Research Paper Antimicrobial Properties Of

Research Paper

Antimicrobial properties of plants of Chungtia village used customarily to treat skin related ailments: From antimicrobial screening to isolation of active compounds

Accepted 14th May, 2018

ABSTRACT

Chungtia villagers of Nagaland, India, have a strong reliance on plants as medicines. Previous studies have shown that 31 Chungtia medicinal plants (and parts therein) used customarily for skin related treatments possess antimicrobial properties against skin pathogens, strongly supporting the use of these plants by the Chungtia villagers. Five plants, namely Albizia lucidior, Begonia picta, Cassia floribunda, Holboellia latifolia and Maesa indica have no previous studies on their antimicrobial properties, while Prunus persica has only antimicrobial activity reported on its fruit, with no reports on its roots, which are used by the Chungtia villagers. The aim of the study was to investigate these six plants for their antimicrobial properties against dermatologically relevant pathogens and undertake phytochemical analysis of the most active species, P. persica. 70% aqueous ethanoic extracts were prepared of leaves of B. picta, C. floribunda, H. latifolia and M. indica and roots of A. lucidior and P. persica. The crude extract of P. persica was successively partitioned between water and n-hexane, dichloromethane (DCM) and ethyl acetate (EtOAc). TLC bioautography guided fractionation using column chromatography of the n-hexane and EtOAc partition afforded five compounds. The structures of the compounds were elucidated by 1D and 2D NMR spectroscopic techniques and comparison with published data. The crude extract and partitioned fractions of P. persica as well as, the isolated compounds were screened against antibiotic sensitive and resistant strains of Staphylococcus aureus and Escherichia coli as well as, antibiotic sensitive Pseudomonas aeruginosa, Streptococcus pyogenes, Salmonella typhimurium and the fungus C. albicans using the MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide) microtitre dilution and disc diffusion assay methods. All of the plant aqueous ethanolic extracts exhibited antibacterial activity against at least two of the tested micro- organism. The most noteworthy activity was shown by P. persica root extract against antibiotic sensitive as well as, resistant (methicillin [MRSA] and multi drug resistant [MDRSA]) strains of S. aureus (0.156 mg/ml for all tested strains). None of the plants were active against the fungus C. albicans. This is the first report of antibacterial activity, with the highest antimicrobial activity for the EtOAc fraction with MIC 312 µg/ml (susceptible as well as, resistant strains of S. aureus), followed by n- hexane MIC 625 µg/ml (susceptible S. aureus) and MIC 312 µg/ml (MRSA and MDRSA). Bioassay guided fractionation of the EtOAc partition resulted in isolation of ent-epiafzelechin- (2α→O→7, 4α→8)-(-)-ent- afzelechin (1), afzelechin (2), Caffeic acid phenylethyl ester (3) and Gallic acid (4). GC-MS analysis of Teresa Malewska1*, Meyanungsang the n-hexane extract showed the presence of the antibacterial compounds palmitic acid, linoleic acid Kichu1, Emma C. Barnes1, Wendy Loa- and oleic acid. Bioassay guided fractionation of the n-hexane partition resulted in isolation of three Kum-Cheung1, Joseph J. Brophy2, antibacterially active compounds: α-cyanobenzyl benzoate (4), MIC 0.78 µg/ml for susceptible and Imchawati Imchen3, Subramanyam resistant S. aureus strains, MIC 312 µg/ml for susceptible and resistant E. coli strains and MIC 625 µg/ml Vemulpad1 and Joanne F. Jamie1 for P. aeruginosa), β-sitosterol (5) MIC 2500 µg/ml for susceptible and resistant S. aureus strains and antibiotic susceptible E. coli, MIC 625 µg/ml for antibiotic susceptible S. typhimurium and MIC 1250 1Indigenous Bioresources Research µg/ml for antibiotic susceptible P. aeruginosa) and stigmast-4- en-3-one (6) MIC 156 µg/ml for Group, Faculty of Science and susceptible and resistant S. aureus strains, MIC 312 µg/ml for antibiotic susceptible E. coli, MIC 625 Engineering, Macquarie University, NSW, µg/ml for antibiotic susceptible S. typhimurium and MIC 1250 µg/ml for antibiotic susceptible P. 2109, Australia. aeruginosa). This is the first report for antibacterial activity for α-cyanobenzyl benzoate and stigmast- 2School of Chemistry, Faculty of Science, 4-en-3-one. Aqueous ethanolic extracts of six Chungtia medicinal plants used for skin related ailments University of New South Wales, NSW, were found to have antibacterial properties against dermatologically relevant bacteria, supporting their 2052, Australia. customary uses by Chungtia villagers. Good antibacterial activity of the EtOAc and n-hexane partitioned 3Chungtia Village, Mokokchung, fractions from the P. persica root extract and the isolated compounds ent-epiafzelechin-(2α→O→7, Nagaland, 79860, India. 4α→8)-(-)-ent-afzelechin, α-cyanobenzyl benzoate, β-sitosterol and stigmast-4-en-3-one as well as, antibacterially active compounds identified from the GC- MS studies further supports the customary *Corresponding author. E-mail: use of roots of this species in treating skin related ailments. [email protected]. Tel: +61 2 98508283; Fax: +61 2 98508313. Keywords: Antimicrobial properties, pathogens, phytochemical analysis, P. persica.

INTRODUCTION

The presence of pathogenic bacteria and fungi can cause skin infections and exacerbate the healing and seriousness Academia Journal of Biotechnology; Malewska et al. 145

of sores and wounds (Edwards and Harding, 2004). Skin (254 and 365 nm), respectively and vanillin-sulphuric acid disease and infections cause a significant global disease spray reagent. UV- visible spectra were recorded on a CARY burden and with the escalating occurrence of multidrug 1 Bio spectrophotometer (Varian, USA). IR spectra were resistant micro-organisms, there is heightened concern that recorded on a NICOLET iS10 (Thermo Scientific, USA). the rates of skin infections will only worsen (Hay et al., Optical rotations were measured using a P- 1010 2014). Much research effort is therefore being focussed on polarimeter (JASCO, Japan). Analytical gas chromatography identifying new antimicrobial compounds, including those (GC) was carried out on a Shimadzu GC-17A gas isolated from nature (Edwards and Harding, 2004; Kirst, chromatograph with an FID detector. Normal phase column 2013). Since the introduction of conventional antibiotics in chromatography was performed using silica gel 60 (0.040 the 1950's, there has been little use of plant derivatives as to 0.063 mm, Merck, Germany). Size exclusion antimicrobials. However, interest in using phytochemicals chromatography (SEC) was carried out using Sephadex LH- (products from secondary plant metabolism) for the 20 (18-111 µm, GE Healthcare Biosciences AB, Sweden). All treatment of microbial infections has increased from the chemical solvents used for extraction and chromatographic late 1990's following the poor efficacy of conventional separations were of analytical HPLC grade from Merck, antibiotics, due in part to their often excessive and Germany. Organic solvents were evaporated using a Buchi inappropriate use in mammalian infections (Cowan, 1999; rotary evaporator (Switzerland). 1H, 13C, HSQC, COSY, Fitzgerald‐Hughes et al., 2012; Khatri et al., 2016). HMBC and NOESY NMR spectra were recorded on Bruker The Chungtia villagers of Nagaland, North East India, Avance AMX 400 and Bruker DRX600K 600 MHz NMR have developed a wealth of knowledge on medicinal flora Spectrometers (Germany) using standard pulse sequences. over many generations (Kichu et al., 2015). In a recent The 1H and 13C chemical shifts were referenced relative to ethnobotanical study (Kichu et al., 2015), we documented the residual chloroform (1H δ 7.24 and 13C δ 77.2), acetone 37 medicinal plants used by Chungtia villagers for the (δH 2.04 and δc 205.8 and 30.6) and methanol (δH 3.31 and treatment of skin related ailments consistent with a δc 49.0) solvent peaks. A Shimadzu 2010 LC-MS system was microbial aetiology. In this study, we provide a literature used for electrospray ionisation mass spectrometry (ESI- review of these plants with a focus on the antimicrobial MS) analyses. A Shimadzu GC-17 system was used for properties of relevance to their applications. Six of these electron impact mass spectrometry (EI-MS) analyses. High plants were then analysed for the first time using resolution mass spectrometry (HR-MS) was determined antimicrobial screening methods against dermatological using a Bruker Apex 3 instrument and circular dichroism relevant microbes. The antimicrobial properties of fractions was measured on Jasco Circular Dichroism and compounds isolated from the roots of the most active Spectropolarimeter. Freeze drying was conducted with a species, Prunus persica, are also reported. CHRIST alpha 1-4 LDplus (Labconco, USA) freeze drier.

