Bioassay-guided isolation and structure elucidation of anti-mycobacterium tuberculosis compounds from Galatella grimmii (Regel & Schmalh.) Sennikov

Abstract

Background

Galatella is a genus in the family Asteraceae, represented by 35-45 species. Considering the high effectiveness of the ethyl acetate (EtOAc) fraction of G. grimmii against Mycobacterium tuberculosis (MIC = 0.5 µg/mL), a bioassay-directed fractionation of this extract was carried out.

Methods

The methanolic extract of the aerial parts of G. grimmii was obtained using maceration, then it was suspended in water and partitioned with petroleum ether, dichloromethane (CH₂Cl₂), EtOAc, and n-butanol (n-BuOH), successively. The most potent fraction (EtOAc), was selected for further isolation by Sephadex LH–20 and semi-preparative HPLC to obtain active compounds.

Results

Fractionation of the EtOAc solvent fraction resulted in the characterization of five compounds, among them, compounds 1 and 2 showed the highest anti-mycobacterial effects with MICs of 0.062 and 1.00 µg/mL against H37Rv M. tuberculosis, respectively, which were higher than those of rifampin (MIC of 1.25 µg/mL) and isoniazid (MIC of 0.31 µg/mL), as positive controls. Also, compound 1 ​​inhibited all tested strains of drug-resistant Mycobacterium (MDR and XDR). Notably, the isolated compounds have been reported for the first time from G. grimmii.

Conclusion

Due to the potent anti-mycobacterial effect of isolated compounds from G. grimmii, this study could pave the way for developing a novel class of natural anti-tuberculosis compounds.

Introduction

Tuberculosis (also known as TB) is an airborne bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) and primarily affects the lungs but can involve other tissues and organs. Based on the World Health Organization (WHO) Global TB Report, approximately 1.8 billion people, or one-quarter of the world’s population, have been infected with M. tuberculosis (but most of them had latent TB) and about 5–10% of this population (equivalent to 10.6 million individuals) will eventually manifest symptoms and progress to active TB disease. In 2021, a total of 1.6 million individuals died from TB. On a global scale, TB ranks as the 13th leading cause of mortality and stands as the 2nd most significant infectious agent causing fatalities, following COVID-19, surpassing both HIV and AIDS [1]. Various treatment regimens are prescribed for tuberculosis disease with durations of 4, 6, or 9 months depending on corresponding protocol. The most common antibiotics used for drug-sensitive (DS) M. tuberculosis strains include isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide which requires stringent adherence to avoid relapse and resistance. Treatment is recommended for both latent and active TB [1, 2]. Tuberculosis that doesn’t respond to standard first-line drugs is called drug-resistant TB, categorized into several types, including monodrug resistance, polydrug resistance, multidrug resistance (MDR), rifampicin resistance (RR), pre-extensively drug-resistant TB (pre-XDR TB), extensively drug-resistant TB (XDR TB) and recently isolated totally drug-resistant (TDR) tuberculosis [3]. Common TB drugs, introduced in the TB control program over 40 years ago, have extensive renal and hepatic side effects and currently lost their efficacy on new resistant strains [4, 5]. Only a few drugs were approved in recent decades for the treatment of TB [2]. So, there is an urgent need to find novel drugs for the treatment of MDR and XDR-TB. As mentioned above, the severity of the tuberculosis threat, the adverse effects of chemical drug regimens, the increasing rates of multidrug resistance, and the emergence of extensively drug-resistant strains, have indeed complicated the issue of tuberculosis control a more complicated challenge. Consequently, it has prompted an urgent need among mankind to develop and produce anti-tuberculosis drugs, especially those derived from plants, with a focus on replacement antimicrobial agents that exhibit greater efficacy and fewer side effects, instead of antimicrobial drugs with lower efficacy and more side effects. Among the discovered biological activities of natural products, some exhibit anti-mycobacterial effects [6]. In our previous research study, the anti-mycobacterial activity of 22 Iranian endemic and rare plant extracts was investigated, among which, the C. grimmii (synonym of G. grimmii) demonstrated one of the most potent inhibitory effects against the growth of MDR and XDR M. tuberculosis [7]. G. grimmii (Asteraceae) is a perennial plant in Iran, Turkmenistan, Afghanistan, and Central Asia. Its stems are branched, 25–25 cm tall, covered with yellowish glands and sparse multicellular hairs [8]. The main aim of the present study is to isolate the potent anti-mycobacterial bioactive components of G. grimmii using the bioassay-guided approach.

