FIRST DESCRIPTION OF ANTIBACTERIAL AND IN VITRO WOUND HEALING PROPERTIES OF COCOS NUCIFERA TOMENTUM

Objective: The objective of the study was to evaluate the antibacterial, antioxidant, and in vitro wound healing property of Cocos nucifera tomentum. Methods: Ethanolic and methanolic extracts of C. nucifera tomentum were prepared using the Soxhlet extraction method and aqueous extract was prepared by the shaker flask method. These extracts were investigated for the phytochemical profile by gas chromatography and mass spectroscopy (GC-MS) and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-ToF MS), for antibacterial activity by microdilution method, for the antioxidant property by 2,2-diphenyl-1-picrylhydrazyl assay, and for in vitro wound healing activity on cell lines. Results: The preliminary phytochemical screening showed the presence of alkaloids, flavonoids, and various bioactive components. GC-MS and LCQ-ToF MS analysis of extracts revealed the presence of bioactive compounds known to have wound healing properties as well as antioxidant and antimicrobial activities. The methanolic extract had better antibacterial activity against Proteus vulgaris and Escherichia coli, and better antioxidant and wound healing activity, when compared to the ethanolic and aqueous extracts. Conclusion: The present study suggests that C. nucifera tomentum might be a potential therapeutic source for topical antibacterial and wound healing applications.


INTRODUCTION
Wounds are breaks in the skin (due to cuts, scrapes, or scratches) or injuries in other body tissues. They often happen due to trauma or due to an accident, but can also be caused due to surgery, or by sutures and stitches. A major problem with wounds is the risk of infections caused by microorganisms. Inhibition or killing of microorganisms is an essential component of the wound healing process and is generally achieved with antimicrobial agents (antibiotics). These can be applied to the wound (topical) or be given systemically (e.g., by oral administration) [1,2].
Cocos nucifera L., commonly known as the Coconut, is a member of the family Arecaceae (Palms). It is highly valued both as a source of food and a source of medicines. The plant is said to have originated from Southeast Asia, or Islands of the Indian and Pacific oceans [3]. India is the third largest coconut producing country [4]. Only recently has modern medical science unlocked the secrets to coconut's amazing healing powers. It is widely used in Ayurveda for various skin problems and microbial infections [5]. Coconut shell charcoal powder is also very effective as a potential medicine for wound healing, for kidney trouble, for ulcers, and other soft tissue diseases [6]. Virgin coconut oil extract contains various phytochemicals and shows high antioxidant activity [7]. The C. nucifera L. leaf petiole possesses a caducous tomentum abaxially, which appears as a closely matted hair or downy covering. The villagers and farmers of Kerala and Tamil Nadu in India, traditionally apply the tomentum of coconut on cuts and wounds. Such ethnomedicines are widely prevalent and they have been the source for the development of new drugs [8,9].

Haritha et al.
of molecular and elemental compositions of unknown organic compounds in a mixture [14]. GC-MS analysis for TE and TM was carried out on the Shimadzu GC-MS (QP2010S). The column used was Rxi-5sil MS (300 mm×0.2 mm×0.25 µm). Helium was the carrier gas at a flow rate of 1.0 mL/min. The instrument was set to an initial temperature of 80°C and maintained for 2 min. At the end of this period, the oven temperature was raised to 280°C (at 5°C/min) and maintained for 5 min. Injection port temperature was 260°C, the injection volume was 1.0 µL, and the helium flow rate was 3.0 mL/min. The total GC-MS running time was 40.8 min. The phytochemical constituents were identified using the National Institute of Standards and Technology Mass Spectral Database (NIST 11) and WILEY 8 library [15,16].
Liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-ToF MS) analysis LC-Q-ToF MS is used to determine the fragmentation and structural information of the known and unknown compounds present in a complex mixture [17]. LC-Q-TOF MS analysis for TA was performed on the Acquity H class (Waters India Pvt. Ltd., Bengaluru, India) ultraperformance LC system connected with Xevo G2 (Waters India Pvt. Ltd., Bengaluru, India) Q-TOF mass spectrometer with LC column BEH C18 column (50 mm×2.1 mm×1.7 µm). The mobile phase was a gradient of water+0.1% formic acid (A), and methanol (B), and the samples were analyzed from 0.1 min to 9.0 min. The flow rate was 0.3 mL/min. Total run time was 9 min. The LC-Q-TOF mass spectrometer analysis was performed with 135°C source temperature and 350°C desolvation temperature. The positive ionization mode was with the energy of 3 kV, with sample cone at 30 V and extraction cone at 1 V, while the negative ionization mode was with the capillary voltage at 2.5 kV, and sample cone at 30 V, and extraction cone at 1 V. The identification of compounds present in the sample was performed by comparison of MS/MS Spectra using the ChemSpider database [18].

Antibacterial activity by microdilution assay
The minimum inhibitory concentrations (MIC) of the extracts against test bacteria were determined using a modified microdilution technique originally described by Eloff [19]. Test solutions (10 mg/mL) of the extracts were prepared with Dimethyl sulfoxide and serial two-fold dilutions were made. 50 µL of the test bacteria (1.0×10 7 CFU/mL) were grown in tryptone soya broth. The covered microplates were incubated at 37°C for 24 h [20]. To indicate bacterial growth 30 µL of thiazolyl blue tetrazolium bromide dye was added to each well, and plates were further incubated for 30 min at 37°C. Formation of blue color indicated the presence of viable cells [21]. The experiments were carried out in triplicate.