MATERIALS AND METHODS Plant material

Ethics Albizia lucidior (roots), Begonia picta (leaves), Cassia floribunda (leaves), Holboellia latifolia (leaves), Maesa This research was approved by the Human Research Ethics indica (leaves) and P. persica (roots) were collected from Committee at Macquarie University (Ref: HE22JUN2007- Chungtia village, Nagaland, India, by the villagers with the R05316 and Ref: 5200700334). It was governed by assistance of Mr. Anungba Jamir, a Chungtia Senso collaborative research agreements that followed the Mokokchung Town (CSMT) representative. The collections principles of the Convention of Biological Diversity (CBD) were done between the months of June and August, 2007 along with the stepwise participatory Action Research and 2009. All plant materials were thoroughly inspected by (PAR) methodology of UNESCO (Tuxill and Nabhan, 2001) Mr. Anungba Jamir for precise identification of the species and was conducted under the framework of best ethical and dried in the shade for 10 to 20 days. Voucher specimens practice, working in partnership with Indigenous people for each plant were deposited at the Botanical Survey of (NHMRC, 2003). India (BSI), Shillong Branch, India. For further processing, A. lucidior, B. picta, C. floribunda, M. indica, H. latifolia were transported to Chennai, India to Dr Velmurugan, while P. General persica was transported to Dr. Udaya Sankar of Central Food Technological Research Institute (CFTRI), located in Preparative TLC (PTLC) was carried out using Uniplate Mysore, India. Upon receipt by Dr. Velmurugan or Dr. preparative TLC plates (Sigma- Aldrich, Australia). Sankar, the plant materials were separated from foreign Analytical normal phase Thin Layer Chromatography (TLC) particles, washed with clean water, dried in the shade for was performed on fluorescent Merck silica gel F254 plates 24 h and then dried in a vacuum drier at 75 to 85°C. After (Germany). The TLC plates were visualized using UV light 48 h of drying under vacuum, the plants were kept in the Academia Journal of Biotechnology; Malewska et al. 146

shade for three days and rechecked for foreign particles. The were freeze dried overnight. plant materials were then chopped and passed through a micro pulveriser for grinding. The process was repeated until 130 to 200 mesh size was obtained. The powders were then Minimum inhibitory concentration (MIC) sieved and dried again under vacuum to ensure that they did not contain any moisture. The plant materials were allowed to The MTT microdilution assay was used to quantify the cool in the shade and packed into plastic bags, which in turn minimum inhibitory concentration (MIC) values of the plant were packed in plastic containers and sealed. The sealed extracts (Awouafack et al., 2013). The plant samples (10 containers were couriered to Macquarie University, Australia, mg/ml) or the antibiotic (1 mg/ml) were dissolved in 200 µl under the import permit IP12012991 from the Department of DMSO and diluted with MH II broth to a final volume of 1 ml Agriculture, Fisheries and Forestry (DAFF). for all bacterial strains, with the exception of S. pyogenes for which TH broth was used. SAB broth was used for C. albicans. Using a 96 well microtitre plate, 100 µl of suitable broth was Antimicrobial activity assays dispensed into wells 1 to 11 (from left to right) for each row, 100 µl of the samples or antibiotic was added to well 1 (in Culture preparation different rows for each sample) and thoroughly mixed, after which 100 µl was taken out and dispensed to the next well The use of all microbial strains was approved by the (that is, well 2). This process of two-fold serial dilution was Macquarie University Biosafety Committee (approval carried out until well 10, and skipping well 11, with the final references 05/14 LAB, TEM170512BHA). All cultures were volume dispensed into well 12. 100 µl each of the microbial provided by Dr. John Merlino (Department of Microbiology, inoculum was dispensed into wells 1 to 11 leaving well 12. Concord Hospital, Sydney). These included a susceptible strain Since well 11 was free of the test sample or the antibiotic, this of Staphylococcus aureus (ATCC 29213), community acquired acted as a positive control for the growth of the inoculum and methicillin resistant (MRSA) S. aureus (ATCC BAA 1026), wild well 12 being free of inoculum served as the sterile control of multidrug resistant (MDRSA) clinical isolate S. aureus, β the assay. 2% DMSO/H2O was also included as a negative lactamase negative – sensitive to common antibiotics E. coli control. The plate was incubated at 37°C for 18 to 20 h. 20 µl of (ATCC 25922), β lactamase positive, resistant to antibiotics E. MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazoliium coli (ATCC 35218), sensitive to common antibiotics P. bromide) solution in methanol (5 mg/ml) was added to each aeruginosa (ATCC 27853), clinical isolates Streptococcus well followed by incubation for 30 min. The MIC values were pyogenes and Salmonella typhimurium as well as, a clinical determined as the lowest concentration of the test sample or isolate Candida albicans. Stock cultures of the bacterial strains antibiotic that showed no visible colour change from yellow to were maintained in Mueller-Hinton II (MH II) broth containing blue. 10% v/v glycerol. The stock culture of the fungus was maintained in Sabouraud Dextrose broth (SAB) containing 10% v/v glycerol. Fresh subcultures were made by inoculating Disc diffusion method the bacterial cultures in MH II broth with the exception of S. pyogenes which was inoculated in Todd Hewitt (TH) broth and For the disc diffusion assay, freeze dried samples (10 mg) were the fungus in SAB broth, followed by an overnight incubation at dissolved in 200 µl DMSO, and then diluted to a final volume of 37°C (bacteria) and 30°C (fungus). For all antimicrobial assays, 1 ml in distilled water. Discs were loaded with the samples and the bacteria were grown overnight in MH II broth and C. albicans dried with a hair dryer to afford a sample concentration of 2 in SAB broth. After overnight incubation the optical density at mg per disc. MH II agar medium was prepared for all bacterial strains (except S. pyogenes) as per the manufacturer’s protocol 600 nm (OD600) was measured and the density adjusted to 0.08 with fresh MH II, TH or SAB broths, as appropriate. The and autoclaved at 121°C for 20 min. Potato dextrose agar was used for C. albicans and horse blood agar for S. pyogenes. The CFU/ml of used bacteria was 1.5 × 106 CFU/ml. Vancomycin and kanamycin (Amresco, USA) were used as positive controls molten media were poured into petri plates (10 to 15 ml) and for S. aureus strains (susceptible S. aureus, MRSA, MDRSA) and allowed to solidify. Using a sterile cotton swab, the diluted gentamycin was used for β-, β+ E. coli, P. aeruginosa and S. culture of the micro-organism was swabbed evenly on the typhimurium and S. pyogenes. Fluconazole was used as a entire surface of the appropriate agar plate to provide a lawn positive control for C. albicans. of microbes. Whatman discs (6 mm) impregnated with the samples were then placed on the inoculated plate and pressed down gently. The plates were incubated at 37°C for 18 h for

bacteria and at 30°C for 24 h for C. albicans. The diameter of the Aqueous ethanoic extracts preparation zones of inhibition was measured with a ruler (including Nagaland plant materials were prepared in Nagaland as earlier the 6 mm disc diameter). Negative control discs were described. Tested plant materials were suspended in 70% prepared with 20% DMSO/H2O. Positive control discs were aqueous ethanol, shaken overnight at room temperature and impregnated with appropriate antibiotics (2 µg per disc). vacuum filtered to collect the filtrates. The materials were re- Vancomycin and kanamycin were used as positive controls extracted as aforementionrd, twice and the combined filtrates for S. aureus strains (susceptible S. aureus, MRSA, MDRSA) were rotary evaporated (Büchi) at 37°C. The water residues and gentamycin was used for β-, β+ E. coli, P. aeruginosa, S. Academia Journal of Biotechnology; Malewska et al. 147

typhimurium and S. pyogenes. Fluconazole was used as a orange spots and steroids as red or blue spots (Wagner, positive control for C. albicans. 1996; Gibbons, 2005; Spangenberg, 2008; Waksmundzka- Hajnos et al., 2008).

TLC Bioautography analysis Extraction and isolation of compounds Rahalison et al. (1991) described the method used for TLC bioautography with modifications. In brief, developed TLC The EtOAc extract of P. persica (8 g) was dissolved in plates were placed face up on sterile petri dishes and methanol (10 ml), mixed with silica gel (5 g) and molten, warm, inoculated agar was rapidly distributed over evaporated to dryness by rotary evaporation at 37°C. The each of them. After solidification of the medium, the TLC sample was then applied to the top of a normal phase silica plates were incubated overnight and stained with MTT (3- column (270 g silica) prepared with 100% chloroform. The [4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium column was eluted with a gradient of chloroform: methanol bromide) dye. An inoculum of test bacteria (susceptible (100: 0 to 0: 100%). 120 fractions (20 ml each) were strain of S. aureus) was prepared in the broth by overnight collected, spotted in duplicate on TLC plates and developed incubation. After incubation, optical density of the culture with chloroform: methanol: water solvent system in was measured and adjusted to 0.16 by diluting with the different proportions (1:8:1 to 8:1:1). One of the TLC plates broth (1:50), thereafter, an equal volume of molten agar was then stained with vanillin-sulfuric acid reagent; the was added, resulting in a final dilution of 0.08 optical other was used for overlay TLC bioautography testing density. Approximately, 10 ml of the inoculum was rapidly against susceptible S. aureus and thereafter, combined into distributed over the TLC plates (10 × 10 cm). After three major sub-fractions according to similar TLC Rf solidification of the medium, the overlayed TLC plate was values (chloroform: methanol: water 7:3: 0.2 and 7: 3: 0.4) incubated overnight at 37°C. The bioautogram was then and TLC bioautography results to give EtOAc-1 [200 mg, gently covered with a methanolic solution (2.5 mg/ml) of yellow solid, Rf 0.75 to 0.9 (chloroform:methanol:water 7:3 MTT with a sterile micropipette, and then incubated for 5 :0.4), two spots on TLC plate, none active], EtOAc-2, [1.8 g, min at 37°C for the visualisation of results. orange solid, four spots of Rf 0.45, 0.60, 0.63 and 0.66

