EVALUATION OF WOUND HEALING POTENTIAL OF CRUDE LEAVE EXTRACTS OF BIXA ORELLANA LINN. IN WISTAR RATS.

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Subhashree et al. SJIF Impact Factor Volume 5, Issue 8, Research Article ISSN EVALUATION OF WOUND HEALING POTENTIAL OF CRUDE LEAVE EXTRACTS OF BIXA ORELLANA LINN. IN WISTAR RATS. 1 *Subhashree Padhi and 2 Sangram K. Panda Jeypore College of Pharmacy, Rondapalli, Jeypore , Koraput, Odisha, India. Article Received on 08 June 2016, Revised on 28 June 2016, Accepted on 18 July 2016 DOI: /wjpps *Corresponding Author Subhashree Padhi Jeypore College of Pharmacy, Rondapalli, Jeypore , Koraput, Odisha, India. ABSTRACT The present study was an attempt to investigate the wound healing potential in two different types of wound models in albino rats viz., incision and excision. of various extracts of Bixa orellana leave. The different crude extracts such as ethanol, ethyl acetate, methanol and petroleum ether are obtained by successive soxhlet extraction. The ethanolic extract showed significant increase in wound contraction and formation of scar in excision wound model. The extract showed significant increase in the breaking strength of resutured incision wound as compared to control group(p<0.05). The result of the present study indicate that ethanolic extract of B. orellana has more significant wound healing property than the other three extracts in excision and incision wound model. KEYWORDS: Bixa orellana L.; Excision and incision wound model. INTRODUCTION Herbal medicine has become an integral part of standard healthcare, Wounds are inescapable events of life, which arise due to physical or chemical injury or microbial infections. Herbal preparations can be more effective than conventional medicines and their non-toxic nature means that they can be administered over long periods. The tribal areas of Koraput (District) of Eastern Orissa(India) due to its unique varieties geographical and climatic factors have had a rich variety of medicinal plant Bixa orellana also known as sindur (Oriya) were frequently distributed and extensively used traditionally by the tribal people for curing their aliments. Bixa orellana L. (Bixaceae), commonly known as annatto in English, Sinduri in Sanskrit and sindur in Odia is indigenous and native to tropical America but now cultivated in many tropical countries including India. [1,3]. Bixa orellana is an evergreen shrub or small tree, 2-8 m high bark light to dark brown, tough, smooth, sometimes. Leaves spirally arranged, simple, Vol 5, Issue 8,

2 stipulate, ovate, x 4-16 cm, shallowly cordate to truncate at base, longley acuminate at apex, green or dark green above, grey or brownish. Flowers in terminal branched panicles, 8-50 flowered, covered with reddishbrown scales; petals 4-7, obovate, 2-3 x 1-2 cm, pinkish, whitish. Fruit a spherical or broadly elongated ovoid capsule, 2-4 x cm, flattened, green, greenish-brown or red when mature; seeds numerous, with bright orange-red fleshy coats [4,6]. Traditionally the plant was used as a colouring agent, it is also used to colour butter, cheese, beverages and fish and meat products. it has been used as an ingredient in weight- loss products and also in the treatment of snakebite. It is also used in the formation of herbal lipstick. Annatto possesses various pharmacological activities like anti-diarrheal, antiinflammatory, antioxidant, hypoglycemic, anti- bacterial. B. orellana is known to have bioactivity, particularly regarding seed and leaf extracts. Scientific evidences show that it possesses antioxidant, antimicrobial, anticonvulsant, antidiabetic and cardio-protective activity [7,10]. The decoction of leaves is used to prevent vomiting and nausea; to treat urinary difficulties and stomach problems. [11] Roots and leaves of the plant are useful for the treatment of sore throat, jaundice, snake bites, dysentery, gonorrhea, liver disease, diuretic and antipyretic agent including malaria [12]. MATERIALS AND METHODS Collection of Plant Material The leaves of Bixa orellana were collected from the Herbal garden of Jeypore college of pharmacy, Jeypore, Koraput district.(india) in the month of December 2015.The plant was identified, confirmed and authenticated by the Biju Patnaik Medicinal Plants Garden and Research Centre, Dr. M. S. Swami Nathan Research Foundation, Jeypore, Koraput (District), Orissa (Letter No. MJ/SS/P-305/15,dated ( ). After authentification leaves were collected in bulk and washed under running tap water to remove adhering dirt. Then the leaves were shade dried. The dried materials were made into coarse powder by grinding in mechanical grinder. and stored in a closed air tight container for further use. Preparation of Extracts The coarse powder was taken in Soxhlet apparatus and extracted successively with ethanol, ethyl acetate, methanol and petroleum ether as solvent. A total amount of 650 g coarse powder was extracted with 1200 ml of each solvent. For each solvent,10 cycles were run to obtain a thick slurry. Each slurry was then concentrated under reduced pressure to obtain the Vol 5, Issue 8,

