Wound healing properties of Aloe Vera-Scratch Assay

Wound healing properties of Aloe Vera-Scratch AssayType of manuscript:Original research.Running Title:Wound healing properties of Aloe Vera-Scratch AssayManya SureshUndergraduate StudentSaveetha Institute of medical and technical sciences,Chennai, India.Lakshmi ThangaveluAssistant Professor Department of PharmacologySaveetha Dental College, Saveetha University, ChennaiAnitha RoyAssistant Professor Department of Pharmacology  Saveetha Dental College, Saveetha University,ChennaiTelephone no:Email:[email protected] : To study the wound healing properties of Aloe Vera by conducting a scratch assay.Materials and Methods:Aloe vera extract used in this study was obtained from Green chemical ,Bengaluru,India. FBS was obtained from Sigma Aldrich.co.india. The normal mouse skin fibroblast line (c147) employed in this study were obtained from a NCCS, Pune. The in vitro scratch assay was carried out to detect the effect of Aloe vera extract on the migration of mouse skin fibroblast cells. Wound closure was examined by the quantity of transferred fibroblasts from the edge of the nick in extract treated wells in comparison to the control wells for 48h in four separate fields.Results:The Aloe vera extract at 50µg/ml, 100µg/ml as well as epidermal growth factor 4ng/ml exhibited progressive wound closure compared to control and DMSO group.Conclusion:Aloe vera extract possess wound healing properties by exhibiting progressive wound closure compared to control and DMSO group.Keywords:Aloe Vera,Scratch assay,Wound healing,extract,epidermal growth factor.IntroductionTreatment of wounds is a frequent indication recorded in ethnopharmacological studies. Many traditional medicines are used for cleaning or treating wounds, but only a few have been tested pharmacologically for their wound healing potentials.[1]The Aloe vera plant has been known and used for centuries for its health, beauty, medicinal and skin care properties. The name Aloe vera derives from the Arabic word “Alloeh” meaning “shining bitter substance,” while “vera” in Latin means “true.” 2000 years ago, the Greek scientists regarded Aloe vera as the universal panacea. The Egyptians called Aloe “the plant of immortality.”[2]Aloe Vera is scientifically known as A. barbadensis Mill.Its healing property is related to a compound that is called glucomannan, which is enriched with polysaccharides like mannose. The glucomannan affects fibroblast growth factor and stimulates the activity and proliferation of these cells and in turn improves collagen production and secretion. The mucilage of aloe vera not only increases amount of collagen on wound site, but also increases transversal connections among these bands rather than creation of change in collagen structure and as a result accelerates wound improvement [3].Using medicinal herbs has been noticed in therapy of types of wound from the very beginning. Due to reduced financial load and its medical effects, these plants are noticed by the people. Several plants are used traditionally in treatment of many skin wounds and burnings in various points of the world [4]. Wound healing property is related to a compound that is called glucomannan, which is enriched by polysaccharides like mannose.The glucomanna affects fibroplast growth factor and stimulates the activity and proliferation of these cells and in turn improves collagen production and secretion.The mucilage of aloe vera not only increases amount of collagen, but also increases transverse connections among these bands rather than creation of change in collagen structure and as a result accelerate wound improvement(6).Results of in Citroen studies on effect of aloe Vera on cell proliferation are contradictory.One explanation is that the sap could have cytotoxic activity while the gel might promote cell growth.(7)Aloe Vera is useful in treating wounds and burns,minor skin infections, cysts, diabetes, and elevated blood lipids in humans, and shows some promise in treating more serious and persistent conditions such as eczema, genital herpes, dandruff, psoriasis, canker sores, skin ulcers and others. Other uses include, provide rich nutrient for good health, acts as moisturiser, treats acne, lessens the visibility of stretch marks,soothes in periodontal disease, and also aids in digestion.  The Egyptians used aloe vera plant for treatment of wounds, burns, and infections for the first time. After them, Greeks, Spanish, and African peoples used aloe vera plant by various techniques for several purposes. According to classic medicine in Iran, aloe vera has hot and dry humor and its extract is used for medicinal purpose. Aloe vera gel contains two hormones namely,Auxin and gibberellins that is responsible for providing wound healing and anti-inflammatory properties which reduce skin inflammation. A. vera inner extract increases Vitamin C and E absorption after application. A. vera is not recommend in combination with antidiabetic, diuretic, laxative drugs, sevoflurane, and digoxin.