(chloroform:methanol:water 7:3:0.2), 2 inhibition zones

merged R 0.63-0.66] and EtOAc-3 [3.0 g, red solid, R Solvent-solvent fractionation of P. persica f f baseline-0.45 (chloroform:methanol:water 7:3:0.4), four The freeze dried crude aqueous ethanolic extract of P. spots on TLC plate, two active]. persica roots (25 g) was resuspended in water (900 ml) and EtOAc-2 (1.8 g) was dissolved in methanol, mixed with successively partitioned with n-hexane (300 ml), DCM (300 silica (1 g) and rotary evaporated at 37°C to dryness. The ml) and EtOAc (300 ml). The process was repeated thrice. solid was applied on top of a normal phase silica column Rotary evaporation of the combined extracts and freeze (200 g silica) prepared with 100% chloroform. Elution with drying afforded four partitions, that is, n-hexane (2 g, green an increasing polarity gradient of 100% chloroform: 100% solid), DCM (6 g, light red solid), EtOAc (8 g, dark red solid) methanol yielded 30 sub-fractions, which were spotted in and water (5 g, blackish red solid). duplicate on TLC plates and developed, one being visualised with vanillin-sulfuric acid reagent, the other tested by TLC bioautography. The fractions were combined based on their Phytochemical analysis TLC Rf values and bioautography results into two sub- fractions: EtOAc-2a [800 mg, red solid, two spots, Rf 0.63 to The n-hexane, DCM, EtOAc and water partitioned fractions 0.66 of merged antibacterial activity (chloroform: (10 mg/ml dissolved in methanol, 20 µl) were applied on methanol: water 7:3:0.2)] and EtOAc-2b [600 mg, red solid, TLC plates in duplicate and developed with petroleum three spots, Rf 0.63, 0.66 and 0.45, spots of Rf = 0.63, 0.66 ether:diethyl ether (7:3) for the n-hexane and DCM showed very faint inhibition zones and spot of partitions and chloroform: methanol: water eluent systems Rf 0.45 showed strong inhibition (chloroform: (7:3:0.4 and 7:3:0.2) for the EtOAc and water partitions, methanol: water 7: 3: 0.2)]. Crystallisation of EtOAc-2b with respectively. Chemical constituents were detected by methanol gave ent- epiafzelechin-(2α→O→7, 4α→8)-(-)-ent- visualisation under visible and ultraviolet light (365 and afzelechin (1) as a yellow solid (1 mg). The same compound 254 nm) and by spraying with vanillin-sulfuric reagent and was later isolated from the EtOAc-3 fraction (5 mg). heating of the plates at 100°C for 1 to 2 min. Vanillin- Sub-fraction EtOAc-2a (800 mg) was dissolved in sulfuric acid reagent was prepared by mixing 6 g vanillin methanol (0.25 ml) and subjected to size exclusion column with 2.5 ml concentrated H2SO4 in 250 ml ethanol. The chromatography (Sephadex LH-20), eluting with methanol. vanillin-sulfuric acid stain indicated the presence of This afforded three fractions, EtOAc-2a1 [220 mg, yellow terpenes as blue, red or violet spots, phenols as pink, red or solid, two spots, base line-Rf 0.35 with faint activity and Rf Academia Journal of Biotechnology; Malewska et al. 148

0.66, no activity ([chloroform: methanol: water 7: 3: 0.2)]. s, H-6’), 7.39 (2H, d, J 8.6 Hz, H-12’, H-16’), 6.89 (2H, d, J 8.6 This subfraction was further subjected to exclusion column 13 Hz, H-13’, H-15’), C NMR (150 MHz, CD3COCD3) δ 100.1 chromatography eluting chloroform: methanol mixture (C-2), 67.1 (C-3), 28.8 (C-4), 156.5 (C-5), 97.8 (C-6), 157.9 (1:1) to afford two fractions base line-Rf 0.35 and Rf 0.66. (C-7), 96.1 (C-8), 153.8 (C-9), 103.7 (C-10), 131.4 (C-11), NMR of the two fructions revealed the presence of more 129.3 (C-12), 115.2 (C-13), 158.5 (C-14), 115.2 (C-15), than one compound so the fractions were further purified 129.3 (C-16), 83.6 (C-2’), 67.7 (C-3’), 29.2 (C-4’), 155.5 (C- on sephadex column eluting with methanol to afford two 5’), 96.4 (C-6’), 151.8 (C-7’), 106.3 (C-8’), 150.6 (C-9’), compounds: Caffeic acid phenylethyl ester (3) and Gallic 102.7 (C-10’), 129.8 (C-11’), 129.8 (C-12’), 116.0 (C-13’), acid (4). EtOAc-2a2 [200 mg, white flecks, one spot, Rf 0.63, 158.5 (C-14’), 116.0 (C-15’), 129.8 (C-16’). no activity (chloroform: methanol: water 7: 3: 0.2)], and EtOAc-2a3 [150 mg, white solid, two spots, no activity, Rf 0.63-0.66 (chloroform: methanol: water 7: 3: 0.2)]. Sub- Afzelechin (2) fraction EtOAc-2a2 was subjected to preparative thin layer chromatography (PTLC) to provide afzelechin (2) [2 mg, Rf This is a creamish amorphous solid. UV (MeOH): λmax 273. 0.63, no antibacterial activity (chloroform: methanol: water ESI-MS m/z 273 [M+H]+, 1 7: 3: 0.2). H-NMR (400 MHz, CD3OD) δ 2.49 (1H, dd, J 8.5, 16.1 Hz, The n-hexane extract (1.5 g) was dissolved in minimal H-4), 2.87 (1H, dd, J 5.5, 16.1 Hz, H-4), 3.97 (1H, m, H-3), methanol (appx 1 ml) and subjected to size exclusion 4.58 (1H, d, J 8.0 Hz, H-2), 5.83 (1H, d, J 2.1 Hz, H-6), 5.91 column chromatography (Sephadex LH-20) eluting with (1H, d, J 2.1 Hz, H-8), 6.77 (2H, d, J 8.5 Hz, H-13, 15), 7.21 methanol. This afforded five fractions, Hex-1 (150 mg, (2H, d, J 8.5 Hz, H-12, 16). 13CNMR (75 MHz, CD3OD) δ yellow oil, three spots, Rf ranging from 0.30 to 0.58, one 82.77 (C-2), 68.87 (C-3), 29.15 (C-4), 157.88 (C-5), 95.48 active Rf 0.45 (petroleum ether:diethyl ether 3:7)], Hex-2 (C-6), 157.8 (C-7), 96.41 (C-8), 157.28 (C-9), 100.8 (C-10), [130 mg, white flecks, four spots, Rf 0.25 to 0.70, one active 131.5 (C-11), 129.58 (C-12), 116.21 (C-13), 156.86 (C-14), R 0.70 (petroleum ether: diethyl ether 3:7)], Hex-3 [60 mg, f 116.21 (C-15), 129.58 (C-16). beige solid, one spot, active, Rf 0.65 (petroleum ether: diethyl ether 3: 7)] and fractions Hex-4 and Hex-5 (15 mg and 8mg, yellow oils, three spot and four spots respectively, Gallic acid (3) Rf = 0.30 to 0.58 and 0.30 to 0.70, one active of Rf = 0.45 [petroleum ether: diethyl ether 3:7]). Separate purification This refers to white needles. ESI-MS m/z 169.1 [M-H]-, 1H- of Hex-1, Hex-4 and Hex-5 by Sephadex LH-20 column NMR (400 MHz, CD3COCD3) δ 6.46 (2H, s, H-1,5). 13C NMR chromatography, eluting with methanol, yielded α- (75 MHz, CD3COCD3) δ 167.90 (C-7), 145.85 (C-2,4), cyanobenzyl benzoate (3, 5, 1.5 and 1 mg, respectively). 138.43(C-3), 109.16 (C-1,5). Recrystallisation with methanol of fraction Hex-3 yielded β- sitosterol (4, 50 mg). Further purification of Hex-2 by Sephadex LH-20 column chromatography, eluting with Caffeic acic phenylethyl ester (4) methanol, yielded stigmast-4-en-3-one (5, 1 mg). These are creamish amorphous solids. ESI-MS m/z 283 [M- - Structure elucidation of isolated compounds H] . ent-Epiafzelechin-(2α→O→7’,4α→8’)-(-)-ent-afzelechin (1) α-Cyanobenzyl benzoate (5)

