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Fırat University Journal of Health Sciences (Veterinary)
2018, Cilt 32, Sayı 3, Sayfa(lar) 173-177
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Propolis ve Hypericum perforatum Krem’in Yanık Yara İyileşmesi Üzerine Etkileri
Ali Said DURMUŞ1, Mehmet Cengiz HAN1, Aydın SAĞLIYAN1, Mustafa ÖZKARACA2, Fatih Mehmet KANDEMİR3
1University of Fırat, Faculty of Veterinary, Department of Surgery, Elazığ, TURKEY
2University of Atatürk, Faculty of Veterinary, Department of Pathology, Erzurum, TURKEY
3University of Atatürk, Faculty of Veterinary, Department of Biochemistry, Erzurum, TURKEY
Özet
Bu çalışmada, Propolis (Pr) ve Hypericum perforatum (HP) kreminin, bir rat modelinde yanık yaralarında bir dizi biyokimyasal ve histopatolojik parametreler üzerindeki etkilerini değerlendirmeyi amaçladık. Kırk iki dişi 3 aylık Wistar Albino rat üç eşit ana gruba ayrıldı. Bu ana grupların her biri, 7 ve 14 günlük postoperatif takipler için iki alt gruba ayrıldı. Genel anestezi altında tüm ratların sırt derisi üzerinde iki adet 10 mm çapında ikinci derece termal yanık oluşturuldu. Birinci, ikinci ve üçüncü gruplardaki yanmış deri bölgeleri sırasıyla plasebo krem (kontrol), %50 Pr krem ve % 50 HP krem ile kaplandı (her grup için n= 14). Postoperatif 7 ve 14 günlük takip sürelerini tamamlayan ratlar ötenazi edildi ve deri örnekleri biyokimyasal ve histolojik olarak incelendi. Sonuçlar Pr'nin diğer gruplara göre daha fazla antioksidan özelliği olduğunu gösterdi. Pr ve HP, epitelizasyon, granülasyon dokusu ve kollajen için teşvik edici özelliği ile yanık yara iyileşme etkisini arttırdı (P<0.05). Sonuç olarak, %50 Pr ve %50 HP kremilerinin topikal uygulamaları yanık yaralarının iyileşmesinde etkilidir.
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    Burned tissues and cells are destroyed by thermal injury and ischemic processes. The indirect destruction is derived in part from disorders in circulation of blood with ischemia, infarctions, stasis, and thrombosis 1,2.

    Propolis (Pr) is a product of the honey bees. Pr is a natural drug commonly used since ancient times. It contains phenolic acids, amino acids, cinnamic acid, flavanoids, terpens and caffeic acid. Topical application of Pr has been found to be efficacious in producing a granulating bed and controlling infection 3-5.

    It is reported that Hypericum perforatum (HP) have sedative, antidepressant, antispasmodic, and antiseptic effects as well as wound-healing effects 6,7. The active contents of HP are hypericin and hyperforin. Hypericin is the most the effective component of HP, which is responsible from the wound healing 8-11.

    This study aimed to evaluate the effects of Propolis and Hypericum perforatum cream on a number of clinical, biochemical and histopathological parameters in burn wounds in a rat model.

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    Chemicals: Placebo cream, Pr extract cream and HP oil cream were used in this study.

    Pr used in this study was obtained from beekeepers in Elazığ, Turkey. Extract of Pr was prepared and used during this study as defined by Isla et al. 12. Frozen Pr was crushed in a mortar. Then, the resulting powder of Pr was extracted with ethanol with continuous stirring for 24 hour at room temperature. The suspension was let to precipitate for 3 days. The supernatant was concentrated in a evaporator and the sediment was blended with equal quantity of placebo cream.

    HP oil (Aksu Vital, 20 mL) was mixed within equal amount of placebo cream, and HP cream was prepared.

    Animals: Forty two, 2-month old, weighing 210-240 g, female Wistar rats were used in the study. This study was approved by The Experimental Animals Local Ethical Committee of the Firat University, Turkey (Protocol No: 2014/127). Animals were anaesthetised with an intramuscular injection of 80 mg/kg ketamine hydrochlorure (100 mg/mL, Ketasol, Richter Pharma AG) and 5 mg/kg xylazine hydrochlorure (23.32 mg/mL, Rompun, Bayer).

