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

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INTRODUCTION
The presence of pathogenic bacteria and fungi can cause skin infections and exacerbate the healing and seriousness of sores and wounds (Edwards and Harding, 2004).Skin disease and infections cause a significant global disease burden and with the escalating occurrence of multidrug resistant micro-organisms, there is heightened concern that the rates of skin infections will only worsen (Hay et al., 2014).Much research effort is therefore being focussed on identifying new antimicrobial compounds, including those isolated from nature (Edwards and Harding, 2004;Kirst, 2013).Since the introduction of conventional antibiotics in the 1950's, there has been little use of plant derivatives as antimicrobials.However, interest in using phytochemicals (products from secondary plant metabolism) for the treatment of microbial infections has increased from the late 1990's following the poor efficacy of conventional antibiotics, due in part to their often excessive and inappropriate use in mammalian infections (Cowan, 1999;Fitzgerald-Hughes et al., 2012;Khatri et al., 2016).
The Chungtia villagers of Nagaland, North East India, have developed a wealth of knowledge on medicinal flora over many generations (Kichu et al., 2015).In a recent ethnobotanical study (Kichu et al., 2015), we documented 37 medicinal plants used by Chungtia villagers for the treatment of skin related ailments consistent with a microbial aetiology.In this study, we provide a literature review of these plants with a focus on the antimicrobial properties of relevance to their applications.Six of these plants were then analysed for the first time using antimicrobial screening methods against dermatological relevant microbes.The antimicrobial properties of fractions and compounds isolated from the roots of the most active species, Prunus persica, are also reported.

Ethics
This research was approved by the Human Research Ethics Committee at Macquarie University (Ref: HE22JUN2007-R05316 and Ref: 5200700334).It was governed by collaborative research agreements that followed the principles of the Convention of Biological Diversity (CBD) along with the stepwise participatory Action Research (PAR) methodology of UNESCO (Tuxill and Nabhan, 2001) and was conducted under the framework of best ethical practice, working in partnership with Indigenous people (NHMRC, 2003).

General
Preparative TLC (PTLC) was carried out using Uniplate preparative TLC plates (Sigma-Aldrich, Australia).Analytical normal phase Thin Layer Chromatography (TLC) was performed on fluorescent Merck silica gel F254 plates (Germany).The TLC plates were visualized using UV light (254 and 365 nm), respectively and vanillin-sulphuric acid spray reagent.UV-visible spectra were recorded on a CARY 1 Bio spectrophotometer (Varian, USA).IR spectra were recorded on a NICOLET iS10 (Thermo Scientific, USA).Optical rotations were measured using a P-1010 polarimeter (JASCO, Japan).Analytical gas chromatography (GC) was carried out on a Shimadzu GC-17A gas chromatograph with an FID detector.Normal phase column chromatography was performed using silica gel 60 (0.040 to 0.063 mm, Merck, Germany).Size exclusion chromatography (SEC) was carried out using Sephadex LH-20 GE Healthcare Biosciences AB,Sweden).All chemical solvents used for extraction and chromatographic separations were of analytical HPLC grade from Merck, Germany.Organic solvents were evaporated using a Buchi rotary evaporator (Switzerland). 1 H, 13 C, HSQC, COSY, HMBC and NOESY NMR spectra were recorded on Bruker Avance AMX 400 and Bruker DRX600K 600 MHz NMR Spectrometers (Germany) using standard pulse sequences.The 1 H and 13 C chemical shifts were referenced relative to the residual chloroform ( 1 H δ 7.24 and 13 C δ 77.2), acetone (δH 2.04 and δc 205.8 and 30.6) and methanol (δH 3.31 and δc 49.0) solvent peaks.A Shimadzu 2010 LC-MS system was used for electrospray ionisation mass spectrometry (ESI-MS) analyses.A Shimadzu GC-17 system was used for electron impact mass spectrometry (EI-MS) analyses.High resolution mass spectrometry (HR-MS) was determined using a Bruker Apex 3 instrument and circular dichroism was measured on Jasco Circular Dichroism Spectropolarimeter.Freeze drying was conducted with a CHRIST alpha 1-4 LDplus (Labconco, USA) freeze drier.

