A new flavonol derivative and other compounds from the leaves of Bauhinia thonningii Schum with activity against multidrug-resistant bacteria

Abstract Investigation of the leaves of Bauhinia thonningii Schum led to the isolation and identification of a new flavonol derivative, 6-C-methylquercetin-3,4'-dimethyl ether (1) together with eleven known compounds (2–13), with two of them (10 and 11) obtained as a mixture. Their structures were established by extensive spectroscopic analyses. Antibacterial activity of compound 1 as well as the reference antibiotic, ciprofloxacin was tested on Gram-negative multidrug-resistant bacteria overexpressing active efflux pumps, and against methicillin-resistant strains of Staphylococcus aureus (MRSA). Samples were tested alone and in combination with an efflux pump inhibitor (EPI), phenylalanine-arginine-β-naphthylamide (PAßN). Results show that when compound 1 was tested alone, its inhibitory effects were obtained on 7/10 tested bacteria with the highest MIC value of 128 µg/mL whilst in the presence of EPI, this activity significantly increase in all the 10 bacteria. An interesting antibacterial activity was obtained with compound 1 against Klebsiella pneumoniae ATCC11296 (MIC of 4 µg/mL), KP55 and Staphylococcus aureus MRSA6 in the presence of the PaßN. Graphical abstract


Introduction
Bauhinia is a genus of flowering plants of the Caesalpiniaceae subfamily, generally in shrub or liana form and is very widespread in all the Sudano-Guinean savannas of tropical Africa (Ake et al. 1980). Bauhinia thonningii is widely used in pharmacopeia in the treatment of several diseases: hemorrhoids, skin infections, dysentery, fever (Malgras 1992;Barata et al. 1999). Previous phytochemical and biological studies on the plants of the genus Bauhinia revealed the presence of several classes of secondary metabolites including terpenoids, flavonoids and other phenolic compounds with various biological activities (Olalekan et al. 2008;Kama-Kama et al. 2017;Ni et al. 2020;da Silva et al. 2021;Monteiro et al. 2022).
Bacterial drug resistance remains a global health concern as many antibacterial agents commonly used result in the selection of resistant phenotypes . Several natural products including flavonoids have been found to be active against multidrug-resistant (MDR) bacteria (Omosa et al. 2016). In the context of the search for novel molecules to combat MDR bacteria, the current study focused on the investigation of bioactive secondary metabolites from the leaves of Bauhinia thonningii Schum. We isolated and characterized one new flavonol derivative, 6-C-methylquercetin-3,4 0 -dimethyl ether (1) together with twelve known compounds including nine flavonoids (2-11), ursolic acid (12) and daucosterol (13) (Figure 1). The newly isolated flavonoid was evaluated on a panel of Gram-positive and Gram-negative bacteria expressing resistant phenotypes.
The antibacterial activity of compound 1 as well as the reference antibiotic, ciprofloxacin was tested on Gram-negative multidrug-resistant bacteria overexpressing active efflux pumps, and against methicillin-resistant strains of Staphylococcus aureus (MRSA). Samples were tested alone and in combination with an efflux pump inhibitor (EPI), phenylalanine-arginine-b-naphthylamide (PAßN). The results are summarized in Table S2. Results show that when compound 1 was tested alone, its inhibitor effects were obtained on 7/10 tested bacteria with the highest MIC value of 128 mg/mL whilst CIP was active on all the ten pathogens, with the MIC values ranging from 8 to 128 mg/mL. In the presence of the EPI, the activity of compound 1 significantly increased in all the 10 bacteria, with MIC ranging from 8 to 4 mg/mL. A significant increase in the activity of ciprofloxacin (CIP) was also noted in the presence of PAbN (MIC ranging from 2 to 8 mg/mL). This is a clear indication that the tested bacteria expressed active efflux pumps, and that compound 1 is a substrate of this bacteria resistance system. In antibacterial screening, it has been established that phytochemicals display significant effects if the recorded MIC value is below 10 mg/mL . Consequently, interesting antibacterial activity was obtained with compound 1 against Klebsiella pneumoniae ATCC11296 (MIC of 4 mg/mL), KP55 and Staphylococcus aureus MRSA6 (MIC of 8 mg/mL) in the presence of the EPI. It is also worth noting that the activity of CIP was restored in the presence of EPI, with significant antibacterial effects being obtained on all the 10 tested pathogens (Table S2). These data suggest that compound 1 might possibly be exploited to fight bacterial drug resistance, especially when it is combined with an EPI.
Several studies have documented the antimicrobial potential of natural occurring flavonoids (Cushnie and Lamb 2005;Ngameni et al. 2013;Wang et al. 2018). The antibacterial effect of quercetin, isolated in the current study, has been also reported (Wang et al. 2018).

