Development And Research On GanJin JieDu YuYang Gargle

Objective:Ganjin Jiedu Yuyang Gargle is composed of Camellia nitidissima C.W.Chi leaves,Saccharum officinarumLeaves and other traditional Chinese medicine.The formula of Ganjin Jiedu Yuyang gargle is based on the Instructor of Academic Experience Inheritance of National Famous TCM Experts,Professor Deng Jiagang’s experience,it has the effect of clearing heat and detoxifying,removing corrosion and healing,and can be used for the treatment of oral burn,flesh erosion and pain after chemotherapy.In this study,according to the declaration requirements of medical institution preparations,the traditional Chinese medicine theory was combined with modern science and technology to optimize the preparation process of the product,develop a set of stable,controllable and reasonable quality standards,and study its pharmacodynamic mechanism and safety,so as to provide experimental data for medical institution preparation declaration,and provide reference for the development of gargles with Chinese medicine characteristics.Methods:1.Preparation process:The content of total polysaccharides in Ganjin Jiedu Yuyang gargle was determined by UV-vis spectrophotometer;A QAMS method was established to determine the contents of protocatechuic acid,caffeic acid,isofraxidin,cichoric acid,Okicamellia and rosmarinic acid in Ganjin Jiedu Yuyang gargle.The single factor experiment was used to investigate the extraction time,extraction times and solid-liquid ratio.Combined with orthogonal design experiment L₉（3）⁴,with the total normalized value（OD value）as the evaluation index,the best preparation process was optimized.2.Quality standard research:according to the 2020 edition of《Chinese Pharmacopoeia》technical quality specification,the perfume,clarity,cold stability,heat stability,PH,relative density and other performance indicators of gargle were investigated;TLC was used to identify the five herbs in Ganjin Jiedu Yuyang gargle,including Camellia nitidissima leaves,Saccharum officinarumLeaves,etc.The optimal TLC conditions were screened from the influencing factors of thin layer plate,developing agent,chromogenic agent,temperature and humidity.HPLC was used to establish the fingerprint of Ganjin Jiedu Yuyang gargle;The contents of protocatechuic acid,caffeic acid,isofraxidin,chicoric acid,Okicamellia and rosmarinic acid in Ganjin Jiedu Yuyang gargle were determined by QAMS method,and the content limits of each component were determined.3.Efficacy study:To observe the effect of Ganjin Jiedu Yuyang gargle on the general state of golden hamsters with chemotherapy-induced oral mucositis by establishing golden hamster model of chemotherapy-induced oral mucositis,and to score the buccal bursa inflammation of golden hamsters;The serum levels of TNF-γ,IL-1,IL-6,IFN-γwere detected by ELISA.Pathological changes of golden hamster cheek pouch were observed by HE staining.The expression levels of MMP-9,COX-2,PARP in the buccal pouch tissue of hamsters were detected by immunohistochemistry.4.Safety evaluation:The acute toxicity test of Ganjin Jiedu Yuyang gargle was carried out in mice and rats,and the intragastric administration toxicity and oral administration toxicity were studied respectively.According to the maximum dose method,the mice were intragastrically administrated with Ganjin Jiedu Yuyang gargle（concentration 1.09 g/ml）,and the intragastric volume was 0.3 mL/10 g.The rats were intragastrically administrated with Ganjin Jiedu Yuyang gargle（concentration 1.09 mg/ml）,0.5 mL/time,4times/d,and the situation before and after administration was observed.In the local toxicity test of Ganjin Jiedu Yuyang gargle,New Zealand white rabbits and guinea pigs were selected for skin irritation test and allergy test of Ganjin Jiedu Yuyang gargle,respectively.Results:1.Study on preparation technology:A method for determination of total polysaccharide in Ganjin Jiedu Yuyang gargle was established.The linear range of total polysaccharide was 0.01147 mg～0.1147 mg（R²=0.99979）.The RSD of precision,repeatability,stability and recovery were 0.45%,0.64%,0.45%and 0.93%,respectively.A QAMS method was established to determine the contents of protocatechuic acid,caffeic acid,isofraxidin,cichoric acid,okicamellia and rosmarinic acid in Ganjin Jiedu Yuyang Gargle.The linear ranges of protocatechuic acid,caffeic acid,isofraxidin,cichoric acid,okicamellia and rosmarinic acid were12.02～120.18μg/ml（R²=0.99995）,25.92～259.20μg/ml（R²=0.99998）,18.40～184.00μg/ml（R²=0.99995）,109.98～1099.80μg/ml（R²=0.99996）,46.65～466.48μg/ml（R²=0.99966）,14.00～140.00μg/ml（R²=0.99997）,respectively.The precision RSD was 0.14%,0.14%,0.18%,0.24%,0.21%,0.44%,the stability RSD was 0.5%,0.93%,1.29%,1.29%,1.52%,1.04%,the repeatability RSD was 0.91%,0.56%,0.94%,0.92%,1.06%,1.08%,and the recovery RSD was1.49%,0.63%,1.61%,1.72%,0.66%,1.76%,respectively.RSDs of relative correction factors of protocatechuic acid,caffeic acid,isofraxidin,okicamellia and rosmarinic acid were 