Plant material
Albizia lucidior (roots), Begonia picta (leaves), Cassia floribunda (leaves), Holboellia latifolia (leaves), Maesa indica (leaves) and P. persica (roots) were collected from Chungtia village, Nagaland, India, by the villagers with the assistance of Mr. Anungba Jamir, a Chungtia Senso Mokokchung Town (CSMT) representative.The collections were done between the months of June and August, 2007 and 2009.All plant materials were thoroughly inspected by Mr. Anungba Jamir for precise identification of the species and dried in the shade for 10 to 20 days.Voucher specimens for each plant were deposited at the Botanical Survey of 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. persica was transported to Dr. Udaya Sankar of Central Food Technological Research Institute (CFTRI), located in Mysore, India.Upon receipt by Dr. Velmurugan or Dr. Sankar, the plant materials were separated from foreign particles, washed with clean water, dried in the shade for 24 h and then dried in a vacuum drier at 75 to 85°C.After 48 h of drying under vacuum, the plants were kept in the shade for three days and rechecked for foreign particles.The 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 sieved and dried again under vacuum to ensure that they did not contain any moisture.The plant materials were allowed to cool in the shade and packed into plastic bags, which in turn were packed in plastic containers and sealed.The sealed containers were couriered to Macquarie University, Australia, under the import permit IP12012991 from the Department of Agriculture, Fisheries and Forestry (DAFF).

Culture preparation
The use of all microbial strains was approved by the Macquarie University Biosafety Committee (approval references 05/14 LAB, TEM170512BHA).All cultures were provided by Dr. John Merlino (Department of Microbiology, Concord Hospital, Sydney).These included a susceptible strain of Staphylococcus aureus (ATCC 29213), community acquired methicillin resistant (MRSA) S. aureus (ATCC BAA 1026), wild multidrug resistant (MDRSA) clinical isolate S. aureus, β lactamase negative -sensitive to common antibiotics E. coli (ATCC 25922), β lactamase positive, resistant to antibiotics E. coli (ATCC 35218), sensitive to common antibiotics P. aeruginosa (ATCC 27853), clinical isolates Streptococcus pyogenes and Salmonella typhimurium as well as, a clinical isolate Candida albicans.Stock cultures of the bacterial strains were maintained in Mueller-Hinton II (MH II) broth containing 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 the bacterial cultures in MH II broth with the exception of S. pyogenes which was inoculated in Todd Hewitt (TH) broth and the fungus in SAB broth, followed by an overnight incubation at 37°C (bacteria) and 30°C (fungus).For all antimicrobial assays, the bacteria were grown overnight in MH II broth and C. albicans in SAB broth.After overnight incubation the optical density at 600 nm (OD600) was measured and the density adjusted to 0.08 with fresh MH II, TH or SAB broths, as appropriate.The CFU/ml of used bacteria was 1.5 × 10 6 CFU/ml.Vancomycin and kanamycin (Amresco, USA) were used as positive controls for S. aureus strains (susceptible S. aureus, MRSA, MDRSA) and gentamycin was used for β-, β+ E. coli, P. aeruginosa and S. typhimurium and S. pyogenes.Fluconazole was used as a positive control for C. albicans.

Aqueous ethanoic extracts preparation
Nagaland plant materials were prepared in Nagaland as earlier described.Tested plant materials were suspended in 70% aqueous ethanol, shaken overnight at room temperature and vacuum filtered to collect the filtrates.The materials were reextracted as aforementionrd, twice and the combined filtrates were rotary evaporated (Büchi) at 37°C.The water residues were freeze dried overnight.

