Quality Control Of Burn Ointment And Its Effect On Scarring In Rats With Deep Ⅱ Degree Burn Wounds

Burn is an invasive disease, and it remains a global public health issue. Many researches have showed that traditional Chinese medicine is effective on treating burn. Burn ointment is a preparation of burn treatment that developed by the First Affiliated Hospital of Anhui Medical University. Burn ointment is composed of rhubarb, burnet, and gall and it has the function of anti-bacterial, anti-inflammatory and convergence. Burn ointment can significantly promote the various types of burn wound healing and show protective effects on patients with severe burn. It can inhibit the formation of scar as well. However, since burn ointment is a traditional Chinese medicine that has complex composition, so its quality control is very important. In this study, we first established the quality standards of burn ointment. Then, researched the effective of burn ointment on scar in rats’ deep Ⅱ degree burn wounds by testing H&E, Masson and the content of HYP and Col-I that are the symbol of scars.Objective:1. To develop an HPLC method for simultaneous determination of gallic acid, aloe-emodin, emodin and chrysophanol in burn ointment.2. Establishing HPLC fingerprint of burn ointment to provide evidence for quality control.3. Researching the effective of burn ointment on scar in rat deep Ⅱ degree burn wounds.Method:1. HPLC was performed on a Diamonsil-C18 chromatographic column and the mobile phase was methanol(A)-0.1%phosphoric acid(B):Omin:10%A; 0～ 15min:10%～50%A; 15～30min:50%～85%A; 30～50min:85% A. The detection wavelength was 254nm, the column temperature was 30℃ and the flow rate was 1.0 ml/min.2. The HPLC method was used, chromatography conditions were Diamonsil C18 (4.6×250mm,5μm) column with gradient mobile phase of methanol(A)-0.1% phosphate(B):0 min,10%A; 0～20 min,10%A→0%A; 20～30 min,30% A→45%A; 30～42 min,45% A→55%A; 42～50 min,55% A→65% A; 50～70 min,65% A→80%A; 70～80 min,80% A→90% A; 80～90 min,90% A→10% A. UV detection wavelength was 254nm and the column temperature was 30℃ with the flow rate of 1.0 mL/min, the sample injection was 20μl.3. We used rat deep Ⅱ degree burn models and the rats were sacrificed on 21th after modeling. Tissues were collected and used to pathological staining, Masson staining and tested content of HYP and Col-I.Results:1. Gallic acid, aloe-emodin, emodin and chrysophanol in Burn Ointment showed the good linear relationships at the range of 0.0611～ 0.4885mg·mL-1 (r=0.9992), 0.0084～0.0675 mg·mL-1 (r=0.9993),0.0109～0.0870 mg·mL-1 (r=0.9997), 0.0739～0.5908 mg·mL-1 (r=0.9994)respectively. Their average recoveries:Gallic acid was 98.22%, aloe-emodin was 98.24%, emodin was 97.28%, chrysophanol was 98.18%.2. HPLC fingerprints of 10 samples of burn ointment were established.11 common peaks were selected as the fingerprint peaks in all samples. Among the obtained fingerprints, most of the detected peaks were separated effectively.10 samples had high similarities.3. Burn ointment reduced the content of HYP and Col-I. Results of HE staining showed that burn ointment could decrease the thickening of the dermis. Masson staining showed that compared with the model group, burn ointment could protect structure of collagen.Conclusion:1. This method was simple, accurate, reliable and repeatable, it can be used for quality control of burn ointment.2. The established HPLC fingerprint has desirable accuracy, repeatability and stability, which can be used for one of the quality control methods of burn ointment.3. Burn ointment could inhibit the form and development of scar in rats.