This refers to yellow oil. EI-MS m/z 51, 77, 105, 115, 237. decomposition at These are yellow crystals having mp. 1 238°C. UV (MeOH): λmax 273. IR (neat) υmax (cm-1): 3391 H-NMR (600 MHz, CD3OD) δ 6.71 (1H, s, H-7), 7.43 (5H, m, (br, strong), 2918, 2849, 1700, 1364, 835. CD (MeOH) J 16.2 Hz, H-1’, 2’, 3’, 4’, 5’), 7.58 (3H, m, J 17.4 Hz, H-2, 3, 4), 13 [θ]232 – 44.87 [α]D – 113.7º (MeOH). ESI-MS m/z 543 [M- 7.96 (2H, d, J 7.6 Hz, H-1, 5). C NMR (150 MHz, CD3OD) δ 64.8 (C-7), 117.6 (C-8), 128.8 (C-2, C-4), 129.6 (C-3’), H]-, HREI MS 567.1266 [M+Na] (calc. 567.1267), consistent 129.8 (C-5, C-1), 130.3 (C-2’, C-4’), 130.8 (C-1’, C-5’), 131.3 C H O . 1H-NMR (600 MHz, CD COCD ) with the formula 30 24 10 3 3 (C-6), 133.7 (C-3), 135.2 (C-6’), 165.9 (C-9). δ 4.22 (1H, d, J 3.5 Hz, H-3), 4.24 (1H, d, J 3.5 Hz, H-4), 5.91 (1H, d, J 2.2 Hz, H-6), 6.06 (1H, d, J 2.2 Hz, H-8), 7.53 (2H, d, J 8.6 Hz, H-12, H-16), 6.85 (2H, d, J 8.6 Hz, H-13, H-15), 4.81 β-Sitosterol (6) (1H, d, J 8.5 Hz, H-2’), 4.13 (1H, m, H-3’), 3.04 (1H, dd, J 5.5, 16.5 Hz, H-4’), 2.61 (1H, dd, J 5.5, 16.5 Hz, H-4’), 6.13 (1H, These are white needle-like crystals having melting point of Academia Journal of Biotechnology; Malewska et al. 149

138°C (lit. 136-139°C) (Kircher and Rosenstein, 1973). IR injector and detector were both programmed at 220°C. GC (neat) υmax (cm-1): 3400 (br strong), 2955, 2918, 2850, integrations were performed on a SMAD electronic 1735, 1463, 1377, 1057. EI-MS m/z 55, 57, 81, 95, 105, integrator. GC- MS was carried out on a Shimadzu GCMS- 119, 133, 145, 159, 173, 185, 199, 213, 231, 241, 255, 273, QP5000 mass spectrometer operating at 70 eV ionisation 288, 303, 315, 329, 341, 354, 367, 381, 396, 414, energy. Mass spectra were recorded in electron impact (EI) fragmentation pattern identical with the authentic sample mode at 70 eV, scanning the 41 to 450 m/z range. (Sigma-Aldrich), HREI-MS m/z 414.3855 (calc. 414.3862 for Compounds were identified by matching GC retention C H O). 1H- NMR (600 MHz CDCl ) δ 0.65 (3H, s, H-18), indices relative to n-alkanes and by comparison of their 29 50 3 mass spectra with either known compounds or published 0.79 (3H, d, J 8.6 Hz, H-26), 0.81 (3H, d, J 8.6 Hz, H-19), 0.83 spectra. (3H, d, J 8.6 Hz, H-29) 0.89 (3H, d, J 6.5 Hz, H-21), 0.91 (1H, m, H-9), 0.91 (1H, m, H-24), 0.94 (1H, m, H-17), 0.94 (1H, m, H-14), 0.98 (3H, s, H-27), 1.13 (2H, m, H-23), 1.13 (2H, RESULTS AND DISCUSSION m, H-1), 1.24 (2H, m, H-28), 1.29 (2H, m, H-22), 1.33 (1H, m,

H-20), 1.47 (2H, m, H-11), 1.49 (2H, m, H-7), 1.55 (2H, m, H- Selection of plants for antimicrobial testing 15) 1.64 (1H, m, H-25), 1.81 (2H, m, H-2), 1.97 (2H, m, H-

12), 1.97 (H, m, H-8), 2.21 (1H, m, H-4), 2.27 (1H, m, H-4), We have previously reported the customary uses by (1H, d, J = 5.3 Hz, H-6). 13C NMR (150 3.50 (1H, m, H-3), 5.33 Chungtia villagers of 38 plants for the topical treatment of MHz, CDCl3) δ 37.22 (C-1), 31.64 (C-2), 71.80 (C- 3), 42.27 skin related ailments of a likely microbial aetiology (Kichu (C-4), 140.73 (C-5), 121.72 (C-6), 31.87 (C-7), 31.89 (C-8), et al., 2015). Following an extensive literature review on 50.09 (C-9), 36.48 (C-10), 21.06 (C-11), 39.74 (C-12), 42.30 the antimicrobial activities and antimicrobially active (C-13), 56.74 (C-14), 24.28 (C-15), 28.23 (C-16), 56.01 (C- extracts/chemical constituents of these plants (Table 1), it 17), 11.84 (C-18), 19.81 (C-19), 36.13 (C-20), 18.76 (C-21), was found that extracts from 21 of the plants, including 33.91 (C-22), 26.01 (C-23), 45.79 (C-24), 29.10 (C-25), relevant plant parts used by the Chungtia villagers, have 19.00 (C-26), 19.38 (C-27), 23.03 (C-28), 11.96 (C-29). previously been analysed for their antimicrobial properties, 12 were reported for possessing antimicrobial activity in a different plant part and compounds with antimicrobial Stigmast-4-en-3-one (7) activities have been isolated from fourteen of them. These findings strongly support the customary uses of these White solid, EI-MS m/z 55, 95, 107, 124, 135, 147, 207, plants by the Chungtia villagers. Five Chungtia plants, 1 229, 271, 288, 327, 370, 397, 412; H-NMR (600 MHz, namely A. lucidior, B. picta, C. floribunda, H. latifolia and M. CD3COCD3) δ 075 (3H, m, H-18), 0.83 (3H, m, H-27), 0.84 indica have no prior studies on their antimicrobial (3H, m, H-26), 0.93 (1H, m, H-9), 0.96 (1H, m, H-24), 1.01 properties, while only fruit of P. persica have been (1H, m, H-7), 1.05 previously studied for antimicrobial activity, with no (1H, m, H-17), 1.15 (1H, m, H-14), 1.22 (3H, m, H-19), 1.27 reports on the roots used by the Chungtia villagers. These (1H, m, H-12), 1.48 (1H, m, H-11), 1.56 (1H, m, H-11), 1.65 six plants were therefore selected for antimicrobial studies. (1H, m, H-1), 1.68 (1H, m, H-25), 1.87 (1H, m, H-7), 2.02 (1H, m, H-1) 2.03 (1H, m, H-12), 2.18 (1H, m, H-2), 2.26 (1H, m, H-6), 2.40 (1H, m, H-2), 2.45 (1H, m, H-6), 5.62 (1H, Antimicrobial activity of selected plants 13 s, H-4). C NMR (150 MHz, CD3COCD3) δ 198.6 (C-3), 171.5 (C-5), 124.2 (C-4), 56.6 (C-14), 56.1 (C-17), 54.9 (C-9), 46.7 The 70% aqueous ethanolic extracts of A. lucidior (roots), B. (C-24), 43.2 (C-13), 40.6 (C-12), 39.3 (C-10), 37.0 (C-20), picta (leaves), C. floribunda (leaves), H. latifolia (leaves), M. 36.4 (C-22), 36.1 (C-1), 34.7 (C-8), 34.6 (C-2), 33.4 (C-6), indica (leaves) and P. persica (roots) were analysed by disc 33.0 (C-7), 29.0 (C-16), 29.0 (C-25), 26.8 (C-23), 24.8 (C-28), diffusion assay against susceptible and resistant strains of 21.8 (C-11), 20.1 (C-26), 19.4 (C-27), 19.2 (C-21), 17.6 (C- S. aureus and E. coli. Susceptible strains of P. aeruginosa, S. 19), 12.3 (C-18), 12.3 (C-29). typhimurium and S. pyogenes as well as, fungal strain of C. All spectral data were in agreement with the published albicans using 2 mg of extract per disc and a microbial results. density of 1.5 × 106 CFU/ml. This is consistent with the ideal concentration of crude plant material for the disc diffusion assay is between 1 to 5 mg per disc and the recommended

GS-MS analysis microbial density being 1.5 × 106 CFU/ml (Jeevan et al., 2004; Kuete et al., 2008). For interpretation of the results, Analytical gas chromatography (GC) was carried out on a zones of inhibition larger than 15 mm in diameter Shimadzu GC17A gas chromatograph with a BP-5 column (including the 6 mm disc) were considered as having high (30 m × 0.25 mm ×25 μm) that was programmed from 35 to activity, between 10 and 15 mm as having moderate 250°C at 3°C/min with helium as the carrier gas. The activity and less than 10 mm as having weak activity. Academia Journal of Biotechnology; Malewska et al. 150

Table 1: Reported antimicrobial activities and antimicrobially active extracts/chemical constituents from plants used by Chungtia villagers for skin related conditions of possible microbial aetiology.