    Thermal Injury: The skins of the backs of the animals were shaved and burn wounds of 10 mm diameter were obtained. Skin burn models were constituted as defined by previous studies 13,14. Animals were exposed to second-degree skin burns with 10 mm diameter brass probe. The probe was put in boiling water until thermal balance was reached. This probe was then put on the skin of back of the animals for 15 s without applying pressure.

    Treatment Protocol: The rats were divided into 3 groups. Immediately after burn, placebo cream (Group control), 50% Pr, and 50% HP cream were performed locally to the burned wounds in the first, second, and third groups respectively (n= 14 for each group). Each main group was separated into 2 subgroups for followed up on the 7th and 14th days. The topical implementations were repeated twice a day. Clinically, the burned wounds were observed in all groups every day. Seven and 14 days later, the burned skin specimens were taken from animals for biochemical and histological examinations.

    Biochemical Analysis: In the skin homogenate, the malondialdehyde (MDA) levels were measured in accordance with the method of Placer et al. 15. The skin catalase (CAT) activity was measured according to the method of Aebi 16. Concentration of protein was measured in accordance with to the method of Lowry et al. 17. The glutathione (GSH) level was measured according to the method of Sedlak and Lindsay 18. The glutathione peroxidase (GSH-Px) activity in skin tissue was evaluated in accordance with the method of Matkovics et al. 19. The superoxide dismutase (SOD) activity in skin tissue was measured in accordance with the method of Sun et al. 20.

    Histological Examination: The skin samples were fixed in 10% neutral buffered formalin, and were cut into 5 μm sections. The sections were stained with haematoxylin and eosin, and examined by light microscopy. The samples were evaluated with regard to epithelisation, collagen deposition and granulation tissue, inflammatory cell infiltration, and angiogenesis. Sections were evaluated semiquantitatively as no (-), mild (+), medium (++) and severe (+++).

    Statistical Analysis: SPSS (22.0 version) was used in statistical analyses. After Normality test, One-Way test ANOVA were used to detect differences among the groups. Significant differences were further compared with Tukey HSD test. Within each group, the Independent Samples t–test was used to calculate statistical differences between the 7th and 14th days. Values are presented as means±SEM. P<0.05 was considered as significant.

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    Mortality was not observed in the animals during this study. Clinically, it was observed that a less redness, swelling and heat in the Pr and HP groups than in the control group.

    The mean tissue levels of GSH, GSH-Px, MDA, CAT and SOD in all groups are shown in Table 1. On day 7th, the local usage of Pr and HP reduced the MDA level (P<0.001). In the Pr group, levels of GSH, CAT, and GSH-Px were increased on the 7th day (P<0.001). On the 14th day, a significant decrease was found in the MDA level in the Pr group (P<0.001), besides an increase in the CAT, GSH, GSH-Px and SOD levels (P<0.001) (Table 1).


    Büyütmek İçin Tıklayın
    Table 1: The mean tissue levels of GSH, GSH-Px, MDA, CAT and SOD (values shown as mean±SEM)

    Histological evaluations of the wounds on 7th and 14th days are shown in Table 2. It was found that the levels of epithelisation, collagen and granulation tissue deposition on the 7th and 14th days were lesser in the control group than in the Pr and HP groups (P<0.05). In addition, it was found that the severe oedema seen in the granulation tissue of the control group decreased in the Pr and HP groups (Figure 1).


    Büyütmek İçin Tıklayın
    Table 2: Histological evaluation of the wounds on 7th and 14th days (values shown as mean±SEM)


    Büyütmek İçin Tıklayın
    Figure 1: The microscopic images of the burned skin wounds on days 7 and 14. Seventh day: Group control: insufficient epithelisation; Group propolis (Pr): more improved epithelisation (arrow); Group Hypericum perforatum (HP): more improved epithelisation (arrow) (H&E). Fourteenth day: Group control: appearance of oedematous immature granulation tissue; Group propolis (Pr): appearance of less oedematous mature granulation tissue; Group Hypericum perforatum (HP): appearance of less oedematous mature granulation tissue (H&E).

