International Journal of Applied Research in Natural Products Vol. 5 (3), pp. 23-29. Directory of Open Access Journals ©2012. IJARNP-HS Publication
Original Research Wound healing activity of the leaves of Wattakaka volubilis (L.f.) Stapf (Asclepiadaceae) Ashoka Babu VL1, Goravanakolla A1, Murali A2*, Madhavan V1,Yoganarasimhan S N 1 Department of Pharmacognosy1, M.S. Ramaiah College of Pharmacy, Bangalore 560054, Karnataka, India. Department of Pharmacology2, M.S. Ramaiah College of Pharmacy, Bangalore 560054, Karnataka, India. Summary: The aqueous and alcohol extracts of the leaves of Wattakaka volubilis (L.f.) Stapf were investigated for wound healing potential by excision, incision and dead space wound models in Wistar rats. Significant wound contraction and skin breaking strength, decrease in epithelization period, increased granulation tissue formation with high breaking strength were observed in animals treated with the extracts. Histopathological sections from the alcohol extract 400 µg/ml treated animals showed predominant collagen formation with scanty macrophages whereas samples from other extract treated groups showed moderate number of macrophages, collagen formation and fibroblast. HPTLC fingerprinting studies indicated the presence of flavonoids in alcohol and aqueous extracts of the leaves of W. volubilis. Industrial relevance: The leaves of Wattakaka volubilis is traditionally used in the treatment of various types of wounds. However there is no scientific data available to support this. Hence we conducted this study to evaluate the wound healing potential of this plant in three different wound models. The phytoconstituents responsible for the wound healing property could be isolated and developed into suitable formulations which would be an effective option as a wound healing drug, on a commercial scale. Keywords: Dead space wound; Excision wound; Incision wound; Wattakaka volubilis.
INTRODUCTION Wound is defined as the disruption of the cellular and anatomic continuity of a tissue; it may be produced by physical, chemical, thermal, microbial or immunological insult to the tissue (Cherry et al., 2000). Wound care and maintenance involve a number of measures including dressing and administration of painkillers, use of antiinflammatory agents, topical and systemic antimicrobial agents and healing drugs (Nayak, 2007). A large number of plants/plant extracts/decoctions or pastes are equally used by tribals and folklore traditions in India for treatment of cuts, wounds, and burns. Plants or chemical entities derived from plants need to be identified and formulated for the treatment and management of wounds (Kumara et al., 2007). In this direction a number of plant drugs are being investigated at present. Many plant drugs have been used in management and treatment of wounds over the years. Plants and their extracts have immense potential in the management and treatment of wounds. The ______________________ *Corresponding Author.
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leaves of W. volubilis are used traditionally in Andhra Pradesh, in a paste form in the treatment of fissures in the feet and in rheumatic pain (Reddy et al., 2008); an ointment known as Hemajeevanti prepared from the leaves was found to be effective in the treatment of wounds, tinea pedis, scabies, and in plantar psoriasis (Thomas et al., 1996). The leaves are applied to boils and abscesses to promote suppuration (Chopra et al., 2002). W. volubilis is distributed throughout the hotter parts of India, Taiwan, Cambodia, Nepal and Sri Lanka (Anonymous, 1978). The chief phytoconstituents reported in the leaves and stems of W. volubilis are glycosides, flavonoids, triterpenoids and saponins (Rastogi, 1985). W. volubilis leaf extract is reported to possess antiinflammatory and analgesic activities (Divya, 2009). Plant is also reported to possess mild CNS depressant, anthelmintic, antispasmodic, cytotoxic, antimutagenic and anticancer properties (Levekar, 2007). The roots of W. volubilis are reported to possess antipyretic activity (Madhavan et al., 2010). Recent studies have shown that phytochemical
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constituents like flavonoids and triterpenoids are known to promote the wound healing process mainly due to their astringent and antimicrobial properties (Nayak, 2006). This study was undertaken to
substantiate the traditional use of the leaves of W. volubilis for the treatment of wounds which has not been reported so far (Gurudeva and Yoganarasimhan, 2009; Iyengar, 1975).
