Studies On The Preparation And Quality Control Methods Of Compound Chihong Ganules

Objective:To establish the production process and the quality control methods for compound Chihong granules(CCGs);to understand the potential molecular mechanism of CCGs in the treatment of rheumatoid arthritis,and to provide reference for its clinical application.Methods:(1)The extraction amount of corresponding component of the main drug and the extraction rate in CCGs were used as the evaluation indexes,solvent volumes,extraction time,and extraction times were chosen as the factors for single-factor experiment,orthogonal experiments was adopted for the optimization of the water extraction parameters for CCGs;the optimal relative density of the thick paste and the mixing time for making soft materials for granulation were investigated;molding rate,fluidity,and solubility of the particles were used as indicators for the evaluation of the molding process of CCGs was investigated.Based on the preparation conditions selected from the previous experiments,the scale-up tests of three different batches of pilot samples of CCGs were completed.(2)TLC was used to qualitatively identify the two monarch drugs of Radix Angelicae and Chuanxiong in CCGs;the particle size,moisture content,solubility,and packaging differences of the granules were determined according to the requirements of General Rule 0104 of the Fourth Section of the Chinese Pharmacopoeia(2020);HPLC methods were used to determine the contents of the main active components of CCGs(i.e.,paeoniflorin,gallic acid,and protocatechuic acid).(3)UPLC-MS/MS method was used to analyze the components of CCGs,and network pharmacology and molecular docking technologies were used to analyze the core components,targets,and related signaling pathways of CCGs in the treatment of rheumatoid arthritis,in order to understand the possible molecular mechanisms of CCGs in the treatment of rheumatoid arthritis.Results:(1)The production parameters of CCGs were optimized as follows: 14 Chinese medicinal herbs were weighed according to the proportion of the prescription,and mixed.After adding 6 volumes of water,the mixture was decocted for two times(each for 2 hours).The extracts were then combined,filtered,and concentrated to a thick paste with a relative density of 1.20～1.30(80 ℃).The pre-gelatinized starch was added to the paste to mix for 15 minutes.The mixture was then granulated,dried,screened,arranged,and packaged.(2)In the TLC qualitative identification studies of Radix Angelicae and Chuanxiong,the spots were clear without interference for the negative control sample and with good reproducibility when compared with those in the control sample;the particle size,moisture,solubility,and loading difference of three different batches of samples were all qualified;paeoniflorin has a good linearity in the range of 5.22 to167.04 μg/m L with the RSD of precision,stability and repeatability being all less than 2%.The average sample recovery rate was 102.84%with the RSD of 1.36%.In the determination of gallic acid and protocatechuic acid,gallic acid has a good linearity in the range of 50 to1600 μg/m L and protocatechuic acid has a good linearity in the range of 2.625 to 84.000 μg/m L.The RSDs of the precision,stability and repeatability tests of gallic acid and protocatechuic acid were all less than 2%.The average recovery rate of gallic acid was 101.56% with the RSD of 1.87%,and the average recovery rate of protocatechuic acid was 100.04% with the RSD of 1.78%.(3)Based on UPLC-MS/MS results,and with the aid of related databases and literatures,31 active components(i.e.,isoliquitigenigenin,beta-aryophylene,chlorogenic acid)were identified and 135 corresponding targets were obtained of the CCGs,which shared with the 5662 RA disease targets 82 common targets(e.g.,TNF,IL-6,TP53).Through regulation of the biological processes such inflammatory reactions and cellular apoptosis,CCGs elicited anti-RA effect through regulating the TNF,NF-κB,MAPK signalling pathways.The docking energy of four main active components(isoliquitigenin,beta-aryophyllene,Myristicin,and osthol)on the five core targets(TNF,IL-6,TP53,CASP3,CAT)were all less than-5k J/mol,indicating that these targets may be the CCGs’ s targets.Conclusions:(1)The preparation parameters of CCGs were well controlled and suited for large-scale production.(2)The established TLC identification methods of Radix Angelicae and Chuanxiong and HPLC content determination methods of paeoniflorin,gallic acid and protocatechuic acid were simple,accurate,reliable,and reproducible,which can provide experimental data for the quality control of CCGs.(3)Through network pharmacology and molecular docking method,it was preliminarily revealed that multiple components in CCGs,including isoliquidigenin,beta-aryophylline,and chlorogenic acid,played a therapeutic role in RA by regulating the signal pathways such as TNF,NF-κB,and MAPK signaling pathways.The work can provide a basis for the clinical application of CCGs.