Food Sci. Biotechnol. 22(S): 99-105 (2013) DOI 10.1007/s10068-013-0054-4
RESEARCH ARTICLE
Wound Healing Effect of Cuttlebone Extract in Burn Injury of Rat Je Kwan Jang, Ok Sang Lee, Tae Jin Kang, and Sung Cil Lim
Received: 4 April 2012 / Revised: 8 August 2012 / Accepted: 22 September 2012 / Published Online: 28 February 2013 © KoSFoST and Springer 2013
Abstract Burn injury, one of the most common diseases in primary care, is also a major cause of death and disability. The aim of this study was to evaluate the effect of cuttlebone (CB) extract in thermal burn wounds in rats and to compare its effects with those of silver sulfadiazine (SSD), the most widely used burn treatment. Burn injury was produced in rats by immersion of the shaved dorsal area to hot water. CB or SSD was applied topically after burn injury. Histological analysis, CBC counts and malonialdehyde (MDA) activities were evaluated 1, 4, 7, and 14 days post-treatment. CB and SSD significantly increased re-epithelialization in burn wounds and decreased WBC levels after 14 days of treatment. These drugs also reduced expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6. By FT-IR, we characterized chitin the main component of CB. Taken together, these results suggest the wound healing effects of CB and its therapeutic value in the treatment of burn injury. Keywords: burn, wound healing, cuttlebone, tumor necrosis factor (TNF)-α, interleukin (IL)-6
Introduction Burn injury is one of the most common diseases in primary care. In the United States, burn accident is the second most common cause of mortality following traffic accidents (1). Burns may be caused by electricity, chemicals, or friction. Je Kwan Jang, Ok Sang Lee, Sung Cil Lim () Department of Pharmacy, College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea Tel: +82-43-261-3590; Fax: +82-43-268-2732 E-mail:
[email protected] Tae Jin Kang Department of Pharmacy, College of Pharmacy, Sahmyook University, Seoul 139-742, Korea
Burn injury activates complicated inflammatory responses and other intermediates related to inflammation, and leads to the development of other complex symptoms. Improved outcomes for patients with burn injury have been attributed to medical advances in nutritional support, burn wound care, pulmonary care, and infection control practices. However, burn wound healing still remains a challenge to modern medicine. Methods such as sap therapy, wound cure, skin graft, and nutrition need simultaneous treatment because burn can lead to serious complications (2-4). Current available ointments for burn injury include silver sulfadiazine (SSD) 1%, mafenide acetate 10% cream, and povidone-iodine (5-8). Skin substitutes composed of artificial materials such as DuoDERM and Opsite are also used for wound closure (9). Cuttlebone (CB), known also as cuttle fish bone, has been used as a traditional medicine for treating sore skin. In Korea, it has been used to improve body circulation and hemostasis, and also to neutralize gastric acid. Moreover, CB has been applied as a strong astringent to treat various ulcerating wound, some hemorrhage symptoms such as intestinal hemorrhage and abnormal uterine bleeding, and to treat anemia, hearing loss, peptic ulcer, amenorrhea, and bolus. Recent studies have demonstrated antibacterial activity (10) and bone healing (11) properties of polysaccharides obtained from CB. However, no study has ever reported its therapeutic effects on burn wounds. To this end, we investigated the effects of CB extract in second degree burn-induced rat model.
