Study On The Preparation And Agents Of Antimicrobial Peptide From Dried Skin Of Rana Temporaria

As the first important line of defense, human skin which against externalattacks played an important role in the evolution of human development.However, due to long exposure in the environment, human skin is one of themost disease-prone sites. Among these multiple diseases, infections caused bypathogenic microorganisms are undoubtedly the most serious, and along with theabuse of antibiotics, a growing number of pathogenic microorganisms produced atenacious resistance, people had to accelerate the speed of antibiotics updates, orto find an antibiotic substitute that does not easily produce resistance.As the unique wildlife resources in northeast, the medicinal value ofChinese forest frog (Rana Chensinensis) was as early as recorded in the"Compendium of Materia Medica”. However, in recent several decades, manyresearchers have found that, even though Rana’s skin long-term exposured to themoist air and microbial aggregation environment, they are not infected by thediseases caused by microorganisms. Studies found that, there is a antimicrobialpeptide in the skin of Rana which can inhibit microorganisms’ survivesignificantly. Compared with those traditional antibiotics, this peptide has a morebroad-spectrum antibacterial activity. More important is that this peptide is noteasy to produce drug resistance, and without a strong irritant of the skin on thehuman body. Therefore, this peptide found in the skin of Rana will be the idealalternative to the traditional antibiotics, and be concerned by more and morepeople.In this paper, using the unique northeast resources—the Rana dry skin—forecperiment materials, we studied the factors that affect the antibacterial activityof Rana skin peptides during its extraction process, such as extraction solvent,extraction solvent concentration, extraction time, extraction temperature, andsolid-liquid ratio. On the basis of study for each impact factors, we optimized theextraction conditions of the Rana skin peptides using response surfacemethodology, the optimal extraction conditions are as follows: the extractionsolvent is6.3%acetic acid solution, extraction time is3.0h,and liquid feed ratiois34.7mL.g-1, rana skin peptides’ antimicrobial activity (inhibition zone) is27.58mm under this conditions. The average inhibition zone of three parallelexperiments for verification is27.31mm, compared with the theoretical prediction relative error is-0.98%.In addition, we studied on the preliminary separation and purification ofRana skin peptides crude extracts in this paper. First, using the SepadexG-25molecular sieve for the initial purification of the skin peptides, we collected theactive part of the collection; then, we used Trcine-SDS-PAGE electrophoresis todetermine the molecular weight and found that these peptides’ molecular weightare about3000-6000Da; third, we used amino bonded column for further purifyand collect the peaks of the various parts using the semi-preparativehigh-performance liquid chromatogram, then, we detected these peptides’ massusing mass spectrometry. Combined with the results of electrophoresisexperiments, we found that these peptides consist of some peptides and theirmolecular weights are very close to5500Da.Based on the good characteristics of topical gel and more advantages thantraditional antibiotics, such as not be easy to produce drug resistance, nostimulation to skin, we used the forest frog skin peptide and the sodiumcarboxymethyl cellulose gel matrix as the main materials make the gelformulations in this paper. Using the bio-mimetic model——response surfaceanalysis——which newly developed, we optimized the gel’s prescription, and theoptimized prescription is as follows: substrate carboxymethyl cellulose content is2.51%, frog skin peptide content is33.34%, and surfactant content is0.09%, thegel’s inhibitory activity (inhibition zone) is30.48mm under this condition. Theaverage inhibition zone of three parallel experiments for verification is30.10mm,compared with the theoretical prediction relative error is-1.25%.On the basis of above, we also researched on the quality standards andstability of the gel in this paper. We initially establish the quality standards of thegel, aiming to provide a theoretical standard for production quality control. At thesame time, the stability experimental results showed that the property, viscosity,pH, concentration and antibacterial activity of the frog skin peptide gel don’tchange significantly under the conditions for6months that temperature is30℃±2℃and relative humidity is65%±5%; even after a long-term of12monthsstay, the results also showed that the frog skin peptide gel’s property, viscosity,pH, concentration and antibacterial activity don’t change significantly in thecondition for12months that temperature is25℃±2℃and relative humidity is 60%±10%. In short, the gel has a good stability.