Materials and methods

General experimental procedures

Type Culture Collection (ATCC), the susceptible strain of M. tuberculosis ATCC 27294 (H37Rv) was provided. From the Tuberculosis Reference Laboratory at Shariati Hospital in Mashhad, Iran, resistant clinical isolates were acquired. All solvents for extraction and fractionation of extract used were analytical grade and purchased from Dr. Mojallali Industrial Chemical Complex Company. Silica gel 230–400 mesh (Merck, Germany) was used for column chromatography (CC). Semi-preparative HPLC was conducted on a KNAUER instrument with a DAD (Smartline DAD 2800) detector (using a C18 column (100 × 10 mm, onyx monolithic, USA). ¹H NMR (300 MHz) and ¹³C-NMR (75 MHz) were measured on a Bruker AV III 300 MHz spectrometer. Purified compounds were dissolved in methanol-d₄ for the spectroscopic analysis, and MestReNova x64-14.3.1-31739 software was applied for the data processing. From the American.

Collection of plant material

The aerial part of G. grimmii were collected and identified in July 2019 from the northern slopes of Zarinkooh Mountain, located in the Northeast of Dargaz County, Khorasan-Razavi Province, Iran by Dr. Amiri (Assistant Professor, Department of Biology, Payame Noor University, Tehran, Iran) following proper guidelines and legislation procedures. A voucher specimen (13154) was deposited in the herbarium of the School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. No specific license was required for the collection of the plants.

Extraction and bioassay-guided fractionation

The air-dried plant material was powdered (100.0 g) and extracted with methanol at room temperature by maceration. The methanolic extract was concentrated under reduced pressure (45 ^ºC), then it was suspended in water (90:10, 500 mL) and partitioned with petroleum ether, CH₂Cl₂, EtOAc, and n-BuOH (1:1, v/v), successively. Each fraction was tested for their anti-Mycobacterium tuberculosis (H37Rv) effects using the resazurin microtiter assay (REMA) [7]. Among them, the EtOAc extract showed potent anti-mycobacterial activity (MIC = 0.5 µg/mL). Taking these observations into account, the EtOAc extract (2.0 g) was fractionated over a Sephadex (LH-20) column chromatography, and eluted by MeOH to obtain eleven fractions (F1-F11). The anti-mycobacterial effects of all fractions were evaluated, and the fraction exhibiting the higher effect (F6, 51.6 mg) was selected for further purification by semi-preparative HPLC using a C₁₈ column with a gradient elution of MeOH and H₂O containing 0.1% TFA as the mobile phase at flow rate 5 mL/min. The gradient was the following: from 20 to 100% methanol in 7 min, 100% methanol in 5 min; from 100 to 20% methanol in 3 min and reequilibration at initial condition for 3 min to yield compounds 1 (10.0 mg, t_R: 7.25 min), 2 (4.1 mg, t_R: 7.15 min), 3 (6.2 mg, t_R: 6.12 min), 4 (8.6 mg, t_R: 6.15 min) and 5 (3.1 mg, t_R: 7.18 min). The bioassay-guided fractionation process are depicted in Fig. 1.

Fig. 1

Flow chart of bioassay-guided fractionation of anti-TB comounds from G. grimmii

Microorganisms and cultivation

In a regional tuberculosis reference laboratory, both the G method and culture were used for validation of these isolates, and through the Levin-Stein Johnson method (the gold standard proportional method), confirmation of their drug resistance to both first and second-line tuberculosis (TB) treatment was conducted. All strains were cultivated in Levin-Stein Johnson (LJ) medium for 2 weeks at 37 °C. By vortexing with glass beads, mycobacterial cells were harvested and de-clumped. The cellular concentration was adjusted to 3 × 10⁸ CFU/mL equal to 1 McFarland standard.

Antibacterial assay

Using the resazurin microtiter assay (REMA) plate method, the growth inhibitory activity of extracts was evaluated [7]. After sterilization of all extracts by syringe filters (0.45 μm, Jet Biofil), they dispersed in Middlebrook 7H9-S medium. This medium composed of Middlebrook broth, 0.5% glycerol, 0.1% casitone, and 10% OADC (oleic acid, albumin, dextrose, and catalase) supplement (Sigma-Aldrich). Subsequently, 100 µL of the diluted extract/compound was added to the first well of each column in a sterile 96-well tissue culture plate (SPL, Korea). The serial dilution was performed twofold with 7H9-S medium, resulting in final concentrations ranging from 50 to 0.015 µg/mL. A bacterial suspension of M. tuberculosis was prepared at a concentration of 10⁶ CFU/mL. To achieve 10⁵ CFU/mL final concentration, 100 µL of this cell suspension was inoculated into each well. Each plate had included three wells with rifampin (2 µg/mL), ethambutol (1.5 µg/mL), and isoniazid (1.25 µg/mL) for standard tests, two wells with no drug to serve as growth controls, and a column with all solutions but devoid of bacterial cells for the blank. The plates were duplicated, sealed with plastic film, and incubated at 37 °C. Following seven days of incubation, 30 µL of resazurin 0.01% (w/v) was treated in each well. To visually observe the color change, the plates were then incubated overnight. Active bacterial cells undergo a reduction process, transforming the non-fluorescent resazurin (blue) into the fluorescent resorufin (pink). This alteration signifies both the reduction of resazurin and bacterial growth. The minimum inhibitory concentration (MIC) value was considered as the lowest concentration that inhibited bacterial growth. Subsequently, the extracts exhibiting noteworthy anti-mycobacterial activity against H37Rv were chosen for further evaluation. These selected extracts were then evaluated against 5 MDR-TB and 2 XDR-TB clinical isolates. Then fractions derived from the most potent compounds against clinical resistant isolates were assessed for their anti-mycobacterial effects and the MIC value was reported.