Figure 1. Flowering twig of Wattakaka volubilis
Figure 2. Fruiting twig of Wattakaka volubilis
Reprostar 3 with 12 bit CCD camera for image capturing, controlled by WinCATS- 4 software. All the solvents used were of HPLC grade, obtained from MERCK. The solvent system used for the detection of flavonoids in alcohol extracts was n-butanol: 27% aq. acetic acid (1:1) (Nair, 1975) and for aqueous extract toluene: ethyl acetate: formic acid: water (2:5:1:1) was used (Wagner and Bladt, 1996). Animals: Albino rats (Wistar strain) weighing 150-250 g of either sex were used for the study. Inbred animals were acclimatized to the experimental conditions in the animal house of M S Ramaiah College of Pharmacy, Bangalore. Animal house was well maintained under standard hygienic conditions, at a temperature (22 ± 2ºC), room humidity (60 % ± 10%) with 12 h day and night cycle, with food and water ad libitum. They were provided with commercial food pellets and purified water. Animal maintenance was in accordance with CPCSEA (Committee for the purpose of Control and Supervision of Experimentation of Animals) guidelines and the study protocol was approved by Institutional Animal Ethics Committee of M S Ramaiah College of Pharmacy (Ref.No:MSRCP/M35/2009). Acute toxicity studies on alcohol and aqueous extracts of the leaves were carried out following OECD guidelines (423). Albino rats of either sex of 8-12 weeks old weighing 170 to 200g was used.
MATERIALS AND METHODS Plant material: Leaves of W. volubilis were collected from the forests of Tirumala hills, Tirupati, Chittoor district of Andhra Pradesh during May 2010 and was authenticated by Dr. K. Madhava Chetty, Taxonomist, Dept of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A voucher herbarium specimen (Anand Goravanakolla 036) along with a sample of the test drug has been deposited at the Department of Pharmacognosy, M S Ramaiah College of Pharmacy, Bangalore (MSRCP). The leaves were shade dried, powdered and passed through sieve no. 60. Preparation of extracts: The shade dried leaf powder of W. volubilis (150 g) was extracted exhaustively with 95% v/v alcohol in a soxhlet apparatus. The alcohol extract was concentrated to a small volume and evaporated to dryness (yield 22 g). The aqueous extract was prepared by maceration of 100 g of powdered leaves with chloroform-water (0.25% v/v in distilled water), for 12 h followed by filtration and concentration of the extract to a small volume and evaporated to dryness (yield 10.2 g). The extracts were made into a suspension in distilled water and used for further studies. The alcohol and aqueous extracts of the plant were subjected to preliminary phytochemical analysis and HPTLC fingerprint studies. HPTLC studies were performed using Camag HPTLC system equipped with a Linomat V applicator, a TLC scanner 3, a 24
Wound healing activity of Wattakaka volubilis leaf
Wound healing activity: Healthy albino Wistar rats weighing 150-250 g were used in the study. Screening for wound healing activity was performed by excision, incision and dead space wound models. Excision wounds: The animals were randomly divided into 4 groups, each consisting of 6 rats. Group I served as untreated control. Group II served as standard group, treated with framycetin sulfate cream (Soframycin, Aventis). Group III was treated with aqueous extract (10% w/v) and Group IV with alcohol extract (10% w/v) of the leaves of W. volubilis, till complete epithelization. The dorsal fur of the animals was removed from the dorsal thoracic region of the rats using a depilator cream. A round seal of 2.5 cm diameter was impressed on the dorsal thoracic central region 5 cm away from the ears on the depilated part of the skin. The entire thickness of the skin from the demarcated area was excised under mild ether anesthesia. The drugs were topically applied once daily, starting from the initial day. The area of the wound was measured on 2nd, 6th, 10th, 14th and 20th day following wounding (Madhavan et al., 2006). Incision wounds: The animals were randomly divided into 4 groups, each consisting of 6 rats. Group I served as untreated control. Group II served as standard