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Antimicrobial, Phytochemical Analysis and Wound Healing Activity of Couroupita guianensis aubl. Abstract Couroupita guianensis is a medical plant, used in several diseases like skin diseases antibiotic, antifungal, antiseptic and analgesic qualities. The juice made from the leaves is used to cure skin diseases. The inside of the fruit can disinfect wounds and young leaves ease toothache. The methanol extract showed high antibacterial activity against Staphylococcus aureus, Bacillus subtilis, E. coli, Klebsiella. The crude methanol solvent extract showed antifungal activity against Aspergillus flavus, A. niger and Pencillium citrium. Four different compounds were obtained from TLC analysis among the four compounds two of them were major fraction, which contains higher Rf values. One of the major compound was partially purified by scrapping and elution method. The wound was created of about 500 mm2 diameter on the dorsal back of testing rats. The C. guianensis water extract ointment treated wounds was compared with control and herbal ointment. The tensile strength indicates better wound healings stimulated by applied herbal formulation. Wherever created the wound, the scar areas were cured with in nine days of period. Key words:Antimicrobial, Couroupita guianensis, HPLC, Wound healing et al., 2007). Since the role of free radicals in the physiology of wound is clearly defined and the plant has been reported to contain tannoid principles with potent antioxidant activity, we have studied the wound healing potential of the Couroupita guianesis extract. Also, since the plant has been used traditionally in the treatment of skin diseases, antimicrobial activity of the extract has been also studied for scientific validation of ethnobotanical claims. Phytochemical analysis was also studied to reveal the mechanism behind the wound healing activity. Materials and MethodsAntibacterial activity Nutrient agar plates were prepared and tested for antibacterial activity. By using sterile swabs the bacterial culture was spread on nutrient agar plates and wells were created by using puncture then appropriate amount of ethanol and methanol sample was added and incubated at 37oC for 24 hours. Antifungal activity The different concentrations (250, 500 and 750 μl) of ethanol extracts were solubilized in DMSO and incorporated in PDA media. Similar concentrations of ketoconzole used as control. The colony diameter measured for every 24 hours for 3 days. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) Methanol extract of compounds from C. guininsis were subjected to thin layer chromatography, showed four major different compounds based on the Rf values. Among four compounds two of them were major fraction Introduction Currently 80% of the world population depends on plant-derived medicine for the first line of primary health care for human alleviation because it has no side effects. In the last century, roughly 121 pharmaceutical products were formulated based on the traditional knowledge obtained from various sources (Alves and Rosa, 2007). Inflammation is often associated with pain and fever, which may be due to release of histamine, kinins, serotonin and prostaglandin (Rang, 1995). Inflammation, which constitutes a part of the acute response, results in a coordinated influx of neutrophils at the wound site. In spite of tremendous advances in the pharmaceutical drug industry, the availability of drugs capable of stimulating the process of wound repair is still limited (Udupa et al., 1995). Moreover, the management of chronic wounds is another major problem due to the high cost of therapy and the presence of unwanted side effects (Suh et al., 1998). Some important proinflammatory roles for O2 include endothelial cell damage and increased microvascular permeability, formation of chemotactic factors such as leukotriene B4, lipidic peroxidation and oxidation and DNA single strand damage (Salvemini et al., 1999). Couroupita guianensis Aubl. (Family: Lecythiadaceae) commonly known as Nagalingam in Tamil and Canaball tree in English, found throughout India in plains. The flowers are used to cure cold, intestinal gas formation and stomachache. Little work has been reported on phytochemical constituents and pharmacological actions (Khan et al., 2003). Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Proanthocyanidins and other tannins facilitate wound healing (Umachigi 38 l Herbal Tech Industry l October 2010 Review was a decrease in the epithelization period, along with a visibly decreased scar area. There was a significant increase in the tensile strength and hydroxyproline content compared to the control group and comparable to the nitrofurazone group. The observations and results obtained in this study indicate that the alcoholic extract of C. guianesis significantly stimulated wound contraction. Since the extract of Couroupita guianesis has been found to contain stigmasterol and Flavonoids are the major constituents (Rane et al., 1994; Umachigi et al., 2007). The significant antimicrobial effect of C. guianensis against pathogens confirmed that the compounds present in the crude extract are responsible for the effective antimicrobial activity. Thin layer chromatography studies indicated the presence of different compounds (Fig. 1), further confirming the synergistic action (Umachigi et al., 2007). Methanol extract of C. guianensis showed four different spots based on Rf values by TLC experiment two of them were obtained form major which contain higher Rf value. The HPLC of C. guianensis showed only the presence of 16 difference peaks with the height (19.8459, 43.7545, 7.8910, 18.2563, 4.5377, 0.7382, 1.0090, 0.6770, 0.2995, 0.1340, 0.4479, 0.4208, 0.6158, 0.5710, 0.4651, 0.3363), all these 16 molecules separated individually or compound, which contains higher Rf values. One of the major compounds was partially purified by scrapping and eluted. By using Shimadzu LC 10AT VP instrument, column Phenomenex C-18, 250 4.60 mm, mobile phase acetonitrile:water (60:40) were used to performance HPLC analysis. The flow rate was at 0.5 ml/min the absorbance used for this study 254 nm. The injected sample volume was about 20 μl. Phytochemical analysis Presence of steroids, terpenoids, alkaloids, flavonoids, tannin, proteins and stigmasterol in C. guininsis were tested. Determination of steroid—Ten percentage of concentrated sulphuric acid in alcohol was added to the sample and tested for the presence of steroids. Determination of terpenoids—The reagent was added to the sample and heated at 1200C. The presence of terpenoids were tested. Determination of carbohydrates—Ten percentage of concentrated sulphuric acid was added to the sample and heated at 120 C. Charring takes place indicating the presence of sugar. Determination of tannins—The saturated solution of ferric chloride was added to the sample and checked for the presence of tannins. Determination of flavonoids—Ten percentage of aluminium chloride in alcoholic solution was added to the sample and heated at 110 C and viewed under 366 nm for the presence of flavonoids. Determination of alkaloids—One millilitre of extract mixed with the Dragendroffs reagent. The appearance of yellow/orange precipitate was observed for the presence of alkaloids (Rang, 1995). Wound healing activity Two-hundred and Two-hundred and fifty grams rats were chosen for this wound healing activity of C. guininsis they were housed individually is standardized environmental condition (Udupa et al., 1995). The wound was created on the dorsal back of rats. The NEBASULF ointment along with standard and control were applied throughout the period, twice daily for 9 days. Results and Discussion Sixty microlitre of ethanol and methanol extract from C. guianensis showed higher inhibitory activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae (Table 1). Ethanol extract of C. guianensis gave a very good antifungal activity with Aspergillus niger, A. flavus, Penicillum citrium tested (Table 2). The results showed that upon administration of C. guianensis extract, there 0 0 38 l Herbal Tech Industry l October 2010 Table 1: Antibacterial activity of ethanol and methanol extract from Couroupita guianensis Organisms Ethanol extract Methanol extract (mm) 20 μl 40 μl 60 μl 20 μl 40 μl 60 μl Size of the inhibition zone (mm) Staphylococcus aureus Bacillus Escherichia coli Klebsiella 1.2 1.1 1.5 1.3 1.8 1.7 1.9 2.0 1.6 1.5 1.7 1.8 1.5 1.3 1.5 1.4 1.8 1.6 2.0 1.8 1.5 1.3 1.7 1.5 Table 2: Antifungal activity of methanol extract from Couroupita guianensis Organisms (mm) Aspergillus Niger Aspergillus Flavus Pencillium citrium Test Control Test Control Test Control 250 μl 500 μl 750 μl 1.0 0.8 0.5 1.4 0.6 0.4 0.2 0.8 0.4 0.6 0.8 1.6 I Day II Day 250 μl 500 μl 750 μl 1.5 1.2 0.8 1.9 0.9 0.6 0.4 1.2 1.3 1.1 1.0 1.8 III Day 250 μl 500 μl 750 μl 1.8 1.5 1.1 2.2 1.2 1.0 0.7 1.6 1.6 1.3 1.4 2.4 Figure 1. Methanol Extract of Couroupita guianensis separated by TLC. Mobile Phase 2.5:2:0.8 ratio chloroform:ethyl acetate: formic acid Figure 2: HPLC of methanol extract of Couroupita guianensis
 * K. Amutha and R. Rangaiah