Results and discussion

In continuation of our project to discover new bioactive compounds from plants [9, 10], five compounds were obtained from the EtOAc extract of G. grimmii through anti-TB bioassay-guided fractionation in this study. The structure of purified compounds was elucidated using 1D and 2D NMR (COSY, HSQC, HMBC) spectroscopy on a Bruker AVANCE Ш-300 spectrometer (Bruker, Germany). Compounds 1–5 were obtained as yellow solids and showed similar NMR profiles. The ¹H-NMR spectroscopic data of 1 (Table 1) showed four methyne protons in the aromatic region at δ_H 6.83 s (1H, H-3), δ_H 7.67 d (1H, H-2’, J = 2.1 Hz), δ_H 7.21 d (1H, H-5’, J = 8.4 Hz), and δ_H 7.77 dd (1H, H-6’, J = 8.4, 2.1 Hz), four signals at δ_H 4.11 s, 4.03 s, 4.00 s, 3.97 s, and 3.92 s (3H), indicating the presence of four methoxy groups which was also confirmed by the signals at δ_C 60.1 ppm (OMe-6), 61.1 (OMe-7), 61.4 (OMe-8), 55.4 (OMe-3’), and 55.4 (OMe-4’) in the ¹³C-NMR spectrum of 1, as well as a characteristic signal at δ_H 12.81 s is related to the OH-5 in the structure. Comparison of the NMR data of 1 with those of 2–5 indicated that all isolated compounds are similar, in 2 a methoxy group is missing at C-4’, 3 contains a hydroxy group at C-7 instead of the methoxy group, and a hydroxy group can be found at C-3’ and a methoxy group at C-4’. The only difference between compounds 2 and 4 was the presence of a hydroxy group instead of a methoxy group at C-7. In addition, compounds 3 and 5 differ only in the presence of a methoxy group instead of the hydroxy group at C-7 in 5. Based on a comprehensive examination of NMR spectra and comparison with previously reported data, the isolated compounds were identified as flavonoids. The compounds were identified as 5-demethylnobiletin (1) [11], 8-methoxycirsilineol (2) [12], 6,8-dimethoxyhesperetin (3) [13], sudachitin (4) [14], and 5,3’-dihydroxy-6,7,8,4’-tetramethoxyflavone (5) [15] (Fig. 2). The ¹H and ¹³C-NMR data of isolated compounds are reported in Tables 1 and 2.

Table 1 ¹H- (300 MHz) spectroscopic data for compounds 1–5 (MeOH, δ in ppm, J in hz)

Fig. 2

Chemical structures of isolated compounds

Table 2 The ¹³C-NMR shifts (ppm) for compounds 1–4 (MeOH, δ in ppm)

Many bacterial pathogens involved in human infectious disease epidemics have developed multidrug resistance (MDR) following antibiotic use. An example of such a pathogen is MDR M. tuberculosis, a significant pathogen, found in both developing and industrialized countries [16]. Medicinal plants have long been recognized as reliable sources for developing new antimicrobial drugs. In the past decade, considerable attention has been devoted to studying phytochemicals for their antibacterial activity, especially against MDR bacteria [17, 18]. In our study, the activity of the isolated compounds was investigated against one drug-sensitive, five MDR, and two XDR strains. Among them, compounds 1 and 2 were found to show anti-mycobacterial effects against the drug-sensitive H37Rv strain with MIC values of 0.062 and 1.0 µg/mL, respectively. Additionally, compound 1 exhibited growth inhibitory effects on all MDR and XDR strains investigated with a MIC range of 8–16 µg/mL. This inhibition potency is notably higher and remarkable when compared to natural compounds. However, compounds 3 to 5 showed no inhibitory effects against any of the investigated strains (MIC > 50 µg/mL) (Table 3). One of the most noteworthy result in this study is the superior effectiveness of compounds 1 and 2 compared to the positive controls, isoniazid and rifampicin against the drug-sensitive H37Rv strain with MIC values of 0.31 and 1.25 µg/mL, respectively.

