 2011). The World Health Organization declared that TB is counted as one of the most contagious disease as well as one of the top 10 leading causes of mortality worldwide; recording 10 million infected people and 1.4 million deaths in 2019 (Chakaya et al. 2021). TB represents a global public health threat because of the prevalent emergence of multiple drug resistant strains subsequently leading to inappropriate response to active therapy viz. isoniazid and rifampicin (Nkenfou et al. 2015).
TB directed drug discovery and development of potent lead compounds against TB have been concerned with discovery of novel and safe new TB drugs to overcome MDR-TB strains (Brinza et al. 2020;Gamal El-Din et al. 2018;Garc ıa et al. 2012;Korany et al. 2021;Moussa et al. 2021;Singab et al. 2014;Taha and Eldahshan 2017;Todirascu-Ciornea et al. 2019;Younis et al. 2022). Recently, the use of natural products for the treatment and prevention of infectious diseases has an emergent conceptual strategy in drug discovery which interestingly has attracted the attention of many researchers nowadays (Ashmawy et al. 2021;Ayoub et al. 2021;Azab et al. 2017;Dong et al. 2017;Gad et al. 2021;Gamal El-Din et al. 2018;Mostafa et al. 2016;Saeed Kotb et al. 2020;Shahat et al. 2017). Vitex genus embraces species viz, Vitex negundo Linn leaves and Vitex trifolia leaves which exhibit promising activity against TB (Ladda and Magdum 2018;Tiwari et al. 2013).
In silico molecular docking study is a virtual screening method aids in exploring the binding affinity of natural products that act as ligand towards different proteins (Ayoub et al. 2022;Younis et al. 2022). KasA enzyme is a pivotal enzyme in Mtb survival which plays an essential role in the formation of mycolic acid that supports the synthesis of Mtb cell wall (Yuanita et al. 2020). Accordingly, the inhibition of KasA enzyme leads to disruption of Mtb cell wall synthesis. Mtb C171Q receptor is one of the influential TB molecular targets that acts as an KasA enzyme inhibitor (Youssef et al. 2021).
Vitex pinnata Linn. (syn: Vitex pubescens Vahl.), commonly known in Malaysia as Halban (Al-Akwaa et al. 2020;Promprom et al. 2020;Thenmozhi et al. 2021), belongs to family Lamiaceae that have a plethora of ethnopharmacological activities (Goh et al. 2017). It is a woody plant with moderately sized tree about 25-30 metre tall with trifoliate leaves, grey to brown bark native to Southeast Asian territorial such as Indonesia, Malaysia, India and Philippines (Thenmozhi and Subasini 2016). The whole plant is used in traditional medicine for treatment of jaundice while the bark is used for treatment of stomach aches, dysentery, wounds and gastric ulcers (Anwar et al. 2015).
The aim of this study is to investigate the chemical profile of the lipophilic extract of Vitex pinnata bark via GC-MS and in vitro anti-TB activity using Micro Alamar Blue Assay (MABA) for the first time also cytotoxic activity was carried out on normal Vero and A-549 cancerous cell lines to ensure its safety. In addition, an in silico molecular docking study was performed on the major identified constituents of VP n-hexane extract on Mtb C171Q receptor as KasA (b-ketoacyl synthases) inhibitor.

GC-MS analysis
A total of 81 compounds were identified representing 86% of all chemical constituents detected. The GC-MS chromatogram was illustrated in Figure S1.

In vitro anti-TB activity
The in vitro anti-TB activity has been evaluated using the Micro Alamar Blue Assay (MABA) in a dose dependent manner with the highest concentration of 125 mg/mL that exhibits a promising minimal inhibitory concentration (MIC) Value (62.5 mg/mL), compared to isoniazid MIC (0.24 mg/mL), as shown in Table S2. Many reviews on the natural products that exhibited activity against TB suggested that the plant extract exhibited MIC value less than 100 mg/mL considered promising against TB (Mohamad et al. 2018). On the other side, according to (Tosun et al. 2004) that considered the plant extract is inactive when exhibited MIC value more than 200 mg/mL. As per the previous data, the n-hexane extract of Vitex pinnata bark exhibited a potential activity against TB. In comparison with other reported anti-TB activity of genus Vitex, the lipophilic extract of Vitex negundo leaves exhibited anti-TB with MIC value (150 mg/mL) using Nitrate Reductase Assay (NRA) owing to b-sitosterol and ursolic acid content (Ladda and Magdum 2018).
Based on the lipophilic nature of the constituents of VP n-hexane extract which aids in the penetration of the lipid and waxed hydrophobic outer membrane of tuberculosis exerting inhibitory activity (Bibi et al. 2010).
It worth to mention, the concomitant use of 50% concentration of the VP n-hexane extract in combination with 50% concentration of isoniazid showed MIC value of 31.25 mg/mL as shown in Figure S3, which showed relevant synergistic combination. This co-administration might have a dual beneficial action as a promising anti-TB drug and minimise the incidence of drug resistance and serious side effects of isoniazid therapy as hepatotoxicity (Nam et al. 2016;Yee et al. 2003).

