Comprehensive Analysis and Assessment of the Role of Major Bioactive Compounds Isolated from Justicia Adhatoda Leaves as Potent Antimycobacterial Agents

Tuberculosis (TB) continues to be one of the world's leading causes of death by the infectious pathogen Mycobacterium tuberculosis , which infects one-third of the global population. The emergence of the COVID-19 pandemic made its spread rapid and the treatment task more daunting. With the havoc of infectious disease expansion, traditional medicines have triggered tremendous interest worldwide. However, less availability of scientific evidence still hinders its practical use. In the present study, we evaluated the potential of the traditional medicinal plant, Justicia adhatoda , which has been used to treat respiratory ailments since ancient times. We have successfully isolated and characterized several bioactive compounds viz- Vasicoline, Vasicolinone, Adhatodine, Adhavasine, Aniflorine, and Vasicinone from J. adhatoda plant leaves, including Vasicine as the principal compound, and showed their anti-tubercular activity on nutrient-starved Mycobacterium smegmatis and Mycobacterium bovis . The study also directs their in-vitro and ex-vivo antimycobacterial potential on THP1 macrophages with internalized Mycobacterium . Our study is one of its first kind, where we assessed the synergistic antimycobacterial effect of the isolated compounds with the first-line drug Isoniazid (INH). Their potential role in promoting phagolysosome fusion and apoptosis of M. bovis infected THP1 macrophages is further evaluated.

The plant possesses anti-in ammatory, antispasmodic (bronchodilator), and expectorant properties. 11 Plant extracts are also traditionally used to treat cold, cough, asthma, and tuberculosis. 12,13 The ancient Indian saying, "No man suffering from phthisis need despair as long as the Vasaka plant exists," proves the importance of J. adhatoda plant in treating respiratory ailments. 8 WHO has recognized the properties of J. adhatoda in their manual "The Use of Traditional Medicine in Primary Health Care." 14 In North-East India, the "Meitei" community uses plant leaves and in orescence to cure fever, cough, asthma, and dysentery.
They also prepare a variety of cuisines for good digestion and health. 15 Recent studies on J. adhatoda L. plant leaf extracts showed that the plant has broad-spectrum antimicrobial and antifungal against several pathogens such as E. coli, S. epidermidis, E. aerogenes, P. aeruginosa, B. subtilis, Rhizopus, Penicillium notatum, Candida albicans, Cryptococcus neoformans, and Aspergillus avus. Studies conducted on J. adhatoda L. leaf extracts showed anti-tubercular activity for M. tuberculosis MDR strains. [16][17][18][19] J. adhatoda L. plant leaves contain alkaloids as major bioactive components, out of which study of vasicine is extensively carried out for drug development purposes. 20,21 Our previous study on the plant leaf extract of J. adhatoda L. proved the presence of several bioactive compounds besides vasicine, possessing antimycobacterial activity with low cytotoxic effects. We further found the components to exhibit a synergistic effect with Isoniazid (INH) on M. smegmatis and M. bovis (BCG). 22 In the present study, we have characterized the isolated active compounds and analyzed their role as antimycobacterial agents on nutrient-starved Mycobacterium smegmatis and Mycobacterium bovis (BCG). The synergistic antimycobacterial effect of the isolated components was further assessed with Isoniazid (INH) on the Mycobacterium. Additionally, we also studied the action of the isolated compounds on BCG-infected THP1 macrophages to analyze their activity on intracellular Mycobacterium.