Table 3 MIC (µg/mL) of isolated compounds against drug-sensitive and drug-resistant M. Tuberculosis strains

Flavonoids have become increasingly prominent in medicinal research. According to reports, these compounds possess numerous beneficial properties, including anti-inflammatory, estrogenic, enzyme inhibition, antimicrobial, anti-allergic, antioxidant, vascular protection, and cytotoxic anti-tumor activities [19]. Notably, among the discovered biological activities of flavonoids, their antibacterial effect and specific anti-mycobacterial effects, have been highlighted [20]. For example, in a study, the growth inhibitory activity of nine flavonoids, isolated from Eriosema chinense were tested on M. tuberculosis. Among them, four compounds, namely dehydrolopinifolinol, phlemicin D, eriosemaquinone A, and luteinifolin exhibited inhibitory activity with a MIC of 12 µg/mL [21].

Polymethoxyflavones are found mainly in the peel of citrus fruits (Rutaceae) and some other plant families, such as Lamiaceae and Asteraceae. They have attracted considerable attention due to their remarkable biological activities including anti-inflammatory, antiviral, antiatherosclerosis, neuroprotective, anticancer, antidiabetic, antioxidative, and anti-lipogenic activities [22, 23]. Our present observations are consistent with the recently published paper in 2023, which reported two flavonoids, 5,7,4` -trimethoxy flavanone and 5‑hydroxy-3,7,4` -trimethoxyflavone with good anti-mycobacterial activities against M. tuberculosis H37Rv with MIC values of 31.0 and 63.0 µg/mL, respectively [24]. It seems that the number and location of the methoxy groups in the structure are important for the anti-mycobacterial activities. Moreover, methoxylation in some phenolic compounds resulted in higher stability and bioavailability with better biological activities compared to the hydroxylated ones [25]. In another study, it was reported that polymethoxylated flavones exerted better lipid lowering activity than those substituted with free hydroxy groups [26]. Kawaii et al., synthesized several polymethoxylated flavones and evaluated their antiproliferative activities, and compared with the hydroxylated ones. They concluded that having more methoxy groups on the A-ring of polymethoxylated flavones resulted in better activity, while more methoxy groups in the B-ring reduced the activity [27]. In our study, although compound 1 with five methoxy groups (more in the B-ring), showed better activity than others, we did not find a clear correlation between the number of methoxy groups in the structures and their activities, as compound 2 with four methoxy groups showed relatively high activity (MIC range 1–32 µg/ mL), while compound 5 with the same number of methoxy groups displayed no activity. This behavior is consistent with another previous study that concluded no obvious correlation between the number of methoxy groups and the antiproliferative activity of isolated polyhydroxy flavonoids [28]. Based on our current findings, we suggest that the presence of methoxy groups at C-6,7,8, and C-3’ are necessary for anti-mycobacterial activity, while hydroxy groups at C-7 and C-3’ resulted in the reduction of the activity. However, more investigations are needed to confirm this structure-activity relationship. Given the extensive side effects of synthetic anti-tuberculosis drugs, the findings of this research could serve as a foundation for the development of new anti-tuberculosis drugs derived directly or indirectly from natural sources that are more effective, better tolerated, and having fewer adverse effects for patients. It is recommended that future studies focus on investigation of the cellular toxicity and possibility of synthesizing these compounds or their derivatives with the aim of developing novel anti-tuberculosis drugs. Molecular and docking studies could also reveal the potential mechanisms of action of these compounds. Considering that in a previous study, the EtOAc and n-BuOH fractions of G. grimmii extract also demonstrated significant anti-mycobacterial effects, further investigations of these fractions are recommended in order to identify other compounds with anti-mycobacterial properties.

Conclusion

In this study, five flavonoids were identified as a result of an anti-mycobacterial activity-guided isolation from G. grimmii. Among isolates, compounds 1 and 2 exhibited significant effects against the drug-sensitive M. tuberculosis strain (H37Rv). Moreover, compound 1 demonstrated the growth inhibition of all investigated MDR and XDR strains. Despite the observed promising activity, further studies are needed to investigate the anti-mycobacterial mechanisms of these compounds and their cellular effects to ensure their non-toxic nature. This will allow for the potential use of these natural compounds or their synthetic derivatives as anti-mycobacterial drugs in clinical settings.