SRM Journal of Research in Dental Sciences

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 11  |  Issue : 4  |  Page : 172--177

Effect of caffeic acid phenethyl ester and Ankaferd Blood Stopper® on palatal wound healing in the diabetic rats


Mehmet Gul1, Ahmet Günay2,  
1 Department of Periodontology, Faculty of Dentistry, Harran University, Sanliurfa, Turkey
2 Department of Periodontology, Faculty of Dentistry, Dicle University, Diyarbakir, Turkey

Correspondence Address:
Dr. Mehmet Gul
Department of Periodontology, Faculty of Dentistry, Harran University, Campus, Sanliurfa 63000
Turkey

Abstract

Background/Aim: Caffeic acid phenethyl ester (CAPE) and Ankaferd Blood Stopper® (ABS) are thought to contribute to wound healing. The aim of this study is to investigate the effect of ABS and CAPE on the secondary wound healing of oral mucosal tissue. Materials and Methods: A total of 63 male Sprague-Dawley rats were used in the study. Rats were randomly divided into three groups: ABS group, CAPE group, and control group. Healthy 63 rats were intraperitoneally injected with streptozotocin (Sigma Chemical Co., St. Louis, MO) 50 mg/kg in 0.2 ml 10 m citrate solution. After a week, if the blood glucose value of the rat is ≥250 mg/dl, the rats are considered to be diabetic. General anesthesia of the rats was conducted with intramuscular ketamine (8 mg/100 g). The excisional palatal wound was formed by using a 4 mm punch biopsy tool. Topical ABS was applied to ABS groups; topical CAPE application was applied to CAPE groups. Animals were sacrificed at 7, 14, and 21 days. The palatal specimens were painted with hematoxylin and eosin. The Mann–Whitney U test was used for comparison of the two groups. Results: The results of the statistical analysis determined the vessel dilatation and hemorrhage to be significantly lower in the diabetic ABS and CAPE group than the control group at 7, 14, and 21 days (P < 0.05). Conclusions: In the present study, it is concluded that ABS and CAPE have a positive effect on the wound healing process.



How to cite this article:
Gul M, Günay A. Effect of caffeic acid phenethyl ester and Ankaferd Blood Stopper® on palatal wound healing in the diabetic rats.SRM J Res Dent Sci 2020;11:172-177


How to cite this URL:
Gul M, Günay A. Effect of caffeic acid phenethyl ester and Ankaferd Blood Stopper® on palatal wound healing in the diabetic rats. SRM J Res Dent Sci [serial online] 2020 [cited 2021 Feb 26 ];11:172-177
Available from: https://www.srmjrds.in/text.asp?2020/11/4/172/308784


Full Text



 Introduction



Wound healing is a biological event in which cellular and molecular processes occur consecutively, including integration into inflammation, granulation tissue formation, and re-epithelialization.[1],[2],[3] The biological processes of wound healing in gingival tissue are the same as in the skin, but healing in the gingival region occurs faster and with less scar formation than in the skin.[4] The reason for this difference is not fully understood, but saliva, leukocytes, growth factors, and specific fibroblast subpopulations are thought to play a role.[5]

Diabetes occurs when insensitivity occurs from insufficient levels of insulin and the effects of hormones related to diabetes. Consequently, it has been described as a systemic disease that causes hyperglycemia. Diabetes causes many complications, including delayed wound healing.[6] In patients with diabetes, the wound healing process is disturbed. In the wound healing process, a number of adverse conditions are observed, including dysfunction in the inflammatory response, reduction in granulation tissue, disruption of angiogenesis.[7]

Caffeic acid phenethyl ester (CAPE) is known as one of the active components of propolis contained in extracts collected from plants by bees. In the past, propolis was used for many reasons, by the public, and in the medical field, primarily for its antibacterial and anti-inflammatory effects.[8] CAPE is known to have antioxidant, anti-inflammatory, and anti-cancer activities.[9]

Ankaferd Blood Stopper® (ABS) is a medicinal plant extract used as a hemostatic agent in Turkish medicine. ABS is obtained by standardizing the extracts from plants and obtaining a mixture of these: 8 g Vitis vinifera, 7 g Alpinia officinarum, 5 g Thymus vulgaris, 7 g Glycyrrhiza glabra, and 6 g Urtica dioica. The effect of each plant in this herbal mixture on vascular endothelium, blood cells, angiogenesis, cell proliferation, and various mediators has been proven in various studies.[10]

The aim of the present study was to research the effects of topical ABS and CAPE on secondary healing of the surfaces of experimental excisional wound areas created in the palatal mucoperiosteum of diabetic rats.