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    Alternative treatment methods have been commonly used in treatment of diseases since ancient times. For many years the effects of herbal medication on wound healing have been reported. Herbal products seem to have effect with less or no toxicity comparing to the synthetic drugs 21-23. In this study, stimulatory effects of Pr and HP on burn wound healing were studied in clinical, biochemical, and histological terms.

    Experimental animals such as rats have been used in wound healing studies 13,24,25. In this study, the burn wound model used is easy and reproducible.

    In accordance with previous studies 2,14, the back skin of the rat seems to be an appropriate model for the study of burn wound healing and we used this model due to its simplicity.

    Appropriate wound healing requires equilibrium between antioxidants and oxidative stress. Increase of reactive oxygen species inhibits wound healing, and also in a reduction in some free radical scavengers to cause tissue damage. Antioxidants increase the healing of wounds by decreasing the damage that is caused by free oxygen radicals 26-28.

    Numerous antioxidants have been used in the treatment of wound both experimentally and clinically. Among them; Vitamins A, C, E, β-carotene, glutathione, SOD, dimethyl sulfoxide, melatonin, selenium, uric acid, ceruloplasmin, coumarine, ubiquinol, glycolic acid, and caffeic acid phenethyl ester could be mentioned 4,14.

    Pr is a natural medication that has been used since ancient times. In the last decades, Pr has attracted investigators’ attention owing to several pharmacological and biological properties 3. It was notified that the antimicrobial 29 and anti-inflammatory activity 5 of Pr is owing to caffeic acid phenethyl ester. Some researchers 12,28, reported that antioxidative effect of Pr.

    The time-dependent reductions in MDA level and statistical increments in all antioxidants on day 14 were determined in both control and Pr groups when compared with the values on day 7 (Table 1). This situation may be explained with the natural increase in wound healing by the time and subsequent improvements in oxidant/antioxidant balance. In addition, results of this study clearly demonstrated that Pr plays a role positively in healing of skin burn wounds by its anti-inflammatory and antioxidant effects.

    It is reported that the HP extract has anti-inflammatory and antioxidant effects 6,7,11,30. In this study, MDA and SOD levels were significantly different in HP group compared to the control group on day 7 (P<0.001). A statistical increase was observed in only SOD activity between days 7 and 14 in HP group (P<0.05) (Table 1). However, it is believed that HP does not have strong antioxidant properties compared with Pr.

    The accumulation of collagen demonstrates wound healing 31. Pr was used as an antiseptic and cicatrizant in burn wound treatment 4. It was reported that Pr extract have important anti-inflammatory properties and has reduced oedema in inflammation via this feature 5.

    It was reported that HP has stimulating effects of fibroblast migration, collagen accumulation, and revascularisation 9,10,32.

    In this study, on the 7th and 14th days, granulation tissue, collagen accumulation and epithelisation in animals in the Pr and HP groups were higher compared with the control group (P<0.05), as shown by the previous studies 9,32,33. In accordance with by the previous studies 3,4, it was found that the serious oedema observed in the control group decreased in the Pr and HP groups.

    It is known that HP increased wound healing 33. Samadi et al. 34 showed that HP aided in healing caesarean wounds.

    The results acquired in this study indicated that the antioxidant property of Pr was more than that of HP. Pr and HP were found to accelerate wound healing probably by increasing the formation of collagen deposition, epithelial and granulation tissue. It is also thought that antioxidant property of Pr contribute to the wound healing.

    Consequently, results of this study remarked that Pr and HP skin creams could be applied in treatment of burn wounds and it may be an alternative to the traditional methods.

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    1) Durmuş AS, Yaman M, Can HN. Effects of extractum cepae, heparin, allantoin gel and silver sulfadiazine on burn wound healing: an experimental study in a rat model. Vet Med Czech 2012; 57: 287-292.

    2) Yaman İ, Durmuş AS, Çeribaşı S, Yaman M. Effects of Nigella sativa and silver sulfadiazine on burn wound healing in rats. Vet Med Czech 2010; 55: 619-624.

