Terpenes from Zingiber montanum and Their Screening against Multi-Drug Resistant and Methicillin Resistant Staphylococcus aureus

Bioassay directed isolation of secondary metabolites from the rhizomes of Zingiber montanum (Fam. Zingiberaceae) led to the isolation of mono-, sesqui-, and di-terpenes. The compounds were characterized as (E)-8(17),12-labdadiene-15,16-dial (1), zerumbol (2), zerumbone (3), buddledone A (4), furanodienone (5), germacrone (6), borneol (7), and camphor (8) by analysing one-dimensional (1D) (1H and 13C) and two-dimensional (2D) (COSY, HSQC, HMBC, and NOESY) NMR data and mass spectra. Among these terpenes, compounds 1 and 2 revealed potential antibacterial activity (minimum inhibitory concentrations (MIC) values 32–128 µg/mL; 0.145–0.291 mM)) against a series of clinical isolates of multi-drug resistant (MDR) and Methicillin resistant Staphylococcus aureus (MRSA).


Introduction
Antimicrobial resistance has increasingly become a major public health issue that currently claims 700,000 lives every year. It is predicted that if no action is taken, there will be approximately 10 million deaths each year globally by 2050, which will be more than the predicted number of deaths by cancer [1] and will cause a cumulative 100 trillion USD of economic output due to the rise of drug-resistant infections [1]. The morbidity due to resistant infection has doubled since 2007, which equals the burden of HIV, influenza, and tuberculosis [1]. As antibiotic resistance occurs naturally, its mishandling and misuse in humans and animals accelerate the process of development of resistant infection [2]. Infectious diseases, like pneumonia, tuberculosis, gonorrhoea, and salmonellosis are becoming difficult to treat because the antibiotics that are used to treat them are becoming less effective [2]. The gram-positive bacterium Staphylococcus aureus relates to an extensive range of infection of skin and soft tissue, pneumonia, endocarditis, sepsis and bacteremia [3] that causes nosocomial infection (resistant to methicillin and vancomycin). Therefore, it is no doubt important to discover new antibiotics to act against multi-drug resistant (MDR) and Methicillin resistant Staphylococcus aureus (MRSA).

General
Analytical solvents, such as n-hexane, chloroform, ethyl acetate, acetone, and methanol were purchased from Fisher Scientific, Loughborough, UK. Dimethyl sulfoxide, sodium chloride, norfloxacin and 3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide (MTT) used during antibacterial assay were purchased from Sigma Aldrich (Dorset, UK). Silica gel 60H used for vacuum liquid chromatography was purchased from Merck Millipore, UK. Sephadex LH 20 used for gel filtration chromatography was purchased from GE healthcare, Uppasala, Sweden. Prepacked silica column (normal phase) used for solid phase extraction (SPE) was purchased from Phenomenex, Cheshire, UK. Analytical and preparative TLC carried out on 0.2 mm silica gel 60 F 254 was purchased from Merck, Darmstadt, Germany. Spots on the TLC plates were visualized under short UV (254 nm) and long UV (366 nm), and also by spraying them with 1% vanillin in concentrated H 2 SO 4 followed by heating at 100 • C for 3-6 min. The NMR spectroscopy was performed with Bruker AMX 600 NMR spectrometer, Coventry, UK (600 MHz for 1 H, and 150 MHz for 13 C) in the Department of Chemistry at University College London (London, UK). High Resolution Mass Spectrometry (HRMS) was performed in Liverpool John Moores University (Liverpool, UK). IR spectroscopy was recorded on Agilent FT-IR (Cary 630, Stockport, UK).

Plant Material
The rhizomes of Zingiber montanum were collected from Bangladesh National Botanical Garden, Dhaka, Bangladesh in September 2016. The plant was identified by the Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh, where a voucher specimen (DACB 43550) of this collection was deposited.

