Dual function of active constituents from bark of Ficus racemosa L in wound healing

Background Different parts including the latex of Ficus racemosa L. has been used as a medicine for wound healing in the Ayurveda and in the indigenous system of medicine in Sri Lanka. This plant has been evaluated for its wound healing potential using animal models. The aim of this study was to obtain an insight into the wound healing process and identify the potential wound healing active substance/s present in F. racemosa L. bark using scratch wound assay (SWA) as the in-vitro assay method. Method Stem bark extracts of F. racemosa were evaluated using scratch wound assay (SWA) on Baby Hamster Kidney (BHK 21) and Madin-Darby Canine Kidney (MDCK) cell lines and Kirby Bauer disc diffusion assay on common bacteria and fungi for cell migration enhancing ability and antimicrobial activity respectively. Dichloromethane and hexanes extracts which showed cell migration enhancement activity on SWA were subjected to bioactivity directed fractionation using column chromatography followed by preparative thin layer chromatography to identify the compounds responsible for the cell migration enhancement activity. Results Dichloromethane and hexanes extracts showed cell migration enhancement activity on both cell lines, while EtOAc and MeOH extracts showed antibacterial activity against Staphylococcus and Bacillus species and antifungal activity against Saccharomyces spp. and Candida albicans. Lupeol (1) and β-sitosterol (2) were isolated as the potential wound healing active compounds which exhibited significant cell migration enhancement activity on BHK 21 and MDCK cell lines (> 80%) in par with the positive control, asiaticoside at a concentration of 25 μM. The optimum concentration of each compound required for the maximum wound healing has been determined as 30 μM and 35 μM for 1 and 2 respectively on both cell lines. It is also established that lupeol acetate (3) isolated from the hexanes extract act as a pro-drug by undergoing hydrolysis into lupeol in the vicinity of cells. Conclusion Different chemical constituents present in stem bark of Ficus racemosa L show enhancement of cell migration (which corresponds to the cell proliferation) as well as antimicrobial activity. This dual action of F. racemosa stem bark provides scientific support for its traditional use in wound healing.


Background
A wound is an injury that results in opening or breaking of the skin [1]. Because the skin is the protective barrier, wounds should be healed rapidly and efficiently within the shortest possible time [2]. Various complications can occur if wound healing does not set forth in an orderly and timely manner [3]. Many of the drugs currently used for wound management are expensive while some shows allergic reactions and drug resistance [4,5]. Phytomedicines have generated much interest for the treatment of skin ailments as they are affordable and supposedly safe [6]. Hence, the possibility of deriving cost effective therapies from plant based traditional medicine has been explored [7,8].
The classical model of wound healing is divided into three sequential, yet overlapping phases, inflammatory, proliferate and remodeling [31]. Immediately after an injury, a fibrin clot is formed to limit the active bleeding (homeostasis). In the inflammatory phase, bacteria and debris are removed by phagocytic action. Proliferate phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction [32]. Later part of the wound healing process involves remodeling the dermal tissues to produce greater tensile strength. The principal cell type involved in this process is the fibroblast [31].
Investigation of wound healing active constituents have been carried out employing both in-vivo methods and human trials in earlier times whereas at present, in-vitro methods based on cell cultures of fibroblasts, keratinocytes, epithelial cells and endothelial cells are used to identify the wound healing active plant secondary metabolites in addition to in-vivo methods [8]. The scratch wound assay has been established as a simple but valuable and inexpensive tool to obtain first insights into how plant preparations or their secondary metabolites can persuade formation of new tissue [33]. Scratch wound assay is particularly suitable for studying unidirectional cell migration and its regulation by intercellular interactions and interactions between cells with the extracellular matrix (ECM) in comparison with other popular in-vitro methods, such as time-lapse microscopy and Boyden chamber assays. During this wound healing assay, scratched cell monolayer responds to the disturbance of cell-cell contacts by increasing the concentration of growth factors and cytokines at the wound edge [34].
Herein we report the identification of lupeol (1) and βsitosterol (2) (Fig. 1) as the potential wound healing active constituents from extracts of stem bark of F. racemosa and that lupeol acetate (3) acts as a prodrug for wound healing, through bioactivity directed fractionation using scratch wound assay (SWA) over Baby Hamster Kidney (BHK 21) and Madin-Darby Canine Kidney (MDCK) epithelial cell lines. 1 It is known that keratinocytes, which produce keratin, are one of the main cell types that initiate cell proliferation and cell migration leading to closure of a wound. MDCK cells are reported to be capable of producing keratin [35]. Hence, although MDCK cell line is a general model of epithelial cells it has been used as an in-vitro model in wound healing assay [36].

