Influence of Flower Head Aqueous Extract of Sphaeranthus indicus Linn. on Wound Healing in Albino Rats

The basic objective of the present work was to assess the wound healing activity of Sphaeranthus indicus flower head by providing better tissue formation and protection against microbial invasion. Various ointments of aqueous extracts in various proportions were prepared and subjected for assessment of wound healing activity in albino rats. Based on the comparison of wound healing activity of various formulations, the formulation comprising of 6% (w/w) aqueous extract of flower head of Sphaeranthus indicus found to be superior to that of control and standard formulation. In addition to greater hydroxyproline content found in healed wounds, the formulation also showed formation of epidermis, keratinization, connective tissue, vascular tissue and collagen at greater level as compared to control and standard formulation. Key word:Sphaeranthus indicus Hydroxyproline Ointment Tensile strength Epithelization Aqueous extract Neomycin INTRODUCTION at the site of injury and they provide key signals are Sphaeranthus indicus Linn belongs to family is the connective tissue cell responsible for collagen Asteraceae. The plant is commonly known as deposition that is needed to repair the tissue injury. Gorakhmundi in Hindi. It is an annual spreading herb, Collagen is the most abundant protein in the animal which grows approximately 15-30 cm in height. The plant kingdom, accounting for 30% of the total protein in the is distributed through out the plains and wet lands in human body [6]. In normal tissues collagen provides India, Sri Lanka and Australia. In folk medicine, the plant strength, integrity and structure. When tissues are is reportedly used in treating epileptic convulsions, disrupted following injury, collagen is needed repair the mental illnesses and hemicranias [1]. The external defect and restores anatomic structure and function. application of a paste of this herb is beneficial in treating Hence we set out to investigate wound contraction, pruritus and edema, arthritis, filariasis, gout and cervical tensile strength measurement and determination of adenopathy [2]. Essential oil, obtained by steam hydroxyproline content and histopathological studies in distillation of the whole herb, contains ocimene, rats. There was no previous report on wound healing terpinene, methyl-chavicol, -citral, geraniol, -ionone, ßactivities of Sphaeranthus indicus in literature to the best ionone, d-cadinene, p-methoxycinnamaldehyde [3] and an of our knowledge and in this paper, we report for the first alkaloid sphaeranthine [4]. time, the efficacy of Sphaeranthus indicus flower head Normal wound healing response begins the aqueous extract in the treatment and management of moment the tissue is injured. The healing cascade wounds. begins immediately following injury when the platelets come into contact with exposed collagen. As platelet MATERIALS AND METHODS aggregation proceeds, clotting factors are released resulting in the deposition of a fibrin clot at the site of Collection and Identification: Sphaeranthus indicus injury. The fibrin clot serves as a provisional matrix flower head were collected from K.C. Jain traders, Lalitpur and sets the stage for the subsequent events of healing. and identified from at the Department of Ethnobiology, The inflammatory cells also arrive along with the platelets Jiwaji University, Gwalior. known as cytokines or growth factors [5]. The fibroblast Am-Euras. J. Sci. Res., 6 (1): 13-18, 2011 14 Preparation of Extract: The flower head was shade dried, the wound were completely healed. To have uniform powdered mechanically and sieved by using a mesh (size parameters for comparison of the effects of different drugs no. 10/44). It was extracted with distill water in a soxhlet was calculated by Litchfield and Wileoxon method [12]. extractor. The concentrated material was reduced to a The time taken for epithelialization was measured in days thick mass at room temperature and water was removed by required for full epithelialization was indicated by fall of placing it in a desiccators. The weight of the dried mass scale leaving no raw wound behind. The progressive was recorded and used for experimental studies [7]. changes in wound area are monitored planimetrically by Preparation of Ointments: The general method of changes in healing of wound measurement of wound area preparation of various ointments of aqueous extract was on graph paper is expressed as unit (mm ) [13]. as follows: Dried extract was taken in glass mortar and triturated first. Then small parts of PEG-400 were added Resutured Incisional Wound Model: Incision wound were with triturating to dissolve or to suspend the drugs. inflicted by the method of Ehrlich and Hunt [14]. Groups Portions of PEG-6000 (melted at 70°C) were added to of animals containing six in each group are anaesthetized above dispersion with triturating to form a homogenous and two paravertebral long incisions of 2.5 cm length are mass of desired consistency [8]. made through the skin and cutaneous muscles at a Evaluation of Ointments for Physicochemical Parameters depilated back of rat. After mopping the wound dry, pH: The pH of all the ointments was determined using intermittent sutures were applied by surgical nylon thread digital pH meter. 0.5 g of the weighed formulation and curved needle No.11, 0.5 cm apart. On the 8 day was dispersed in 50 ml of distilled water and the pH was sutures were removed and on 10 day, the tensile strength noted [9]. was measured by the method of Lee [15]. Homogeneity: All the developed ointments were tested Tensile Strength Measurement: Tensile strength (the for homogeneity by visual inspection. They were tested force required to open the healing skin) was used to for their appearance with no lumps [10]. measure the extent of healing. The model used for this Skin Irritation Test: The test for irritation was performed fixed in one side of edge of board. Two Allis forceps, one on human volunteers. For each cream, five volunteers is fixed to the opposite side of pulley edge and another is were selected and 1 g of weighed formulation was applied tied with and hanged with rope that is attached to the pan on an area of 2 sq. inch to the back of the hand and through pulley on which the weights are placed. The covered with cotton. The volunteers were asked to report weights are increased slowly till it breaks the healed after 24 hours to observe for any reaction or irritation [10]. wound. One day before performing this experiment the Evaluation of Wound Healing Activity of Various recovery [16]. Prepared Formulation Experimental Animals: Male Albino rats of wistar strain Determination of Hydroxyproline Content in Granular (150-250 g) were housed under standard conditions of Tissue by Colorimetry: Hydroxyproline is an amino acid temperature, 12 hour light / dark and fed with standard present in the collagen fibers of granulation tissue. Its pellet diet and water ad libitum. Animals were estimation helps clinically to understand progress rate at acclimatized to laboratory conditions at least 24 hours which the healing process is going on in the connective before conducting the experiments (CPCSEA Registration tissue of the wound. The hydroxyproline contents of No.-915/ac/05/CPCSEA). the granulation tissue were calculated from standard Wound Models Excision Wound Model Histopathological Studies: On the 10 post wounding Wound Contraction Studies: A circular piece (300 mm in day, small pieces of skin were excised from the rats under 2 area) of full thickness skin was excised from the dorsal light ether anesthesia in such a way that each piece interscapular region [11]. Wound contractions were represented the skin surrounding the incision originally monitored by measuring wound area, on alternate days till made. The sections of the skin were stained with eosin tracing the wound margin on graph. To determine the