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Syzygium mundagam bark methanol extract restores skin to normal in diabetic wounded rats
Biomedicine & Pharmacotherapy 94 (2017) 781–786
Available online at
ScienceDirect www.sciencedirect.com
Original article
Syzygium mundagam bark methanol extract restores skin to normal in diabetic wounded rats Rahul Chandrana,* , Heidi Abrahamsea , Thangaraj Parimelazhaganb , Gowtham Duraib a b
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, 17011, South Africa Bioprospecting Laboratory, Department of Botany, Bharathiar University, Coimbatore, Tamil Nadu 641046, India
A R T I C L E I N F O
Article history: Received 21 March 2017 Received in revised form 13 July 2017 Accepted 24 July 2017 Keywords: Diabetic wound Epithelialization Neutrophils Fibroblasts Syzygium mundagam
A B S T R A C T
Diabetic wounds have been identified as one of the major complications associated with diabetes. This study features the use of Syzygium mundagam bark methanol (SMBM) extract in the treatment of wounds in Streptozotocin-Nicotinamide induced diabetic rats. The extract ointment base, at 1 and 2% respectively, was applied to the wounded areas on the rats and monitored for 21 days. The wound closure, epithelialization period and histopathology of the wounds were evaluated during the study. Both the concentrations of the extract (1% and 2%) healed the wounds even under diabetic conditions induced in rats on day 21 (99.69% and 100% respectively). The 2% SMBM treated animals showed a higher rate of epithelialization of the wound (15 0.49 days). The histopathology of the wounded skin on day 10 revealed that the rats treated with SMBM extract could initiate the healing and re-epithelialization. This was evident from the migration of neutrophils and proliferation of fibroblasts. On the 21st day, complete healing of the skin could be observed in the rats treated with 2% extract which was evident from the newly formed epidermis, collagen fibers and fibroblast. The results compared well with those treated with betadine (5%). The results of this study will support the use of this plant extract for diabetic healing over the use of commercially available synthetic drugs. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Incidence of Diabetes Mellitus (DM) is increasing throughout the world with an immediate appeal to researchers to find a reliable therapeutic modality. Diabetic foot ulcers is one of the major complications among diabetic patients. Wounds, under diabetic conditions show delayed healing due to microbial infection, generation of reactive oxygen species and reduced blood flow. In the past decades, an increase in such complications associated with DM is being observed all over the world [1,2]. It is predicted that under chronic conditions, patients will suffer from diabetic ulcers, especially at the proximal regions of the limb [3]. Undeniably, it can lead to limb infection, decay of tissues, limb amputation, and even death if not treated properly [4]. Diabetic wounds are also associated with multiple risk factors which have to be taken into consideration before treatment [5,6]. The risk
* Corresponding author at: Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, South Africa. E-mail addresses: [email protected] (R. Chandran), [email protected] (H. Abrahamse), [email protected] (T. Parimelazhagan), [email protected] (G. Durai). http://dx.doi.org/10.1016/j.biopha.2017.07.114 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
factors include: longer duration, high Body Mass Index, ageing, and other issues such as; diabetic peripheral neuropathy, diabetic retinopathy, high glycated hemoglobin level (HbA1C), foot deformity, high plantar pressure, peripheral vascular disease etc. [7,8]. Peripheral sensorimotor and autonomic neuropathy leads to high foot pressure, foot abnormalities, and gait instability. These pathways advance foot complications in diabetic patients, which accelerates the chances of ulcer progression [9]. A control over the blood glucose along with alternative therapies would be an ideal measure to treat diabetic foot ulcers and wounds. The search for cost-effective medication with maximum healing properties and minimal to no side effect has led scientists to investigate plants as an alternative source of medicinal products. The family Myrtaceae is estimated to contain more than 5500 species. Among them, many spp. of Syzygium have been studied extensively for various properties including the treatment of diabetes. The fruits of S. mundagam (Bourd.) Chitra are eaten by the Paniya and Kuruma tribes of Kerala, India [10]. Chandran et al. [11] have reported the anti-hyperglycaemic property of bark methanol extract of S. mundagam. However, earlier reports revealed that this plant was given less attention in view of its anti-diabetic properties. Hence, this study was focused on the wound healing property of S. mundagam in diabetic rats.
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2. Materials and methods 2.1. Collection of plant material S. mundagam bark was collected during October 2011 from Chanthanathodu, Wayanad, Kerala, India. The authenticity was confirmed (Voucher no: CMPR 7932) by Dr. M. Prabhukumar, Scientist and Head-in-Charge, Plant Systematic and Genetic Resources Division, Centre for Medicinal Plant Research, Arya Vaidya Sala, Kottakkal, Kerala, India. The freshly collected bark was cleaned, shade dried and ground into a fine powder before extraction.
base alone); Group III: Diabetic wound rats with SMBM extract (1%); Group IV: Diabetic wound rats with SMBM extract (2%); Group V: Diabetic wound rats with standard drug betadine (5%). A standardized wound area (2 cm2 and 0.2 cm depth) was created on the shaved dorsal skin of the diabetic rats under anesthesia with diethyl ether. The therapeutic property was analysed by percentage wound shrinkage on 5th, 10th, 15th and 21st day and period of epithelialization. The granulomatous tissue from the wounded area was taken for histopathological analyses [12,13]. The wound tissues from the treatment groups were cut into 2 mm sections using a microtome. The sections were then fixed in 10% formalin and stained with haematoxylin and eosin.