 Materials and Methods



Sixty-three male Sprague-Dawley rats were used in this research. The study was conducted at the Health Institution Research Centre, Dicle University, Diyarbakir, Turkey (ethics committee approval no: 2015/17). During the experiment, all rules determined by the ethics committee were followed. During anesthesia and surgical procedures, the rules specified by the ethics committee were completely followed. They were randomly divided into three groups: a control group, CAPE group, and ABS group, and each group was divided into groups on 7, 14, and 21 days (n = 7). Palatal mucosal defects were created in the three groups. The rats were topically treated with 0.10 ml of ABS solution in the ABS group, 100 mmol/kg of CAPE in the CAPE group, and 0.9% NaCl isotonic in the control group.

Diabetes formation procedure in rats

Sixty-three healthy rats were treated with streptozotocin (Sigma Chemical Co., St. Louis, MO) 50 mg/kg in 0.2 ml of 10 mM citrate solution, and intraperitoneal injection was performed 1 week later (Hannover, Germany). If the blood glucose value was ≥250 mg/dl, the rats were considered diabetic (OK Biotech Co., Ltd., Taiwan).[11] All rats were made diabetic. Those with a blood glucose level of less than 250 mg/dl and those with a weight <250 g were not included in the study. Groups were determined randomly.

Creation of palatal mucosal defects

Before the formation of palatal secondary mucosal defects (4 mm), anesthesia was performed using intramuscular ketamine HCl (Alfamine 10%: Netherlands) at a dose of 45 mg/kg and xylazine HCl (Alfazyne 2%) at 2.5 mg/kg. Rats were prepared for surgical procedures in accordance with standard disinfection and sterilization rules. Instruments used in surgical procedures were sterilized and used. A full-thickness excisional wound on the palatal mucosa of rats was created with a 4 mm punch biopsy tool [Figure 1].{Figure 1}

The wound areas were allowed to heal secondarily. On the 7th day, 21 rats were sacrificed. Then, 21 rats were sacrificed on the 14th day. Finally, 21 rats were sacrificed on the 21st day. The rats in each group were sacrificed by a single dose of sodium thiopentone (60 mg/kg). The application was performed intraperitoneally. The tissues were obtained, including excision of the tissue around the wound site. The tissues obtained were stored in 10% formaldehyde containers for histological evaluation.

Histopathological method

The palatal pieces were fixed directly in neutrally buffered formalin fixative. The specimens were dehydrated through a graded alcohol series for 12 h. Xylol was embedded immediately in paraffin blocks. Parts of paraffin-embedded tissues 6 μm thick were created and painted with hematoxylin and eosin dye for routine staining. The sections of paraffin-embedded tissues were accommodated on poly-L-lysine-coated slides. They were placed in an incubator at 60°C overnight. After cooling, the sections were kept in xylene for 2–5 min, then passed through 96%, 80%, 70%, and 60% ethyl alcohol for 5 min, respectively. They were kept in distilled water for 5 min. Sections were placed in ethylenediaminetetraacetic acid solution to melt gum tissue. Samples were placed on slides and then surrounded by Dako Pen (Huiyou, China). The samples were kept in citric acid (pH 6.0) in a 700 W microwave oven for 7 + 5 min to remove antigen masking samples were cooled at room temperature for 20 min and washed with phosphate-buffered saline (PBS) solution. For endogenous peroxide blockade, it was placed in 3% hydrogen peroxide for 20 min. Sections were washed with PBS and placed in an incubation vessel. Finally, for histopathological analysis, the sections were evaluated using a photomicroscope (Nikon Eclipse i50; Nikon, Tokyo, Japan) for blind evaluation.

Histological evaluation

Scoring of histological sections was used with values ranging from 0 to 4 as described by Mehrabani et al.[12] The evaluation was made according to the following situations.