Materials
Solvents used for the chemical extraction were of either analytical grade or general purpose reagent grade purified by fractional distillation. Chemicals and regents were purchased from Merck, Sigma-Aldrich, Fluka, Himedia and Invitrogen. Silica gel 60 G (Fluka − 60741) was used as solid phase, for gravity columns. Analytical and preparative Thin Layer Chromatography (TLC) was performed on pre-coated 0. 25   Cultures were maintained at 37°C in a 5% CO 2 humidified incubator. Cell cultures were screened periodically for mycoplsama, bacterial and fungal contamination. A batch of seed samples in cryopreserving ampoule was stored in liquid nitrogen vessel for future use. Cells which grow at confluent stage were harvested from tissue culture vessel by centrifugation. Healthy cells were seeded in clear bottom 12 well plates in 10% DMEM growth medium at high density (3 × 10 4 cells per well). Plates were incubated at 37°C for 24 h in humidified incubator. Cultures were observed with an inverted microscope, equipped with digital camera for the formation of monolayer.
A scratch (wound) was performed on monolayer of cells along the vertical axis of each well under the microscope. The monolayer with wound was washed with 750 µL of phosphate buffer saline (PBS). Each test well was filled with 990 µL of DMEM and added 10 µL of DMSO containing appropriate amount of test sample. Two negative controls, 1% DMSO in DMEM and 100% DMEM were used in this experiment. In addition, asiaticoside (25µM), a potent wound healing agent [37] was used as the positive control when pure compounds were assayed. Plates were incubated for 24 h at 37°C with 5% CO 2 . The widths of the wounds at different time intervals (0 h, 12 h, 18 h, and 24 h) were measured and the cell migration enhancement is presented as the percentage wound closure. Images at each stage were photographed. All the experiments were carried out in three replicates and three measurements were taken for each wound.
Anti-bacterial assay: Kirby-Bauer disk diffusion method Preparation of discs All the extract and fractions were applied at a dose of 500 µg / disc. Test samples were dissolved in MeOH (10 µL) and adsorbed on to filter paper discs (Diameter = 6 mm). The negative control was prepared by adding MeOH (10 µL) to a disc. Discs containing samples and control discs were dried in a vacuum oven maintained at 30°C for 24 h in order to remove all traces of solvents. A disc containing 25 µg of Amoxycillin (Himedia) were used as positive control [38].

Culture preparation
Antibacterial assay was carried out using against Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) (Bacterial culture collection, Department of Botany, The Open University of Sri Lanka) in the preliminary investigation and the fractionation was selectively guided by the antibacterial assay against most effective pathogens. Bacterial species were grown as pure cultures on nutrient agar (NA) (Himedia-M001) plates and incubated for 24 h at 35°C. Each culture was made into a suspension in sterilized distilled water up to a certain density, which was measured as a predetermined optical density (OD value); 0.05 OD at λ = 500 nm for Bacillus subtilis, 0.1 OD at λ = 500 nm for E. coli, S. aureus and P. aeruginosa. From each suspension 0.1 mL aliquots were transferred on to different NA plates and were spread using a sterilized spreader to prepare a uniform lawn of each bacterial species under study. The positive control, negative control and sample discs were placed on each plate. All experiments were done twice in duplicate and plates were incubated at 35°C for 24 h. The zone of inhibition if present was Anti-fungal assay: Kirby-Bauer disc diffusion method Preparation of discs All the extract and fractions were applied dose of 500 µg / disc. Test samples were dissolve in MeOH (10 µL) and adsorbed in to filter paper discs. The negative control was prepared by adding MeOH (10 µL) to discs. All the discs were dried in a vacuum oven maintained at 30°C for 24 h in order to remove trace of solvents. Disc containing 300 µg of Polymyxin B (Himedia) were used as positive control [38].