2.2. Methanol extraction of bark 2.6. Ointment preparation The fine powders of bark were packed into thimbles (100 g) and extracted in Soxhlet apparatus with methanol (300 mL). The solvent extract was concentrated using a rotary vacuum evaporator (Equitron Ev11-ABS.051) and then air dried. 2.3. Animals and ethics Healthy female Swiss albino mice weighing 20–25 g and Wistar albino rats (100–150 g) of approximately the same age, were used for the acute toxicity and diabetic wound studies respectively. The animals were fed with water (ad libitum) and a standard chow diet throughout the study period. The animals were maintained in clean polypropylene cages at room temperature. The experimental protocol was examined and approved by the institutional animal ethics committee (KMCRET/PhD/04/2012-13). 2.4. Acute dermal toxicity
The ointment was prepared by the trituration method as mentioned in British Pharmacopoeia [14]. Briefly, cetostearyl alcohol (0.5 g) and hard paraffin (0.5 g) were melted and was stirred well with yellow soft paraffin (8.5 g) and wool fat (0.5 g) until all the ingredients were melted. The mixture was stirred until uniform mixing and cold. The unwanted particles were removed by decantation. Then, 100 and 200 mg/kg of SMBM extract were added to the ointment base to get 1 and 2% respectively. 5% betadine was used as a standard. 2.7. Statistical analyses The results were analysed and shown as a mean SEM. The data obtained from the study were analysed statistically using oneway ANOVA followed by Dunnett’s t-test (SPSS version 17.0). Values at p < 0.05, p < 0.01 and p < 0.001 were considered statistically significant.
Acute dermal toxicity was performed as per the OECD guidelines 402 (1987) to determine any allergic reaction and adverse effects after applying a concentration of the test substance on the skin. Here, the test was done using Syzygium mundagam bark methanol (SMBM) extract to determine the therapeutic dose. The ointment containing SMBM extract with the highest concentration of 5% (w/w) was uniformly applied on the shaved dorsal skin of a rat and observed for any sign of toxic or allergic reactions for 48 h.
SMBM extract was found to be non-toxic, and treated rats did not show any sign of allergic reactions when applied with 5% extract. Based on the safety test, 1% and 2% of the extract were fixed for the diabetic wound study.
2.5. In vivo diabetic wound healing activity
3.2. Diabetic wound
To induce diabetes, rats were administered intraperitoneally (i. p.) with 120 mg/kg nicotinamide. After 15 min, 60 mg/kg streptozotocin (STZ) was injected (i.p.) and monitored. The elevated blood glucose and hyperglycemia were confirmed in rats at 72 h and on 10th day after injection. The rats with no change in blood glucose level (>250 mg/dL) on the 10th day were used for the diabetic wound study. Animals were divided into five groups of six rats each and treated for 21 days. Group I: Untreated diabetic control rats with wound; Group II: Diabetic control rats with wound (ointment
The topical application of SMBM extracts (1% and 2%) demonstrated a significant reduction in the wound areas. Both the concentrations of the extract (1% and 2%) could treat the wound completely under severe diabetic conditions of the rats on day 21 (99.69% and 100% respectively). The wound contraction and healing were evident from day 10. Table 1 and Fig. 1 depicted that 2% SMBM treated animals have faster epithelialization at the wound site (15.49 days) than the animals treated with betadine (5%) (17.40 days).
3. Results 3.1. Dermal toxicity study
Table 1 Diabetic wound healing activity of SMBM extract. Groups and Dose
Wound contraction (%) Day 5
Day 10
Day 15
Day 21
(Days)
Ointment base only Control SMBM (1%) SMBM (2%) Betadine (5%)
28.31 8.38 23.94 6.06 24.56 8.35 25.88 8.37 22.81 11.69
67.19 10.15 52.31 6.91 84.44 1.10* 83.13 2.17* 57.56 10.03
74.81 14.28 64.06 3.36 94.94 0.74* 96.88 0.83** 86.94 4.51*
89.50 6.10*** 69.12 2.21 99.69 0.31*** 100.00 0.00*** 99.25 0.48***
23.60 6.34 23.61 1.69 15.80 0.12 15.49 0.13 17.40 0.95
Epithelialization period
The data represents mean SEM (n = 6). Significantly different at * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to control. SMBM- S. mundagam bark methanol extract.
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Fig. 1. The figure shows the progression of wound healing from day 1 to 21. The wound closure can be clearly observed at the end of the study in extract treated rats compared to diabetic control.
3.3. Histopathology On day 10, the wound section (Fig. 2) showed full thickness of ulceration on the epidermis with the proliferation of fibroblasts and neutrophil infiltrations forming granulation at the wound site of both untreated and ointment base treated rats. The reduction in neutrophil population and increased migration of fibroblast clearly indicates the shift from inflammatory phase to epithilialization phase. These changes are visible in extract and ointment treated groups. The rats treated with betadine (5%) also had the similar cellular architecture with ulceration of epidermis and focal
fibroblast proliferation with formation of granulation on the dermis. The histopathologic evaluation of rats treated with SMBM 1% expressed a normal morphology of the epidermal skin. The dermis indicated dense proliferation of fibroblasts and thin walled congested vessels with very few scattered neutrophils and lymphocytic infiltrates. The rats treated with SMBM 2% revealed signs of re-epithelialisation. The proliferation of fibroblasts, capillaries and neutrophils were observed as in 1% extract treated rats. Formation of blood vessels is evidence of angiogenesis. The fibroblasts and collagen fibers are well organised in SMBM treated groups compared to untreated and ointment base treated rats.