Results And Discussion
Bioactive compound isolation and characterization Alkaloids isolated from J. adhatoda majorly constituted quinazoline alkaloids, as depicted from our study of the 1 HNMR spectrum. We isolated ~99% pure Vasicine, a white-colored powder, and the highest content (~27%) amongst the total isolated alkaloids. The purity of Vasicine was con rmed via HPLC, HRMS, 1 HNMR, and FTIR results. (Table 1) Besides Vasicine, several other fractions of alkaloids were also isolated.
They were all subsequently tested for their in-vitro antimycobacterial activity (Data already published). Six of the best fractions, labeled as 2A, 2B, 3A, 3B, 5A, and 5B, were selected and subjected to HPLC   Figure 2) The isolated compounds were quinazoline compounds, con rmed by observing the 1HNMR spectrum, having signature peaks in nitrogen, aromatic, and sugar region. Signature peaks in the 1 HNMR spectrum are identi ed and depicted in Table 2. Minor peaks observed in the spectrum were considered trace impurities often seen during isolation. Several functional groups like -OH, -N-H, C-H, C=O, and others were recognized from the FTIR results. The molecular weight of the isolated fractions was obtained from HRMS in (M + H) + form, further con rming our ndings. (  in the presence of INH was recorded (6.50±0.004), which was signi cantly lower than that of the normal one (31.65±0.016). (Figure 1) The isolated alkaloid fractions obtained from J. adhatoda plant leaves showed signi cant growth inhibition on nutrient-starved M. smegmatis and M. bovis (BCG). The fractions signi cantly increased the lag phase and decreased the log phase of the bacterial growth curve. In the standard growth curve of nutrient-starved M. smegmatis, the lag phase was observed for 6 hours, followed by the log phase, which continued beyond Day 2, subsequently transforming to the stationary phase. While, most of the fractions decrease the log phase to Day 1, making it less steep with time progress. Maximum inhibition was observed in 5A, followed by 5B, Vasicine, 2A, and 2B. 3A and 3B showed lower inhibition but the maximum synergy with INH. The bacterial CFU reached >10 11 CFU/mL for the assay at Day 2, from initial inoculum 1.5×10 6 CFU/mL. While total CFU counted for the bacteria treated with the fractions was signi cantly lower than the control, indicating the bactericidal activity of the fractions on the bacteria. 2A, 5B, and Vasicine showed bactericidal activity as log 10 CFU decreased to 4-5 for these fractions, even without INH. Whereas 3A, 3B, and 5A showed bacteriostatic effect having log 10 CFU more than 6, i.e., >10 6 CFU/mL. However, they exhibited bactericidal activity when incubated with INH, as log 10 CFU was reduced to 3 (10 3 CFU/mL). The cumulative effect of the fractions and INH showed synergy in 3A, 3B, 5A, and 2B, where 3A showed the best synergy and additive effect for Vasicine, 2A, and 5B. (Figure 2) For M. bovis (BCG), the lag phase was observed till Day 2, followed by the log phase from Day 2 to Day 7. The stationary phase started from Day 8 to Day 28, with little growth from Day 21. In M. bovis (BCG), the maximum growth inhibition was observed on Day 7, followed by Day 21, and total CFU was counted on Day 7. For BCG, the fractions showed the bactericidal property in general, except 3A and 3B. 5A and 5B showed the highest antimycobacterial activity, followed by Vasicine. Co  Mycobacterium bacilli, i.e., M. bovis (BCG) and M. tuberculosis, can prevent the host cell from forming lysosomes, making it their temporary resident. Upon treatment with the isolated fractions, the BCG-infected THP1 macrophage cells produce lysosomes targeted explicitly towards the bacilli. GFP-tagged BCG and Lysotracker Red-stained LAMP-1 protein, expressed on the lysosomes, were colocalized and analyzed by using the software. Yellow coloration indicates colocalization, obtained due to the superimposition of red and green colors. Percent colocalization, measured by the software, was recorded highest in 2A, 2B, and 5B. 5A and Vasicine showed moderate, while no colocalization was found for 3A, 3B, and INH. For the fractions showing signi cant colocalization, the percent green overlap was signi cantly higher than the red overlap, except 5A and 5B.
The above result implies that other fractions promote lysosome production spread across the cytoplasm, while 5A and 5B direct lysosomes towards the internalized bacteria. In Vasicine and 2B, profound lysosome production was noted. (Figure 6) Macrophages expressed a signi cantly higher number of lysosomes directed towards the ingested bacilli when co-administered with the fractions and INH. All fractions except 3A showed an increase in the percent colocalization when co-administered with INH. Fractions 2B and 5B showed synergy with INH, while VAS, 2A, 3B, and 5A showed additive effect with INH for phagolysosome fusion. (Figure 7) The above result shows that the alkaloid fractions isolated from J. adhatoda leaf help macrophages kill internalized BCG by promoting phagolysosome fusion.

