Plant material and extract preparation
The flowers of C. roseus were collected locally in April 2006 and identified by the plant taxonomist and curator, National Herbarium of Trinidad and Tobago, The University of the West Indies, St. Augustine, Trinidad and a voucher specimen was also deposited at the herbarium (specimen number: 36458). The fresh flowers were shade dried and ground into a powder using an electric blender. The fine powder (50 g) was suspended in 100 ml of ethanol for 20 hours at room temperature. The mixture was filtered using a fine muslin cloth followed by filter paper (Whatman No: 1). The filtrate was placed in a water bath to dry at 40°C and the clear residue was used for the study. The extract was subjected to preliminary phytochemical tests.
The study was approved by the Ethics Committee for animal experimentation (AHC06/07/1), The Faculty of Medical Sciences, The University of the West Indies, St. Augustine
Healthy inbred gender-matched Sprague Dawley rats weighing 200–220 g were used for the study. They were individually housed and maintained on normal food and water ad libitum. Animals were periodically weighed before and after the experiment. The rats were anaesthetized prior to and during infliction of the experimental wounds. The surgical interventions were carried out under sterile conditions using ketamine anaesthesia (120 mg/kg). Animals were closely observed for any infection and those which showed signs of infection were separated and excluded from the study and replaced.
An acute toxicity study was conducted for the extract by the stair-case method . The animals were fed with increasing doses of (1, 2, 4, and 8 g/kg body weight).
Excision, incision and dead space wound models were used to evaluate the wound-healing activity of C. roseus
Excision wound model
Animals were anaesthetized prior to and during creation of the wounds. The rats were inflicted with excision wounds as described by Morton and Malon . The dorsal fur of the animals was shaved with an electric clipper and the anticipated area of the wound to be created was outlined on the back of the animals with methylene blue using a circular stainless steel stencil. A full thickness of the excision wound of 2.5 cm (circular area = 300mm2) in length and 0.2 cm depth was created along the markings using toothed forceps, a surgical blade and pointed scissors. The entire wound was left open . The animals were divided into two groups of 6 each. Group 1 animals were topically treated with the carboxymethyl cellulose (100 mg/kg/day) as a placebo control. The animals of group 2 were topically treated with the ethanol extract of C. roseus at a dose of 100 mg/kg/day) till complete epithelization. The wound closure rate was assessed by tracing the wound on days 1, 5 and 15 post-wounding using transparency paper and a permanent marker. The wound areas recorded were measured using a graph paper. Number of days required for falling of eschar without any residual raw wound gave the period of epithelization.
Incision wound model
As with the above model rats were anaesthetized prior to and during creation of the wound. The dorsal fur of the animals was shaved with an electric clipper. A longitudinal paravertebral incision, six centimeters in length was made through the skin and cutaneous muscle on the back as described by Ehrlich and Hunt et al. . After the incision, surgical sutures were applied to the parted skin at intervals of one centimetre. The wounds were left undressed. The rats were given flower extract (dissolved in drinking water) orally at a dose of 100 mg kg-1 day-1. The controls were given with normal saline. The sutures were removed on the 8th post wound day and the treatment was continued. The skin-breaking strength was measured on the 10th day by the method described by Lee .
Dead space wound model
Dead space wounds were inflicted by implanting two sterilized cotton pellets (10 mg), one on either side of in the lumbar region on the ventral surface of each rat. On the 10th postwounding day, the granulation tissue formed on the implanted cotton pellet was carefully removed. The wet weight of the granulation tissue was noted. These granulation tissues were dried at 60°C for 12 hours, and weighed, and the weight was recorded. To the dried tissue added 5 ml 6 N HCl and kept at 110°C for 24 hours. The neutralized acid hydrolysate of the dry tissue was used for the determination of hydroxyproline .
Determination of wound breaking strength
The anesthetized animal was secured to the table, and a line was drawn on either side of the wound 3 mm away from the line. This line was gripped using forceps one at each end opposed to each other. One of the forceps was supported firmly, whereas the other was connected to a freely suspended light weight metal plate. Weight was added slowly and the gradual increase in weight, pulling apart the wound edges. As the wound just opened up, addition of weight was stopped and the weights added was noted as a measure of breaking strength in grams. Three readings were recorded for a given incision wound, and the procedure was repeated on the contralateral wound. The mean reading for the group was taken as an individual value of breaking strength. The mean value gives the breaking strength for a given group
Estimation of Hydroxyproline
Hydroxyproline present in the acid hydrolysate of granulation tissue oxidized by sodium peroxide in the presence of copper sulfate, when complexed with para-dimethylaminobezaldehyde, develops a pink color that was measured at 540 nm using colorimetry
Phytochemical screening methods
Test for saponins: Boiled 300 mg of extract with 5 ml water for two minutes. Mixture was cooled and mixed vigorously and left it for three minutes. The formation frothing indicates the presence of saponins.
Test for tannins: To an aliquot of the extract added sodium chloride to make to 2% strength. Filtered and mixed with 1% gelatin solution. Precipitation indicates the presence of tannins.
Test for Triterpenes: 300 mg of extract mixed with 5 ml chloroform and warmed for 30 minutes. The chloroform solution is then treated with a small volume of concentrated sulphuric acid and mixed properly. The appearance of red color indicates the presence of triterpenes.
Test for alkaloids: 300 mg of extract was digested with 2 M HCl. Acidic filtrate was mixed with amyl alcohol at room temperature, and examined the alcoholic layer for the pink colour which indicates the presence of alkaloids.
Test for flavonoids: The presence of flavonoids was determined using 1% aluminum chloride solution in methanol, concentrated HCl, magnesium turnins, and potassium hydroxide solution.
The thin layer chromatography of the ethanol extract was done using following medium as mobile phase:
Petroleum ether: ethyl acetate (4:1 × 1 vol/vol)
Chloroform: methanol (4:1 × 1 vol/vol)
Chloroform: ethanol (1:1 × 1 vol/vol)
Pseudomonas aeruginosa, Beta-hemolytic streptococci, Enterobacter agglumerans and Staphylococcus aureus were the organisms tested. The bacterial strains were obtained from fresh colonies grown on Mac Conkey and blood agar plates. The sensitivity testing was done using Muller Hinton Agar plates. Known volume of bacterial suspension was transferred to each microplate well. Ten microlitres of C. roseus extract dissolved in deionised water (200 μg/ml) was added to the microplate wells and incubated at 35–37°c for 18–20 h. Results were analyzed visually on the basis of turbid zone of inhibition. [+ = bacterium colonies deposited in the bottom of the well, ++ = turbidity with bacterium colonies being deposited, +++ = light turbidity, and ++++ = total growth inhibition].
Results, expressed as mean ± SD were evaluated using Student's t-test and significance was set at p < 0.05.