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Fig. 2. The histopathology of wound skin on day 10 display the initial stages of healing with migration of neutrophils and fibroblast formation.
On the 21st day, the wound section of ointment base treated rats (Fig. 3) displayed skin with final stages of healing with dense fibroblast proliferation and scattered inflammatory cells. However, incomplete healing was observed with migrated neutrophils and inflammatory cells at the wound site of untreated rats. Thin walled capillaries and congested vessels forming granulation tissues can also be seen. Betadine (5%), SMBM (1%) and SMBM (2%) groups show profuse proliferation of fibroblasts and collagen fibers indicating complete re-epithelialization. A prominent epidermal layer seen in the betadine treated group is a good indication of healing. The tissue cells are more compact and dense in SMBM and betadine treated groups compared to loosely arranged cells of diabetic control and ointment base treated group. The thin walled capillaries and mature granulation tissue in the wound area indicate healed wound.
4. Discussion The dermal toxicity study showed that the extracts are devoid of any substances, or levels of compounds, that may cause an allergic reaction to the skin. Hence, the result recommends the safety level of the extract and its use in biological studies. Activation of macrophages, endothelial cells and fibroblasts are the major cellular responses involved in wound healing. In a diabetic wound, delayed healing was observed due to certain physiological conditions including diminished fibroblast proliferation [15], impaired angiogenesis [16] and increased inflammatory responses [17]. It has been reported that increased blood glucose inhibits the proliferation of primary dermal fibroblast in patients and could not reverse the growth inhibition even after the addition of epidermal growth factor and insulin-like growth factor-I [15]. Increased
R. Chandran et al. / Biomedicine & Pharmacotherapy 94 (2017) 781–786
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Fig. 3. On day 21, the skin depicts densely proliferated fibroblast, collagen fibers, reduced neutrophils, and epidermis formation, indicating the efficient healing capacity of SMBM extract.
inflammatory cytokine, tumor necrosis factor-a (TNF-a) expression and poor vasculatures of diabetic wounds were also observed in diabetic mice [16,17]. However, the treatment with SMBM extract might have reversed these conditions to faster healing of wound. Pain and inflammation are the major initial phases which come into action to initiate wound healing. But the inflammatory mediators like bradykinins and prostaglandins can worsen the condition if the wounds are deep and accompanied with diabetes. SMBM might have controlled the further progression of these mediators and accelerated the formation of fibroblast and collagen fibers for faster healing. Lack of inflammation at the wound site towards the end of the study is an evidence for the effect. Immediately after injury, an increase in collagen fiber proliferation is observed in the wound area. This extracellular protein is abundantly found in the granulation tissue of a healing wound. Collagen is significantly involved in providing strength and integrity to a tissue matrix. In addition, collagen also plays an
important role in epithelialization process [18]. In the study, it was clearly observed that t treatment with the extract activated the fibroblast cells and collagen fibers, which might be the reason for complete healing on 21st day. It can also be assumed that one of the factors which induced faster healing of wound is the suppression of TNF-a by the extract. Other mechanisms would be angiogenesis and promotion of fibroblast growth. The wound site is rich in free radicals like superoxide which elevate the severity of a wound. Hence the removal of this radical either by the antioxidant property of extract [11] or by the stimulation of SOD production at wound site would help in faster healing of wound. Neutrophils are the innate immune cells which initiate the inflammatory response in wound healing process and are designated as the initial line of cells involved in defence. The neutrophils play an important role at the wound site by destructing the invading microbes and decontaminating the open wound. The immediate response of neutrophils reduces the chance of
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infectious complication at the injury site [19]. However, there are studies that highlight the negative role of neutrophils [20], where they activate protease enzyme which increases the severity of the wound. The cellular architecture of the wound tissue is in agreement with both the statements. On day 10 neutrophil migration to the wound site can be observed which could be due to its protective action against microbial infections. The lower number of neutrophils on the 21st day could be an indication of improved healing. The histopathology of the wounded tissue on 10th and 21st day showed an increase in the collagen fiber and fibroblast formation in the treated groups compared to untreated control, which signifies the healing efficiency of the SMBM extract. This impairment might have delayed the proliferation and remodelling phases in untreated control rats. Fernandes et al. [21] studied the wound healing property of Psidium guajava hydroalcoholic extract of leaf and reported the enhanced proliferation of fibroblast during wound healing. Similar observation was made in a study by Palanimuthu et al. [22], where complete healing was observed in the rats treated with bark ethanol extract of Eugenia jambolana. 5. Conclusion The results of diabetic wound study revealed that 1% and 2% SMBM extract could heal the wound completely under diabetic conditions in the rats after 21 days of treatment. The histopathology of the wounded skin depicted that the rats treated with SMBM (2%) extract could initiate the healing and re-epithelialization at a faster rate. The study has proved the potential of bark methanol extract to heal the diabetic wound, where wound healing is usually prolonged due to several complications. The phytoconstituents of SMBM extract might have accelerated the rate of healing by reducing microbial infection, antioxidative property and decreased neutrophil migration. Hence, the plant, S. mundagam, can be recommended for the use in the treatment of diabetic wounds and further research is underway to evaluate the role of extract and its chemical compounds in other diabetic complications. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors are grateful to Department of Science and Technology, Govt. of India (DST/INSPIRE Fellowship 2010/ IF10618) and Innovation in Science Pursuit for Inspired Research (INSPIRE) program for providing financial support for carrying out the work. This work is also based on the research supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (Grant No. 98337).