(19) Today, the aloe vera plant is used for various purposes. In this study,we are doing a scratch assay to assess the wound healing properties of aloe Vera.(8)Materials and MethodsPlant materials:   Aloe vera extract used in this study was obtained from Green chemical ,Bengaluru,India.Chemicals used:      FBS was obtained from Sigma Aldrich.co.india.All the other chemicals used in the study were up to analytical gradeCell cultureThe normal mouse skin fibroblast line (c147) employed in this study were obtained from a NCCS, Pune and were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Biosera, France) + fetal bovine serum (FBS) 10% (Invitrogen, USA) medium, 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, USA). Cells were kept under standard culture conditions at 37°C and 5% CO2. All cells were used between passages 5 and 6. Trypsin 0.025%-ethylene diamine tetra acetic acid 0.02% (Sigma-Aldrich, USA) in phosphate-buffered saline was used to separate fibroblast cells from the flasks.Cell migration assayThe in vitro scratch assay was carried out to detect the effect of Aloe vera extract on the migration of mouse skin fibroblast cells. Fibroblasts were seeded at high density on a 24-well plate in RPMI 1640 medium containing 100 U/mL penicillin and 100 µg/mL streptomycin and 10% FBS. After 24 h, fibroblasts were attached and spread to form a confluent monolayer. Cell monolayer was scraped with a tip. Parted cells were removed and the attached cells were incubated with 500 ?L of RPMI medium containing 5% FBS, 50?g/mL and 100µg/mL of Aloe vera, epidermal growth factor (4ng/ml) and incubated at 37°C, 5% CO2 and 90% humidity. Wound closure was examined by the quantity of transferred fibroblasts from the edge of the nick in extract treated wells in comparison to the control wells for 48h in four separate fields.Results The figure shows the images of scratch assay on mouse fibroblast cell lines after 48h injury with and without treatments. The Aloe vera extract at 50µg/ml, 100µg/ml as well as epidermal growth factor 4ng/ml exhibited progressive wound closure compared to control and DMSO group. The enhanced migration and wound closure exhibited by aloe vera extract indicates the wound healing property of aloe vera extract, compared with that of growth factor. The images were micrographed at magnification of 10X. Discussion  From the above results of the present study it is evident that The Aloe vera extract at 50µg/ml, 100µg/ml as well as epidermal growth factor 4ng/ml exhibited progressive wound closure compared to control and DMSO group. The enhanced migration and wound closure exhibited by aloe vera extract indicates the wound healing property of aloe vera extract, compared with that of growth factor.Despite its widespread popularity, scientific evidence on aloe vera remains sparse. Aloe vera gel is regarded as safe  if applied topical with only a few allergic reactions being reported.9 The efficacy of aloe vera gel to treat burn wounds, genital herpes, and seborrheic dermatitis have been shown in clinical trials, but other indications such as psoriasis or internal application for the treatment of type 2 diabetes remain inconclusive. The major application of aloe vera gel remains as a skin moisturizer in cosmetics and as an après treatment for sunburns, for which it has proven its effectiveness.10,11In a similar study “Effectiveness of Aloe Vera Gel compared with 1% silver sulphadiazine cream as burn wound dressing in second degree burns”,conducted by Muhammad Naveed Shahzad and Naheed Ahmed they stated that “aloe vera gel promoted wound healing in second degree burn patients better than SSD cream. The mechanism of the remarkable efficacy of aloe cream in the healing of burn injuries may be explained by its cell proliferation, and anti-inflammatory effects. It also relieves pain better than SSD and is cost effective.”(12)The gel of the aloe vera plant has a long folk history in the treatment of skin conditions. There is some evidence from human and animal studies that aloe might be helpful for wound healing, (13-14) but one study found that aloe gel actually slowed the healing of surgical wounds. 15 Also, a review of 7 trials involving 347 people did not find evidence that aloe can improve wound healing. 16 But ,In another study there were twenty-seven patients with partial thickness burn wound, they were treated with aloe vera gel compared with vaseline gauze. It revealed the aloe vera gel treated lesion healed faster than the vaseline gauze area. The average time of healing in the aloe gel area was 11.89 days and 18.19 days for the vaseline gauze treated wound. Statistical analysis by using t-test and the value of P < 0.002 was statistically significant. In histologic study, it showed early epithelialization in the treated aloe vera gel area. Only some minor adverse effects, such as discomfort and pain were encountered in the 27 cases. This study showed the effectiveness of aloe vera gel on a partial thickness burn wound, and it might be beneficial to do further trials on burn wounds.