Compounds from Vernonia arborea Buch.-Ham. Inhibit Microbes that Impair Wound Healing

Aims: To identify the antimicrobial potency of the leaf fractions of Vernonia arborea against selected wound microbes viz., Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Stenotrophomonas ranging from 15.62 µg/mL to 500 µg/mL for the different microbes. Quantitative High-Performance Thin Layer Chromatography (HPTLC) revealed two compounds (a and b) in the bioactive fraction10 with yields of 633 mg (63%) and 97 mg (9.7%) per gram of the extract. Conclusion: The findings suggest the potential use of the bioactive compound in chronic infectious wound management therapy.


INTRODUCTION
Vernonia arborea, the tree species of the family Asteraceae is found in Western Ghats of India and Sri Lanka [1]. Apart from the traditional decoctions used for wound healing, the methanol and aqueous leaf extracts of this species showed wound healing activity in excision, incision and dead space wound models of Wistar rats [1][2][3][4][5].
Wounds require a complex repertoire of cellular events to heal. The major reason for impairment in wound healing in most cases has been wound infection caused by a poly-microbial community [6]. Some of these opportunistic strains maybe resistant to regular antibiotics while the others impact the wound microenvironment by producing digestive enzymes like proteases and hyaluronidase, contributing to the increasing surface area and depth of the wound [7].
In the ordered series of events, the early onset and resolution of wound inflammation is a kick start for the actual healing process, apparently delayed by the microbial load at the wound site. Notwithstanding a wide range of the microbes in the polymicrobial community, the most commonly found microbes in clinical cases were chosen for this study, viz., Escherichia coli ( Among the various classes of phytocompounds, sesquiterpenes showed promising antimicrobial potency and happens to be a major phytocompound reported to occur in V. arborea species [1,2]. Sesquiterpenes from other plant species like, Cnicin, Cynaropicrin, Ferusinol, Secodesma showed activity against E. coli, S. aureus, P. aeruginosa and K. pneumoniae respectively [9,10].
Guaianolide and Germacranolide class of sesquiterpene lactones were found active against S. aureus [11]. Isabelin, a sesquiterpene lactone was reported to be active against S. maltophilia [12].
Reports on the wound healing potency of V. arborea in wound models of Wistar rats, prompted us to investigate the antimicrobial activity of hexane leaf extracts [1,3,5]. Zaluzanin D, a sesquiterpene from the hexane leaf extract of V. arborea was reported to possess anti-fungal activity [10,12]. Considering the polymicrobial community colonising a wound and impairing the healing process, novel compounds active against a wide range of wound microbes are vital in wound management [7][8][9][10]13,14].
In this study we report fractionation of V. arborea leaf extracts, the HPTLC profiles and the bioactivity of selected fractions against the five wound microbes using agar well plate assays, the minimum inhibitory and bactericidal concentrations.

Plant Material Collection and Authentication
The Vernonia arborea leaves were collected from Anaimalai Hills, Pollachi District (10°22′N 77°07.5′E) in the month of October. The collected material was identified and authenticated and a voucher specimen maintained in the department (PARC/2012/1392).

Extraction and Column Fractionation of the Crude Extract
The dried, ground leaf material (1 kg) was extracted by exhaustive percolation for three days in n-hexane with intermittent stirring for every 18 hours at room temperature.

Antimicrobial Activity of the Column Fractions
The column fractions were tested against five wound microbes to screen for their antimicrobial activity using agar well diffusion assay. Thin layers of (1% Muller Hinton) agar seeded with the test strains (0.5 MacFarland Standard concentrations) were added along with the indicator, tetrazolium chloride (0.1%) [18]. The column fractions were added to the agar wells at 1000 μg concentration in triplicates. Ampicillin at the inhibitory concentrations for the respective bacteria was used as positive control. Ampicillin has been studied to inhibit the growth of the test strains by mechanisms including induction of cell wall damage by action on autolysin inhibitor, inhibition of cell wall penicillin-binding proteins and β-lactamase inhibition [19][20][21][22]. After incubation for 18 h at 37 ºC, the mean diameter of inhibition zones as (mm) ± SD were determined. Reproducibility of these assays was tested at least five times. The fractions that had antimicrobial activity against all of the five bacterial strains were selected for further analysis.

TLC bioautography
TLC of the bioactive fractions were developed and bioautography was performed by overlaying agar containing the test organism on the developed chromatogram. The zone of inhibitions at the specific Rf were observed.

MIC microbroth dilution method
The minimum inhibitory concentration (MIC) of the selected bioactive fractions were determined using resazurin microtiter assay (REMA) plate method individually against all the five bacterial strains as per the standard reference method (CLSI 2000). The selected bioactive fractions were serially diluted from 1mg/mL concentration with 100 µL of uninoculated broth in the microtiter plate. Ten microlitres of the standard inoculum (1:100 dilution of the McFarland standard) was added and incubated overnight.
After overnight incubation, 30 µL of 0.01% resazurin was added to all the wells and observed for color change after one hour of incubation.
Uninoculated broth control, inoculated broth control and with ampicillin drug (at inhibitory concentration for the respective strain) were controls used for comparison. The MIC was identified to be the least concentration of the fraction that showed absence of growth (i.e., inhibition) indicated by no change in color of the dye. Growth was seen as change from purple to pink color due to reduction of the indicator dye [15,16]. The assay was independently performed twice for reproducibility and duplicates for each concentration were maintained.