References [1] M. Aalaa, O.T. Malazy, M. Sanjari, M. Peimani, M. Mohajeri-Tehrani, Nurses’ role in diabetic foot prevention and care; a review, J. Diabetes Metab. Disord. 28 (11) (2012) 540–543. [2] A. Alavi, R.G. Sibbald, D. Mayer, L. Goodman, M. Botros, D.G. Armstrong, K. Woo, T. Boeni, E.A. Ayello, R.S. Kirsner, Diabetic foot ulcers: part II. Management, J. Am. Acad. Dermatol. 70 (1) (2014) 21.e1–21.e4. [3] S. Leone, R. Pascale, M. Vitale, S. Esposito, Epidemiology of diabetic foot, Infez. Med. 20 (1) (2012) 8–13. [4] R.J. Snyder, J.R. Hanft, Diabetic foot ulcers-effects on QOL, costs and mortality and the role of standard wound care and advanced-care therapies, Ostomy Wound Manage. 55 (11) (2009) 28–38. [5] M.S. Bortoletto, S.M. de Andrade, T. Matsuo, C. Haddad Mdo, A.D. González, A. M. Silva, Risk factors for foot ulcers – a cross sectional survey from a primary care setting in Brazil, Prim. Care Diabetes 8 (1) (2014) 71–76. [6] R. Waaijman, M. de Haart, M.L. Arts, D. Wever, A.J. Verlouw, F. Nollet, S.A. Bus, Risk factors for plantar foot ulcer recurrence in neuropathic diabetic patients, Diabetes Care 37 (6) (2014) 1697–1705. [7] B. Iraj, F. Khorvash, A. Ebneshahidi, G. Askari, Prevention of diabetic foot ulcer, Int. J. Prev. Med. 4 (3) (2013) 373–376. [8] L.N. McEwen, K.R. Ylitalo, W.H. Herman, J.S. Wrobel, Prevalence and risk factors for diabetes-related foot complications in translating research into action for diabetes (TRIAD), J. Diabetes Complic. 27 (6) (2013) 588–592. [9] C. Formosa, A. Gatt, N. Chockalingam, Diabetic foot complications in Malta: prevalence of risk factors, Foot (Edinb.) 22 (4) (2012) 294–297. [10] M.K.R. Narayanan, N. Anilkumar, V. Balakrishnan, M. Sivadasan, A.H. Ahmed, A. A. Alatar, Wild edible plants used by the Kattunaikka: Paniya and Kuruma tribes of Wayanad District, Kerala, India, J. Med. Plants Res. 5 (15) (2011) 3520– 3529. [11] R. Chandran, T. Parimelazhagan, B.P. George, Antihyperglycemic activity of the bark methanolic extract of Syzygium mundagam in diabetic rats, Alex. J. Med. (2017), doi:http://dx.doi.org/10.1016/j.ajme.2016.12.001. [12] P.K. Mukherjee, Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals, 1st ed., Business Horizons, New Delhi, 2002, pp. 816. [13] V.R. Bharathi, Antioxidant and wound healing studies on different extracts of Stereospermum colais leaf, Int. J. Res. Pharm. Sci. 1 (4) (2010) 435–439. [14] British Pharmacopoeia, British Pharmacopoeia Commission, Vol. 2, HMSO Publication Centre, London, 1993 pp. 1096. [15] K. Hehenberger, A. Hansson, High glucose-induced growth factor resistance in human fibroblasts can be reversed by antioxidants and protein kinase C inhibitors, Cell. Biochem. Funct. 15 (3) (1997) 197–201. [16] R.D. Galiano, O.M. Tepper, C.R. Pelo, K.A. Bhatt, M. Callaghan, N. Bastidas, S. Bunting, H.G. Steinmetz, G.C. Gurtner, Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells, Am. J. Pathol. 164 (6) (2004) 1935–1947. [17] G. Naguib, H. Al-Mashat, T. Desta, D.T. Graves, Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation, J. Invest. Dermatol. 123 (1) (2004) 87–92. [18] B.C. Nwomeh, H.X. Liang, I.K. Cohen, D.R. Yager, MMP-8 is the predominant collagenase in healing wounds and non-healing ulcers, J. Surg. Res. 81 (2) (1999) 189–195. [19] V.D. Julia, M.S. Anna, L.A. DiPietro, Neutrophil function in the healing wound: adding insult to injury? Thromb. Haemost. 92 (2004) 275–280. [20] G.S. Ashcroft, K. Lei, W. Jin, G. Longenecker, A.B. Kulkarni, T. Greenwell-Wild, H. Hale-Donze, G. McGrady, X.Y. Song, S.M. Wahl, Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing, Nat. Med. 6 (10) (2000) 1147–1153. [21] K.P.S. Fernandes, S.K. Bussadori, M.M. Marques, E. Bach, N. Wadt, E.M. Santos, V.C. Pavesi, M.D. Martins, Healing and cytotoxic effects of Psidium guajava (Myrtaceae) leaf extracts, Braz. J. Oral. Sci. 9 (4) (2010) 457–466. [22] D. Palanimuthu, S. Nandagopal, M.D. Jalaludeen, K. Subramonian, A. Saravana Ganthi, S. Sankaram, Wound healing activities of Eugenia jambolana lam. Bark extracts in albino rats, Int. J. App. Biol. Pharmaceut. Technol. 2 (1) (2011) 112– 116.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Syzygium mundagam bark methanol extract restores skin to normal in diabetic wounded rats Rahul Chandrana,* , Heidi Abrahamsea , Thangaraj Parimelazhaganb , Gowtham Duraib a b
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, 17011, South Africa Bioprospecting Laboratory, Department of Botany, Bharathiar University, Coimbatore, Tamil Nadu 641046, India
A R T I C L E I N F O
Article history: Received 21 March 2017 Received in revised form 13 July 2017 Accepted 24 July 2017 Keywords: Diabetic wound Epithelialization Neutrophils Fibroblasts Syzygium mundagam
A B S T R A C T