(17)Conclusion  Aloe vera extract possess wound healing properties by exhibiting progressive wound closure compared to control and DMSO group. In recent times, there has been an increase in demand for alternative medicine. Medicinal plants, such as Aloe vera, have natural phytochemicals and have proven to be better than synthetic drugs(20) .In conclusion, the use of aloe vera extract and its components for the treatment of a wounds needs further clinical evidence through well-designed studies with defined aloe extracts .In (June 2012), 5 national and international clinical studies were listed by the United States National Institutes of Health clinical trial database with a major emphasis on the use of aloe vera in the treatment of wounds.18 This indicates the scientific significance of aloe vera  and the need to establish it as a valid treatment option for wounds. However, the use of aloe vera in topical applications has widely been confirmed in the clinical studies as safe.References1.Freiesleben, Sara H. et al. “Determination of the Wound Healing Potentials of Medicinal Plants Historically Used in Ghana.” Evidence-based Complementary and Alternative Medicine?: eCAM 2017 (2017): 9480791. PMC. Web. 17 Dec. 2017.2. Hashemi, Seyyed Abbas, Seyyed Abdollah Madani, and Saied Abediankenari. “The Review on Properties of Aloe Vera in Healing of Cutaneous Wounds.” BioMed Research International 2015 (2015): 714216. PMC. Web. 17 Dec. 2017.3. Boudreau M. D., Beland F. A. An evaluation of the biological and toxicological properties of Aloe barbadensis (Miller), Aloe vera. Journal of Environmental Science and Health—Part C: Environmental Carcinogenesis and Ecotoxicology Reviews. 2006;24(1):103–154. doi: 10.1080/10590500600614303.  4. Muthu C., Ayyanar M., Raja N., Ignacimuthu S. Medicinal plants used by traditional healers in Kancheepuram District of Tamil Nadu, India. Journal of Ethnobiology and Ethnomedicine. 2006;2, article 43 doi: 10.1186/1746-4269-2-43.  5. Chen HH, Zhou HJ, Fang X. Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro. Pharmacol Res. 2003;48:231–6.6.Boudreau MD, Beland FA.An evaluation of the biological and toxicological properties of Aloe barbadensis (miller), Aloe vera.J Environ Sci Health C Environ CarcinogEcotoxicol Rev. 2006 Apr;24(1):103-54.[PMD].7.Yagi A, Egusa T, Arase M, et al. Isolation and characterisation of the glycoprotein fraction with a proliferation-promoting activity on human and hamster cells in vitro from Aloe vera gel. Planta Med 1997; 63: 18-21. 8. Sandhya1, Dr. Gowri2  , Effects of Aloe Vera on Skin and on Wound Healing - A Review,International journal of science and research,Volume 6 Issue 4,April 2017.9.  Thornfeldt C. Cosmeceuticals containing herbs: fact, fiction, and future. Dermatol Surg. 2005;31:873-80.10. Diehl B, Teichmueller EE. Aloe vera, Quality inspection and identification. Agro Food Ind HiTech. 1998;9:14-6.11. Thamlikitkul V, Bunyapraphatsara N, Riewpaiboon W, Theerapong S, Chantrakul C, Thanaveerasuwan T. Clinical trial of aloe vera Linn. for treatment of minor burns. Siriraj Hosp Gaz. 1991;43(5):313-316.12.Shahzad MN, Ahmed N,Effectiveness of Aloe Vera gel compared with 1% silver sulphadiazine cream as burn wound dressing in second degree burns, J Pak Med Assoc. 2013 Feb;63(2):225-30.13. Chithra P, Sajithlal GB, Chandrakasan G. Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats. Mol Cell Biochem. 1998;181:71-76.14. Fulton JE Jr. The stimulation of postdermabrasion wound healing with stabilized aloe vera gel-polyethylene oxide dressing. J Dermatol Surg Oncol. 1990;16:460-467.15. Schmidt JM, Greenspoon JS. Aloe vera dermal wound gel is associated with a delay in wound healing. Obstet Gynecol . 1991;78:115-117.16. Jull AB, Walker N, et al. Honey as a topical treatment for wounds. Cochrane Database Syst Rev . 2013 Feb 28;2:CD005083.17.Visuthikosol, V & Chowchuen, Bowornsilp & Sukwanarat, Y & Sriurairatana, S & Boonpucknavig, V. (1995). Effect of Aloe vera gel to healing of burn wound– a clinical and histologic study. Journal of the Medical Association of Thailand = Chotmaihet thangphaet. 78. 403-9. 18. ClinicalTrials.gov. National Institues of Health (NIH), National Library of Medicine (NLM), Department of Health and Human Service (HHS); 2008. Accessed 08/03/2008.19.K.Deepthi,R.Pradeep Kumar , Aloe Vera -Nature’s healer, JPSR journals,vol:12 issue:1.20. Arundhathy A Nair et al / The Comparison of the Antiplaque Effect of Aloe Vera, Chlorhexidine and Placebo Mouth Washes on Gingivitis Patients, J. Pharm. Sci. & Res. Vol. 8(11), 2016, 1295-1300