| Objective:Recurrent oral ulcer is one of the most common diseases of the oral mucosa,which annoyed greatly to the people's normal lives.Propolis has a significant effect on the treatment of oral ulcer.Small molecule nano hydrogel is a new material,which can be utilized as the extended-release drug carrier.This research work is based on the original study in the laboratory to prepare a new generation of small molecule pharmaceutical carrier gel material on the basis of the principle of supramolecular self-assembly.By using a new generation of nano-gel formulation to carry propolis,a new propolis preparation was intended to be developed for the treatment of oral ulcers.The In addition,it is also aimed to clarify the action meachnism of the self-assembly of the selected two components,as well as the influence ofexternal conditionsto the self-assembly behavior,to study and regulate the optimal gel formation,and to investigate the action mechanism of herbal drug-carrying small molecue nano hydrogel.Through research on the quality standard,pharmaceutics and pharmacodynamic evaluation of the newpreparation,scientific evidence will be provided for the clinical application of nano-hydrogels propolis small molecules and new formulations of traditional Chinese medicine treatment of oral ulcers.Methods:1.Construction of small molecules nano-hydrogel matrix and property research(1)Using Sol-gel method,take a certain mass of riboflavin derivative and the triazine ring with a volume of pure water,heated at 120?,the mixture was completely dissolved and then rapidly cooled to room temperature,to obtain small molecule nano hydrogel matrix.(2)Adopting NMR,IR to study the hydrogen bonding position,using scanning electron microscopyto observe the aggregation behavior of microscopic gel under X-ray diffraction.(3)The critical concentration is determined by measuring the gel into a gel capacity,investigated the effects of different pH values on the AB hydrogel small molecules,determination of the gel matrix of the gel to study the properties of sol-gel matrix transition temperature,pH.Results:(1)Riboflavin derivative and a triazine ring were mixed in a molar rati oof 1:1,1:2,2:1,3:1,4:1,respectively,to form a stable yellow opaque gel in water,and then to record AB11,AB12,AB21,AB31,AB41,all gel processes are thermally reversible with good stability.(2)The critical concentration:critical concentration of the small molecules of nano water gel formed by AB11(c = 0.12 mol·L-1)with AB12(c= 0.11mol-L-1)is larger.All ratios of gels have thermal reversible property and good stability,which have no deteriozation or crystallization under the sealed condition in normal temperature.(3)Observed under a scanning electron microscope,AB 1 1 xerogels microstructure is composed of fiber bundles with many nodes linked between these fiber bundles,forming a spherical structure AB 12 xerogels microstructure is composed of stacked-together fibers with length of 2-4?m and sheet-shaped.Such sheet's width and length could reach up to 10-20?m and hundreds of these sheets can be stacked higher dimensional sheet assembled aggregates.AB21,AB31,AB41 xerogels showed rod-like structure disorganized.There are the short rods with length of 3-5?m as well as the long rods with length of 10-20?m.(4)Certain proportions of A and B were self-assembled through intermolecular hydrogen bonding interaction.(5)Critical concentration:The critical concentration of AB 11(c = 0.12 mol·L-1)small molecules nano hydrogel is relatively large when compared with AB12(c = 0.11 mol·L-1).Sol-gel transition temperature:When AB11 concentration increased from 0.06 mol·L-1 to 0.13 mol·L1 Sol-gel transition temperature rose from 72? to 98?.When the concentration of AB12 increased from 0.06 mol·L-1 to 0.12 mol·L-1,sol-gel transition temperature rose from 90? to 98?.The pH values of AB 11,AB12,AB21,AB31,AB41 were 6.73,6.92,7.05,7.35,7.33.Different pH values of hydrogel nano affect AB small molecule:AB small molecules gel at solution of pH 2,4,6,7,8,10,12 are more stable.Conclusion:Riboflavin and triazine ring derivatives can self-assemble to form small molecule nano-hydrogels by intermolecular hydrogen bonding interactions.AB 11 and AB12 can be selected as the drug matrix,which has good thermal stability,insensitive to acid and alkali solution with pH of 6.7-7.02.Performance study of small molecule nano hydrogel carrier propolis fine powderMethods:(1)To adopt sol-gel method for the preparation of propolis small molecule nano hydrogel,and to determine the optimal drug loading and maximum drug loading the gel through drug loading capacity test.