Culture preparation
Antifungal assay was carried out against two Saccharomyces cervisiae (NCYC 2401, NCYC 2402), a locally isolated Saccharomyces sp. (Fungal culture collection of the Department of Botany, Sri Jayewardenepura University, Sri Lanka) and Candida albicans (ATCC 10231). Pure cultures of above fungi were grown as pure cultures on Yeast Malt Agar (YMA) (Himedia-M) plates and incubated for 24 h at 35°C. Suspensions of these cultures were prepared in sterile distilled water having predetermined cell densities measured as OD value: 0.05 OD at λ = 500 nm for Saccharomyces sp., 0.1 OD at λ = 500 nm for Candida albicans. A uniform lawn of each fungal species was prepared by spreading 0.1 mL aliquots from each suspension on different YMA plates. The positive control negative control and sample discs were placed on each plate. All experiments were done twice in duplicate and plates were incubated at 35°C for 24 h. The zones of inhibition if present were measured and the mean with the standard errors were calculated.

Extraction of the plant material
Dried powdered bark of F. racemosa L. was provided by Link Natural Products (Pvt) Ltd. Sri Lanka, in May 2011. Plant specimens were identified by Mr. T. M. S. G. Tennakoon, Head R & D section, Link Natural Product Ltd., Sri Lanka comparing F. racemosa L. specimens deposited in the Herbarium at Peradeniya Royal Botanical gardens, Sri Lanka and consulting the relevant literature [39]. A voucher specimen (68-002-01) was deposited at the herbarium of Link Natural Products Ltd. Sri Lanka. Plant material (500 g) was sequentially extracted with hexanes (9.32 g, 1.864%), dichloromethane (CH 2 Cl 2 ) (1.35 g, 0.264%), ethyl acetate (EtOAc) (1.25 g, 0.250%), and methanol (MeOH) (1.21 g, 0.242%), in a Soxhlet apparatus and the solvents were removed under vacuum at 40°C to obtain the respective extracts.

Bioassay guided fractionation
The hexanes and CH 2 Cl 2 extracts which showed cell migration enhancement activity was subjected to further fractionation using silica gel column chromatography. Column fractions were subjected to SWA at a concentration of 50 mg dm − 3 . The percentage wound closure was calculated at 24 h. Further fractionation of the cell migration enhancement active fractions was carried out and the fractions were subjected to SWA at 25 mg dm − 3 and the fractions which showed wound healing activity were subjected to further fractionation to isolate potential cell migration enhancing compounds.

Fractionation of the CH 2 Cl 2 extract
The CH 2 Cl 2 extract (1.0 g) of F. racemosa bark was chromatographed over a column of silica gel (30.0 g) made up in hexanes and eluted with hexanes containing increasing amounts of CH 2 Cl 2 , CH 2 Cl 2 and CH 2 Cl 2 containing increasing amounts of MeOH and finally washed with MeOH yielding 77 fractions. The column fractions were combined according their TLC patterns to give 18 major fractions (DF 1 -DF 18 ) and were subjected to scratch wound assay. Column fractions which showed > 65% cell migration enhancement activity were further fractionated to isolate the active constituents using column and preparative thin layer chromatography.

Fractionation of the hexanes extract
The hexane extract (2.5 g) of F. racemosa bark was chromatographed over a column silica gel (60 g) made up in hexanes and eluted with hexanes, hexanes containing increasing amounts of CH 2 Cl 2 , CH 2 Cl 2 and CH 2 Cl 2 containing increasing amounts of MeOH and finally washed with MeOH yielding 52 fractions. These column fractions were combined according their TLC patterns to give 11 major fractions (HF 1 -HF 11 ) and were subjected to scrath wound assay. Column fractions which showed > 65% wound healing activity were further fractionated to isolate the active constituents using column and preparative thin layer chromatography.

Statistical analysis
All the results are presented in this paper as mean ± standard error (n = 3 for SWA and n = 4 for antimicrobial assay). Mean comparisons, were performed using ANOVA/LSD post hoc test. P-values of less than 0.05 were considered to be significant. All statistical tests were carried out using the computer programme SPSS Version 20.0 computer programme.