Plant and parts used by Chungtia Antimicrobial activity (Plant part: microbe against which active) Antimicrobial compounds villagers Reported active Reported not active (Plant part: compound:microbe) 1 Albizia lucidior, Roots None found None found None found Bulbs S. aureus ; Whole plant : S. aureus (Yu et al., 2013; 2 Allium chinense, Bulbs None found No active compounds isolated Lanzotti et al., 2014) WR Leaves: S. aureus, MRSA, E. coli, S. typhimurium, P. aeruginosa, P. mirabilis, B. subtilis, B. cereus, L. monocytogenes, S. enterica, E. faecalis, A. flavus (Thapa et Fruit: cycloartenone: E. coli, S. aureus, K. pneumoniae, al., 2016) (Baliga et al., 2011); seeds: S. aureus, E. coli, P. A. niger, A. flavus (Jain et al., 2001), cycloartenol: E. coli, aeruginosa, B. megaterium, B. subtilis, B. cereus, M. luteus, S. P. aeruginosa (Ragasa et al., 2004; Momo et al., 2011), epidermitis, S. faecalis, S. pneumoniae, C. freundii, E. artocarpesin: S. aureus, E. coli, P. aeruginosa, S. typhi aerogenes, K. pneumoniae, N. gonorrhoeae, P. mirabilis, P. (Kuete et al., 2009; Manuel et al., 2012); wood: vulgaris, S. marcescens (Nair et al., 2013); Stem, root heart- cycloartocarpin: S. aureus, E. coli, P. aeruginosa, S. Artocarpus heterophyllus, Sap 3 wood, leaves, fruits, seeds: P. aeruginosa, E. coli, S. aureus, None found pyogenes (Septama and Panichayupakaranant, 2015), 69596 C. albicans, S. typhymurium, S. typhi, S. pyogenes, B. cereus, artocarpin: S. aureus, MRSA, E.coli, P. aeruginosa (Dej- B. coagulans,B. megaterium, B. subtilis, M. luteus, M. roseus, adisai et al., 2014; Septama, 2016), artocarpanone: S. S. albus, S. epidermidis, S. faecalis, S. pneumoniae, C. aureus (Dej-adisai et al,. 2014), cyanomaclurin: S. freundii, E. aerogenes, K. pneumoniae, N. gonorrhoeae, P. aureus, E. coli, S. pyogenes (Septama and mirabilis, P. vulgaris, S. marcences (Khan et al., 2003, Baliga Panichayupakaranant, 2015) artocarpin: MRSA, E.coli, et al., 2011, Septama and Panichayupakaranant 2015); P. aeruginosa (Septama, 2016) whole plant: MRSA, E.coli, P. aeruginosa (Septama, 2016); seeds: E. coli, S. typhi (Shah et al., 2017) Leaves, seeds: S. aureus, E. coli, B. subtilis (Lentz et al., Asclepias curassavica, Leaves Leaves, seeds: P. aeruginosa (Lentz et al., 1998); fruit: S. 1998); aerial parts: S. 4 No active compounds isolated 69617 aureus (Sundararajan and Koduru, 2014) aureus, E. coli, P. aeruginosa (Facey et al., 1999, Sundararajan and Koduru, 2014); aerial parts: S. aureus, S. pyogenes, C. albicans (Neto et al., 2002, Kavitha and Satish, 2016); roots: S.

aureus, P. aeruginosa, C. albicans (Kurdekar et al. , 2012) 5 Begonia picta, Leaves 69642 None found None found None found

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Table 1 Contd: Reported antimicrobial activities and antimicrobially active extracts/chemical constituents from plants used by Chungtia villagers for skin related conditions of possible microbial aetiology.

Plant and parts used by Chungtia Antimicrobial activity (Plant part: microbe against which active) Antimicrobial compounds villagers Reported active Reported not active (Plant part: compound:microbe) WR Flowers: S. aureus, E. coli, P. aeruginosa, S. Flowers: anhydrosophoradiol-3- acetate: S. aureus, E. typhimurium, B. subtilis, B. megaterium, S. lutea, S. sonnei, S. Roots, leaves: S. aureus, E. coli, S. typhi, S. typhimurium, P. aeruginosa, B. subtilis, S. Calotropis gigantea, Leaves shiga, S. dysenteriae, C. albicans (Habib and Karim 2009, coli, P. aeruginosa 6 lutea, S. sonnei, S. shiga, S. dysenteriae, A. flavus, di-(2- 69691 Chand et al. 2016); stem, roots, leaves, seed: S. aureus, E. (Rajakaruna et al., 2002); ethylhexyl)phthalate: S. aureus, E. coli, S. lutea (Habib coli, S. typhimurium, S. typhi, V. cholera, P. aeruginosa flowers: B. megaterium and Karim 2009, Zangeneh et al., 2017) (Radhakrishnan et al., 2013; Chand et al., 2016) 7 Cassia floribunda, Leaves 69535 None found None found None found Whole plant : K. Flowers, seeds: S. aureus, C. albicans, B. subtilis (Yun et al. pneumoniae, S. aureus, E. 8 Celosia cristata, Leaves 69520 2008); aerial parts: S. aureus, E. coli (Akhtar et al. 2015); coli, S. typhi, P aeruginosa, No active compounds isolated whole plant: B. subtilis (Bazzaz and Haririzadeh 2003) C. albicans (Bazzaz and Haririzadeh, 2003) Seeds: Gallic acid: S. aureus. E. coli, P. aeruginosa, S. WR Flowers: S. aureus, E. coli, B. subtilis, P. vulgaris, typhi, C. albicans (Chanwitheesuk et al., 2007, He et al., 2016), caffeic acid : P. aeruginosa, E. coli (Aziz et al., Flowers: S. aureus, E. coli, Chrysanthemum indicum, Leaves S. typhi, K. pneumoniae, E. faecalis, P. mirabilis, Candida sp 1998, Rauha et al., 2000), Luteolin-7-O-β-D-glucoside: 9 Candida sp (Shunying et 69529 (Shunying et al., 2005); aerial parts: S. aureus, S. pyogenes, S. aureus: (Musa et al., 2011), α- pinene, Camphor, 1,8- al., 2005) E. coli, S. epidermidis, S. mutans, S. anginosus, S. gordoniiP. Cineole, Terpinen-4-ol, Borneol, β- Caryophyllene: S. gingivalis (Jung, 2009) aureus, S. pyogenes, E. coli, S. epidermidis, S. mutans, S. anginosus, S. gordonii, P. gingivalis (Jung, 2009) WR Tuber: E. coli, S. pyogenes, P. vulgaris, P. mirabilis (Raja et al. 2011); whole plant: E. coli, P. aeruginosa, S. aureus, S. Tuber: S. aureus, S. typhi 10 Cyclea peltata, Leaves 69650 No active compounds isolated pyogenes, K. pneumoniae, P. vulgaris (Abraham and (Raja et al., 2011) Thomas, 2012; Singh and Nishteswar, 2013) Leaves: S. aureus, B. Leaves: E. coli, P. aeruginosa, E. aerogenes (Tanti et al., Dendrocnide sinuata, Stem subtilis, M. luteus, C. 11 2010); roots: S. aureus, E. coli, B. subtilis, S. typhi (Subba et No active compounds isolated 69508 albicans (Tanti et al., al., 2016) 2010) Aerial parts: cerebroside: S. aureus, E. coli, M. Drymaria cordata, Whole plant Aerial parts: S. aureus, E. coli, P. aeruginosa, B. subtili, B. tuberculosis, C. albicans (Cateni et al., 2003); 12 pumilis (Mukherjee et al., 1997; Pulok et al., 1997; Nono et None found monogalactosyldiacylglycerol: S. aureus, MRSA, E. coli, 69530 al., 2014) S. enterica, E. faecalis, M. tuberculosis (Martins et al., 2011) Whole plant: S. aureus, E. coli, S. epidermidis, B. cereus, B. Duabanga grandiflora, Stem subtilis (Othman et al., 2011); leaves: MRSA (Santiago et None found No active compounds isolated bark 69686 13 al., 2015)

