Antimicrobial and Wound Healing Potential of Canthium coromandelicum Leaf Extract-A Preliminary Study

MATERIALS AND METHODS

Plant materials: The plants were authenticated by Dr. S. Singaravadivel, Senior Scientist, Head of the Department, Department of Microbilogy, Indian Institute of Crop Processing Technology, Thanjavur, Tamil Nadu, India. The plant Canthium coromandelicum leaves were dried and extracted with ethanol using soxhlet apparatus and prepared as paste.

Phytochemical analysis: Chemical tests were carried out on the aqueous extract and on the powdered specimens using standard procedures to identify the constituents as described by Mohan et al. (2012a).

Microorganisms: Staphylococcus aureus (Gram positive), Escherichia coli (Gram negative) and Bacillus subtilis (Gram positive) and Candida albicans were the microorganisms used and they were obtained from the Microbiology Laboratory of the Thanjavur Medical College Hospital, Thanjavur. These microorganisms were identified and confirmed by Microbiologists, Department of Microbiology, Thanjavur Medical College, Thanjavur.

Antimicrobial assay: Antibiogram was done by disc diffusion method (NCCLS, 1993; Awoyinka et al., 2007) using plant extracts. Petri plates were prepared by pouring 30 mL of NA/PDA medium for bacteria/fungi. The test organism was inoculated on solidified agar plate with the help of micropipette and spread and allowed to dry for 10 min. The surfaces of media were inoculated with bacteria/fungi from a broth culture. A sterile cotton swab is dipped into a standardized bacterial/fungi test suspension and used to evenly inoculate the entire surface of the Nutrient agar/PDA plate. Briefly, inoculums containing Staphylococcus aureus, Escherichia coli and Bacillus subtilis were spread on Nutrient agar plates for bacteria and Candida albicans was spread on potato dextrose agar for fungus strains. Using sterile forceps, the sterile filter papers (6 mm diameter) containing the crude extracts (50, 100 and 150 μL) were laid down on the surface of inoculated agar plate. The plates were incubated at 37°C for 24 h for the bacteria and at room temperature (30±1°C) for 24-48 h for yeasts strains. Each sample was tested in triplicate.

Measurement of zone of inhibition: The antimicrobial potential of test compounds were determined on the basis of mean diameter of zone of inhibition around the disc in millimeters. The zones of inhibition of the tested microorganisms by the extracts were measured using a millimeter scale.

Animals: Male albino rats of Wistar strain approximately weighing 180-200 g were used in this study. Male albino rats were purchased from Indian Institute of Science, Bangalore. The animals were housed in spacious polypropylene cages bedded with rice husk. The animal room was well ventilated and maintained under standard experimental conditions (Temperature 27±2°C and 12 h light/dark cycle) throughout the experimental period. All the animals were fed with standard pellet diet and water were provided ad libitum. Before one week to the experiment, animals were accustomed to new climate and surroundings. The animal feed composition is crude protein (22.3%), crude oil (4.01%), crude fiber (4.02%), ash (8.02%) and sand silica (1.02%).

Method of ointment preparation: In this preparation 1 g of suitable extract is mixed with 10 g of ointment base (10%). Then it is stirred well until homogenous base is obtained.

Wound healing experiments: The wound site was prepared following the excision wound model (Opara, 1999). The animals were anaesthetized with diethyl ether and the hairs on the skin of the back, shaved with sterilized razor blades. The dorsal fur was shaved and a full thickness of the excision wound of 1.5 cm in width and 0.2 cm depth was done. After complete wounding, wound was washed and cleaned out with normal saline solution and the medicated cream was applied.

Experimental protocol: Animals bearing partial thickness wound were distributed into various groups such as Group I served as control, Group II served as extract and Group III served as standard treated groups. Each group had six animals. Standard drug povidone iodine (Commercially called as CIPLADINE) was selected as standard drug for the comparison of wound healing actions in experimental animals.