Epithelium regeneration: 0 = Absence of epithelial proliferation ≥70% of tissue, 1 = Poor epidermal organization ≥60% of tissue, 2 = Incomplete epidermal organization ≥40% of tissue, 3 = Moderate epithelial proliferation ≥60% of tissue, 4 = Complete epidermal remodeling ≥80% of tissue. Inflammation: 0 = 13–15 İnflammatory cells per histological field, 1 = 10–13 İnflammatory cells per histological field, 2 = 7–10 İnflammatory cells per histological field, 3 = 4–7 İnflammatory cells per histological field, 4 = 1–4 İnflammatory cells per histological field. Vascular dilatation and hemorrhage: 0 = Absence of angiogenesis presence of congestion, hemorrhage, edema, 1 = 1–2 vessels per site edema, hemorrhage, congestion, 2 = 3–4 vessels per site, moderate edema, congestion, 3 = 5–6 vessels per site slight edema congestion, 4 = More than 7 vessels per site vertically disposed toward the epithelial surface. Fibrosis: 0 = İmmature and inflammatory tissue, 1 = Thin immature and inflammatory tissue, 2 = Moderate remodeling, 3 = Thick granulation layer and well-formed collagen matrix, 4 = Complete tissue organization.

Statistical analysis

The statistical analysis of the data was performed using the SPSS (version 21.0, IBM Corp., New York, USA) for Windows statistical program. Means of numerical values and standard deviation values of histological data are shown. Mann–Whitney U test was used to compare the two groups. A Mann–Whitney U test with Bonferroni correction was used to compare more than two groups. A P < 0.05 was considered significant in all statistical tests.

 Results



Histopathological findings

The diabetic ABS group showed hypertrophy of cells in the basal and spinosum layer and regeneration of the cells toward the papillary region, dilatation of the vessels, and hemorrhage in the 7-day group. In the diabetic 7-day CAPE group, degenerative changes were observed in some cells in the basal layer and spinosum layer, while desquamative cells were not observed in lamina propria. In the diabetic 7-day control group, hypertrophy, regeneration of the basal layer cells, mononuclear cell infiltration under the basal layer, increasing in fibrous tissue, and thinning of the vessel wall were observed [Figure 2].{Figure 2}

Diabetic ABS showed regeneration of cells in the basal layer, desquamatic cell clusters in the papillary region, collagen fibers in the lamina propria, and diffuse cell infiltrations, in the 14-day group. Diabetic CAPE showed hyperplasia in basal cells and pycnosis in the nucleus in cells near the surface of the spinosum layer, in the 14-day group. Thinning of collagen fibers in the lamina propria and diffuse mononuclear cells were observed. In the diabetic 14-day control group, regenerated epithelial cells in small groups under the basal layer marked dilatation of the vessels, and small areas of hemorrhage and increased mononuclear leukocyte infiltration around the vessel were observed [Figure 2].

Diabetic ABS showed regeneration in basal cells, thinning of collagen fibers, and decreased cell infiltration in the 21-day group. Diabetic CAPE showed increased hyperplasia in the basal layer cells in the 21-day groups. In the diabetic 21-day control group, cell clusters regenerating to the papillary region of the palate epithelium increased. Intense leukocyte infiltration was found around the vessel, which started to increase dilatation in the vessel structure [Figure 2].

Results of the statistical analysis of the working groups

Day 7

In comparison between the control group and ABS group, a decrease in epithelial regeneration was observed. In the control group and the CAPE group, epithelial regeneration decreased. In the control group and the ABS group, fibrosis decreased. In the control group and the CAPE group, fibrosis decreased. The control group and the ABS group showed a decrease in vascular dilatation and hemorrhage. The control group and the CAPE group showed a decrease in vascular dilatation and hemorrhage and were statistically significant [Table 1].{Table 1}

Day 14

In comparison between the control group and the CAPE group and the ABS group and the CAPE group, there was a decrease in epithelial regeneration. In the control group and the CAPE group and the ABS group and the CAPE group, inflammation decreased. In the comparisons between the control group and the ABS group and between the control group and the CAPE group, there was a decrease in fibrosis. Vascular dilatation and hemorrhage were decreased between the control group and other groups, while an increase was observed between the ABS group and CAPE, a statistically significant difference was found in all groups [Table 2].{Table 2}

Day 21

In the comparisons between the control group and the ABS group and between the control group and the CAPE group, fibrosis, inflammation, vascular dilatation, and hemorrhage decreased; the differences were found to be statistically significant. There was a decrease in the comparison between the control group and the CAPE group that was found to be a statistically significant difference [Table 3].{Table 3}

