Preparation and extraction of C. asiatica
Collected aerial parts of C. asiatica were oven-dried at 50°C and then powdered using a milling machine. The powdered plant (1 kg) was macerated with n- hexane (3 × 4 L, 3 days each) at room temperature. The pooled filtrates were dried under reduced pressure to give the n-hexane extract (HexE) at a constant weight of 17.8 g (0.18% yield of fresh plant). The marc was air-dried before it was further macerated with ethyl acetate and methanol, respectively, using the procedure described above to give the ethyl acetate (EtAcE, 32.7 g, 0.33% yield) and methanol (MeE, 267.5 g, 2.68% yield) extracts. Subsequently, a portion (300 g) of the dried marc was boiled in distilled water (2 L) for 3 h, then filtered and dried to give 95.1 g of the hot aqueous extract (AqE, 3.17% yield). All solvents used were of commercial grade and were obtained from U & V Holding Ltd., Bangkok, Thailand.
Tween 20® (polyoxyethylene (20) sorbitan monolaurate) as a 10% solution in distilled water was used as vehicle for preparation of a 10% (w/v) of each extract for topical application. Ten grams of the dried extract was triturated with 9 mL of vehicle. Vehicle was then added gradually until the final concentration of the extract was 10%.
Thin layer chromatography (TLC) profiling of the extracts
Characterization of the extracts was performed by TLC with a silica gel 60 F254 plate. The solvent systems used for analysis of HexE, EtAcE, MeE, and AqE were as follows: chloroform: acetate (9:1), chloroform: methanol (9:1), ethyl acetate: methanol: water (4:0.4:0.2) and ethyl acetate: methanol: water (4:0.4:0.3), respectively. The TLC plate for each extract was sprayed with 10% sulfuric acid in ethanol and then heated on a hot plate at a constant temperature of 100°C for 15 min.
A total of 112 male Sprague–Dawley rats weighing 250–300 g obtained from the National Laboratory Animal Center, Mahidol University, Nakornpathom, Thailand were used in this study. The rats were caged in special rooms with a temperature of 25 ± 1°C, free access to commercial pellet diet and water ad libitum. The rats were used 7 days after arrival in the animal facility. This 7-day period allowed the animals to acclimatize to the laboratory environment. Animal experiments in this study were carried out in accordance with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines  and the Guide for the Care and Use of Laboratory Animals of the National Research Council of Thailand. All care was taken to minimise the suffering of the animals. The experimental protocol was approved by the Ethics Committee of the Faculty of Pharmaceutical Sciences, Chulalongkorn University.
The animals were randomly divided into incision and burn wound groups with each group having 56 animals. Each group was further divided into seven subgroups composed of eight animals per subgroup: 1) untreated group, 2) NSS-treated group, 3) Tween 20®-treated (vehicle control) group, 4) HexE-treated group, 5) EtAcE-treated group, 6) MeE-treated group, and 7) AqE-treated group.
Effect of C. asiatica extracts on healing of incision wounds
The effect of Centella asiatica extracts on the healing of incision wounds was investigated by using the model of Baie and Shiekh . The animals were anesthetized intraperitoneally with sodium pentobarbital at 60 mg/kg BW during induction of the wound. The right side on the back of each animal was shaved and depilated. Next, a 3 cm long, midline incision was made through the skin with a sharp scalpel and was closed with a 0.5 cm spaced interrupted sutures with black silk no.3-0 to secure the edges. After creating the wound, 0.5 mL of the test substance was topically applied to the affected area once daily. On the seventh day after inflicting the wound, the animals were sacrificed with intra-peritoneal injection of sodium pentobarbital at 100 mg/kg BW. Sutures were removed and tissues were isolated from the healed wound to assess its tensile strength, as described below.
Assessment of the tensile strength of incision wounds
Healing of incision wounds was evaluated by measuring the tensile strength on day 7 after inflicting the wound. Sutures were removed and the skin tissue was cut 1 cm away from each side of the wound. The isolated wound tissues were used to measure the load (force) required to break the tissue with a computerized tensiometer (EZ-TEST I 30804100798, Shimadzu Corp., Japan). Tensile strength was calculated using the following formula: tensile strength (N/cm2) = breaking force (N)/area (cm2); where area (cm2) = thickness (cm) × width (cm) .
Effect of C. asiatica extracts on healing of burn wounds
The effect of C. asiatica extracts on the healing of the burn wounds was investigated using the method described by [Somboonwong et al.], which was modified from Zawacki . The animals were anesthetized with intra-peritoneal injection of sodium pentobarbital at 60 mg/kg BW during induction of the wound. The back of the animal between the lower parts of both scapulas were shaved and depilated. Next, a partial thickness burn was made by putting a hot plate (3.5 × 4.6 cm) at a temperature of 75°C on the prepared area for 10 s. The burnt area was about 10% of the total body surface area. After burning, 0.5 ml of the test substances was topically applied to the burnt area once daily. The burnt area was measured immediately after the burn and on Days 3, 7, 10 and 14 after burn injury using millimeter-scale graph paper. The degree of wound healing was calculated as described below. On day 14 after burn injury, the animals were sacrificed with intra-peritoneal injection of sodium pentobarbital at 100 mg/kg BW. Tissue of the healed wound (0.5 × 0.5 cm) was collected from each animal for histological examination.
Gross examination of the burn wound lesion
The wound was grossly examined on Days 3, 7, 10 and 14 after burn injury. The lesion of the wounds was examined using the following criteria: wound bed, color, exudates, swelling of the wound surface, and the consistency of tissues surrounding the wound.
Assessment of the degree of healing of burn wounds
On Days 3, 7, 10 and 14 after burn injury, color photographs of the wounds were taken by digital camera. The areas of the wound were measured by tracing the wound boundaries using millimeter-scale transparent graph paper with a permanent marker. The degree of wound healing was calculated using the following formula: degree of wound healing (%) = 1-[wound area on the corresponding day (cm2)/wound area on day zero (cm2)] × 100 .
Histopathological examination of burn wounds
A specimen of the skin (0.5 × 0.5 cm) was taken from the middle of the burnt area. The tissue was preserved in a 10% fresh, neutral buffered solution of formaldehyde for at least 24 h. Sections were stained with hematoxylin and eosin dyes and examined using a light microscope (Nikon 516609) with × 20 and × 40 objective lens.
Results are presented as mean ± standard error of mean (SEM). Differences among experimental groups were compared by one-way analysis of variance (ANOVA), followed by the least significant different test (LSD), and were considered significant when P was less than 0.05.