Diabetic wounds have been identified as one of the major complications associated with diabetes. This study features the use of Syzygium mundagam bark methanol (SMBM) extract in the treatment of wounds in Streptozotocin-Nicotinamide induced diabetic rats. The extract ointment base, at 1 and 2% respectively, was applied to the wounded areas on the rats and monitored for 21 days. The wound closure, epithelialization period and histopathology of the wounds were evaluated during the study. Both the concentrations of the extract (1% and 2%) healed the wounds even under diabetic conditions induced in rats on day 21 (99.69% and 100% respectively). The 2% SMBM treated animals showed a higher rate of epithelialization of the wound (15 0.49 days). The histopathology of the wounded skin on day 10 revealed that the rats treated with SMBM extract could initiate the healing and re-epithelialization. This was evident from the migration of neutrophils and proliferation of fibroblasts. On the 21st day, complete healing of the skin could be observed in the rats treated with 2% extract which was evident from the newly formed epidermis, collagen fibers and fibroblast. The results compared well with those treated with betadine (5%). The results of this study will support the use of this plant extract for diabetic healing over the use of commercially available synthetic drugs. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Incidence of Diabetes Mellitus (DM) is increasing throughout the world with an immediate appeal to researchers to find a reliable therapeutic modality. Diabetic foot ulcers is one of the major complications among diabetic patients. Wounds, under diabetic conditions show delayed healing due to microbial infection, generation of reactive oxygen species and reduced blood flow. In the past decades, an increase in such complications associated with DM is being observed all over the world [1,2]. It is predicted that under chronic conditions, patients will suffer from diabetic ulcers, especially at the proximal regions of the limb [3]. Undeniably, it can lead to limb infection, decay of tissues, limb amputation, and even death if not treated properly [4]. Diabetic wounds are also associated with multiple risk factors which have to be taken into consideration before treatment [5,6]. The risk
* Corresponding author at: Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, South Africa. E-mail addresses: [email protected] (R. Chandran), [email protected] (H. Abrahamse), [email protected] (T. Parimelazhagan), [email protected] (G. Durai). http://dx.doi.org/10.1016/j.biopha.2017.07.114 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
factors include: longer duration, high Body Mass Index, ageing, and other issues such as; diabetic peripheral neuropathy, diabetic retinopathy, high glycated hemoglobin level (HbA1C), foot deformity, high plantar pressure, peripheral vascular disease etc. [7,8]. Peripheral sensorimotor and autonomic neuropathy leads to high foot pressure, foot abnormalities, and gait instability. These pathways advance foot complications in diabetic patients, which accelerates the chances of ulcer progression [9]. A control over the blood glucose along with alternative therapies would be an ideal measure to treat diabetic foot ulcers and wounds. The search for cost-effective medication with maximum healing properties and minimal to no side effect has led scientists to investigate plants as an alternative source of medicinal products. The family Myrtaceae is estimated to contain more than 5500 species. Among them, many spp. of Syzygium have been studied extensively for various properties including the treatment of diabetes. The fruits of S. mundagam (Bourd.) Chitra are eaten by the Paniya and Kuruma tribes of Kerala, India [10]. Chandran et al. [11] have reported the anti-hyperglycaemic property of bark methanol extract of S. mundagam. However, earlier reports revealed that this plant was given less attention in view of its anti-diabetic properties. Hence, this study was focused on the wound healing property of S. mundagam in diabetic rats.
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R. Chandran et al. / Biomedicine & Pharmacotherapy 94 (2017) 781–786
2. Materials and methods 2.1. Collection of plant material S. mundagam bark was collected during October 2011 from Chanthanathodu, Wayanad, Kerala, India. The authenticity was confirmed (Voucher no: CMPR 7932) by Dr. M. Prabhukumar, Scientist and Head-in-Charge, Plant Systematic and Genetic Resources Division, Centre for Medicinal Plant Research, Arya Vaidya Sala, Kottakkal, Kerala, India. The freshly collected bark was cleaned, shade dried and ground into a fine powder before extraction.
base alone); Group III: Diabetic wound rats with SMBM extract (1%); Group IV: Diabetic wound rats with SMBM extract (2%); Group V: Diabetic wound rats with standard drug betadine (5%). A standardized wound area (2 cm2 and 0.2 cm depth) was created on the shaved dorsal skin of the diabetic rats under anesthesia with diethyl ether. The therapeutic property was analysed by percentage wound shrinkage on 5th, 10th, 15th and 21st day and period of epithelialization. The granulomatous tissue from the wounded area was taken for histopathological analyses [12,13]. The wound tissues from the treatment groups were cut into 2 mm sections using a microtome. The sections were then fixed in 10% formalin and stained with haematoxylin and eosin.
