Four new N-phenethylbenzamide derivatives from the stems of piper betle and their antimicrobial activity

Abstract Four new N-phenethylbenzamide derivatives, named piperbetamides A-D (1–4), and six allylbenzene derivatives (5–10) were isolated from the stems of Piper betle L. Their structures were determined by HR-ESI-MS and NMR spectroscopic methods. Compounds 1–10 were evaluated for their inhibitory effects on the growth of nine microorganisms including five Gram-negative (Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, Pseudomonas aeruginosa, and Extended-spectrum beta-lactam resistant Klebsiella pneumoniae), three Gram-positive (Listeria monocytogenes, Methicilin-resistant Staphylococcus aureus, Vancomycin-resistant Enterococcus faecalis), and one yeast (Candida albicans) strains. Compounds 1, 3, 4, 6 and 10 exhibited potential antimicrobial activity against S. flexneri, L. monocytogenes, methicillin-resistant S. aureus and vancomycin-resistant E. faecalis with minimum inhibitory concentration (MIC) values in the range of 16–32 µg/mL. Graphical Abstract


Introduction
Piper betle is a climbing shrub and popularly cultivated in the tropical countries. Its leaves, roots, and fruits have long been used in traditional medicines due to anti-bacterial activity, wound healing property, promoting blood circulation, and prevention of blood bleeding (Salehi et al. 2019). The roots and fruits are used to treat asthma and malaria. The roots of P. betle is also documented famous with female contraceptive effects. The leaves of P. betle is the most widely used and studied both on the chemical constituents and pharmacological activities (Madhumita et al. 2020;Nayaka et al. 2021). A broad range of P. betle medicinal uses have been listed against many diseases such as cough remedy, nosebleed, rheumatism, mastitis, halitosis, ringworm, digestive diseases, headache, and conjunctivitis (Madhumita et al. 2020). Over the past decades, numerous studies have focused on anti-bacterial and antifungal properties and suggested valuable anti-microbial products from P. betle leaves (Nayaka et al. 2021). Phenolic compounds in term of allylbenzene derivatives such as eugenol, isoeugenol, chavicol were reported to be major active components from the leaves of P. betle (Kavitha et al. 2019;Atiya et al. 2022). Additionally, other bioactive compounds like flavonoids, terpenes, alkaloids, and fatty acids were also identified from this plant (Parmar et al. 1997). To date, almost phytochemical and anti-microbial studies focused on the leaves of P. betle while the stem parts would be interesting in the liana plant. Ongoing to find new and anti-microbial constituents of P. betle, herein, we describe the isolation and identification of four new N-phenethylbenzamide derivatives and six known allylbenzene derivatives from the stems of P. betle. The isolated compounds were evaluated for their inhibitory effects on the growth of nine pathogens by broth microdilution assay.
Fresh or dried P. betle materials have long been used for their antimicrobial property (Salehi et al. 2019;Nayaka et al. 2021). Numerous studies on the antimicrobial activity of essential oil, chloroform, ethyl acetate, methanol, and aqueous extracts of P. betle were highlighted in a recent review (Biswas et al. 2022). Therefore, compounds 1-10 were evaluated for their effects on the growth of microbial strains including Gram-negative bacteria (Escherichia coli ATCC25922, Salmonella enterica serovar Typhimurium ATCC14028, Shigella flexneri ATCC12022, Pseudomonas aeruginosa ATCC27853, Extended-spectrum beta-lactam resistant Klebsiella pneumoniae ATCC700603), Gram-positive bacteria (Listeria monocytogenes ATCC19115, Methicilinresistant Staphylococcus aureus ATCC12493, Vancomycin-resistant Enterococcus faecalis ATCC51299), and yeast (Candida albicans ATCC 10231). As shown in the Table S2, all tested compounds 1-10 showed anti-microbial activity towards tested pathogens, except P. aeruginosa. In general, the compounds 1-10 exhibited stronger antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria. Among isolated compounds, compound 10 exhibited the best antimicrobial activity against the tested pathogens. Particularly, it inhibited the growth of L. monocytogenes, Methicillin-resistant S. aureus and Vancomycin-resistant E. faecalis strains with the same and lowest MIC value (16 mg/mL). Compounds 3 and 4 were strongly active against Methicillinresistant S. aureus (MIC ¼ 16 mg/mL). Compounds 1, 3, 4, 6 and 10 showed antimicrobial activity towards all tested Gram-positive bacteria (MIC in the range of 16-32 mg/ mL). However, these compounds potentially inhibited only S. flexneri strain (MIC ¼ 32 mg/mL) among five tested Gram-negative bacterial strains. Except benzamide derivatives (1-4), allylbenzene derivatives (5-10) inhibited the growth of yeast, C. albicans strain. This result is in agreement with previously reported on potential antifungal activity of compound 5 against a series of fungal strains (Ali et al. 2010). A mixed antibiotics (chloramphenicol, tetracycline, and kanamycin, 1:1:1) and amphotericin were used as positive control against bacteria and yeast, respectively, showing their MIC values of 2 mg/mL. The obtained mixed antibiotic was 8 times more active than compound 10 against Gram-positive bacteria. To the best of our knowledge, compounds 1-4, 6-10 have not been previously investigated for antimicrobial activity against human pathogens. Therefore, the compounds 1, 3, 4, 6 and 10 exhibited potential antimicrobial activity towards pathogenic bacteria including multidrug-resistant strains which could be helpful as new antimicrobial agents from natural source as well as the stems of P. betle.