Results
Evaluation of the wound healing effect of extracts and fractions of stem bark of F. racemosa Results indicated that the hexanes and dichloromethane extracts of the stem bark of F. racemosa has statistically significant effect on enhancing the wound healing (p ≤ 0.05) and the percentage of wound closure has been increased over both tested cell lines at 24 h (Table 1). Images of the SWA upon MDCK and BHK cells using the DCM extract are shown in Fig. 2. . These fractions were subjected to SWA at a concentration of 50 mg dm − 3 and the fraction DF 7 was found to be the most active fraction (Fig. 3). Further fractionation of fraction DF 7 (4.0 mg) by preparative TLC (20 cm × 10 cm: 1 plate eluent: CH 2 Cl 2 ) led to the isolation of the wound healing active compound which was identified as lupeol (1) (2.9 mg; 0.004%) by spectroscopic data and comparison with an authentic sample, m. p. 213-215°C) (lit. 215°C) [40] by spectroscopic data and comparison with an authentic sample (TLC, Co-TLC and mixed m. p.). 1  TLC comparison of fractions DF 4 , DF 8 , DF 9 -DF 12 with lupeol (1) indicated that lupeol is present in fractions DF 4 and DF 8 while fractions DF 9 -DF 12 did not contain detectable amounts of lupeol. Fraction DF 9 (35.0 mg) was further fractionated by column chromatography over a column of silica gel (1.0 g) made up in hexanes/ CH 2 Cl 2 (75:25) and eluted with hexanes/CH 2 Cl 2 (65:35), hexanes/CH 2 Cl 2 (50:50), hexanes/CH 2 Cl 2 (25:75) and CH 2 Cl 2 to give 4 major sub fractions [DF 9 1 (7.7 mg), DF 9 2 (3.9 mg), DF 9 3 (7.1 mg), DF 9 4 (6.6 mg)]. These fractions were subjected to SWA at a concentration of 25 mg dm − 3 and fractions DF 9 2 and DF 9 3 were found to be active. Fraction DF 9 3 was found to be constituted of (90%) a single compound and purification of this fraction (4 mg) on preparative TLC (20 cm × 10 cm: 1 plate eluent: CH 2 Cl 2 ) led to the isolation of β-sitosterol (2) (3.4 mg; 0.002%) which was identified by spectroscopic data and comparison with an authentic sample. 1
All three pure compounds, lupeol (1), β-sitosterol (2), and lupeol acetate (3) were subjected to SWA at 25 µM concentration along with asiaticoside as the positive control and the percentage closure of wound at 24 h was obtained (Fig. 5).
Optimum concentrations of lupeol (1) and β-sitosterol (2) required for the maximum cell migration enhancement activity at 24 h were determined by carrying out the SWA for these two compounds with asiaticoside as the positive control at different concentrations (10-50 µM) and the results are shown in Fig. 6. It was found that optimal concentrations of 1, 2, and positive control are 30 µM, 35 µM and 25 µM respectively against both cell lines.

Role of lupeol acetate (3) in wound healing
Although lupeol acetate (3)  The mean value followed by the standard error of the mean within the parentheses in BHK cell line and 72% in MDCK cell line at 48 h (Fig. 7). This observation prompted us to investigate whether lupeol acetate acting as a pro-drug and undergo hydrolysis in the vicinity of the wound to produce lupeol (1). In order to test this hypothesis, we incubated lupeol acetate in DMEM (10 mg dm − 3 ) in the presence of cells with a wound and absence of cells and tested the medium for the presence of lupeol (by TLC) with time while simultaneously monitoring the cell migration enhancement activity. Comparative TLC examination of the two assay media revealed that, lupeol acetate (3) has been hydrolyzed to give lupeol (1) in the presence of the cells while no change has occurred in the absence of cells (Fig. 8), revealing that lupeol acetate act as a pro-drug in the vicinity of cells. Anti-bacterial activity of extracts of F. racemosa

Sample
It was found that the EtOAc and the MeOH extracts were active against the two gram positive bacteria B. subtilis and S. aureus, at a dose of 500 µg/disc but inactive against the gram negative bacteria species E. coli and P. aeruginosa (HPLC traces of EtOAc and MeOH extracts are shown in Fig. 9). The hexanes and CH 2 Cl 2 extracts did not show activity against any of the tested organisms (TLC profiles of hexanes and CH 2 Cl 2 extracts of F. racemosa are shown in Fig. 10). Disc containing Amoxicillin 25 µg served as the positive control. Results obtained against the four different bacterial strains tested are tabulated in Table 3. However it was not possible to isolate the active constituents because they were inseparable on normal and reversed phase TLC.
Anti-fungal activity of extracts of F. racemosa It was found that the EtOAc and the MeOH extracts were active against the all four fungi species at a dose of 500 µg/disc (HPLC traces of EtOAc and MeOH extracts are shown in Fig. 9). The hexanes and CH 2 Cl 2 extracts did not show activity against any of the tested organisms (TLC profiles of hexanes and CH 2 Cl 2 extracts of F. racemosa are shown in Fig. 10). Disc containing Polymyxin B 300 µg served as the positive control. Results obtained against three different Saccharomyces spp. and Candida albicans are tabulated in Table 4.

