Plant material and extraction
Blechnum orientale Linn. was obtained from Putrajaya Botanical Garden, Kuala Lumpur. The identity was confirmed by plant taxonomist Anthonysamy S., formerly from University Putra Malaysia and currently a consultant with the landscape consulting firm, Aroma Tropic Limited, Kuala Lumpur. A voucher specimen (LAA007) was deposited at the Herbarium of Monash University Sunway Campus.
The extracts were prepared as previously described [7]. Briefly, powdered leaves of B. orientale were extracted with methanol at room temperature. The extract solution was filtered and the solvent was evaporated under reduced pressure. After freeze-dried, the dark green mass obtained (17% yield based on dry leaves) was suspended in distilled water (1:10, w/v) and partitioned successively with petroleum ether 40-60°C, chloroform, ethyl acetate and n-butanol. In the final partitioning with butanol, the lower water layer was removed and concentrated under reduced pressure. It was then freeze-dried to obtain a dry brown powder mass labelled as the water extract (6.5% yield based on dry leaves). The sample was stored at -70°C until used.
Quantitation of total tannins
Total tannins were determined as previously described [16]. Tannins were distinguished from nontannins by using polyvinylpolypyrrolidone (PVPP) which has a high affinity for tannins. Total phenolics content (TPC) was measured using the Folin-Ciocalteau method [7] before and after treatment with PVPP. Treatment with PVPP was conducted as follows. Distilled water (1 mL) was added to PVPP (100 mg) before adding 1 mL extract. The mixture was vortexed and centrifuged at 3000 g for 10 min. Supernatant was collected and TPC was determined as before. The standard curve was prepared using 2 - 10 μg/mL tannic acid. Tannin content was calculated as the difference between total phenolics (before PVPP treatment) and the nontannin phenolics (after PVPP treatment). Results were expressed as grams tannic acid equivalent (TAE) per 100 g dry weight.
Animals model
This study was approved by the University Ethics Committee of the Monash University for animal experimentation (SOBSB/MY/2009/46). Sprague-Dawley rats (200 - 250 g) of either sex were purchased from the Animal House of Monash University Sunway campus. Each animal was caged individually and acclimatized for 7 days, under a climate-controlled environment (22.0 ± 3°C) and relative humidity 30-70%), 12-h dark and light cycles. Standard rodent chow pellets were given ad libitum with free access to water.
Materials
Aqueous cream (manufactured by Pharmaniaga Manufacturing Bhd, Malaysia) and povidone-iodine 10% solution (manufactured by Polylab Sdn. Bhd, Malaysia) were purchased from a local pharmacy store. 4-dimethylaminobenzaldehyde and chloramines T were purchased from Acros, citric acid and sodium citrate from Fischer and hydroxyproline from Sigma. All other chemicals were of extra-pure grade and used as received.
Two concentrations of the water extract cream was formulated using aqueous cream base as the vehicle. The aqueous cream consisted of emulsifying wax (9%), white soft paraffin (15%), liquid paraffin (6%), chlorocresol (0.1%), glycerin (5%) and purified water. For 1% (w/w) extract cream, 1 g of the dry water extract was incorporated in 100 g of aqueous cream and warmed at 50-55°C, with constant stirring until a homogeneous extract-cream formation was obtained. For 2% (w/w) extract cream, 2 g of the dry water extract was used in place of 1 g of the extract. The extract cream was weighed into eppendorf tubes (approximaely 0.20 g per tube) and left to equilibriate at room temperature for 3 days, before use.
Wound healing activity
The procedure described by Nayak et al. (2009) [13] was followed with slight modifications. The animals were divided into four groups with six animals per group. Animals were anaesthetized by intraperitoneal injection of ketamine/xylazine (ketamine at 100 mg/kg and xylazine 10 mg/kg). An area (150 mm2) was marked using a frame and marker pen. The required area (approximately 5 mm bigger than the marked area) of the dorsal fur of the animals was shaved with an electric clipper. The area was sterilized by spraying with 70% ethanol. A full thickness skin (150 mm2) was excised from the predetermined area by removing the epidermis and dermis layer until the subcutaneous fat (avoiding panniculus carnosus and the muscle layer). Carprofen at 5 mg/kg was injected subcutaneously every day for 5 days as analgesia.
Group I was applied topically with aqueous cream (negative control), group II with povidone-iodine 10% (Polylab®, positive control), groups III and IV with 1% and 2% (w/w) water extract cream respectively. The reference (povidone-iodine), extract cream and the base cream were applied topically (dose approximately 0.20 g/wound) once a day until the wound was completely healed or to a maximum of 14 days. Special care was taken to avoid variation in the dose given.
Animals were monitored every day. An animal monitoring sheet was used to record all observations e.g. its activity, alertness, body condition, body weight, breathing, its coat condition, signs of dehydration, drinking, eating, conditions of its eyes, feces, nose, urine, its movement and vocalization.
The wound area contractions were measured on the 1st (wounding day) and thereafter every alternate day until completely healed. The wound margin was traced on a sterile autoclaved transparent paper (3 times to get an average area) and then placed on a graph paper to determine the area. Wound contraction was calculated as percentage reduction of initial wound area. Wounds were considered closed (completely healed) if moist granulation tissue was no longer apparent and the wound was covered with new epithelium.
After complete healing, rats were killed using carbon dioxide gas [17]. The healed skin was excised. A small piece of tissue was fixed in 10% formalin for histopathological examination. The remaining tissue was used for the determination of total collagen in the hydroxyproline assay.
Determination of total collagen - Hydroxyproline assay
The procedure used for the hydrolysis of the granulation tissue is as described by Nayak et al. (2009) [13]. The wet weight of the granulation tissue was recorded. The tissue was dried at 60°C for 12 h and the dry tissue weight recorded. To the dried tissue, 5 mL 6 N HCl was added and autoclaved at 120°C for 20 min. The neutralized acid hydrolysate of the dry tissue was used for the hydroxyproline assay.
Total collagen was determined following the method described by Jorge et al. (2008) [14]. Hydrolyzed samples (20 μL) were added to 96-well plate and incubated for 20 min at room temperature with 50 μL/well of chloramines T solution (282 mg chloramines T, 2 mL n-propanol, 2 mL distilled water, and 16 mL citrate acetate buffer). Then 50 μL/well of Erlich's solution (2.5 g 4-dimethylamino benzaldehyde, 9.3 mL n-propanol, and 3.9 mL 70% perchloric acid) was added and incubated for 15 min at 65°C. Absorbance was measured at 550 nm with a microplate reader. Hydroxyproline concentrations from 0 to 20 μg/mL were used to make a standard curve. Results were expressed as mg of hydroxyproline per g of dry tissue.
Histopathological studies
Skin specimens were immediately fixed in 10% (v/v) neutral-buffered formalin with the fixative solution replaced every 2 days until the tissues hardened. Each specimen was embedded in a paraffin block and thin sections (3 μm) were prepared and stained with Masson trichrome (for detection of collagen fibers) and haematoxylin and eosin (H&E) (for general morphological observations). Slides were examined qualitatively under a light microscope, for collagen formation, fibroblast proliferation, angiogenesis, epithelization and granulation tissue formation, employing light to intense scale (+ to +++) [15, 18].
Statistical analysis
All data were expressed as mean ± SD. Statistical analyses were evaluated by one-way ANOVA followed by Tukey HSD test. Values with P < 0.001 were considered statistically significant.