General
HR-ESI-MS was acquired on a Sciex X500 Q-TOF LC/MS system. Optical rotations were obtained on a Jasco P2000 polarimeter. NMR spectra were measured on a Bruker AVANCE III 500 MHz spectrometer. Column chromatography was performed using silica gel (40-63 mm), ODS (150 mm), or diaion HP-20 resins. Thin layer chromatography was carried out on pre-coated plates. Semi-reparative HPLC was acquired on an Agilent 1260 infinity II including binary pump, autosampler, DAD detector and equipped with YMC J'sphere ODS-H80 column (20 Â 250 mm, 4 mm). Flow rate was set at 3 mL/min.

Plant material
The stems of Piper betle L. were collected at Thai Binh province, Vietnam in October 2020. Its scientific name was identified by botanist Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. Voucher specimen (sample number: NCCT-P108) was kept at the Institute of Marine Biochemistry.

Acid hydrolysis and confirmation of D-glucose residue
Compounds 1-4 (each 10 mg) were separately dissolved in 1 mL solution of HCl 1 M in dioxan/water (1/1, v/v) and heated to 80 C in a water bath for 2 h. The acidic solution was neutralized with silver carbonate, the precipitated silver chloride was removed and the solution was concentrated thoroughly under nitrogen atmosphere. After extraction with ethyl acetate, the aqueous layer was concentrated to dryness using nitrogen gas and monosaccharide (1.3 mg) was purified by preparative TLC (CHCl 3 / MeOH/H 2 O, 4/3/0.3, v/v/v). The specific optical rotation of the monosaccharide was recorded after dissolving in water for 24 h and compared to the authentic D-glucose prepared in a similar manner, ½a 25

Antimicrobial assay
The minimum inhibitory concentrations (MICs) of pure compounds were determined separately by broth microdilution assay with the range of 2 and 256 mg/mL against 9 pathogens including Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028, Shigella flexneri ATCC 12022, Pseudomonas aeruginosa ATCC 27853, Listeria monocytogenes ATCC 19115, Candida albicans ATCC 1023, Extendedspectrum beta-lactam resistant Klebsiella pneumoniae ATCC 700603, Methicilin-resistant Staphylococcus aureus ATCC 12493 and Vancomycin-resistant Enterococcus faecalis ATCC 51299 (Mogana et al. 2020). Briefly, for each pathogen, suspension culture was freshly prepared in Mueller Hilton broth (MHB) to obtain a final concentration of 10 8 CFU/mL. Then, 180 mL of the suspension culture was transferred into each 96-microplate well followed by adding 20 mL of tested compound solution with final concentrations in range of 2 mg/mL to 256 mg/mL. The plates were incubated at 37 C, and the optical density (OD) was measured at 600 nm after 24 h of incubation using a microplate reader SpectraMax iD5 (Molecular Devices, USA). The suspension culture without tested compound solution served as negative control. A mixed antibiotics of chloramphenicol, tetracycline and kanamycin (1:1:1, w/w/w) was used as positive control for bacterial test and amphotericin was used as positive control against yeast C. albicans. All experiments were performed in triplicate under the same conditions. The MIC value was determined as the lowest concentration showing 95% inhibition of bacterial growth.

Conclusions
Phytochemical study on the stems of P. betle afforded four new N-phenethylbenzamide derivatives, named piperbetamides A-D (1-4), and six known allylbenzene derivatives (5-10). Compounds 1, 3, 4, 6 and 10 showed potential antimicrobial activity towards Gram-positive bacteria and selective inhibitory activity on Gram-negative bacteria. Especially, compound 10 showed antimicrobial activity towards all tested microbial strains (MIC in the range of 16-128 mg/mL), except Pseudomonas aeruginosa. Allylbenzene derivatives exhibited antifungal activity meanwhile benzamide derivatives were inactive. Further studies should be warned on anti-bacterial activity of benzamide derivatives and anti-fungal activity of allylbenzene derivatives. Additionally, our results suggest that anti-microbial products could be developed not only from the leaves of P. betle but also from its stems.