Antioxidant activity
The antioxidant activity of the tomentum extracts was determined in terms of hydrogen donating or radical scavenging ability using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [22]. Samples and standard (ascorbic acid) were taken in various concentrations, and the volume was adjusted to 100 µL with methanol. About 3 mL of 0.1 mM methanolic solution of DPPH was added to the aliquots of samples and mixed well. Negative control was prepared by adding 100 µL of methanol in 3 mL of 0.1 mM methanolic solution of DPPH. The tubes were allowed to stand in the dark for 30 min at room temperature. The absorbance of the sample was measured at 517 nm against a Blank [23]. The radical scavenging activity was calculated using the following formula [24]:

DPPH scavenging activity % = [(Control−Test sample)/Control]*100
Scratch wound healing assay Mouse fibroblast cells (L929) were grown in 24-well plates at a density of 1×10 5 cells/mL and cultured to 80% confluency. A small linear scratch was created in the confluent monolayer by gently scraping with a sterile cell scrapper [25]. Cells were thoroughly rinsed with phosphate-buffered saline to remove cellular debris and treated with different concentrations of the methanolic or ethanolic extracts of C. nucifera tomentum. Cell proliferation was monitored at different time points (0 h, 4 h, 18 h, and 24 h), and images of migrated cells were taken at different time points using a digital camera (Nikon, Tokyo, Japan) connected to an inverted phase contrast microscope (Radical Instruments, India).

Preliminary phytochemical screening
The phytochemical analysis of the tomentum of C. nucifera L. showed the presence of alkaloids in TE and TM, while it was absent in TA. Tannins, phenols, flavonoids, and sterols were present in varying concentrations in all extracts, as indicated by the intensity of the colored solution and precipitates. Terpenoids were absent in TE and TA but present in TM ( Table 1).

LC-Q-ToF MS analysis
The total ion chromatogram of TA is represented in Figs. 3 and 4. Although several bioactive compounds have been identified, further studies are needed to get complete profiling of components present in the sample [35,36].  Haritha et al.

The MIC of tomentum extracts
The MIC -determined as the lowest concentration of the crude extract that showed no microbial growth -was carried out against three Gram-negative and one Gram-positive bacteria using various extracts of C. nucifera (

Reference antibiotics
Chloramphenicol for Gram-positive bacteria and gentamicin for Gramnegative bacteria were used.

Antioxidant activity
Comparison of the antioxidant activity of the tomentum extracts and ascorbic acid by DPPH method is shown in Fig. 5. The various extracts TM, TE, and TA exhibited significant dose-dependent inhibition of DPPH activity. Among these, TM showed the highest potential to scavenge DPPH when compared to TE and TA. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm which is induced by antioxidants [37]. At 400 µg/mL concentration, the percentage of inhibition of TM, TE, and TA is 49%, 15.8%, and 38%, respectively, when compared to ascorbic acid as the standard (39%). The IC 50 value of TM was 400 µg/mL, TA was 800 µg/mL, and the TA was 1000 µg/mL. In our study, the highest antioxidant activity was found in TM, followed by TA and TE.

Scratch assay
Proper healing of wounds is necessary for the restoration of the skin [5]. For our in vitro wound-healing assay, the L929 mouse fibroblast cell line was used for the surrogate "Scratch assay." A small linear scratch was created in a confluent monolayer of L929 cells by scraping with a sterile cell scraper. An image analyzer was then used to calculate the time required to close the gap at different concentrations of extracts.
The time taken to close the gap using various extracts was plotted and compared with untreated cells (Fig. 6). The results showed that at a concentration of 75 ug/mL, TM closed the gap in the scratch at the 18 th h when compared the TE, TA, and control (Fig. 7). In the present study, formulation of at the concentration of 50 µg/mL and 75 µg/mL of TM showed significant mobilization of L929 cells and closed the gap, when compared to the control without the addition of formulation. Photographs indicating comparative cell migration in normal control at the 0 th h, non-treated control, TM, TE, and TA at the 18 th h time intervals.

DISCUSSION
C. nucifera is a well-known plant, and its parts (including the milk and oil) are used both as a food, as well as in herbal medicines [38]. Earlier studies have reported the antipyretic, wound healing activity, and anti-hypertensive effects of other parts of C. nucifera [39,40].  The presence of phenolic compounds such as sterols and terpenoids contributes to the antioxidant properties of the tomentum [41]. GC-MS is the best technique to identify the bioactive constituents of long-chain hydrocarbons, alcohols, acids, esters, alkaloids, steroids, amino acid, and nitro compounds [33]. In the present study, all the compounds identified are known to possess biological activities. However, for the LC-Q-ToF MS analysis, further studies are needed to obtain the complete profile of compounds.
Our MIC studies show that test organisms are more sensitive to methanolic extracts of the coconut tomentum and that this antibacterial activity could very well be due to the phytoconstituents present (such as the phenolic compounds) [42][43][44]. It has earlier been reported that C. nucifera oil with silver sulfadiazine is useful in treating burns and wounds [45].
However, our present study is the first to demonstrate the wound healing effect of the hitherto un-studied C. nucifera tomentum. The wound healing property of tomentum may be due to either a rapid cell proliferation stimulated by growth-promoting phytochemicals and antioxidants present, or by prevention of infection due to antimicrobial activity.

CONCLUSION
This work is the first report to identify compounds from the extracts of C.nucifera tomentum, through qualitative and quantitative phytochemical analyses. This study also tested the antioxidant, antibacterial, and in vitro wound healing activities of alcoholic and aqueous extracts of C. nucifera tomentum. The methanolic extract of the coconut tomentum has significant antimicrobial, antioxidant, and wound healing properties. This study reveals the potential source of useful drugs from C. nucifera tomentum.

ACKNOWLEDGMENT
The authors are thankful for the Head and Staff, Department of Microbiology, Nehru Arts and Science College, Coimbatore, India, for their encouragement to carry out this study.