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Plant and parts used by Chungtia Antimicrobial activity (Plant part: microbe against which active) Antimicrobial compounds villagers Reported active Reported not active (Plant part: compound:microbe) Aerial parts: essential oils - 1,8- cineole, R-(+)-α- Aerial parts, leaves: MRSA (Zuo et al. 2008, Abdallah and Whole plant: E. coli (Li et phellandrene, bornyl acetate, camphene, linalool, α- 14 Elsholtzia blanda, Leaves 69524 Abdallah 2016); Whole plant: S. aureus al., 2008) terpinene, α-pinene: S. aureus, E. coli (Fang et al., 1990, Guo et al., 2012) Roots: maackiaflavanone B, alpinumisoflavone, lupalbigenin, cristacarpin, erystagallin A, erythrabyssin: S. aureus, E. coli, P. aeruginosa Bark: S. aureus, MDRSA, MRSA, C. albicans, E.coli (Akter et Erythrina stricta, Stem bark (Rukachaisirikul et al., 2007; Hussain et al., 2011); 15 al., 2016); S. aureus (Sharma, 2013); stem bark: S. aureus, 69629 whole plant: ethanol extract (Sharma 2013); bark: E. coli, P. aeruginosa, C. albicans (Hussain et al., 2011) erynone, wighteone, alpinum isoflavone,, luteone, obovatin, erythrinassinate, isovanillin: S. aureus, MRSA, MDRSA (Akter et al., 2016) WR Whole plant: MRSA, S. aureus (Chomnawang et al., Eupatorium odoratum, Leaves Whole plant: S. aureus 16 2009); leaves: S. aureus, E. coli, C. albicans (Inya-Agha et No active compounds isolated 69523 (Chomnawang et al., 2009) al., 1987; Maity et al., 2010; Sobrinho et al., 2017) Whole plant: S. aureus, C. albicans (Shi et al., 2008); Whole plant: ent-11-hydroxyabieta- 8(14),13(15)- rhizomes: S. aureus, E. coli, K. pneumoniae, B. subtilis, P. 17 Euphorbia royleana, Sap 69684 None found dien-16,12R-olide, helioscopinolide A, helioscopinolide aeruginosa, S. marcenses, S. typhi, Shigella sp (Pratush et B: S. aureus (Shi et al. 2008, Wang et al., 2016) al., 2013) Leaves, stem: E. coli, S. 18 Eurya acuminata, Leaves 69612 Leaves, stem: S. aureus (Grosvenor et al., 1995) cerevisiae, F. oxysporum No active compounds isolated (Grosvenor et al., 1995) Leaves: emodin: S. aureus, E. coli (Coopoosamy and Magwa 2006), morin: S. aureus, MRSA, E. coli, P. aeruginosa, B. cereus (Kopacz et al. 2005, Panhwar and Memon 2011, Amin et al. 2015), rutin: S. aureus, E, Leaves: P. aeruginosa, B. cereus (Almahy et al., 2003); bark Leaves: S. aureus (Parekh coli, S. typhimurium, MRSA, P. aeruginosa, B. cereus 19 Ficus elastica, roots 69619 of aerial roots: S. aureus, E. coli (Mbosso et al., 2012) and Chanda, 2008) (Almahyl et al. 2003, Lou et al. 2010, Amin et al. 2015); bark of aerial roots: ficusamide: E. coli, S. aureus (Dongfack et al. 2012, Mbosso et al., 2012; Teinkela et al., 2017), elasticoside: E. coli, S. aureus (Mbosso et al., 2012) WR Fruits: S. aureus, P. aeruginosa, B. subtilis (Nayak et al., 2012); Roots: S. aureus, E. coli, P. aeruginosa, E. faecalis, A. baumiannii, C. freundii, E. aerugenes, K. pneumoniae, P. 20 Gmelina arborea, Drupe 69518 mirabilis (Mishra and Padhy, 2013); leaves, stem bark: E. None found No active compounds isolated coli, K. pneumoniae, P. mirabilis, S. dysenteriae, S. typhi (El- Mahmood et al., 2010); Bark: E. coli (Chugh and Bharti, 2012) Academia Journal of Biotechnology; Malewska et al. 153

Plant and parts used by Chungtia Antimicrobial activity (Plant part: microbe against which active) Antimicrobial compounds villagers Reported active Reported not active (Plant part: compound:microbe) Hedyotis scandens, Leaves Stem: S. aureus, E. coli, K. pneumoniae, P. vulgaris (Subba Leaves: Rosmarinic acid: S. aureus, P.aeruginosa 21 None found 69539 and Basnet, 2014) (Abedini et al., 2013,; Amoah et al., 2016) Holboellia latifolia, Leaves 22 None found None found None found 69500 Ipomoea nil, Flowers and leaves Whole plant, seeds, leaves: S. aureus, E. coli, B. pumilus, S. Leaves: hydroxybenzoic acid: S. aureus, E. coli, P. 23 pneumoniae, C. freundii, K. pneumoniae, A. niger, C. albicans None found aeruginosa, S. typhimurium, C. albicans (Cho et al., 69678 (Hussain et al., 2014; Isaka et al., 2017) 1998) WR Leaves: S. aureus, E. Leaves: Quercetin: E. coli (Vaquero et al., 2007, Nitiema coli, P. aeruginosa, C. WR Leaves: S. aureus, E. coli, P. aeruginosa, C. albicans (El et al., 2012), rutin: E. coli (Vaquero et al., 2007); aerial albicans (Wiart et al., Abdellaoui et al. 2010, de Souza et al. 2016); whole plant: parts: caffeic acid, p-coumaric acid: E. coli, S. aureus, Kalanchoe pinnata, Leaves 2004); whole plant: S. 24 P. aeruginosa, (Muthuvelan and Raja 2008); leaves, stem: protocatechuic acid: E. coli (Herald and Davidson 69515 aureus, E. coli, S. S. aureus, E. coli, P. aeruginosa, B. subtilis,S. dysenteriae, V. 1983, Aziz et al., 1998, Verma, 2016), 4-hydroxy-3- pneumoniae, B. cereus cholerae (Biswas et al., 2011) methoxycinnamic acid: E. coli, S. aureus (Herald and (Muthuvelan and Balaji, Davidson, 1983) 2008) WR Peel and mesocarp: S. aureus, E. coli, P.aeruginosa, B. subtilis, C. albicans (Dar et al., 2014); seeds: S. aureus, P. aeruginosa, E. coli, S. pyogenes, B. megaterium, E. Lagenaria siceraria, Leaves aerogenes, S. typhi (Chaudhery et al., 2014); leaves: S. WR Fruits: E. coli; leaves: Seeds: palmitic acid: S. aureus, S. typhi (Chaudhery et 25 69521 aureus. E. coli, P. aeruginosa, C. albicans (Badmanaban, E. coli (Goji et al., 2006) al., 2014) 2010; Nagaraja et al., 2011; Roopan et al., 2016); Fruits: P. aeruginosa, S. pyogenes, S. aureus (Nagaraja et al., 2011; Roopan et al., 2016; Dash and Ghosh, 2017) Leaves: S. aureus, E. coli, P. aeruginosa, B. cereus, B. Lasia spinosa, Leaves and stem subtilis, S. lutea, S. paratyphi, S. typhi, S. boydii, S. 26 No active compounds isolated 69655 dysenteriae, V. mimicus, V. parahemolyticus, C. albicans, A. niger (Goshwami et al., 2013) 27 Maesa indica, Leaves 69514 None found None found None found Flowers: ursolic acid: S. aureus, B. cereus, B. subtilis, S. typhi; 2α- hydroxyursolic acid: S. aureus, P. aeruginosa, B. cereus, asiatic acid: S. aureus, B. cereus, K. pneumoniae, β-sitosterol 3-O-β-D- glucopyranoside: S. Leaves: S. aureus, P. aeruginosa (Anbu et al., 2008); aureus, P. aeruginosa, B. cereus, S. typhi, Kaempferol: S. Leaves and flowers: S. aureus, E. coli, P. aeruginosa, B. Whole plant: E. coli, K. Melastoma malabathricum Fruits aureus, E. coli, B. cereus; kaempferol 3-O-α-L- 28 cereus, B. subtilis, S. typhi, K. pneumoniae (Wong et al., pneumoniae (Alnajar et al., and leaves 69652 rhamnopyranoside: S. aureus, B. cereus, K. pneumoniae; 2012); Whole plant: S. aureus, S. agalactiae (Alnajar et al., 2012) kaempferol 3-O-(2”,6”-di-O-E-p-coumaryl)-β- D- 2012) galactopyranoside: S. aureus, quercetin: S. aureus, E. coli, P. aeruginosa, B. subtilis, B. cereus, S. typhi, K. pneumoniae; ellagic acid: S. aureus, B. subtilis, P. aeruginosa (Wong et al., 2012) Academia Journal of Biotechnology; Malewska et al. 154

Plant and parts used by Chungtia Antimicrobial activity (Plant part: microbe against which active) Antimicrobial compounds villagers Reported active Reported not active (Plant part: compound:microbe) Leaves: 5,6,4'-trihydroxy-7,8,3'- trimethoxyflavone, Leaves: P. aeruginosa, B. subtilis, C. albicans, A. niger Leaves: S. aureus and E. coli 29 Mentha cordifolia, Leaves 69683 piperitenone epoxide: P. aeruginosa (Ragasa et al., (Ragasa et al., 2001) (Ragasa et al., 2001) 2001) Mikania cordata, Leaves and Stem: S. aureus, E. coli, S. dysenteriae (Uddin et al. 2014); Leaves: S. aureus, S. epidermidis, S. pyogenes, P. aeruginosa, 30 stem None found No active compounds isolated E. coli, S. typhi, S. sonnie, C. albicans (Hamill et al., 2003, 69534 Nayeem et al., 2011) Leaves: S. aureus, E. coli, P. aeruginosa, B. cereus, B. megaterium, B. subtilis, S. lutea, S. paratyphi, S. typhi, S. Mussaenda roxburghii, Leaves Aerial parts: shanzhiol: S. aureus, E. coli (De Utpal et al., 31 boydii, S. dysenteriae, V. mimicus, V. parahemolyticus None found 69502 2012) (Hossain et al., 2013); aerial parts: S. aureus, E. coli (De Utpal et al., 2012) WR Stem bark: S. aureus, E. coli, P. aeruginosa, B. pumilis, S. Myrica esculenta, Fruits and epidermidis, A. niger, S. cerevisiae, C. albicans, (Mahato and 32 None found No active compounds isolated leaves 69522 Chaudhary, 2005, Agnihotri et al., 2012); leaves: S. aureus, ,E. coli, S. epidermidis (Bamola et al., 2008) Fronds, whole plant: E. coli, P. aeruginosa, S. typhimurium, P. mirabilis, E. aerogenes, K. pneumoniae, B. subtilis, B. Nephrolepis cordifolia, Tubers 33 cereus, S. faecalis Aerial parts: S. aureus, S. pneumoniae, E. None found No active compounds isolated 69623 faecalis, S. typhimurium, K. pneumoniae, S. flexneri, P. vulgaris (Rani et al,. 2010; El-Tantawy et al. , 2016) WR Leaves: S. aureus, E. coli, P. aeruginosa, K. pneumoniae, B. cereus, S. typhimurium, B. subtilis, S. epidermidis (Madduluri et al., 2013; Saxena et al., 2014), S. aureus, E. coli, P. aeruginosa, S. typhimurium, S. epidermidis, S. Leaves: E. coli, P. 34 Piper betel, Leaves 69697 subflava, B. cereus, B. subtilis, B. megaterium, M. flavus, S. aeruginosa (Saxena et al. No active compounds isolated faecalis, S. cremoris, S. agalactiae, P. mirabilis, P. vulgaris, P. 2014) morganii, A. faecalis, E. aerogenes, K. pneumoniae, C. freundii, C. tropicalis, C. albicans (Nair and Chanda, 2008, Row and Ho, 2009; Akter et al., 2014; Valle et al., 2016); WR Roots: S. aureus, E. coli, P. aeruginosa, B. subtilis, B. Leaves: Confertifolin: S. aureus, E. coli, P. aeruginosa, K. Polygonum hydropiper, Leaves megaterium, E. aerogenes, S. typhi, S. sonnei, C. albicans pneumoniae, S. epidermidis, B. subtilis, E. faecalis, P. 35 None found 69641 (Hasan et al. 2009); leaves: S. aureus, E. coli, P. aeruginosa vulgaris (Duraipandiyan et al., 2010; Waqas et al., (Duraipandiyan et al. 2010, Waqas et al. 2016) 2016) 36 Prunus persica, Roots 69503 Fruit: S. aureus, E. coli, C. albicans (Belhadj et al., 2016) None found No active compounds isolated