 Discussion



Kosger et al. formed a 10 mm full-thickness mucosal wound surface in the palatal mucosa of rats and applied Arnebia densiflora extopically to the wound surface. Rats were sacrificed on the 4, 7, 14, and 21 days and evaluated histologically.[13] Oda et al. created a 4 mm defect under the periosteum in the palatal region of rats and injected basic fibroblast growth factor into the defect.[14] Cornelissen et al. investigated wound closure and scar formation after palatal cleft surgery that disrupted the growth of the maxilla. They injected interferon-β into the palatal mucosa of rats.[15] Kozlovsky et al., the effects of various antimicrobial agents on wound healing were evaluated by creating a 5 mm diameter soft-tissue defect in the palatal mucosa of rats.[16] Yamashita et al., zoledronate was used in the rat palatal area as an experimental wound model to evaluate wound healing in bone and mucosa.[17] In the present study, rat palatal mucosa was used to create an excisional wound model that was left to secondary healing. During our study, to evaluate the effects of CAPE and ABS on wound healing in the oral mucosal tissue, tissue samples were taken at 7, 14, and 21 days in light of the oral mucosal healing process of rats and similar studies in the literature. Full-thickness defects 4 mm in diameter were created with punches.

Diabetic individuals often develop wounds that do not heal. Fibroblasts and polymorphonuclear cells use glucose as an energy source in their activities. Changes in glucose balance in individuals with diabetes have a negative impact on metabolism. Due to insufficient fibroblasts and polymorphonuclear cells involved in wound healing, delayed healing occurs.[18] In the present study on the 7th, 14th, and 21st days in diabetic rats, there was a decrease in vascular dilatation and hemorrhage in both the ABS and CAPE groups. This decrease was statistically significant in both groups. This can be interpreted as having a positive effect on wound healing and inflammation in both materials. The anti-inflammatory, antimicrobial, and antioxidant components contained in both materials has a positive effect on wound healing. The decrease in hemorrhage may be due to the anti-bleeding effect of ABS.

Freeman et al. suggested that diabetes stimulates vascular inflammation and that inflammation can cause diabetes. In the case of the occurrence of diabetes, increased C-reactive protein mass index, triglyceride, and glucose levels should be considered. Their increase exacerbates the risk of diabetes.[19] In our study, both the ABS and CAPE groups showed a decrease in inflammation. Inflammation is more common in people with diabetes and is undesirable because it adversely affects the degree of disease and systemic status. In one study, it was emphasized that CAPE is beneficial in oral health. In the study, it was emphasized that it supports bone healing, protects against bone resorption caused by systemic cortisol and periodontitis. It has also been emphasized that it reduces inflammation occurring in periodontal diseases.[20] Bulut et al. In their study, they created calvarium defects on rats that were made diabetic with streptozotocin and applied ABS. As a result of the study, they emphasized that ABS positively affected wound healing.[21] Similarly, in our study, it was concluded that CAPE and ABS positively affect wound healing. Using topical in surgical procedures may not benefit. More comprehensive studies are needed to fully determine the effects of ABS and CAPE. The effects of both ABS and CAPE are due to the anti-inflammatory effects of the components that have positive effects. This shows us the anti-inflammatory and antimicrobial activities of ABS and CAPE.

 Conclusions



In this study, the effects of ABS and CAPE on the healing process of experimentally created secondary wounds in the palatal mucosa of diabetic rats were evaluated histologically and biochemically. ABS and CAPE positively affect the short-term re-epithelization process and their use in wound healing in periodontal surgery will be beneficial. It would be useful to conduct postoperative and long-term examinations to evaluate the clinical effects of ABS and CAPE on the healing of wounds occurring in mucosal tissue.

Acknowledgments

This study was supported by the Dicle University Scientific Research Projects Coordinator (project number: DİS.15.011). The study was conducted at the Health Institution Research Centre, Dicle University, Diyarbakir, Turkey (ethics committee approval no: 2015/17) Histological analysis was done by professor doctor Engin Deveci.

Financial support and sponsorship

This study was supported by the Dicle University Scientific Research Projects Coordinator (project number: DİS.15.011).

Conflicts of interest

There are no conflicts of interest.