Minimum inhibitory concentration (MIC)
The MTT microdilution assay was used to quantify the minimum inhibitory concentration (MIC) values of the plant extracts (Awouafack et al., 2013).The plant samples (10 mg/ml) or the antibiotic (1 mg/ml) were dissolved in 200 µl DMSO and diluted with MH II broth to a final volume of 1 ml 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 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 different rows for each sample) and thoroughly mixed, after which 100 µl was taken out and dispensed to the next well (that is, well 2).This process of two-fold serial dilution was carried out until well 10, and skipping well 11, with the final volume dispensed into well 12. 100 µl each of the microbial inoculum was dispensed into wells 1 to 11 leaving well 12.Since well 11 was free of the test sample or the antibiotic, this acted as a positive control for the growth of the inoculum and well 12 being free of inoculum served as the sterile control of the assay.2% DMSO/H2O was also included as a negative control.The plate was incubated at 37°C for 18 to 20 h.20 µl of -2,5-diphenyltetrazoliium bromide) solution in methanol (5 mg/ml) was added to each well followed by incubation for 30 min.The MIC values were determined as the lowest concentration of the test sample or antibiotic that showed no visible colour change from yellow to blue.

Disc diffusion method
For the disc diffusion assay, freeze dried samples (10 mg) were dissolved in 200 µl DMSO, and then diluted to a final volume of 1 ml in distilled water.Discs were loaded with the samples and dried with a hair dryer to afford a sample concentration of 2 mg per disc.MH II agar medium was prepared for all bacterial strains (except S. pyogenes) as per the manufacturer's protocol and autoclaved at 121°C for 20 min.Potato dextrose agar was used for C. albicans and horse blood agar for S. pyogenes.The molten media were poured into petri plates (10 to 15 ml) and allowed to solidify.Using a sterile cotton swab, the diluted culture of the micro-organism was swabbed evenly on the entire surface of the appropriate agar plate to provide a lawn 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 zones of inhibition was measured with a ruler (including the 6 mm disc diameter).Negative control discs were prepared with 20% DMSO/H 2 O. Positive control discs were impregnated with appropriate antibiotics (2 µg per disc).Vancomycin and kanamycin were used as positive controls for S. aureus strains (susceptible S. aureus, MRSA, MDRSA) and gentamycin was used for β-, β+ E. coli, P. aeruginosa, S. typhimurium and S. pyogenes.Fluconazole was used as a positive control for C. albicans.Rahalison et al. (1991) described the method used for TLC bioautography with modifications.In brief, developed TLC plates were placed face up on sterile petri dishes and molten, warm, inoculated agar was rapidly distributed over each of them.After solidification of the medium, the TLC plates were incubated overnight and stained with MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) dye.An inoculum of test bacteria (susceptible strain of S. aureus) was prepared in the broth by overnight incubation.After incubation, optical density of the culture was measured and adjusted to 0.16 by diluting with the broth (1:50), thereafter, an equal volume of molten agar was added, resulting in a final dilution of 0.08 optical density.Approximately, 10 ml of the inoculum was rapidly distributed over the TLC plates (10 × 10 cm).After solidification of the medium, the overlayed TLC plate was incubated overnight at 37°C.The bioautogram was then gently covered with a methanolic solution (2.5 mg/ml) of MTT with a sterile micropipette, and then incubated for 5 min at 37°C for the visualisation of results.

Solvent-solvent fractionation of P. persica
The freeze dried crude aqueous ethanolic extract of P. persica roots (25 g) was resuspended in water (900 ml) and successively partitioned with n-hexane (300 ml), DCM (300 ml) and EtOAc (300 ml).The process was repeated thrice.Rotary evaporation of the combined extracts and freeze drying afforded four partitions, that is, n-hexane (2 g, green solid), DCM (6 g, light red solid), EtOAc (8 g, dark red solid) and water (5 g, blackish red solid).

Phytochemical analysis
The n-hexane, DCM, EtOAc and water partitioned fractions (10 mg/ml dissolved in methanol, 20 µl) were applied on TLC plates in duplicate and developed with petroleum ether:diethyl ether (7:3) for the n-hexane and DCM partitions and chloroform: methanol: water eluent systems (7:3:0.4and 7:3:0.2) for the EtOAc and water partitions, respectively.Chemical constituents were detected by visualisation under visible and ultraviolet light (365 and 254 nm) and by spraying with vanillin-sulfuric reagent and heating of the plates at 100°C for 1 to 2 min.Vanillinsulfuric acid reagent was prepared by mixing 6 g vanillin with 2.5 ml concentrated H 2 SO 4 in 250 ml ethanol.The vanillin-sulfuric acid stain indicated the presence of terpenes as blue, red or violet spots, phenols as pink, red or orange spots and steroids as red or blue spots (Wagner, 1996;Gibbons, 2005;Spangenberg, 2008;Waksmundzka-Hajnos et al., 2008).