General experimental procedures
The proton and carbon (1 D & 2 D)-NMR (600/500 MHz) spectra were measured on Bruker AMX machine. The chemical shifts of proton and carbon were recorded base on the internal reference TMS (Tetramethylsilane) in d (ppm). Moreover, coupling constants (J) were measured in Hz. High resolution mass spectra were obtained on QTOF Spectrometer equipped with a HESI source. The spectrometer was operated in positive mode (mass range: 100-1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 0.40 ppm deviation using Na formate as calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200 C. Nitrogen was used as sheath gas (10 l/min). The spectrometer was attached to an Ultimate 3000 (Thermo Fisher, USA) UHPLC system consisting of LC-pump, Diode Array Detector (DAD) (k: 190-600 nm), auto sampler (injection volume 5 ml) and column oven (40 C). The separations were performed using a Synergi MAX-RP 100 A (50 Â 2 mm, 2.5 m particle size) with a H 2 O (þ0.1% HCOOH) (A)/acetonitrile (þ0.1% HCOOH) (B) gradient (flow rate 500 mL/min, injection volume 5 mL). Samples were analyzed using a gradient program as follows: 95% A isocratic for 1.5 min, linear gradient to 100% B over 6 min, after 100% B isocratic for 2 min, the system returned to its initial condition (90% A) within 1 min, and was equilibrated for 1 min. UV/Visible spectroscopic data of 1 were recorded on an Evolution 300 spectrophotometer (Thermo Scientific). The purity of compounds and the monitoring of fractions were based on pre-coated silica gel TLC (Thin Layer Chromatography) plates supported on either plastic or aluminum sheets (E. Merck, F 254 ). Spots were visualized on TLC with UV light (254 nm & 365 nm) on CN-6 UV spectrometer then sprayed with ceric sulphate and heated at about 90 C.

Plant material
The leaves of P. thonningii were collected in December 2020 in Bandjoun, West Region-Cameroon and identified at the National Herbarium of Cameroon on voucher number 33258/HNC

Extraction and purification
2.0 kg of air-dried of P. thonningii leaves were crushed and extracted with 15 L of methanol for 72 hours to yield 200.4 g of crude extract. 194.0 g were triturated with EtOAc to afford 96.2 g of EtOAc extract. 96.0 g of the resulted extract were subjected to column chromatography (CC) unsing silica gel and eluted with a gradient of n-hexane-EtOAc (100:0 to 0:100, v/v) and EtOAc-CH 3 OH (100:0 to 0:100, v/v) to afford 168 fractions of 300 mL each, which were combined into seven major fractions (A-G) based on their TLC profiles. Fractions A and B were found to contain mainly fatty acids and were not further investigated. Compounds 5 (15.1 mg) and 12 (19.9 mg) precipitated from fraction C (2.1 g), while compound 13 (25.0 mg) precipitated from fraction E (0.2 g). Fraction D (30.7 g) was subjected to repeated silica gel CC using a gradient elution of n-hexane-EtOAc (100:0 to 0:100, v/v), followed by CC over Sephadex LH-20 eluting with CHCl 2 -CH 3 OH 50:50, v/v to yield compound 1 (9.9. mg), 2 (12.1 mg), 4 (14.7 mg), 9 (6.1 mg), and a mixture of 10 and 11 (19.8 mg).
The residue (78.3 g) from the trituration was subjected to repeated silica gel CC using a gradient elution of n-hexane-EtOAc (100:0 to 0:100, v/v) and EtOAc-CH 3 OH 3.4.2. Bacterial susceptibility testing MICs of compound 1 and ciprofloxacin were determined by the broth micro-dilution methods using INT colorimetric assay (Eloff 1998) as slightly modified lately (Nielsen et al. 2012). Briefly, the two samples were dissolved in 10% DMSO/MHB and a serial two-fold dilution was performed in a 96-well microplate. Then, 100 mL of inoculum (2 Â 10 6 colony forming units (CFU)/mL) prepared in MHB was added to each well. The control drug used was ciprofloxacin (positive control) while a well containing DMSO 2.5% (the highest concentration in a well) served as control (no antibacterial growth was observed with DMSO at 2.5%). The final concentration of the tested samples ranged from 1 to 128 lg/mL. The plates were further covered in a sterile condition, shaken, and incubated at 37 C for 18 h. The MIC of each sample, defined as the lowest sample concentration that completely inhibited bacteria growth was detected following the addition of 40 lL INT (0.2 mg/mL), followed by additional 37 C incubation for 30 min. Visibly, living bacteria reduced the yellow INT dye to pink (Touani et al. 2014). To evaluate the role of efflux pumps in the bacterial resistance to compound 1 as well as ciprofloxacin, both samples were tested in the presence of PAßN (at 30 lg/ mL) against the investigated pathogens (Lorenzi et al. 2009). The activity improvement factor (AIF) or fold increase of the activity was determined as ratio of MIC (sample alone)/MIC (sample þ PAbN) (Demgne et al. 2021). Each assay was performed in duplicate and repeated thrice.

Conclusion
Investigation of the leaves of Bauhinia thonningii Schum led to the isolation of a new flavonol derivative, 6-C-methylquercetin-3,4 0 -dimethyl ether, as well as eleven known compounds. The results of the present study indicate that the newly isolated flavonoid may have a potential to combat bacterial drug resistance, especially in combination with an efflux pump inhibitor