2.2. Methanol extraction of bark 2.6. Ointment preparation The fine powders of bark were packed into thimbles (100 g) and extracted in Soxhlet apparatus with methanol (300 mL). The solvent extract was concentrated using a rotary vacuum evaporator (Equitron Ev11-ABS.051) and then air dried. 2.3. Animals and ethics Healthy female Swiss albino mice weighing 20–25 g and Wistar albino rats (100–150 g) of approximately the same age, were used for the acute toxicity and diabetic wound studies respectively. The animals were fed with water (ad libitum) and a standard chow diet throughout the study period. The animals were maintained in clean polypropylene cages at room temperature. The experimental protocol was examined and approved by the institutional animal ethics committee (KMCRET/PhD/04/2012-13). 2.4. Acute dermal toxicity
The ointment was prepared by the trituration method as mentioned in British Pharmacopoeia [14]. Briefly, cetostearyl alcohol (0.5 g) and hard paraffin (0.5 g) were melted and was stirred well with yellow soft paraffin (8.5 g) and wool fat (0.5 g) until all the ingredients were melted. The mixture was stirred until uniform mixing and cold. The unwanted particles were removed by decantation. Then, 100 and 200 mg/kg of SMBM extract were added to the ointment base to get 1 and 2% respectively. 5% betadine was used as a standard. 2.7. Statistical analyses The results were analysed and shown as a mean SEM. The data obtained from the study were analysed statistically using oneway ANOVA followed by Dunnett’s t-test (SPSS version 17.0). Values at p < 0.05, p < 0.01 and p < 0.001 were considered statistically significant.
Acute dermal toxicity was performed as per the OECD guidelines 402 (1987) to determine any allergic reaction and adverse effects after applying a concentration of the test substance on the skin. Here, the test was done using Syzygium mundagam bark methanol (SMBM) extract to determine the therapeutic dose. The ointment containing SMBM extract with the highest concentration of 5% (w/w) was uniformly applied on the shaved dorsal skin of a rat and observed for any sign of toxic or allergic reactions for 48 h.
SMBM extract was found to be non-toxic, and treated rats did not show any sign of allergic reactions when applied with 5% extract. Based on the safety test, 1% and 2% of the extract were fixed for the diabetic wound study.
2.5. In vivo diabetic wound healing activity
3.2. Diabetic wound
To induce diabetes, rats were administered intraperitoneally (i. p.) with 120 mg/kg nicotinamide. After 15 min, 60 mg/kg streptozotocin (STZ) was injected (i.p.) and monitored. The elevated blood glucose and hyperglycemia were confirmed in rats at 72 h and on 10th day after injection. The rats with no change in blood glucose level (>250 mg/dL) on the 10th day were used for the diabetic wound study. Animals were divided into five groups of six rats each and treated for 21 days. Group I: Untreated diabetic control rats with wound; Group II: Diabetic control rats with wound (ointment
The topical application of SMBM extracts (1% and 2%) demonstrated a significant reduction in the wound areas. Both the concentrations of the extract (1% and 2%) could treat the wound completely under severe diabetic conditions of the rats on day 21 (99.69% and 100% respectively). The wound contraction and healing were evident from day 10. Table 1 and Fig. 1 depicted that 2% SMBM treated animals have faster epithelialization at the wound site (15.49 days) than the animals treated with betadine (5%) (17.40 days).
3. Results 3.1. Dermal toxicity study
Table 1 Diabetic wound healing activity of SMBM extract. Groups and Dose
Wound contraction (%) Day 5
Day 10
Day 15
Day 21
(Days)
Ointment base only Control SMBM (1%) SMBM (2%) Betadine (5%)
28.31 8.38 23.94 6.06 24.56 8.35 25.88 8.37 22.81 11.69
67.19 10.15 52.31 6.91 84.44 1.10* 83.13 2.17* 57.56 10.03
74.81 14.28 64.06 3.36 94.94 0.74* 96.88 0.83** 86.94 4.51*
89.50 6.10*** 69.12 2.21 99.69 0.31*** 100.00 0.00*** 99.25 0.48***
23.60 6.34 23.61 1.69 15.80 0.12 15.49 0.13 17.40 0.95
Epithelialization period
The data represents mean SEM (n = 6). Significantly different at * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to control. SMBM- S. mundagam bark methanol extract.
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Fig. 1. The figure shows the progression of wound healing from day 1 to 21. The wound closure can be clearly observed at the end of the study in extract treated rats compared to diabetic control.
3.3. Histopathology On day 10, the wound section (Fig. 2) showed full thickness of ulceration on the epidermis with the proliferation of fibroblasts and neutrophil infiltrations forming granulation at the wound site of both untreated and ointment base treated rats. The reduction in neutrophil population and increased migration of fibroblast clearly indicates the shift from inflammatory phase to epithilialization phase. These changes are visible in extract and ointment treated groups. The rats treated with betadine (5%) also had the similar cellular architecture with ulceration of epidermis and focal
fibroblast proliferation with formation of granulation on the dermis. The histopathologic evaluation of rats treated with SMBM 1% expressed a normal morphology of the epidermal skin. The dermis indicated dense proliferation of fibroblasts and thin walled congested vessels with very few scattered neutrophils and lymphocytic infiltrates. The rats treated with SMBM 2% revealed signs of re-epithelialisation. The proliferation of fibroblasts, capillaries and neutrophils were observed as in 1% extract treated rats. Formation of blood vessels is evidence of angiogenesis. The fibroblasts and collagen fibers are well organised in SMBM treated groups compared to untreated and ointment base treated rats.