GS-MS analysis
Analytical gas chromatography (GC) was carried out on a Shimadzu GC17A gas chromatograph with a BP-5 column (30 m × 0.25 mm ×25 μm) that was programmed from 35 to 250°C at 3°C/min with helium as the carrier gas.The injector and detector were both programmed at 220°C.GC integrations were performed on a SMAD electronic integrator.GC-MS was carried out on a Shimadzu GCMS-QP5000 mass spectrometer operating at 70 eV ionisation energy.Mass spectra were recorded in electron impact (EI) mode at 70 eV, scanning the 41 to 450 m/z range.Compounds were identified by matching GC retention indices relative to n-alkanes and by comparison of their mass spectra with either known compounds or published spectra.

Selection of plants for antimicrobial testing
We have previously reported the customary uses by Chungtia villagers of 38 plants for the topical treatment of skin related ailments of a likely microbial aetiology (Kichu et al., 2015).Following an extensive literature review on the antimicrobial activities and antimicrobially active extracts/chemical constituents of these plants (Table 1), it was found that extracts from 21 of the plants, including relevant plant parts used by the Chungtia villagers, have previously been analysed for their antimicrobial properties, 12 were reported for possessing antimicrobial activity in a different plant part and compounds with antimicrobial activities have been isolated from fourteen of them.These findings strongly support the customary uses of these plants by the Chungtia villagers.Five Chungtia plants, namely A. lucidior, B. picta, C. floribunda, H. latifolia and M. indica have no prior studies on their antimicrobial properties, while only fruit of P. persica have been previously studied for antimicrobial activity, with no reports on the roots used by the Chungtia villagers.These six plants were therefore selected for antimicrobial studies.
The crude extracts of the six plants were also assayed against test microbes susceptible as well as, drug resistant strains of S. aureus and E. coli and drug susceptible strains of S. typhimurium, P. aeruginosa and S. pyogenes using the MTT microdilution assay.For interpretation of the results, partitions with MIC values < 312 µg/ml were considered as having good activity, between 612 and 1250 µg/ml as having moderate activity and > 1250 µg/ml as having poor activity (Rios and Recio, 2005).Unlike in the disc diffusion assays, all of the plants showed activity against the susceptible S. aureus strain as well as, the resistant MRSA and MDRSA strains (Table 2).P. persica extract showed strongest inhibitory activity against the MRSA and MDRSA strains (MIC 156 µg/ml), and its inhibition was more potent than that of the control antibiotic vancomycin (MIC 312 µg/ml for MRSA and MDRSA).
H. latifolia exhibited high antimicrobial potential against the susceptible S. aureus strain (MIC 156 µg/ml) and moderate activity against the MRSA and MDRSA strains (MIC 1250 µg/ml for both strains).
H. latifolia also moderately inhibited the growth of S. typhimurium with an MIC value of 625 µg/ml.M. indica inhibited the growth of all S. aureus strains (MICs 312 to 2500 µg/ml).This plant extract also weakly inhibited S. typhimurium with an MIC of 2500 µg/ml.B. picta showed moderate activity against the susceptible and MRSA S. aureus strains with MIC values of 612 µg/ml and 1250 µg/ml, respectively.A. lucidior was moderately active against both susceptible and MRSA S. aureus with MIC values of 1250 µg/ml against both strains.C. floribunda showed moderate to weak activity against all the bacterial strains (MIC 612 to 2500 µg/ml).None of the plant extracts exhibited activity against C. albicans (Table 2), while all the tested plant extracts showed activity against at least two micro-organisms in the MTT microdilution assay, only the P. persica extract was active when tested using the disc diffusion method.The discrepancy in results between these two methods is not uncommon (Tan and Lim, 2015), while the disc diffusion assay is a commonly used method for the antimicrobial screening of medicinal plants (Das et al., 2010;Soković et al., 2010;Raja et al., 2011) and the activity measured as the zone of inhibition is influenced by numerous factors including the size and polarity of the compounds present (Valgas et al., 2007).Moreover, Whatman filter paper discs, which are commonly used and were utilised in this study, can also influence results (Valgas et al., 2007).
Paper discs are composed of cellulose, which possesses many free hydroxyl groups which render the surface of the discs hydrophilic (Burgess et al., 1999).Therefore, polar compounds can adsorb to the surface of the discs and not diffuse into the medium.As a consequence, some polar compounds that possess antimicrobial activity may not show a zone of inhibition in the disc diffusion assay (Valgas et al., 2007).Non-polar compounds would not be influenced by the hydroxyls on the surface of the paper, but because of their hydrophobic nature may not diffuse through the aqueous medium resulting in false negatives (Tan and Lim, 2015).Large molecules also often diffuse poorly.Thus, some antimicrobial compounds may not be identified using a disc diffusion assay.On the other hand, the accuracy of the MTT microdilution assay can be compromised by samples that are coloured (such as plant extracts), redox active and/or samples that are not soluble in the medium, which is predominantly aqueous.Although more toxic solvents such as methanol or acetone can be used for water-insoluble compounds (no more that 2% final concentration) (Tan and Lim, 2015), DMSO is a popular alternative due to its comparatively lower toxicity (Tan and Lim, 2015).
Regardless of the solvent used, some of the compounds might still precipitate which will reduce interaction between the sample tested and the bacteria and as a result limit the sample activity (Tan and Lim, 2015).Therefore, a combination of the disc diffusion assay with at least one other assay is often preferred for screening (Valgas et al., 2007).