group, treated with framycetin sulfate cream (Soframycin, Aventis). Group III was treated with aqueous extract (10% w/v) and Group IV with alcohol extract (10% w/v) of the leaves of W. volubilis. The dorsal fur of the animals was removed with a depilator cream prior to the wounding. A longitudinal 6 cm long and 2 mm deep paravertebral incision, was made with a sterile scalpel through the skin and cutaneous muscle on the dorsal surface. The wounds were closed with surgical sutures at intervals of 1 cm. The extracts were topically applied once daily, starting from the initial day for 10 days. Sutures were removed on the 8th day and the tensile strength of the healed wound was measured on the 10th day, by the ‘Continuous Water Pouring Technique’ (Lee, 1970). Dead space wounds: The animals were divided into 5 groups, each consisting of 6 rats. Group I served as untreated control, Group II was treated with aqueous extract 200 mg/kg (p.o.), Group III was treated with alcohol extract 200 mg/kg (p.o.), Group IV was treated with aqueous extract 400 mg/kg (p.o.) and Group V was treated with alcohol extract 400 mg/kg (p.o.), for 10 days. The dorsal fur of the animal was removed as described earlier. A sterile polypropylene tube (2.5×0.3cm) was implanted subcutaneously beneath the dorsal paravertebral lumbar skin. Granulation tissue formed on the polypropylene tube was harvested by careful dissection on day 10 and breaking strength of the
granulation tissue was measured by ‘Continuous Water Pouring Technique’ (Lee, 1970). The granulation tissue was dried in an oven at 60°C overnight to a constant weight and the dry weights were recorded (Shanbag et al., 2006). Histopathological studies were performed on the granulation tissue in one animal of each group, to evaluate effect of the plant extracts on collagen formation (Singh, 2006). Statistical analysis: The data were expressed as Mean values ± S.E.M and were tested with One Way ANOVA followed by Tukey-Kramer multiple comparison test. RESULTS Preliminary phytochemical studies on the leaf extracts of W. volubilis revealed the presence of carbohydrates, glycosides, flavonoids, saponins, starch, phenolic compounds, gums and mucilage. HPTLC fingerprint profiles for flavonoids in alcohol and aqueous extracts were obtained and the R f values recorded. Alcohol extract revealed 10 phytoconstituents having R f values 0.05, 0.24, 0.33, 0.48, 0.54, 0.65, 0.70, 0.92, 0.94, 0.96 under the wavelength of 366 nm (Figure 3). Aqueous extract revealed 10 phytoconstituents having R f values 0.07, 0.11, 0.17, 0.23, 0.33, 0.50, 0.55, 0.73, 0.84, 0.95 under the wavelength of 366 nm (Figure 4).The R f values obtained were comparable with those available for flavonoids in the literature (Wagner and Bladt, 1996). Alcohol and aqueous extracts of the leaves did not exhibit any sign of toxicity nor lethality up to 2000 mg/kg when tested on female albino Wistar rats. In the excision wound model, significant contraction of wounds was observed in the animals treated with aqueous (P< 0. 05) and alcohol (P< 0.001) extracts from 14th day onwards, whereas in the standard group significant wound contraction (P< 0.001) was observed from 2nd day onwards. There was 97.6, 95.2, and 96.2% wound contraction respectively for standard, aqueous and alcohol extract treated animals on the 20th day. The period of epithelization was reduced significantly (P< 0.01and P< 0.001) for the extract groups compared with the control group. These observations indicate wound healing potential of the leaves of W. volubilis (Table.1). In the incision wound model, significant (P< 0.01and P< 0.001) tensile strength was observed in both the aqueous (1358.33g ± 71.20), and alcohol (1508.33g ± 134.42) extract treated groups when compared with the control group (758.33g ± 67.59).The results of alcohol extract treated group was comparable with that of the standard group (Table.2). 25
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In the dead space wound model, significant breaking strength for the granulation tissue formed was observed in animals treated with 200, 400 mg/kg of aqueous extract and alcohol extract 200 mg/kg (P