Cell viability and cytotoxicity assay
The VP n-hexane extract was evaluated for its toxicity to Vero and A-549 cell lines using water-soluble tetrazolium (WST-1) assay. Results showed that the 50% inhibitory concentration (IC 50 ) values were greater than 100 mg/mL and 200 mg/mL, respectively ( Figure S4). It was reported that plant extracts exhibit IC 50 above 50 mg/mL are considered non-toxic (Phongpaichit et al. 2007). Therefore, n-hexane extract was found to be safe to the normal cells and did not show any cytotoxicity on cancerous cells. In comparison with a previous study in the genus Vitex, n-hexane and dichloromethane fractions of Vitex trifolia leaf extract exhibited IC 50 values of 38.1 and 21.1 mg/mL, respectively, using WST-1 assay in U937 macrophage cells (Wee et al. 2020). These results comparatively were more cytotoxic than VP n-hexane extract. The selectivity index (SI) values were determined as the ratio of cytotoxicity over the MIC through the following formula (SI ¼ IC 50 /MIC). The higher value indicates that less toxicity and higher degree of selectivity to TB than mammalian cells (Lawal et al. 2011). The SI of the VP n-hexane extract on Vero cells was found to be > 1.6 and on A-549 cells > 3.2, thus a modest selectivity could be concluded.

In silico molecular docking study
In silico molecular docking of the major identified constituents in n-hexane extract of V. pinnata bark was performed within the active site of Mtb C171Q receptor as KasA inhibitor. Most of the docked compounds exhibited favourable binding within the active site of the selected receptor. n-Nonane showed the highest fitting with free binding energies (DG) equals to À33.34 Kcal/mol followed by n-undecane and 2-methyldecane exhibiting free binding energies of À32.51 and À31.96 Kcal/mol, respectively, as displayed in Table S3 and Figure S5. The favourable binding affinity observed herein within the active binding site of Mtb C171Q receptor could be attributed to the low internal ligand strain energy encountered by the low molecular weight hydrocarbon compounds expressed by high free binding energy. Moreover, the hydrophobic interactions with the amino acids residues within the active site exhibited by n-undecane which formed two p-alkyl bonds with His345and Phe237 also 2-methyldecane formed three p-alkyl bonds His345, Phe237 and Pro280 amino acids. Isoniazid, the standard anti-TB and thiolactomycin, co-crystalized ligand exhibited DG equals to À20.85 and À10.59 Kcal/mol, respectively. The validation of the docking study was performed by calculating the RMSD value of thiolactomycin that docked within the pocket of the active centre and the original co-crystallized inhibitor. RMSD of 1.2874 was observed indicating the validity of the docking study Figure S6.

Conclusion
From this study, we can conclude that the n-hexane extract of Vitex pinnata bark exhibited a potential anti-TB activity with MIC value of 62.5 mg/mL, which was evaluated through in vitro MABA with safety profile to the normal Vero cells and A-549 cancerous cells. This activity is owing to the active phytochemical constituents and the lipophilic nature of the extract. The compounds were analysed using GC-MS data analysis. Hydrocarbons (72.04%) were the dominating class exemplified in n-undecane (14.5%). Therefore, the present study offers a source of a new and a promising candidate against TB. Besides, it sheds the light on the contribution of natural products in the treatment of infectious diseases.

Disclosure statement
The authors declared that there is no conflict of interest relevant to this study.

Funding
The author(s) reported there is no funding associated with the work featured in this article.