Isolation and antimicrobial activity of extracts and compounds of P. persica
As the extract of the roots of P. persica demonstrated good activity in both the disc diffusion and MTT microdilution assays, it was selected for further chemical analyses.The Chungtia villagers consume the liquid from fresh roots of P. persica soaked in water to treat typhoid and the seed endosperm is eaten to treat dysentery and diarrhoea.The liquid from the roots and aqueous decoctions of the leaves are also used to treat skin related infections (Kichu et al., 2015).Except for the roots, all plant parts from this species have been reported for various pharmacological properties, such as antioxidant, anti-inflammatory activities and hepato-and cardio-protective properties.To the best of our knowledge, the only antibacterial activity reported of this species is for the roots, in a recent study (Belhadj et al., 2016).The 70% aqueous ethanolic extract of P. persica was resuspended in water and successively partitioned with nhexane, DCM and EtOAc.These partitions were then tested using the MTT microdilution assay (Table 3).The EtOAc fraction was found to be the most active with MIC values of 312 µg/ml against all tested strains of S. aureus.The nhexane partition MIC value was 625 µg/ml against the susceptible strain of S. aureus and 312 µg/ml against both drug resistant strains.The EtOAc and n-hexane extracts were therefore deemed worthy of further analysis.None of the partitions were active against the strains of E. coli, P. aeruginosa, S. typhimurium, S. pyogenes and C. albicans.
The antimicrobial activities of compounds 1-5 were tested against susceptible as well as, resistant strains of S. aureus and E. coli, susceptible strains of P. aeruginosa, S. typhimurium, S. pyogenes and the fungus C. albicans by the MTT microdilution assay.α-Cyanobenzyl benzoate (3) showed excellent antimicrobial properties (MIC 78 µg/ml) against all tested S. aureus strains, good activity against β lac-E.coli (MIC 312 µg/ml) and moderate activity against P. aeruginosa (MIC 612 µg/ml).The antimicrobial properties of this compound are reported here for the first time.Stigmast-4-en-3-one (4) showed very good antimicrobial properties against susceptible and resistant S. aureus (MIC 156 µg/ml), good antimicrobial activity against antibiotic susceptible E. coli (MIC 312 µg/ml) and moderate antimicrobial properties against P. aeruginosa (MIC 1250 µg/ml) and S. typhimurium (MIC 612 µg/ml).ent-Epiafzelechin-(2α→O→7',4α→8')-(-)ent-afzelechin (1) was found to possess very good antibacterial activity against the susceptible (MIC 156 µg/ml) and resistant strains of S. aureus (MIC = 312 µg/ml) and weak activity against the susceptible strains of E. coli, S. typhimurium and P. aeruginosa.This compound was inactive against the resistant strain of E. coli as well as, the antibiotic susceptible strains of S. pyogenes and C. albicans.This is the first report of compound 1 possessing antimicrobial properties against the antibiotic susceptible as well as MDR bacterial strains.Afzelechin (2) showed no antibacterial activity in the MTT microdilution assay.Compound 2 is a structural subunit of ent-epiafzelechin-(2α→O→7',4α→8')-(-)entafzelechin (1) and as such it is possible that either other compounds were responsible for the activities and were lost during purification process or this compound arises from the degradation of 1 which could explain the loss in activity seen as fraction EtOAc-2 purified.All compounds were previously isolated and the spectral data was in agreement with the results previously reported (Kolodziej 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 compounds 1, 3, 4 and 5 against susceptible as well as, resistant strain of S. aureus.Compound 2 was not active by the TLC bioautography assay against all the tested strains of S. aureus despite spots at its Rf value (0.63) showing activity for the partly purified compound 2 in the TLC bioautography assay.