3 crude extract. All crude extracts were kept in closed air tight containers under cool and dark place for further study [13,14]. phytochemical investigation The crude ethanol, ethyl acetat, methanol and petroleum ether extracts of the leaf of Bixa orellana were subjected to preliminary phytochemical analysis in order to detect the presence of various groups of phytoconstituents by carrying out the chemical analysis [14,15] Animals Healthy adult wisterstrain of albino rats weighing approximately 180 to 250gms were used They were housed in standard conditions of temperature (25±2 C), 12 hours light per day cycle, relative humidity of % in animal house of Jeypore College of Pharmacy. They were fed with standard pellets of food and water. Animals were kept and all operation on animals was done in aseptic condition. All the studies conducted were approved by the Institutional Animal Ethical Committee (1200/ac/08/CPCSEA), Dadhichi college of pharmacy, Vidya vihar, Cuttack, according to prescribed guide-lines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Wound healing Activity Wound model Excision wound For the excision wound study, animals were divided into 5 groups of six rats in each group. Group-I served as control and applied with Vaseline, Group-II, Group-III, Group-IV and Group-V were treated with ethanol, ethyl acetate, methanol and petroleum ether extracts respectively. An impression was made on the dorsal thoracic central region 5mm away from the ears, by using a round seal of 2.5 cm diameter as described by Morton and Malone [16]. The skin of the impressed area was excised to the full thickness to obtained area of about 500 mm2 under light ether anaesthesia in aseptic condition. The animals were housed indivisualy. The methanol extracts in simple ointment base (5% w/w) were applied on the wound once a day for 18 days starting from the day of wounding. The percentage wound closure was observed on 4th, 8th, 12th, 16th, 18th post wounding day. Epithelization time (in days) and size of the scar area was noted. Vol 5, Issue 8,

4 Incision wound Incision wound model was performed according to Ehrlich and Hunt [17]. The animals were divided into 5 groups of six rats in each group, and kept in separate cage. Group-I served as control, received only 2% gum acacia suspension (1 ml/kg,p.o), ethanol, ethyl acetate, n- butanol and petroleum ether extracts (250 mg/kg) were given orally once a day to group-ii, III, IV and V respectively for 10 days. Under light ether anesthesia, the animals were secured to operation table in its natural position. Two paravertebral straight incisions of 6 cm each were made through the entire thickness of the skin, on either side of the vertebral column with help of sharp blade. Removal of the sutures was done on 8th post wounding day. Tensile strength was determined on both wounds by continuous constant water flow technique of Lee [18]. Statistical analysis The results are reported as Mean±SE. Statistical analysis was done using ANOVA (Tukey- Multiple Copmarision Test).When probability (p) was less than 0.05 was considered as significant. [19] Wound model Excision wound For the excision wound study, animals were divided into 5 groups of six rats in each group. Group-I served as control and applied with Vaseline, Group-II, Group-III, Group-IV and Group-V were treated with ethanol, ethyl acetate, methanol and petroleum ether extracts respectively. An impression was made on the dorsal thoracic central region 5mm away from the ears, by using a round seal of 2.5 cm diameter as described by Morton and Malone [16]. The skin of the impressed area was excised to the full thickness to obtained area of about 500 mm2 under light ether anaesthesia in aseptic condition. The animals were housed indivisualy. The methanol extracts in simple ointment base (5% w/w) were applied on the wound once a day for 18 days starting from the day of wounding. The percentage wound closure was observed on 4th, 8th, 12th, 16th, 18th post wounding day. Epithelization time (in days) and size of the scar area was noted. Incision wound Incision wound model was performed according to Ehrlich and Hunt [17]. The animals were divided into 5 groups of six rats in each group, and kept in separate cage. Group-I served as Vol 5, Issue 8,

5 control, received only 2% gum acacia suspension (1 ml/kg,p.o), pet.ether, ethyl acetate, methnol, ehanol extracts (250 mg/kg) were given orally once a day to group-ii, III, IV and V respectively for 10 days. Under light ether anesthesia, the animals were secured to operation table in its natural position. Two paravertebral straight incisions of 6 cm each were made through the entire thickness of the skin, on either side of the vertebral column with help of sharp blade. Removal of the sutures was done on 8th post wounding day. Tensile strength was determined on both wounds by continuous constant water flow technique of Lee [18]. Statistical analysis The results are reported as Mean±SE. Statistical analysis was done using ANOVA (Tukey- Multiple Copmarision Test).When probability (p) was less than 0.05 was considered as significant. [19] Table-1: Effect of extracts of Bixa orellana leave on the breaking strength in incision wound Sl no. Group Breaking strength 1 Control ± petroleum ether ± ethyl acetate ± methanol ± ethanol ± 16.24* Values are mean ± SE (n 6)* p<0.05 vs control Figure-1 4th 8th 12th 16th 18 th Vol 5, Issue 8,