(2)Morphology was observed under a scanning electron microscope before and after the gel matrix vacuum freeze-dried powder drug inquiry microscopic structural changes.(3)Properties of propolis small molecule nano hydrogel were investigated by thermal stability test,cold test,heat test,light test,measurement of pH value and pH range of usage.Result:(1)The AB11s maximum drug loading was 0.3mg(c = 0.12 mol·L-1),AB12's maximum drug loading could reach up to 0.45 mg(c = 0.11 mol·L-1).(2)In SEM figure,single small spheres exsited after loading drug AB11,with 20?m sticks and some bars are embedded in the network structure.Their morphology is the network structure of perforated and there are connected globulars,which are similar with those of no drug loading.AB12 does not contain the drug SEM graph showing sheet of large area.The pieces are stacked after loading drug with the shape of thick sheet and some films overlapping to form petals.The petal-like structure is composed of a series of curling of the sheet with a diameter of 5-10?m.(3)Thermal stability:propolis small molecule nano hydrogel and gel matrix will turn into the solution to flow down when the temperature is between 90-98 ? and 80-98 ?.pH:6.7-7.0?pH range:more stable in solution of pH7?8.Cold test:At a temperature of-19?,the appearance has cracks when taken out after 24h,while at 3?,of the appearance will have no change with the same treatment.Heat test:When taken out from the 55? of incubator after 24h,the gel matrix and propolis small molecule nano hydrogel appearance remain unchanged.Light experiment:removed under 5000 ± 450 1x after 24h,the gel matrix and propolis small molecule nano hydrogel appearance remain unchanged.Summary:AB12 is the best gel matrix with the maximum carrying capacity of propolis of 0.45 mg(c = 0.11 mol·L-1).The gel structure is more stable after the gel matrix carrys propolis.The pH value of propolis small molecule nano hydrogel is between 6.7 and 7.0 with pH7?8 as the optimum for stability.The gel is stable under the temperature of 0 ?-55 ? and also stable to light.3.Pharmaceutical research of propolis small molecule nano hydrogel formulation3.1 Quality Standard of propolis small molecule nano hydrogel Methods:(1)Respectively using TLC,UV spectrophotometry to identify and determinate gel in the propolis;(2)Following all the technical requirements stipulated under the appendix I Q item for gelling agents in Chinese Pharmacopoeia of 2010 edition to perform all the experiments;According to the methods under appendix ? G to determine the pH of propolis small molecule nano-hydrogels;To check the microbial limits following the appendix XII I C;for heavy metals,appendix ? E methods were used;for arsenic salt test,Appendix IX F method is followed.Results and conclusions:(1)Using chloroform-methanol-butanone(9.4:0.3:0.3)as developing solvent,and,aluminum chloridesolution as coloring agent,thin layer chromatography was developed and observed under UV light(365nm)for detection of propolis.The concentration of total propolis flavonoids calculated in rutin(C17H30O16)shall be not less than 30.0%,(2)All test results are in line with the requirements stipulated under the appendix IQ of Chinese Pharmacopoiea for gels.The pH values of three batches are6.9,7.2 and 7.1 respectively;the limits for heavy metals and arsenic salts did not exceed ten per million;for microbial limits,the total number of bacteria detected is 289-31 1 cells/g,total number for fungi(yeast)is less than 10/gPseudomonas aeruginosa,Staphylococcus aureus,Clostridium,Candida albicans,and live mites were not detected.3.2 Pharmaceutics evaluation of propolis small molecule nano hydrogelMethods:The viscosity of gel was tested using viscometer;,adhesion was measured using the displacement method,Koike diffusion was applied for studying the mucosal permeability of gel;and in vitro release behavior was investigated by vitro dissolution method.Results and conclusion:The viscosity,adhesion,permeability and its release of propolis small molecule nano hydrogel are in accordance with the relevant provisions of the 2010 edition of "Chinese Pharmacopoeia".3.3 Pharmacodynamic evaluation and mechanism study of propolis small molecules and nano-hydrogelMethods;(1)Using diffusion method to determine the inhibition rate of propolis small molecule nano hydrogel for the inhibition of seven bacteria to evaluate its inhibitory effect.