Materials and Methods Materials Cuttlebone (Dong-ui Oriental Pharmaceutics), silver sulfadiazine (Il-dong Pharmaceutical Co., Ltd., Seoul, Korea), white petroleum (Sungkwang Co., Ltd., Cheonan, Korea), 2,4,6-trinitrophenol, formaldehyde solution (Junsei Chemical Co., Ltd., Nihonbashi-honcho, Japan), HCl,
100
acetic acid, NaOH, ethyl alcohol (Merck, Damstadt, Germany), and EDTA tube (BD VACUTAINE; BD, Franklin Lakes, NJ, USA) were used in this study. Preparation of cuttlebone (CB) extract CB extract was prepared following the procedures outlined by Hackman (12) and Hackman and Goldberg (13) with some modifications. In brief, CB powder was de-mineralized with 6 N HCl for 24 h at room temperature. Additional de-mineralization was carried out by treating with 2 N HCl until there was no foam. After washing with distilled water (DW), samples were collected and precipitated with 4% NaOH solution for 24 h. CB was washed with DW and ethanol, and dried (12,13). Ointment mixed with currently available white petroleum (vaseline, Sungkwang Co., Ltd.) was also used. The composition of CB ointment was white petroleum: active ingredient=6:4. Analysis for CB components X-ray diffraction was used to check the form of the components of CB after grinding CB powder in the range of 10-100, 40 KV, and 40 mA. CB components were also identified using FT-IR study. The FT-IR spectra were recorded using an IFS-66s FT-IR/Raman spectrometer (Bruker Optik GMBH, Ettlingen, Germany). Both Stokes and anti-Stokes spectral regions were collected. Prior to Fourier transformation, the interferograms were apodized with a Blackman-Harris 4point filter and zero-filled. All the Rama spectra were collected at room temperature. Induction of burn injury and treatment of CB extract Male Sprague-Dawley (7 weeks, 200-250 g) rats were obtained from SamTako Bio Korea Inc. (Osan, Korea). Prior to experiments, they were acclimatized for 1 week in an animal room with 12 h-rotation of light and shade, 24±3oC temperature and , 50±5% humidity. Rats were anesthetized with Zoletil® (tiletamin-zolazapam, 2 mL/kg) and shaved with auto-clippers to expose the entire dorsal surface. Thereafter, the exposed area was disinfected using a 3×3 cm sterilized gauze soaked in ethanol, and exposed to boiling water (100oC) for 10 min to produce a second degree burn. After draining off excess water, gauze was placed on the exposed part for 20 s without additional pressure (14). Rats were randomized to each group and white petroleum (0.5 g), silver sulfadiazine (SSD, 0.5 g), and CB (0.5 g/cm2) were applied twice daily in each treatment group (n=12 animals/group). Clinical signs and burn condition in burn-injured rats were observed and also the survival rate of rats. The weight of the rat was measured at the start of experiment and prior to sacrifice. All experiments were approved by the Research Committee of Chungbuk National University.
Jang et al.
Histological examination For histological examination, animals were sacrificed at indicated time points (1, 4, 7, and 14 day after burn injury). Tissues of interest were dissected and fixed in 10% neutral buffer formalin. Thereafter, tissues were embedded using the paraffin embedding machine (Leica EG1150-H; Leica, Wetzlar, Germany), microtomed (Leica RM2245; Leica), and tissue slices were stained using Hematoxylin-Eosin. The nucleus, cytoplasm, and extracellular matrix were observed using a microscope (Leica DM2500; Leica). The general structure and morphological changes of the tissues were also assessed. Blood test Blood was removed from rats by cardiac puncture and collected in a tube containing EDTA-Na salt. White blood cell (WBC), red blood cell (RBC), hemogloblin (Hb), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) levels were measured in the blood. Platelet, lymphocyte, neutrophil, monocyte, eosinophil, and basophil were also measured as differential counts of leukocyte (Advia 2120; Simens Co., Washington, DC, USA). Lipid peroxidation assay MDA assay was carried out using MDA activity index kit (OxisResearch, Beverly Hills, CA, USA) following the manufacturer’s instruction. Briefly, liver tissue was homogenized using a homogenizer and supernatants were collected for MDA activity. Absorbance of the supernatants was measured at optical density 586 nm. Measurement of cytokines Plasma was assayed for TNF-α and IL-6 using DuoSet ELISA kit (R&D system; Minneapolis, MN, USA) according to the manufacturer’s instruction. The detection limits of TNF-α and IL-6 were 5.0 and 20.0 pg/mL, respectively. Statistical analysis All data were expressed as the mean ±standard error mean (SEM). One-way analysis of variance (ANOVA) was used for statistical analysis. When significant differences were found, Newman Keuls test was performed as a post-hoc test. Significance was set at p