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Fig. 2. The histopathology of wound skin on day 10 display the initial stages of healing with migration of neutrophils and fibroblast formation.
On the 21st day, the wound section of ointment base treated rats (Fig. 3) displayed skin with final stages of healing with dense fibroblast proliferation and scattered inflammatory cells. However, incomplete healing was observed with migrated neutrophils and inflammatory cells at the wound site of untreated rats. Thin walled capillaries and congested vessels forming granulation tissues can also be seen. Betadine (5%), SMBM (1%) and SMBM (2%) groups show profuse proliferation of fibroblasts and collagen fibers indicating complete re-epithelialization. A prominent epidermal layer seen in the betadine treated group is a good indication of healing. The tissue cells are more compact and dense in SMBM and betadine treated groups compared to loosely arranged cells of diabetic control and ointment base treated group. The thin walled capillaries and mature granulation tissue in the wound area indicate healed wound.
4. Discussion The dermal toxicity study showed that the extracts are devoid of any substances, or levels of compounds, that may cause an allergic reaction to the skin. Hence, the result recommends the safety level of the extract and its use in biological studies. Activation of macrophages, endothelial cells and fibroblasts are the major cellular responses involved in wound healing. In a diabetic wound, delayed healing was observed due to certain physiological conditions including diminished fibroblast proliferation [15], impaired angiogenesis [16] and increased inflammatory responses [17]. It has been reported that increased blood glucose inhibits the proliferation of primary dermal fibroblast in patients and could not reverse the growth inhibition even after the addition of epidermal growth factor and insulin-like growth factor-I [15]. Increased
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Fig. 3. On day 21, the skin depicts densely proliferated fibroblast, collagen fibers, reduced neutrophils, and epidermis formation, indicating the efficient healing capacity of SMBM extract.
inflammatory cytokine, tumor necrosis factor-a (TNF-a) expression and poor vasculatures of diabetic wounds were also observed in diabetic mice [16,17]. However, the treatment with SMBM extract might have reversed these conditions to faster healing of wound. Pain and inflammation are the major initial phases which come into action to initiate wound healing. But the inflammatory mediators like bradykinins and prostaglandins can worsen the condition if the wounds are deep and accompanied with diabetes. SMBM might have controlled the further progression of these mediators and accelerated the formation of fibroblast and collagen fibers for faster healing. Lack of inflammation at the wound site towards the end of the study is an evidence for the effect. Immediately after injury, an increase in collagen fiber proliferation is observed in the wound area. This extracellular protein is abundantly found in the granulation tissue of a healing wound. Collagen is significantly involved in providing strength and integrity to a tissue matrix. In addition, collagen also plays an
important role in epithelialization process [18]. In the study, it was clearly observed that t treatment with the extract activated the fibroblast cells and collagen fibers, which might be the reason for complete healing on 21st day. It can also be assumed that one of the factors which induced faster healing of wound is the suppression of TNF-a by the extract. Other mechanisms would be angiogenesis and promotion of fibroblast growth. The wound site is rich in free radicals like superoxide which elevate the severity of a wound. Hence the removal of this radical either by the antioxidant property of extract [11] or by the stimulation of SOD production at wound site would help in faster healing of wound. Neutrophils are the innate immune cells which initiate the inflammatory response in wound healing process and are designated as the initial line of cells involved in defence. The neutrophils play an important role at the wound site by destructing the invading microbes and decontaminating the open wound. The immediate response of neutrophils reduces the chance of
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infectious complication at the injury site [19]. However, there are studies that highlight the negative role of neutrophils [20], where they activate protease enzyme which increases the severity of the wound. The cellular architecture of the wound tissue is in agreement with both the statements. On day 10 neutrophil migration to the wound site can be observed which could be due to its protective action against microbial infections. The lower number of neutrophils on the 21st day could be an indication of improved healing. The histopathology of the wounded tissue on 10th and 21st day showed an increase in the collagen fiber and fibroblast formation in the treated groups compared to untreated control, which signifies the healing efficiency of the SMBM extract. This impairment might have delayed the proliferation and remodelling phases in untreated control rats. Fernandes et al. [21] studied the wound healing property of Psidium guajava hydroalcoholic extract of leaf and reported the enhanced proliferation of fibroblast during wound healing. Similar observation was made in a study by Palanimuthu et al. [22], where complete healing was observed in the rats treated with bark ethanol extract of Eugenia jambolana. 5. Conclusion The results of diabetic wound study revealed that 1% and 2% SMBM extract could heal the wound completely under diabetic conditions in the rats after 21 days of treatment. The histopathology of the wounded skin depicted that the rats treated with SMBM (2%) extract could initiate the healing and re-epithelialization at a faster rate. The study has proved the potential of bark methanol extract to heal the diabetic wound, where wound healing is usually prolonged due to several complications. The phytoconstituents of SMBM extract might have accelerated the rate of healing by reducing microbial infection, antioxidative property and decreased neutrophil migration. Hence, the plant, S. mundagam, can be recommended for the use in the treatment of diabetic wounds and further research is underway to evaluate the role of extract and its chemical compounds in other diabetic complications. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors are grateful to Department of Science and Technology, Govt. of India (DST/INSPIRE Fellowship 2010/ IF10618) and Innovation in Science Pursuit for Inspired Research (INSPIRE) program for providing financial support for carrying out the work. This work is also based on the research supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (Grant No. 98337).