Phytochemical and GS-MS analyses of n-Hexane extracts
Following its promising antibacterial activity, the n-hexane partition of P. persica was selected for further analysis to identify components responsible for its bioactivity.p-Anisaldehyde treatment of the TLC chromatogram of the nhexane partition indicated the possible presence of terpenes (Wagner, 1996).GC-MS analysis of the extract was done using DB-5 column.Eight phytochemicals were identified (Table 4 and Figure 1) by comparing their GC retention times relative to n-alkanes (C5-C26) and also by comparison of their mass spectra (m/z values) with either known compounds or published spectra (Omata et al., 1991;Gherman et al., 2000;Yff et al., 2002;Hayyan et al., 2011).The major constituents identified were hexadec-1ene, octadec-1-ene, palmitic acid, ethyl palmitate, linoleic acid, oleic acid, stearic acid and ethyl stearate.
According to the literature, palmitic acid (Kurtulmus et al., 2009), linoleic acid (Dilika et al., 2000) and oleic acid (Dilika et al., 2000) possess good activity against S. aureus.The antimicrobial activity of the n-hexane partition could therefore be at least partly ascribed to the presence of these bioactive compounds.

Conclusions
Antimicrobial screening of Chungtia medicinal plants used customarily for skin related ailments, that is, A. lucidior (roots), B. picta (leaves), C. floribunda (leaves), H. latifolia (leaves), M. indica (leaves) and P. persica (roots) against dermatologically relevant micro-organisms showed that all  Many doubts still persist even today when it comes to selection of the solvents for extracting the active constituents from various Indian medicinal plants.This study was aimed at assessing and establishing the best solvent for extracting the active constituents from ten (10) plant extracts.Thin layer chromatography (TLC) was used to separate and establish the active constituents present in each of the medicinal plants.Active constituents from each plant was extracted using three different solvent systems namely diethyl ether, chloroform and hexane and were tested against three species of gram negative and three species of gram positive bacteria (Escherichia coli, Pseudomonas aureginosa, Streptococcus pneumoniae, Aeromonas hydrophila, Staphylococcus aereus and Bacillus cereus) by means of agar well diffusion assay.
Studies on the antioxidant activity studies were also carried out for these plant extracts using Diphenylpicrylhydrazyl (DPPH) method.For the antimicrobial activity, the study revealed that among the selected plants, Azadiracta indica, Pongamia pinnata and Aloe barbadensis had maximum antibacterial activity.Among the extraction procedures, diethyl ether was found to be the best solvent that could be used for the extraction procedure.On the antioxidant activity part, Coleus amboinicus and Calotropis procera were found to have high antioxidant activity of 91.64% and 88.72%, respectively.

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.

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.

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.

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.

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.

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.

Table 2 :
MTT microdilution assay results.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.

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

and compounds Minimum inhibitory concentration (MIC, µg/ml) and minimum bactericidal concentration (MBC, µg/ml)
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.

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