Discussion
Plants are the inexhaustible resource of natural products used as folk medicines since humanity. Although the popularity of synthetic drugs increased due to their cost effectiveness, easy quality control, and immediate effects, the rise of resistant bacterial strains and severe side effects makes their use questionable. A vast number of natural product-derived compounds in the pipeline highlights the signi cance of plants as sources of new drug candidates. 8, 9,23 Justicia adhatoda, a well-known indigenous Indian subcontinental plant, is commonly used to treat respiratory ailments in folk medicine. Our study explored the antimycobacterial potential of the plant leaf extracts and successfully isolated and characterized bioactive compounds, besides Vasicine, responsible for the activity. The isolated fractions, labeled as 2A, 2B, 3A, 3B, 5A, and 5B, were identi ed as alkaloids, constituting Vasicoline, Vasicolinone, Adhatodine, Adhavasine, Ani orine, and Vasicinone, respectively. Our previous study proved that the compounds showed excellent antimycobacterial activity against M.
smegmatis and M. bovis, better than the rst-line drugs Isoniazid (INH). 22 The fractions also showed synergy against both bacilli with INH. The present study analyzed the potential antimycobacterial activity of the isolated fractions on nutrient-starved M. smegmatis and M. bovis (BCG). We found that the fractions inhibit bacterial growth and possess a profound bactericidal effect when co-administered with INH. Vasicine is a well-known bronchodilator used for cough treatment. Several in-silico studies and a few invitro studies show its antimicrobial, antioxidant, and cytotoxic potential. 24,25 Vasicine is also known as a moderate anti-tubercular agent. 26 Some in-silico studies on the derivatives of Vasicine, such as Vasicinone, Vasicoline, and Vasicolinone, suggest the anti-TB activity along with anti-proliferative and hepatoprotective effects, where the activity of Adhatodine, Adhavasine, and Ani orine has not yet been assigned. 27,28 Our study is one of its rst kind, where we assessed the in-vitro antimycobacterial activity of these derivatives on nutrient-starved M. smegmatis and M. bovis (BCG). We support our ndings with the study conducted on BCG-infected macrophages. The compounds showed effective in-vitro antimycobacterial activity on both bacilli and showed synergy with the rst-line drug INH. The alkaloids also showed antiproliferative activity on BCG-infected macrophages and proved more effective than INH and Vasicine. They also promote phagolysosome fusion which shows their effect targeted towards the internalized bacilli.
Ani orine, Vasicoline, Vasicolinone, and Vasicinone showed excellent in-vitro antimycobacterial effects and good anti-proliferative activity. They also showed phagosome-lysosome fusion, better than the other compounds. Vasicoline and Vasicolinone showed additive effects, while Ani orine and Vasicinone showed synergy with INH. Although Vasicine shows excellent in-vitro antimycobacterial activity, its impact on internalized M. bovis (BCG) was poor. Neither it showed the anti-proliferative effect nor showed phagolysosome fusion in BCG-infected THP1 macrophages. However, Vasicine showed synergy with INH for phagolysosome fusion.
Our study proves that J. adhatoda is rich in bioactive compounds responsible for the anti-tubercular activity, which should be pursued further for anti-TB drug development. The study also directs the in-vitro and exvivo antimycobacterial potential of quinazoline alkaloids in J. adhatoda and suggests their effectiveness more than vasicine, the principal alkaloid in the plant. The compounds need to be analyzed further on M. tuberculosis and also on the animal model. In-silico and in-vitro study of the isolated compounds, especially Ani orine, Adhavasine, and Adhatodine, should be conducted in perspective of phagosome-lysosome fusion in M. tuberculosis internalized in macrophages to understand the mechanism of action of the compounds.

Methods
Bacterial samples, cell lines, and chemical procurement

Plant Material Collection, Extract Preparation, And Alkaloid Isolation
Plant leaves, acquired in October from Kamala Nehru Ridge, New Delhi, were washed, shade dried, and powdered. The methanol extract was prepared by dissolving powdered leaves in methanol (1:10 leaf: solvent ratio). The mixtures were stirred overnight on the magnetic stirrer at room temperature. The solution was ltered after 72 hours of incubation and concentrated by a rotary evaporator. Alkaloids were isolated via the acid-base extraction method by treating the extract with an excess of 1% citric acid solution. The mixture was stirred overnight, ltered, and suspended in chloroform. The aqueous and chloroform layer was separated and concentrated. The aqueous layer was basi ed to pH 9.5 using NH 4 OH and further extracted using chloroform and treated with Na 2 SO 4 (in excess), ltered, and again concentrated. The obtained compound mixture was measured with Acetone and Petroleum ether (1:1 ratio; 40ml each) and ltered to get puri ed alkaloid -Vasicine. The qualitative tests for the rst fraction con rmed the identity as an alkaloid.

Alkaloid Fractionation And Isolation
The alkaloid fraction obtained was subjected to TLC analysis to standardize the mobile phase for further fractionation. After standardization, Prep TLC was performed using 45% ethyl acetate, 35% methanol, and 20% chloroform as mobile phase at pH 9.5 adjusted by adding NH 4 OH. Different bands were obtained and re-fractionated to varying polarities of the mobile phase adjusted accordingly. Fractions were subjected to antimycobacterial activity assay, and out of several, the best fractions were selected for further analysis.
(Antimycobacterial activity data has been published) Compound Characterization HPLC analysis: Isolated fractions were rst subjected to HPLC analysis, C18 column, 95% Methanol; 4% Acetonitrile; 1% Tetrahydrofuran as mobile phase isocratic solvent. The injection volume was 20µl, and the running time was 20 minutes. Mass Spectrometry: 5mg of the isolated fractions were submitted to the Q-Tof HRMS facility of SAIF CDRI Lucknow for analysis. NMR analysis: Isolated compounds were subjected to H + NMR analysis. 7-8mg/ml of the samples were dissolved in 500 µl of Deuterated Chloroform and subjected for NMR analysis at USIC, North Campus, University of Delhi. FTIR spectrometry: To determine the functional groups present, 5mg of the samples were dissolved in methanol (HPLC grade solvent) and submitted for FTIR analysis to AIRF, JNU.