Academia Journal of Biotechnology; Malewska et al. 155

Plant and parts used by Chungtia Antimicrobial activity (Plant part: microbe against which active) Antimicrobial compounds villagers Reported active Reported not active (Plant part: compound:microbe) Stem bark, whole plant: S. aureus, E. coli, P. aeruginosa, C. albicans, S. typhimurium, B. cereus, B. megaterium, B. Stereospermum chelonoides, Stem bark: stereochenol A, stereochenol B, caffeic acid : 37 subtilis, S. lutea, S. paratyphi, S. typhi, S. boydii, S. None found Stem 69610 E. coli (Haque et al., 2006; Kaisa 2011) disenteriae, V. mimicus, V. parahemolyticus, A. niger, S. cerevacae (Haque et al., 2007; Panda et al. ,2016) Whole plant: MRSA (Chomnawang et al., 2009); roots: S. aureus, E. coli, P. aeruginosa, C. albicans (Chomnawang et Tagetes erecta, Whole plant 38 al., 2009; Chatterjee and Ali, 2010); leaves: S. aureus, E. No active compounds isolated 69670 coli, P. aeruginosa (Dasgupta et al., 2012); flowers: S. aureus, E. coli (Jain et al., 2012; Padalia and Chanda, 2016)

WR - Widely referenced. Bacteria: E. coli - Escherichia coli, P. aeruginosa - Pseudomonas aeruginosa, S. typhimurium - Salmonella typhimurium, S. aureus - Staphylococcus aureus, MRSA - methicillin resistant Staphylococcus aureus, MDRSA - multi drug resistant Staphylococcus aureus, S. pyogenes - Streptococcus pyogenes, Fungi: C. albicans - Candida albicans.

The P. persica extract showed very good activity against the susceptible S. aureus strain (MIC 156 uncommon (Tan and Lim, 2015), while the disc against all three strains of S. aureus. The other plant µg/ml) and moderate activity against the MRSA and diffusion assay is a commonly used method for the extracts were all inactive in the disc diffusion assay MDRSA strains (MIC 1250 µg/ml for both strains). antimicrobial screening of medicinal plants (Das et against all microbial strains. H. latifolia also moderately inhibited the growth of S. al., 2010; Soković et al., 2010; Raja et al., 2011) and The crude extracts of the six plants were also typhimurium with an MIC value of 625 µg/ml. M. the activity measured as the zone of inhibition is assayed against test microbes susceptible as well as, indica inhibited the growth of all S. aureus strains influenced by numerous factors including the size drug resistant strains of S. aureus and E. coli and drug (MICs 312 to 2500 µg/ml). This plant extract also and polarity of the compounds present (Valgas et susceptible strains of S. typhimurium, P. aeruginosa weakly inhibited S. typhimurium with an MIC of al., 2007). Moreover, Whatman filter paper discs, and S. pyogenes using the MTT microdilution assay. 2500 µg/ml. B. picta showed moderate activity which are commonly used and were utilised in this For interpretation of the results, partitions with MIC against the susceptible and MRSA S. aureus strains study, can also influence results (Valgas et al., values < 312 µg/ml were considered as having good with MIC values of 612 µg/ml and 1250 µg/ml, 2007). activity, between 612 and 1250 µg/ml as having respectively. A. lucidior was moderately active Paper discs are composed of cellulose, which moderate activity and > 1250 µg/ml as having poor against both susceptible and MRSA S. aureus with possesses many free hydroxyl groups which render activity (Rios and Recio, 2005). Unlike in the disc MIC values of 1250 µg/ml against both strains. C. the surface of the discs hydrophilic (Burgess et al., diffusion assays, all of the plants showed activity floribunda showed moderate to weak activity 1999). Therefore, polar compounds can adsorb to against the susceptible S. aureus strain as well as, against all the bacterial strains (MIC 612 to 2500 the surface of the discs and not diffuse into the the resistant MRSA and MDRSA strains (Table 2). P. µg/ml). None of the plant extracts exhibited activity medium. As a consequence, some polar compounds persica extract showed strongest inhibitory activity against C. albicans (Table 2), while all the tested that possess antimicrobial activity may not show a against the MRSA and MDRSA strains (MIC 156 plant extracts showed activity against at least two zone of inhibition in the disc diffusion assay (Valgas µg/ml), and its inhibition was more potent than that micro-organisms in the MTT microdilution assay, et al., 2007). Non-polar compounds would not be of the control antibiotic vancomycin (MIC 312 only the P. persica extract was active when tested influenced by the hydroxyls on the surface of the µg/ml for MRSA and MDRSA). using the disc diffusion method. The discrepancy in paper, but because of their hydrophobic nature may H. latifolia exhibited high antimicrobial potential results between these two methods is not not diffuse through the aqueous medium resulting Academia Journal of Biotechnology; Malewska et al. 156

Table 2: MTT microdilution assay results.

MIC (µg/ml) Plant name Yields (g) SA MRSA MDRSA ST E.C β- E.C β+ PA SP M. indica leaves 0.9 625 625 625 2500 na na na na H. latifolia leaves 0.5 156 1250 1250 625 na na na na P. persica roots 2.3 156 156 156 na na na na na B. picta leaves 1.5 612 1250 na na na na na na A. lucidior roots 2 1250 1250 na na na na na na C. floribunda leaves 2 2500 2500 2500 2500 1250 2500 1250 612 Kanamycin 156 nt nt nt nt nt nt nt Gentamycin nt nt nt 312 156 312 312 156 Vancomycin nt 312 312 nt nt nt nt nt

na - not active at 2500 µg/ml, nt - not tested. SA – Staphylococcus aureus, MRSA – methicillin resistant S. aureus, MDRSA – multi drug resistant S. aureus, ST – Salmonella typhimurium, E.C β- - Escherichia coli β lactamase negative, E.C β+ - Escherichia coli β lactamase positive, PA – Pseudomonas aeruginosa, SP – Streptococcus pyogenes. MIC values were determined as the wells with the lowest sample concentration that showed no change from yellow to dark blue colour after addition of MTT.

