The Study On Against HPV (Human Papilloma Virus) Of Arnebia Euchroma (Royle) Johnst

Purpose of Research:To seek and find out one or several compounds of Arnebia euchroma (Royle) Johnst that can kill the human papilloma virus (HPV) which could cause Condyloma acuminatum (CA), and try to find out mechanism of action initially. These could provide the base on treating CA with Arnebia euchroma (Royle) Johnst, and we could take this compound as fundamental basis on Pharmaprojects later.Method of Research:According to my tutor' s foundation of research, he found there was probably anti-HPV material existing Arnebia euchroma (Royle) Johnst, and it displayed the water-solubility fraction in Arnebia euchroma (Royle) Johnst mainly. He carried out initial identification with chemical methods, and found out this material presented the masculine reaction of polysaccharide. I continue the following researchs on this basis: The extracting of Crude Polysaccharide in Arnebia euchroma (Royle) Johnst, and put the identification, purification and abstraction techniques on it; and take the Crude Polysaccharide in Arnebia euchroma (Royle) Johnst extracted and the Polysaccharide in Arnebia euchroma (Royle) Johnst purified and abstracted to do the experiment of extraorgan Pharmacodynamic action against HPV(human papilloma virus); then before determining the molecular weight of Polysaccharide in Arnebia euchroma (Royle) Johnst. with the method of gel-permeation chromatograph (GPC), I abstracte Crude Polysaccharide in Arnebia euchroma (Royle) Johnst with SephadexG-100gelatum column chromatography and prove the effect of againsting HPV-DNA according to the experiment of extraorgan Pharmacodynamic. The concrete methods are following. 1. The extraction, identification, purification and abstraction techniques of Crude Polysaccharide in Arnebia euchroma (Royle) JohnstAt first, I determine the factors and levels which infiuence the extraction of Crude Polysaccharide in Arnebia euchroma (Royle) Johnst on the basis of preliminary test, and extract Crude Polysaccharide according to the table of direct cross test and the method of recirculation. Secondly, I put Crude Polysaccharide to identify initially with the a-naphthyl phenol test(Molish response) and the phenylic alcohol-concentrated sulfuric acid test, then determine the content of Crude Polysaccharide in Arnebia euchroma (Royle) Johnst. So it optimizes the best extracting conditions of Crude Polysaccharide in Arnebia euchroma (Royle) Johnst. Thirdly, as there are many micromolecular pigment materials and vegetable proteins existing Crude Polysaccharide in Arnebia euchroma (Royle) Johnst, I adopt decolorization with activated charcoal and deproteinization with savage method, then I abstracte Crude Polysaccharide in Arnebia euchroma (Royle) Johnst with Sephadex G-100 gelatum column chromatography.2. The experiment of extraorgan pharmacodynamicsWe collect the fresh samples of Condyloma Accuminatum(CA) from clinic at first, then homogenate them with homogenizer and dilute them to different proportion with isotonic Na chloride. It is to extract HPV-DNA respectively, then amplificate HPV-DNA with Fluorescent Quantitation PCR equipment(FQ-PCR), and determine the concentration of HPV-DNA according to amplification positive results. Thirdly, we add the samples of Condyloma Accuminatum(CA) prepared to the supplied trial articals of different concentrations, and take the distilled water as negative control. After acting 24 hours, we extract HPV-DNA, and detect the content of HPV-DNA with Fluorescent Quantitation PCR equipment(FQ-PCR), so to make sure the extraorgan pharmacodynamic action of Polysaccharide in Arnebia euchroma (Royle) Johnst.3. The method in detecting molecular weight of Crude Polysaccharide in Arnebia euchroma (Royle) Johnst with Gel-Permeation Chromatograph(GPC)We make standard curve with the nominal sample by means of different units of molecular weight, the X-axis is the retention time(RT) of nominal sample in the polysaccharide, Y-axis is the logarithm of molecular weight in the polysaccharide, then to obtain the equation of linear regression. The sample of Polysaccharide in Arnebia euchroma (Royle) Johnst is dissolved in Na₂HPO₄-NaH₂PO₄ (0.05mol/L) to prepare the solution(10mg/m L). After passing the filter membrane(0.45μm), we take the solution to analysis with High Performance Liquid Chromatogram(HPLC) in the same method of make standard curve