Extraction and Isolation of Compounds
The rhizomes of Z. montanum were sun dried for 2-3 days, followed by drying in the oven at a temperature of 30-35 • C for 30 min prior to grinding. Subsequently, the plant materials were ground into fine powders using a grinder. The ground plant material (242 g) was Soxhlet extracted with solvents of increasing polarity: n-hexane, chloroform, and methanol (approximately 700 mL, 10-15 cycles each). Each of the extracts was concentrated using rotary evaporator under reduced pressure at a maximum temperature of 40 • C to yield 9.38 g, 10.22 g, and 23.0 g of n-hexane, chloroform, and methanol extracts, respectively. The antibacterial screening was performed on these crude extracts against clinical isolates of MRSA strains. Hexane (MICs 128-256 µg/mL) and chloroform (MICs 64-256 µg/mL) extracts appeared to be active and they were further fractionated by vacuum liquid chromatography (VLC). A portion of n-hexane (6.5 g) or chloroform (7.6 g) were adsorbed into silica gel (70-230 mesh) and loaded into VLC column, which was uniformly packed with VLC grade silica gel (60H), followed by eluting with stepwise gradient of mobile phase initially with mixture of n-hexane and ethyl acetate and then with EtOAc and MeOH mixtures. The eluted fractions (200 mL each) were evaporated using rotary evaporator and analysed by TLC. Based on TLC results, the similar fractions were bulked together for further purifications by solid phase extraction (SPE), column chromatography over Sephadex LH20, and/or preparative TLC. The basic principle of SPE is similar to VLC, but SPE was used in smaller scale fractionation or further purification of compounds from the VLC fractions or pooled fractions from Sephadex LH20 column chromatography. For column chromatography over Sephadex LH20, the glass column was packed with the slurry of Sephadex LH-20, which was soaked in the solvent (50% chloroform in n-hexane or 100% chloroform) half an hour prior to the packing of the column. The sample was dissolved in a small amount of appropriate solvent and then applied onto the top of the adsorbent. The column was eluted with 50-75% chloroform in n-hexane, followed by 100% chloroform and then CHCl 3 + MeOH mixtures of increasing polarity. During preparative TLC, the sample was applied uniformly as band in the sample application zone (2 cm above from the bottom edge of TLC plate) on commercially available TLC aluminium plates (pre-coated silica gel 60 PF 254 ). The TLC plates were developed with appropriate mobile phase up to the upper edge of plates. In addition, the multiple development technique was also adapted for a better accomplishment of separation of compounds of very similar polarity.
VLC fraction eluted with 5-10% of EtOAc in n-hexane of n-hexane extract was subjected to column chromatography over Sephadex LH20. PTLC (mobile phase 15% EtOAc in hexane) on Sephadex column eluted with 50% CHCl 3 in n-hexane yielded compounds 2 (3 mg) and 3 (10 mg), whereas compound 4 (9 mg) was obtained from sephadex column eluted with 100% chloroform. SPE on the VLC fraction eluted with 15% of EtOAc in n-hexane of n-hexane extract provided six sub-fractions. Preparative TLC (mobile phase 4% EtOAc in hexane plus two drops glacial acetic acid) on SPE sub-fraction eluted with 4% EtOAc in hexane yielded compounds 5 (7 mg) and 6 (4 mg), whilst preparative TLC (mobile phase 4% EtOAc in hexane plus two drops glacial acetic acid) on SPE sub-fraction eluted with 4% EtOAc in hexane led to the isolation of compounds 6 (5 mg) and 7 (7 mg). Similarly, VLC fraction of chloroform extract was subjected to SPE and preparative TLC for the purification of compounds. SPE on VLC fraction eluted with 15% EtOAc in n-hexane followed PTLC (mobile phase 4% EtOAc in hexane) on SPE sub-fraction eluted with 4% EtOAc in hexane yielded 8 (4 mg), while compound 1 (6.5 mg) was isolated from the VLC fraction eluted with 25% EtOAc in n-hexane, followed by PTLC (mobile phase 15% EtOAc in hexane) on SPE sub-fraction eluted with 10% EtOAc in hexane.

Antibacterial Assay against Clinical Isolates of Multi-Drug Resistant and Methicillin Resistant Staphylococcus Aureus
The antibacterial activity of crude extracts and the isolated compounds were tested against clinical isolates of MRSA strains by microtitre assay using 96 well plates to determine the minimum inhibitory concentrations (MICs). Mueller-Hinton broth (MHB) used in this study was purchased from Oxoid, Hamshire, UK and prepared as instructed by the supplier; however, MHB was adjusted to contain 20 mg/L and 10 mg/L of Ca 2+ and Mg 2+ , respectively. The clinical isolates of S. aureus strains used in this study included ATCC25923, SA1199B, RN4220, XU212, EMRSA15, MRSA340702, and MRSA274829. A standard laboratory strain, ATCC25923, was also used in this study, which is sensitive to antibiotics, like tetracycline [31]. SA1199B over-expresses the NorA MDR efflux pump [32], RN4220 possesses the MsrA macrolide efflux protein [33], XU212 is a Kuwaiti hospital isolate that is an MRSA strain possessing the TetK tetracycline efflux pump [31], whilst the EMRSA15 strain [34] is epidemic in the UK. All S. aureus strains were subcultured on nutrient agar (Oxoid) and incubated for approximately 24 h at 37 • C prior to MIC determination. All of the bacterial strains were prepared in 9 g/L saline water with an inoculum density of 5 × 10 5 colony forming unit (cfu/mL) by comparison with the 0.5 MacFarland turbidity standard.
The stock solution of control positive (Norfloxacin) was prepared by dissolving the antibiotic (2.0 mg) in DMSO (244 µL) and diluting 16 fold with MHB to obtain the desired concentration of the antibiotic stock solution (512 µg/mL). Similarly, stock solutions of crude extract (2048 µg/mL) and isolated compounds (256-512 µg/mL) were prepared by dissolving in required amount of DMSO, followed by 16 fold dilution with MHB.
During the experiment, using a multi-channel pipette an aliquot of 100 µL of MHB was dispensed into each well of 96-well plate except those in the last column. Then 100 µL of stock solution of crude extract or isolated compounds and antibiotic was added in duplicate to the wells of the first column of 96-well plate (total content 200 µL), followed by mixing the content thoroughly and transferring 100 µL of this content to the wells of the second column of 96-well plate using a multi-channel pipette. This two-fold serial dilution process was continued to the 10th well, followed by the addition of the final 100 µL solution to the empty wells of 12th column of 96 well-plate. The inoculum (100 µL) of each bacterium at a density of 5 × 10 5 cfu/mL was added to all wells, except those in the final (12th) column. The contents of the wells in the 11th and 12th columns represented growth control (bacteria, but no antibiotic, extract, or compounds) and sterility control (antibiotic, extract, or compounds but no bacteria), respectively. Every assay was performed in duplicate. The plates were incubated for 18 h at 37 • C. For the measurement of MIC, 20 µL of a 5 mg/mL methanolic solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide (MTT; Sigma) was added to each of the wells, followed by incubation for 20-30 min at 37 • C. Bacterial growth was indicated by a colour change from yellow (colour of MTT) to dark blue. The MIC was recorded as the lowest concentration at which no growth (yellow color) was observed [13]. If no growth was observed at any of the concentrations tested, the assay was repeated starting from a stock solution of lower concentration. If growth was observed at all of the concentrations tested, the assay was repeated, starting with a stock solution of higher concentration.