in false negatives (Tan and Lim, 2015). Large molecules species is for the roots, in a recent study (Belhadj et al., also often diffuse poorly. Thus, some antimicrobial 2016). compounds may not be identified using a disc diffusion The 70% aqueous ethanolic extract of P. persica was assay. On the other hand, the accuracy of the MTT resuspended in water and successively partitioned with n- microdilution assay can be compromised by samples that hexane, DCM and EtOAc. These partitions were then tested are coloured (such as plant extracts), redox active and/or using the MTT microdilution assay (Table 3). The EtOAc samples that are not soluble in the medium, which is fraction was found to be the most active with MIC values of predominantly aqueous. Although more toxic solvents such 312 µg/ml against all tested strains of S. aureus. The n- as methanol or acetone can be used for water- insoluble hexane partition MIC value was 625 µg/ml against the compounds (no more that 2% final concentration) (Tan and susceptible strain of S. aureus and 312 µg/ml against both Lim, 2015), DMSO is a popular alternative due to its drug resistant strains. The EtOAc and n-hexane extracts comparatively lower toxicity (Tan and Lim, 2015). were therefore deemed worthy of further analysis. None of Regardless of the solvent used, some of the compounds the partitions were active against the strains of E. coli, P. might still precipitate which will reduce interaction aeruginosa, S. typhimurium, S. pyogenes and C. albicans. between the sample tested and the bacteria and as a result Bioassay guided isolation of the n-hexane and EtOAc limit the sample activity (Tan and Lim, 2015). Therefore, a partitioned fractions were performed, using a combination combination of the disc diffusion assay with at least one of normal phase silica gel column chromatography, size other assay is often preferred for screening (Valgas et al., exclusion column chromatography (Sephadex LH-20) and 2007). recrystallization, along MTT microdilution and TLC bioautography assays against susceptible and resistant strains of S. aureus. This afforded ent- epiafzelechin- Isolation and antimicrobial activity of extracts and (2α→O→7’, 4α→8’)-(-)ent-afzelechin (1) and afzelechin (2) compounds of P. persica from the EtOAc partition and from the n-hexane fraction, α- cyanobenzyl benzoate (3), stigmast-4-en-3-one (4) and β- As the extract of the roots of P. persica demonstrated good sitosterol (5). activity in both the disc diffusion and MTT microdilution The antimicrobial activities of compounds 1-5 were assays, it was selected for further chemical analyses. The tested against susceptible as well as, resistant strains of S. Chungtia villagers consume the liquid from fresh roots of P. aureus and E. coli, susceptible strains of P. aeruginosa, S. persica soaked in water to treat typhoid and the seed typhimurium, S. pyogenes and the fungus C. albicans by the endosperm is eaten to treat dysentery and diarrhoea. The MTT microdilution assay. α-Cyanobenzyl benzoate (3) liquid from the roots and aqueous decoctions of the leaves showed excellent antimicrobial properties (MIC 78 µg/ml) are also used to treat skin related infections (Kichu et al., against all tested S. aureus strains, good activity against β 2015). Except for the roots, all plant parts from this species lac- E. coli (MIC 312 µg/ml) and moderate activity against P. have been reported for various pharmacological properties, aeruginosa (MIC 612 µg/ml). The antimicrobial properties such as antioxidant, anti-inflammatory activities and of this compound are reported here for the first time. hepato- and cardio-protective properties. To the best of our Stigmast-4-en-3-one (4) showed very good antimicrobial knowledge, the only antibacterial activity reported of this properties against susceptible and resistant S. aureus (MIC Academia Journal of Biotechnology; Malewska et al. 157

Table 3: Antimicrobial activity of P. persica partitions and compounds by MTT microdilution assay.

Minimum inhibitory concentration (MIC, µg/ml) and minimum bactericidal concentration (MBC, µg/ml) Fractions and SA MRSA MDRSA EC β- ST PA CA, SP, EC β+ compounds MIC MBC MIC MBC MIC MBC MIC MIC MIC MIC n-Hexane 625 625 312 625 312 312 na na na na 3 78 nt 78 nt 78 nt 312 na 625 na 4 2500 nt 2500 nt 2500 nt 2500 625 1250 na 5 156 nt 156 nt 156 nt 312 625 1250 na DCM 625 625 625 625 625 625 na na na na

EtOAc 312 625 312 625 312 625 na na na na 1 156 nt 312 nt 312 nt 2500 2500 2500 na 2 na nt na nt na nt na na na na Water 1250 na 1250 na 1250 na na na na na Vancomycin 156 nt 312 nt 312 nt nt nt nt nt Gentamycin nt nt nt nt nt nt 156* 156 156 156*, 312** Fluconazole nt nt nt nt nt nt nt nt nt 312***

MIC values were determined as the wells with the lowest concentration of the samples that displayed no yellow to blue change of the MTT colour. *MIC values for E. coli (β-, β+), P. aeruginosa and S. typhimurium. **MIC value for S. pyogenes. ***MIC value for C. albicans. NT: not tested; na: not active; DCM: dichloromethane; EtOAc: ethyl acetate; MeOH: methanol; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

156 µg/ml), good antimicrobial activity against antibiotic Phytochemical and GS-MS analyses of n-Hexane susceptible E. coli (MIC 312 µg/ml) and moderate extracts antimicrobial properties against P. aeruginosa (MIC 1250 µg/ml) and S. typhimurium (MIC 612 µg/ml). ent- Following its promising antibacterial activity, the n-hexane Epiafzelechin-(2α→O→7’,4α→8’)-(-)ent-afzelechin (1) was partition of P. persica was selected for further analysis to found to possess very good antibacterial activity against the identify components responsible for its bioactivity. p- susceptible (MIC 156 µg/ml) and resistant strains of S. Anisaldehyde treatment of the TLC chromatogram of the n- aureus (MIC = 312 µg/ml) and weak activity against the hexane partition indicated the possible presence of susceptible strains of E. coli, S. typhimurium and P. terpenes (Wagner, 1996). GC-MS analysis of the extract was aeruginosa. This compound was inactive against the done using DB-5 column. Eight phytochemicals were resistant strain of E. coli as well as, the antibiotic susceptible identified (Table 4 and Figure 1) by comparing their GC strains of S. pyogenes and C. albicans. This is the first report retention times relative to n-alkanes (C5-C26) and also by of compound 1 possessing antimicrobial properties against comparison of their mass spectra (m/z values) with either the antibiotic susceptible as well as MDR bacterial strains. known compounds or published spectra (Omata et al., Afzelechin (2) showed no antibacterial activity in the 1991; Gherman et al., 2000; Yff et al., 2002; Hayyan et al., MTT microdilution assay. Compound 2 is a structural sub- 2011). The major constituents identified were hexadec-1- unit of ent-epiafzelechin- (2α→O→7’,4α→8’)-(-)ent- ene, octadec-1-ene, palmitic acid, ethyl palmitate, linoleic afzelechin (1) and as such it is possible that either other acid, oleic acid, stearic acid and ethyl stearate. compounds were responsible for the activities and were According to the literature, palmitic acid (Kurtulmus et al., lost during purification process or this compound arises 2009), linoleic acid (Dilika et al., 2000) and oleic acid (Dilika from the degradation of 1 which could explain the loss in et al., 2000) possess good activity against S. aureus. The activity seen as fraction EtOAc-2 purified. All compounds antimicrobial activity of the n-hexane partition could were previously isolated and the spectral data was in therefore be at least partly ascribed to the presence of these agreement with the results previously reported (Kolodziej bioactive compounds. et al., 1991; Okimoto et al., 1996; Alexander‐ Lindo et al., 2004; Chaturvedula and Prakash, 2012). In the bioautography method clear zones were observed for Conclusions compounds 1, 3, 4 and 5 against susceptible as well as, resistant strain of S. aureus. Compound 2 was not active by the Antimicrobial screening of Chungtia medicinal plants used TLC bioautography assay against all the tested strains of S. customarily for skin related ailments, that is, A. lucidior aureus despite spots at its Rf value (0.63) showing activity for (roots), B. picta (leaves), C. floribunda (leaves), H. latifolia the partly purified compound 2 in the TLC bioautography (leaves), M. indica (leaves) and P. persica (roots) against assay. dermatologically relevant micro-organisms showed that all Academia Journal of Biotechnology; Malewska et al. 158

Table 4: GS-MS analysis of P. persica n-hexane partition using DB-5 column.

Compounds Retention time (min) m/z value References Hexadec-1-ene 15.433 224 (Oros and Simoneit 2001) Octadec-1-ene 17.800 252 (Oros and Simoneit, 2001) Palmitic acid 19.617 256 (Hayyan et al. 2011) Ethyl palmitate 19.933 284 (Politi et al. 2011) Linoleic acid 21.333 280 (Hayyan et al., 2011) Oleic acid 21.383 282 (Hayyan et al., 2011) Stearic acid 21.600 284 (Hayyan et al., 2011) Ethyl stearate 21.867 312 (Politi et al., 2011)

Figure 1: The structures of compounds 1-5.

of the plants possess various levels of antibacterial activity. and good activity of compound 4 against a range of This supports the customary use of these species. dermatologically relevant bacteria supports the traditional Investigation of the n-hexane and EtOAc fractions of the application of this plant to treat skin related ailments. This root extract of P. persica using bioassay-guided isolation is the first report of the isolated compounds 1 and 3 techniques led to the isolation of ent-epiafzelechin- possessing antimicrobial activities as well as, the first (2α→O→7’,4α→8’)-(-) ent-afzelechin (1, S. aureus MIC 156 report of ent-epiafzelechin-(2α→O→7’,4α→8’)-(-)ent- µg/ml, MRSA and MDRSA MIC 312 µg/ml, E. coli β-, S. afzelechin and α- cyanobenzyl benzoate being isolated from typhimurium and P. aeruginosa MIC 2500 µg/ml), genus Prunus. afzelechin (2, not active), α-cyanobenzyl benzoate (3, S. Many doubts still persist even today when it comes to aureus, MDRSA and MRSA 78 µg/ml, E. coli β- MIC 312 selection of the solvents for extracting the active µg/ml and P. aeruginosa MIC 625 µg/ml), β-sitosterol (4, S. constituents from various Indian medicinal plants. This aureus, MDRSA, MRSA and E. coli β- MIC 2500 µg/ml, S. study was aimed at assessing and establishing the best typhimurium MIC 625 µg/ml, P. aeruginosa MIC 1250 solvent for extracting the active constituents from ten (10) µg/ml) and stigmast-4-en-3-one (5, S. aureus, MDRSA and plant extracts. Thin layer chromatography (TLC) was used MRSA MIC 156 µg/ml, E. coli β- MIC 312, S. typhimurium MIC to separate and establish the active constituents present in 625 µg/ml and P. aeruginosa MIC 1250 µg/ml). Very good each of the medicinal plants. Active constituents from each antimicrobial activity of the isolated compounds 1 and 3 plant was extracted using three different solvent systems Academia Journal of Biotechnology; Malewska et al. 159

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