6 Figure-2 4th 8th 12th 16th 18 th RESULT AND DISCUSSION The preliminary phytochemical screening showed that the different solvent extracts of B. orellana contains alkaloids, flavonoids, terpenoids, saponins, glycoside, steroids and tannins in all the solvent extracts & carbohydrates absent in all the extracts. The ethanol extract yielded strongly, all the phytochemicals followed by petroleum ether, methanol and ethyl acetate. The pet.ether extract also yielded only flavonoid, glycoside and tannin at the poor presence. In the study using excision wound model, animals treated with ethanol extract of B. orellana leave showed significant decrease in epithelization period as evidenced by shorter period for fall of eschar as compared to control group(p<0.05) (Fig-1). The extract also facilitated the increase in rate of wound contraction than control group. The petroleum ether extract treated animal (Group-II) showed wound contraction by %. The ethyl acetate extract treated animals (Group-III) showed wound contraction by 73.33%. The methanol extract treated animal (Group-IV) showed wound contraction by 77.23%,The ethanol extract treated animal (Group-V) showed wound contraction by % as compared with the control (Group-I) by 63.27%.in all the extract.( Fig-2 )The result of present study reveals that ethanolic leave extracts of B. orellana possess a prominent prohealing activity in incision wound model. This was demonstrated by significant increase in the skin tensile strength in methanol extract treated groups (p<0.05) on 10th post wounding day are presented in (Table- 1). Vol 5, Issue 8,

7 CONCLUSION In the present study, wound healing activity of Bixa orellana was studied and the results of the present study suggest that local application and systemic administration of ethanol extract of the leaf has shown more significant wound healing activity in excision and incision wound models and support the popular use of plant to open wound in folk medicine. The wound healing property of B.orellana has been attributed to its antimicrobial effects. The presence of phytoconstituents like flavonoids, terpenoids, saponins, phenols, steroids and tannins either individually or combined together may exhibit the synergistic effect towards healing of wounds. However, further investigation employing isolation of constituents and screening models are needed for further confirmation of wound healing potential of Bixa orellana leave.thus The traditional use has been pharmacologically validated. ACKNOWLEDGEMENTS Authors wish to thank to local people of south eastern Odisha and the Biju Patnaik Medicinal Plants Garden and Research Centre. Jeypore, Koraput (District), Orissa, India. for providing valuable information about the plant and its identification. The author wish to express their gratitude to Jeypore College of Pharmacy, Rondapalli, Jeypore, Koraput, Odisha. REFERENCES 1. Kirtikar KR, Basu BD. Indian medicinal plants. Periodical Experts Books Agency, Delhi 1935; 4(2): Cooke T. The Flora of Presidency of Bombay. Botanical Survey of India, Calcutta. Vol. II Naik VN. Flora of Marathwada. Amrut Prakashan, Aurangabad, Vol. 1, Anon. The cultivation of annatto. Farmer Jamaica 1962; 67(5): Anon. The useful plants of India. Publications & Information Directorate, CSIR, New Delhi, India Dendy DAV. Annatto the pigment of Bixa orellana. East African Agriculture and Forestry Journal 1996; 32(2): Patnaik S, Mishra SR and Choudhury GB. Phytochemical investigation and simultaneously study on anticonvulsant, antidiabetic activity of different leafy extracts of Bixa orellana Linn. International Journal of Pharmaceutical and Biological Archives 2011; 2(5): Vol 5, Issue 8,

8 8. Selvi AT, Dinesh MG, Satyanet RS, Chandrasekaran B and Rose C. Leaf and seed extracts of Bixa orellana L. exert anti-microbial activity against bacterial pathogens. Journal of Applied Pharmaceutical Science 2011; 1(9): Asokkumar K, Jagannath P, Umamaheswari M, Sivashanmugam T, Subhadradevi V and Madeswaran A. Cardioprotective activity of Bixa orellana L. on isoproterenol induced myocardial necrosis in rats. Journal of Pharmacy Research 2012; 5(4): Viuda-Martos M, Ciro-Gomez GL and Ruiz-Navajas Y. In vitro antioxidant and antibacterial activities of extracts from annatto (Bixaorellana L.) leaves and seeds. Journal of Food Safety 2012; 32(4): Giorgi A, De Marinis P, Granelli G, Chiesa LM and Panseri S. Secondary Metabolite Profile, Antioxidant Capacity, and Mosquito Repellent Activity of Bixa orellana from Brazilian Amazon Region. Journal of Chemistry 2013; Castello MC, Phatak A, Chandra N and Saron M, Antimicrobial activity of crude extracts from plant parts and corresponding calli of Bixaorellana L. Indian J Exp. Biol 2002; 40: CK Kokate; AP Purohit; SB Gokhale. Pharmacognosy, Nirali Prakashan, Pune, 2007; JB Harbone. Phytochemical methods, London:Chapman and Hall, Ltd. 1973; GE Trease; WC Evans; Trease and Evans. Pharmacognosy, A Physician s Guide to Herbal Medicine, 13 th ed., Bailliere Tindall, London, 1989; J.P Morton, M.H Malon. Arch Int Pharmacodyn Ther, 1972; 196: H.P Ehrlich, T.K Hunt. J Ann of Surg, 1969; 170: K.H Lee. J Pharmacol Sci, 1968; 57: , OECD, Guideline for the Testing of Chemicals, Guidance document on acute toxic class method, Vol 5, Issue 8,