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    4) Han MC, Durmuş AS, Karabulut E, Yaman İ. Effects of Turkish propolis and silver sulfadiazine on burn wound healing in rats. Revue Med Vet 2005; 156: 624-627.

    5) Martinotti S, Ranzato E. Propolis: A new frontier for wound healing? Burns Trauma 2015, 3: 1-7.

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    13) Durmuş AS, Han MC, Yaman İ. Comparative evaluation of collagenase and silver sulfadiazine on burned wound healing in rats. FÜ Sağ Bil Vet Derg 2009; 23: 135-139.

    14) Hosnuter M, Gurel A, Babuccu O, et al. The effect of CAPE on lipid peroxidation and nitric oxide levels in the plasma of rats following thermal injury. Burns 2004; 30: 121-125.

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    19) Matkovics B, Szabo I, Varga IS. Determination of enzyme activities in lipid peroxidation and glutathione pathways. Laboratoriumi Diagnos 1988; 15: 248-249.

    20) Sun Y, Larry WO, Ying L. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497-500.

    21) Atiba A, Marzok M, Ghazy A. Comparison of Aloe vera and silver sulfadiazine in the treatment of deep second-degree burn in dogs. Glob Vet 2014; 13: 733-737.

    22) Bedi MK, Shenefelt PD. Herbal therapy in dermatology. Arch Dermatol 2002; 138: 232-242.

    23) Farahpour MR, Habibi M. Evaluation of the wound healing activity of an ethanolic extract of Ceylon cinnamon in mice. Vet Med Czech 2012; 57: 53-57.

    24) Gal P, Kilik R, Mokry M, et al. Simple method of open skin wound healing model in corticosteroid-treated and diabetic rats: Standardization of semi-quantitative and quantitative histological assessments. Vet Med Czech 2008; 53: 652-659.

    25) Sabol F, Dancakova L, Gal P, et al. Immunohistological changes in skin wounds during the early periods of healing in a rat model. Vet Med Czech 2012; 57: 77-82.

    26) Akhoondinasab MR, Khodarahmi A, Akhoondinasab M, Saberi M, Iranpour M. Assessing effect of three herbal medicines in second and third degree burns in rats and comparison with silver sulfadiazine ointment. Burns 2015; 41: 125-131.

    27) Daryabeigi R, Heidari M, Hosseini SA, Omranifar M. Comparison of healing time of the 2 degree burn wounds with two dressing methods of fundermol herbal ointment and 1% silver sulfadiazine cream. Iran J Nurs Midwifery Res 2010; 15: 97-101.

    28) Al-Waili N, Hozzein WN, Badr G, et al. Propolis and bee venom in diabetic wounds; a potential approach that warrants clinical investigation. Afr J Tradit Complem 2015; 12: 1-11.

    29) Kartal M, Yildiz S, Kaya S, Kurucu S, Topcu G. Antimicrobial activity of propolis samples from two different regions of Anatolia. J Ethnopharmacol 2003; 86: 69-73.

    30) Ocak T, Duran A, Ozyalvacli G, et al. Protective effects of montelukast and Hypericum perforatum against intestinal ischemia-reperfusion injury in hamsters. Turk J Med Sci 2014; 44: 381-386.

    31) Guo S, DiPietro LA. Factors affecting wound healing. Journal of Dental Research 2010; 89. 219-229.

    32) Dikmen M, Öztürk Y, Sagratini G, et al. Evaluation of the wound healing potentials of two subspecies of Hypericum perforatum on cultured NIH3T3 fibroblasts. Phytother Res 2011; 25: 208-214.

    33) Mukherjee PK, Verpoorte R, Suresh B. Evaluation of in vivo wound healing activity of Hypericum patulum (family: Hypericaceae) leaf extract on different wound model in rats. J Ethnopharmacol 2000; 70: 315-321.

    34) Samadi S, Khadivzadeh T, Emami A, et al. The effect of Hypericum perforatum on the wound healing and scar of cesarean. J Altern Complement Med 2010; 16: 113-117.

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