(2)Using the modified model of Pang rabbits for mouth ulcers model and taking Guilin Watermelon preparation as the positive control,the daily changes in rabbit ulcer diameter were observed after treatment.The ulcer healing time was recorded and histopathological examination of ulcer area specimens are carried out under the light microscope.(3)The anti-inflammatory effect of propolis small molecules Nano-hydrogels was evaluated by two animal models names xylene-induced ear edema in mice and egg-induced rat paw edema.(4)Using the modified model of Pang rabbits mouth ulcers,the serum IL-6 and TNF-? levels of experimental rabbit were measured after administrating the drug for 0 day,four days and eight days.Results:(1)Inhibition rates are measured for each bacteria with the following results:Staphylococcus aureus 89%,Enterococcus 92%,E.coli 88%,Pseudomonas aeruginosa 90%,Proteus 88%,Staphylococcus epidermidis 88%,and Salmonella 87%.(2)Propolis group of oral ulcers rabbits model after the treatment with propolis small molecule nano hydrogelfor 4 days and 8 days,the oral ulcer diameters were significantly lower than the control group with the P value less than 0.001;its anti-ulcer effect is comparable with watermelon preparation group with P value larger than 0.05;the average healing time of propolis group for mouth ulcers are 9.5 ± 1.29)days,which is significantly less than the control group with P<0.05;the average healing time is comparable with watermelon frost group with P>0.05.Under the visible light microscope with 4 days of treatment,each group has significant anti-inflammatory effect but the control group remained visible epidermal tissue ulcers,and a lack of organization,acute inflammatory cell infiltration between muscle and part tissue necrosis.Propolis group still has visible epidermal tissue ulcers but the number of inflammatory cell infiltration in the ulcer was significantly reduced.Watermelon preparation group still possessed visible tissue necrosis and abscess.After eight days of treatment,the control group was observed that the number of inflammatory cells was reduced between muscles,but muscle tissue edema still exist.After 8 days of treatment with Propolis,the ulcer surface tend to heal except for a small amount of tissue with inflammatory cell infiltration,fibroblast proliferation,which was surrounded by some of the collagen fibers.(3)Propolis small molecule nano hydrogel can significantly inhibit the xylene-induced ear edema and egg-induced rat paw edema in mice.(4)The IL-6 and TNF-a values in Propolis group and watermelon preparation group were significantly lower than that in the control group with P<0.001.Conclusion:(1)Propolis small molecule nano-hydrogels has antibacterial effect,and its inhibitory effect is related to the concentration.The positive control Guilin Watermelon preparation and propolis solution is quite similar with P>0.05.It has the strongest inhibitory effect for Staphylococcus aureuswith P<0.05.(2)Propolis small-molecule nano-hydrogel has significant therapeutic effect for improving oral ulcer in Pang rabbits model with the equivalent effect as Guilin watermelonpreparation.(3)Anti-inflammatory tests proved that the product has certain anti-inflammatory effect.(4)propolis small molecules gel formulation can promote the healing of ulcers by reducing peripheral blood TNF-? levels of inflammatory cytokines such as IL-6.3.4 Preliminary stability test of propolis small molecule nano hydrogelMethods:Light acceleration,heat,cold,freezing,centrifugal stress tests were performed for Propolis small molecule gel to determine the preliminary three month stability of three batches of samples.Results and conclusions:The test results showed that propolis small molecule nano hydrogel glue was stable at temperature of 4-55 ?,relative humidity of 85%± 5%,dentrifugation,and at 5000 ± 450 lx light conditions with no change in physical form and content of active ingredients;Under the freezing conditions below 0? the gel turned into solid to give the hint that this formulation can be stored at room temperature.Preliminary tests showed the three batch samples to be stable at room temperature of 20 ?,dark,humidity of 75%for 3 months with no significant change in macroscopical character and content. |
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