and chromatographic condition. According to the calibration trace of GPC and the retention time(RT) of sample, we calculate the molecular weight of each constituent in sample with the GPC software.Results of Research:1. The results of a-naphthyl phenol test(Molish response) and the phenylic alcohol-concentrated sulfuric acid test hint: the presentation of extractive is positive reaction of Polysaccharide, and we identify this material as Polysaccharide in irnebia euchroma (Royle) Johnst initially.2. We optimize the best extracting condition on Crude Potysaccharide in irnebia euchroma (Royle) Johnst with the orthogonal experiment is A₃B₂C₃, it is to Said the number of times in extraction is 3 times, the time in extraction is one hour, and the proportion of solid and liquid in extraction is 1:15. We conclude that there is statistical significance in the number of times in extraction through analysis of variance(iNOVA). Then we prove the effect of againsting HPV-DNA in Crude Polysaccharide in irnebia euchroma (Royle) Johnst according to the experiment of extraorgan Pharmacodynamic.3. The best condition on decolorization with activated charcoal is, 40 minutes, the temperature of water is 80℃, the weight of activated charcoal is 3g, the number of times in decolorization is 2. And in the process of deproteinization, the optimized condition is the proportion of trichlormethlane and n-butanol is 4:1, the mixed liquor agitates on the alternator thermally in 30 minutes, and the number of times in deproteinization is 2 at least.4. We abstracte different molecular weight of Crude Polysaccharide in Arnebia euchroma (Royle) Johnst with SephadexG-100 gelatum column chromatography, and obtain Zcdt-1,Zzcdt-2,Zcdt-3,Zcdt-4,Zcdt-5,Zcdt-6,Zcdt-7,Zcdt-8,Zcdt-9,Zcdt-10,Zcdt-11,Zcdt-12 tubes of Polysaccharide solution. After the identification of extraorgan pharmacodynamics, we conclude the effect of againsting HPV(human papilloma virus) existing the Zcdt-4,Zcdt-5,Zcdt-6 tube.5. The result of determining the molecular weight of Polysaccharide in Arnebia euchroma (Royle) Johnst with gel permeation chromatography is, the test samples appear two peaks in 14.6 minutes and 20 minutes. According to the calibration trace of GPC and the retention time(RT) of sample, we calculate the weight averare molecular weight(Mw) and the number-average molar mass(Mn) of each constituent in sample with the GPC software. In the retention time of 14.6 minutes, the Mv of the peak of Polysaccharide in Arnebia euchroma (Royle) Johnst is 28746 and the Mn of it is 27336. In the retention time of 20 minutes, the My of the peak of Polysaccharide in Arnebia euchroma (Royle) Johnst is 4877 and the Mn of it is 1152.Conclusions of Research:In this experiment, we optimize the best extracting condition in Crude Polysaccharide in irnebia euchroma (Royle) Johnst by Orthogonal Design, and extract Crude Polysaccharide in irnebia euchroma (Royle) Johnst according to this conditions. Then We adopt chemical method to identify the Crude Polysaccharide in Arnebia euchroma (Royle) Johnst, including a-naphthyl phenol test(Molish response), phenylic alcohol-concentrated sulfuric acid test initially, the results hint the presentation of extractive is positive reaction of Polysaccharide, and we identify this material as Polysaccharide in Arnebia euchroma (Royle) Johnst initially. Third we adopt the decolorization with activated charcoal and deproteinization with savage method. Fourthly, we abstracte Crude Polysaccharide in irnebia euchroma (Royle) Johnst with Sephadex G-100 gelatum column chromatography, and take the different distinct fractions to do the experiment of extraorgan pharmacodynamics. We find out there is a part of Polysaccharide againsting HPV-DNA, so we adopt the method in detecting molecular weight of this part with Gel-Permeation Chromatograph(GPC). And we conclude there are two kinds Polysaccharide of different molecular weight existing this part of Polysaccharide in irnebia euchroma (Royle) Johnst, the weight averare molecular weight(Mw) respectively is 28746 and 4877. But, which specific molecular weight of Polysaccharide in irnebia euchroma (Royle) Johnst could kill HPV-DNA, or their joint action, there is still further research. This finding identifies initially the material foundation on treating Condyloma acuminatum (CA) with Arnebia euchroma (Royle) Johnst, and it provides the fundamental basis on clinical Pharmaprojects in CA.