Discussion
In the treatment of wounds, shortening of the healing time and protecting the wound from bacterial infections are two main objectives. The open blood vessels and tissues in a wound area is a favorable place for multiplication of microbes because of provision of proper environment needed for efficient growth of microbes. As such, in addition to enhancing cell proliferation, prevention of microbial invasion of the wound is a paramount important requirement in the effort of wound healing    [41]. The main objective of the antimicrobial therapy in wound care is to control microbial colonization and simultaneously increase cell proliferation, in order to enhance the wound healing [42]. It has been reported that herbal medicines have an array of beneficial properties such as efficacy, nontoxicity, availability, affordability as they are cheaper than the conventional synthetic drugs [43]. Some herbal extracts either directly or indirectly promote wound repair or exhibit antimicrobial properties there by prevent wound infestation. It is important to note that, the antimicrobial property of a drug also plays an important role in wound care. In our experimental work, the biological activities shown by the extracts of F. racemosa were found to be playing a dual role in the healing of wounds. According to the antimicrobial assay results both methanol and ethyl acetate extracts of stem bark of F. racemosa showed antimicrobial activity against gram positive bacteria in addition to the enhanced cell migration activity of hexanes and dichloromethane extracts of the same over both cell lines tested at the 24 h. Based on the results of bioassay guided fractionation, it is revealed that lupeol (1) and β-sitosterol (2) are the cell migration enhancing compounds present in dichloromethane extract of the bark of F. racemosa. It was reported that lupeol (1) isolated from leaves of Celastrus paniculaates has shown potential wound healing activity on excision, incision and dead space wounds in albino rats [29] while β-sitosterol isolated from Aloe vera gel has shown a potential angiogenic activity in the chorioallantoic membrane of chick embryo and stimulates the migration of human umbilical vein endothelial cells [44]. It is also reported that lupeol possesses antiinflammatory activity which too would have an impact on its wound healing activity [45].
It is also revealed, that, both lupeol (1) and β-sitosterol (2) exhibit a significant cell migration enhancement in both cell lines at 24 h which is comparable with that of the positive control, asiaticoside (Fig. 5). Both hexanes and dichloromethane extracts contain lupeol (1) while lupeol acetate (3) present only in hexanes extract and β-sitosterol (2) found only in the dichloromethane extract. The observed cell migration enhancement activity of 3 could be attributed to its hydrolysis into 1 in the vicinity of wound area. As lupeol acetate (3) enhances the cell migration with time by undergoing slow hydrolysis to give lupeol (1) in the presence of the cells while no hydrolysis of 3 occurred in the absence of cells (Fig. 6), it is revealed that lupeol acetate act as a pro-drug in the vicinity of cells. Lupeol acetate which co-exists with lupeol in the hexane extract may improve and assist the wound healing process with time. As different extracts of bark of F. racemosa exhibit both cell migration enhancing as well antimicrobial properties, it could be concluded that a drug developed from this plant extract may have dual action in wound healing.

Conclusion
In the present study we have investigated the wound healing potential of F. racemosa bark extractives using SWA as an in-vitro wound healing model and were able to identify the possible cell migration enhancing constituents as lupeol and β-sitosterol via bio activity directed fractionation procedures. The cell migration enhancement activity of each of these two compounds is concentration dependent and exhibits an optimum value comparable to that of the positive control, asiaticoside. It is also found that lupeol acetate (3) which is present in F. racemosa bark hydrolyzes to form lupeol (1) in the presence of cells and thereby acts as a pro-drug in enhancing the cell migration. The ethyl acetate and methanol extracts of the plant exhibited anti-microbial activity against Staphyllococus, Bacillus and Saccharomyces species as well as Candida albicans.