0.44%,0.10%,0.66%,0.93%and 0.99%,respectively;According to the total evaluation value and considering the actual cost,the extraction time of 1,1.5,2 h,extraction times of 2,3,4,solid-liquid ratio of 1:15,1:20,1:25 were selected by single factor experiment.The orthogonal experiment analysis of variance showed that each factor had no significant effect on the extraction effect（P>0.05）.The comprehensive score showed that the best preparation process was 1.5 h,4 times of extraction,solid-liquid ratio of 1:15.The validation results showed that the normalized average value of three repeated experiments was 0.91,RSD was 2.71%,indicating that the preparation process was stable and reasonable.2.Quality standard research:Through the investigation of the performance indexes of Ganjin Jiedu Yuyang gargle,it can be seen that the Gargle has mint flavor,clarification,pH（25℃）range of 3.8–4.2,relative density（20℃）of1.0012–1.0032,and it is stored at 4℃ for 24 h with a slight freezing;Storage at 40℃ for 24 h without turbidity,color stability;TLC was used for the qualitative identification of five medicinal materials in Ganjin Jiedu Yuyang gargle,including Camellia nitidissima leaves,Saccharum officinarumLeaves,etc.The TLC methodology of each medicinal flavor was established.The results showed that the method was specific.The fingerprint of 10 batches of Ganjin Jiedu Yuyang gargle was established by high performance liquid chromatography,and 17 common peaks were calibrated.The similarity between the fingerprint of 10 batches of Ganjin Jiedu Yuyang gargle and the control fingerprint was greater than 0.97,indicating that the fingerprint was reproducible.The contents of protocatechuic acid,caffeic acid,isofraxidin,cichoric acid,Okicamellia and rosmarinic acid in Ganjin Jiedu Yuyang gargle were determined by QAMS method,and the content limits were determined as follows:protocatechuic acid should not be less than 50μg,caffeic acid should not be less than 100μg,isofraxidin should not be less than 50μg,cichoric acid should not be less than90μg,okicamellia should not be less than 50μg,rosmarinic acid should not be less than 30μg.3.Efficacy study:The model of chemotherapy-induced oral mucositis was used to study the efficacy of Ganjin Jiedu Yuyang gargle.The results showed that Ganjin Jiedu Yuyang gargle had a good therapeutic effect on chemotherapy-induced oral mucositis.Compared with the model group,Ganjin Jiedu Yuyang gargle high,medium and low dose groups could significantly reduce the symptom score of cheek pouch mucositis caused by modeling（P<0.05 or P<0.01）,significantly reduce the levels of TNF-α,IL-1and IL-6 in serum of golden hamsters（P<0.01）,significantly increase the level of IFN-γin serum of golden hamsters（P<0.05 or P<0.01）,improve the pathological structure of cheek pouch mucosa,and significantly reduce the expression of MMP-9,COX-2 and PARP protein in cheek pouch mucosa of golden hamsters（P<0.01）.4.Safety evaluation:In the acute toxicity test,10 min after the end of intragastric administration,the mice showed uncoordinated movement,and then prone did not move,but there was no spasm,local paralysis or rigidity,and the mice were breathing normally.After 20 min,the mice returned to normal activity.During the observation period,the animals’diet,water intake and activity were normal.According to the calculation of clinical adults,the above situation would occur only when the oral intake reached 32.7 g/kg in vivo.However,the clinical application of this product was oral and topical gargle with a concentration of 0.163 g/ml and a daily dosage of about 30 ml.The dosage and the amount that could be absorbed to the whole body were less.The results of pathological section showed that there was no obvious pathological change in the morphological structure of each organ（heart,liver,spleen,lung and kidney）in the Ganjin Jiedu Yuyang Gargle Group.There was no abnormal general condition and oral mucosa in rats after oral administration.At the same time,the pathological section results showed that therewas no obvious pathological change in the morphological structure of each organ（heart,liver,spleen,lung and kidney）in the Ganjin Jiedu Yuyang Gargle Group.Therefore,it can be considered that Ganjin Jiedu Yuyang Gargle has no irritation to oral mucosa,and is safe and low toxic for external use.In local toxicity test,skin irritation test showed that single administration and multiple administration of Ganjin Jiedu Yuyang gargle did not appear erythema,edema and other irritation reactionsin intact and damaged skin areas;In the allergy test shows that Ganjin Jiedu Yuyang gargle use in the skin,do not cause allergic reaction.Conclusion（s）:This research showed that the preparation technology of Ganjin Jiedu Yuyang gargle was reasonable,feasible and stable.The quality of the preparation is controllable and stable;It has a good therapeutic effect on chemotherapy-induced oral mucositis.At the same time,it is safe to use Ganjin Jiedu Yuyang gargle locally in the mouth.