References

1Clark RA. Fibrin is a many splendored thing. J Invest Dermatol 2003;121:xxi-xxii.
2Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008;16:585-601.
3Liu R, Desta T, He H, Graves DT. Diabetes alters the response to bacteria by enhancing fibroblast apoptosis. Endocrinology 2004;145:2997-3003.
4Rinastiti M, Harijadi , Santoso AL, Sosroseno W. Histological evaluation of rabbit gingival wound healing transplanted with human amniotic membrane. Int J Oral Maxillofac Surg 2006;35:247-51.
5van Beurden HE, Von den Hoff JW, Torensma R, Maltha JC, Kuijpers-Jagtman AM. Myofibroblasts in palatal wound healing: Prospects for the reduction of wound contraction after cleft palate repair. J Dent Res 2005;84:871-80.
6Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738-46.
7Desta T, Li J, Chino T, Graves DT. Altered fibroblast proliferation and apoptosis in diabetic gingival wounds. J Dent Res 2010;89:609-14.
8Hepşen İF, Tilgen F, Er H. Propolis: Tıbbi özellikleri ve oftalmolojik kullanımı. Turgut Özal Tıp Merkezi Dergisi 1996;3:386-91.
9Günay A, Arpağ OF, Atilgan S, Yaman F, Atalay Y, Acikan I. Effects of caffeic acid phenethyl ester on palatal mucosal defects and tooth extraction sockets. Drug Des Devel Ther 2014;8:2069-74.
10Goker H, Haznedaroglu IC, Ercetin S, Kirazli S, Akman U, Ozturk Y A, et al. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper®. J Int Med Res 2008;36:163-70.
11Andrade Cetto A, Wiedenfeld H, Revilla MC, Sergio IA. Hypoglycemic effect of Equisetum myriochaetum aerial parts on streptozotocin diabetic rats. J Ethnopharmacol 2000;72:129-33.
12Mehrabani D, Rezaee A, Azarpira N, Fattahi MR, Amini M, Tanideh N, et al. The healing effects of Teucrium polium in the repair of indomethacin-induced gastric ulcer in rats. Saudi Med J 2009;30:494-9.
13Kosger HH, Ozturk M, Sokmen A, Bulut E, Ay S. Wound healing effects of arnebia densiflora root extracts on rat palatal mucosa. Eur J Dent 2009;3:96-9.
14Oda Y, Kagami H, Ueda M. Accelerating effects of basic fibroblast growth factor on wound healing of rat palatal mucosa. J Oral Maxillofac Surg 2004;62:73-80.
15Cornelissen AM, Von den Hoff JW, Maltha JC, Kuijpers-Jagtman AM. Effects of locally injected interferon-β on palatal mucoperiosteal wound healing. J oral Pathol Med 2002;31:518-25.
16Kozlovsky A, Artzi Z, Hirshberg A, Israeli-Tobias C, Reich L. Effect of local antimicrobial agents on excisional palatal wound healing: A clinical and histomorphometric study in rats. J Clin Periodontol 2007;34:164-71.
17Yamashita J, Koi K, Yang DY, McCauley LK. Effect of zoledronate on oral wound healing in rats. Clin Cancer Res 2011;17:1405-14.
18Atalar Ö, Sönmez M, Yüce A, Türk G, Çeribaşı A, Kavaklı A, et al. Diyabet, yara iyileşmesi ve sperm kalitesi üzerine akupunkturun önemi (The Importance of Acupuncture on Diabetes, Wound Healing and Sperm Quality). F U Vet J Health Sci 2013;27:109-13.
19Freeman DJ, Norrie J, Caslake MJ, Gaw A, Ford I, Lowe GD, et al. C-reactive protein is an independent predictor of risk for the development of diabetes in the West of Scotland coronary prevention study. Diabetes 2002;51:1596-600.
20Stähli A, Maheen CU, Strauss FJ, Eick S, Sculean A, Gruber R. Caffeic acid phenethyl ester protects against oxidative stress and dampens inflammation via heme oxygenase 1. Int J Oral Sci 2019;11:6.
21Bulut E, Baş B, Altunkaynak BZ, Bekçioğlu B, Erdem Koç G, Gönülol E, et al. Efficacy of Ankaferd Blood Stopper on bone healing in diabetic rats: A stereological and histopathological study. Biotech Histochem 2014;89:535-43.