References [1] M. Aalaa, O.T. Malazy, M. Sanjari, M. Peimani, M. Mohajeri-Tehrani, Nurses’ role in diabetic foot prevention and care; a review, J. Diabetes Metab. Disord. 28 (11) (2012) 540–543. [2] A. Alavi, R.G. Sibbald, D. Mayer, L. Goodman, M. Botros, D.G. Armstrong, K. Woo, T. Boeni, E.A. Ayello, R.S. Kirsner, Diabetic foot ulcers: part II. Management, J. Am. Acad. Dermatol. 70 (1) (2014) 21.e1–21.e4. [3] S. Leone, R. Pascale, M. Vitale, S. Esposito, Epidemiology of diabetic foot, Infez. Med. 20 (1) (2012) 8–13. [4] R.J. Snyder, J.R. Hanft, Diabetic foot ulcers-effects on QOL, costs and mortality and the role of standard wound care and advanced-care therapies, Ostomy Wound Manage. 55 (11) (2009) 28–38. [5] M.S. Bortoletto, S.M. de Andrade, T. Matsuo, C. Haddad Mdo, A.D. González, A. M. Silva, Risk factors for foot ulcers – a cross sectional survey from a primary care setting in Brazil, Prim. Care Diabetes 8 (1) (2014) 71–76. [6] R. Waaijman, M. de Haart, M.L. Arts, D. Wever, A.J. Verlouw, F. Nollet, S.A. Bus, Risk factors for plantar foot ulcer recurrence in neuropathic diabetic patients, Diabetes Care 37 (6) (2014) 1697–1705. [7] B. Iraj, F. Khorvash, A. Ebneshahidi, G. Askari, Prevention of diabetic foot ulcer, Int. J. Prev. Med. 4 (3) (2013) 373–376. [8] L.N. McEwen, K.R. Ylitalo, W.H. Herman, J.S. Wrobel, Prevalence and risk factors for diabetes-related foot complications in translating research into action for diabetes (TRIAD), J. Diabetes Complic. 27 (6) (2013) 588–592. [9] C. Formosa, A. Gatt, N. Chockalingam, Diabetic foot complications in Malta: prevalence of risk factors, Foot (Edinb.) 22 (4) (2012) 294–297. [10] M.K.R. Narayanan, N. Anilkumar, V. Balakrishnan, M. Sivadasan, A.H. Ahmed, A. A. Alatar, Wild edible plants used by the Kattunaikka: Paniya and Kuruma tribes of Wayanad District, Kerala, India, J. Med. Plants Res. 5 (15) (2011) 3520– 3529. [11] R. Chandran, T. Parimelazhagan, B.P. George, Antihyperglycemic activity of the bark methanolic extract of Syzygium mundagam in diabetic rats, Alex. J. Med. (2017), doi:http://dx.doi.org/10.1016/j.ajme.2016.12.001. [12] P.K. Mukherjee, Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals, 1st ed., Business Horizons, New Delhi, 2002, pp. 816. [13] V.R. Bharathi, Antioxidant and wound healing studies on different extracts of Stereospermum colais leaf, Int. J. Res. Pharm. Sci. 1 (4) (2010) 435–439. [14] British Pharmacopoeia, British Pharmacopoeia Commission, Vol. 2, HMSO Publication Centre, London, 1993 pp. 1096. [15] K. Hehenberger, A. Hansson, High glucose-induced growth factor resistance in human fibroblasts can be reversed by antioxidants and protein kinase C inhibitors, Cell. Biochem. Funct. 15 (3) (1997) 197–201. [16] R.D. Galiano, O.M. Tepper, C.R. Pelo, K.A. Bhatt, M. Callaghan, N. Bastidas, S. Bunting, H.G. Steinmetz, G.C. Gurtner, Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells, Am. J. Pathol. 164 (6) (2004) 1935–1947. [17] G. Naguib, H. Al-Mashat, T. Desta, D.T. Graves, Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation, J. Invest. Dermatol. 123 (1) (2004) 87–92. [18] B.C. Nwomeh, H.X. Liang, I.K. Cohen, D.R. Yager, MMP-8 is the predominant collagenase in healing wounds and non-healing ulcers, J. Surg. Res. 81 (2) (1999) 189–195. [19] V.D. Julia, M.S. Anna, L.A. DiPietro, Neutrophil function in the healing wound: adding insult to injury? Thromb. Haemost. 92 (2004) 275–280. [20] G.S. Ashcroft, K. Lei, W. Jin, G. Longenecker, A.B. Kulkarni, T. Greenwell-Wild, H. Hale-Donze, G. McGrady, X.Y. Song, S.M. Wahl, Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing, Nat. Med. 6 (10) (2000) 1147–1153. [21] K.P.S. Fernandes, S.K. Bussadori, M.M. Marques, E. Bach, N. Wadt, E.M. Santos, V.C. Pavesi, M.D. Martins, Healing and cytotoxic effects of Psidium guajava (Myrtaceae) leaf extracts, Braz. J. Oral. Sci. 9 (4) (2010) 457–466. [22] D. Palanimuthu, S. Nandagopal, M.D. Jalaludeen, K. Subramonian, A. Saravana Ganthi, S. Sankaram, Wound healing activities of Eugenia jambolana lam. Bark extracts in albino rats, Int. J. App. Biol. Pharmaceut. Technol. 2 (1) (2011) 112– 116.