Wound treatment: The wounds of the animals were treated topically using the extracts. Group 2 was treated with the plant extract of Canthium coromandelicum. Group 3 (positive group) was treated with the standard drug purchased from medical store. It is a broad spectrum antibiotic used in the treatment and prevention of local infections of the teat, hoof and skin diseases in animals. While group I which was serve as the negative control group was left untreated.

Wound measurement: The measurement of the wound areas was taken from the day of the excision of the wound and every two or three days interval until the epithelization of the wound was completed. The area of the wound contraction was measured in different treated and control group on 3rd, 6th ,9th and 12th day. Wound contraction which contributes to wound closure was studied by tracing of the site. A meter ruler was placed over the wound and the estimate taken to produce a scale calculation. The percentage of wound closure was calculated as follows using the initial and final area drawn on glass slides during the experiments (Wall et al., 2002):

Where n is a No. of days (3rd, 6th ,9th and 12th day).

RESULTS AND DISCUSSION

In the present study the phytochemical screening (Table 1), antimicrobial activity and the rate of wound contraction by excision wound model was studied. The diameter sizes in mm of the zone of inhibition are shown in the Table 2. The area of wound healing expressed in percentages given in the Table 3.

Table 1:	Phytochemical screening of Canthium coromandelicum

Table 2:	Antimicrobial activity of Canthium coromandelicum extract
Values were expressed as Mean±SD

Table 3:	Percentage of wound healing activity of Canthium coromandelicum
Values were expressed as Mean±SD for six rats in each group, *Significantly different from control (p<0.001). # Significantly different from standard (p<0.01)

Fig. 1:	Percentage of wound healing activity of Canthium coromandelicum

The graphical representation of area of wound healing present in Fig. 1 and the photographical representation in Fig. 2(a-f). The percentage of wound contraction includes by recording the changes in wound area at fixed intervals of time, viz., 3rd, 6th ,9th and 12th day after treated with extract. However, on 12^th post wounding day, Group I animal showed 78.07% of healing which may be due to self immunity of animal whereas the standard treated group (Group II) showed 98.97% healing and the extract treated group (Group III) showed 90.46% healing. When obtained result compared with standard and control, the activity of the extract was found to be significant (p<0.01; p<0.001). Wound healing normally involves an initial inflammatory phase followed by fibroblast proliferation, formation of collagen fibers and shrinking, occurring concurrently but independent of one another. Several plants are having wound healing potential. Phytoconstituents such as flavonoids, glycosides and tannins are reported to have wound healing property (Raina et al., 2008; Venkatanarayana et al., 2010). Wound healing effect is also attributed to free radical scavenging activity of flavonoids.

Fig. 2(a-f):	Wound healing activity of Canthium coromandelicum, (a) Control-0th day (b) Control-12th day, (c) Plant extract-0th day, (d) Plant extract treated rat-12th day, (e) Standard drug 0th day and (f) Standard drug treated rat 12th day

Lipid peroxidation refers to the oxidative degradation of lipids, the final products of lipid peroxidation may produce cancer (Mohan et al., 2012b). The process of lipid peroxidation is important in burns, wounds and skin ulcers. It is believed that the increase in strength of collagen fibers by any drug that stops lipid peroxidation. Flavonoids, glycosides and tannins are known to promote wound healing process mainly by their astringent and antimicrobial property (Vinothapooshan and Sundar, 2010; Venkatanarayana et al., 2010).

CONCLUSION

Preliminary phytochemical analysis of the leaves of Canthium coromandelicum revealed presence of flavonoids, tannins, phenolic compounds and glycosides. Presence of flavonoids and tannins in extracts of leaves of Canthium coromandelicum may be responsible for its wound healing activity. The present study demonstrated that Canthium coromandelicum leaves extract was capable of promoting wound healing activity. Enhanced wound contractions suggest that Canthium coromandelicum leaves extract has potential in the management of wound healing when compared to standard.