Minimum bactericidal concentration
The minimum bactericidal concentration (MBC) of the fractions were determined by plating the sample from the REMA plates after determining the MIC through visual observation. A loop full of the suspension from the wells at which MIC was noted and with no apparent color change was streaked onto Muller Hinton agar plates and incubated overnight. The concentration of each fraction inhibiting 99.9% of bacterial growth (yielding no bacterial colonies) on the solid medium was taken as MBC.

HPTLC Quantification
Quantification of the F10 fraction using the photo-densitometric HPTLC method with a stock solution of the column purified compound (5 mg/mL) dissolved in chloroform was done. A CAMAG Automatic TLC Sampler 4 (ATS4) was used to spot (2 µl, 4 µl, 6 µl and 8 µl) and developed using hexane: ethylacetate (95:5, v/v) and derivatized with methanol: sulphuric acid (90:10, v/v) reagent and chromatograms scanned at 254 nm. The hexane crude leaf extract (5 mg/mL) was (4 µl and 6 µl) spotted on the HPTLC plates precoated with Si-gel Si60F254 (E. Merck). The calibration curve for linearity was obtained by plotting peak area vs. concentration of compound (ng/spot). The data analysed by the linear least square regression method was used to estimate the percentage of the compound (F10) in the HLE of V. arborea sample. Limit of detection and limit of quantification were estimated statistically for three determinations of the sample [19,23-27].

RESULTS AND DISCUSSION
Wound microbes contribute to major impairment in healing process, altering the wound microenvironment by prolonging inflammation, inducing excessive exudate formation, digesting wound tissue with extracellular enzymes, exacerbating devascularization caused by the wound, impeding re-epithelialization [28,29]. Among the listed mechanisms, prolonged inflammation is the primary stumbling block that expedites the repertoire of the other challenging events.
Chronic inflammation due to polymicrobial wound infection, is characterised by increased expression of proinflammatory mediators, altering the infiltration of inflammatory cells like neutrophils and macrophages, secretion of tissue degrading enzymes like protease and hyaluronidase.
The early resolution of inflammation is an indispensable factor to progress with the actual healing phase, including reepithelialisation and tissue remodelling [30,31].
Hence, clinical wound management predominantly entails destruction of the microbial load. Though wound management strategies including mechanical debridement and regular dressing of the wound microenvironment attempts to clear the necrotized, avascular tissue with exudates and bacterial load, wound palliation in immunocompromised patients warrants the need for a topical exogenous drug that can keep a check on the wound microbes and accelerate the healing process as well. Drugs which are claimed to possess wound healing activity fail to do so either due to the lack of inherent antimicrobial property or due to surging resistance to antibiotic agents among the wound microbes [32][33][34][35].

Antimicrobial Activity of the Column Fractions
The 790 elutions obtained were grouped based on their TLC characteristics. They were pooled into 30 fractions and tested for antimicrobial activity in agar well assays.

Agar well diffusion assay
The 30 fractions tested (at 1mg/mL) against the wound microbes, showed varying inhibition zones as compared to the standard ampicillin drug at inhibitory concentrations for the respective microbe. Six of them showed activity against all five or four bacteria with over 10 mm diameter of zones [ Table 1, Fig. 1].

Minimum inhibitory concentration and minimum bactericidal concentration
The minimum inhibitory concentrations of the six fractions were determined using resazurin dye reduction method and recorded as shown in Table 2.
As shown in Fig. 3 Six selected samples of the 30 column fractions of the leaf extract showed activity at their minimum inhibitory and bactericidal concentrations as comparable with the standard antibiotic, ampicillin [49,50]. A few of these samples were found to be mixture of two phytocompounds thus leaving behind a possibility of synergistic role in inhibiting or killing the bacteria.

Quantification of the Partially Purified Bioactive Fraction in HLE by HPTLC Analysis
The partially purified column fraction, F10 which showed good activity (in terms of MIC and MBC) was selected as the standard for HPTLC analysis and the quantity of the same in the HLE was deduced. The densitogram in Fig. 4 showed two prominent peaks in the putative standard and the HLE with the linear regression correlation shown for the two peaks (a and b) [38]. From the regression equation, the compounds a and b were quantified to be 633mg/g (63%) and 97mg/g (9.7%) of the HLE respectively [ The quantification of the bioactive compounds in HLE revealed good yield from the leaf material. Further structural elucidation would open way to QSAR studies and understanding the class of phytoconstituent(s) that could be a lead compound to develop an effective drug. The fraction with potent anti-wound microbial activity could be an effective constituent of medications used in topical management of infectious wounds and is the future objective of this study.

CONCLUSION
The hexane leaf extracts were fractionated with at least two fractions being antimicrobial against the five wound microbes tested in this study.
Fractions 10 showed MICs of 31.5-125 µg/mL while fraction 30 showed a low MIC of 15.62 µg/mL against S. maltophilia. Wound healing by methanol and aqueous leaf extracts of this species in wound models of Wistar rats were reported. The antibiosis against the polymicrobial community impairing wound healing was however not reported and this study demonstrated this in the hexane leaf extracts for the first time.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.