Optimization Of Quality Analysis Method And Improvement Of Quality Standard For Jianwei Xiaoshi Pills

Jianwei Xiaoshi pills(JXP)is a common Chinese traditional medicine for the treatment of functional dyspepsia caused by spleen and stomach weakness,which is composed of Pseudostellariae Radix,Dioscoreae Rhizoma,Crataegi Fructus,Hordei Fructus Germinatus and Citri Reticulatae Pericarpium.The quality analysis items of JXP in the Chinese Pharmacopoeia2020,only the TLC identification of Pseudostellariae Radix and Crataegi Fructus and the determination of hesperidin are included except for the convention inspection items of pills.The current quality evaluation standard indicators are difficult to comprehensively evaluate the quality of JXP.Therefore,there are certain differences in the quality of JXP produced by different manufacturers.Based on the theory of traditional Chinese medicine,this study improved the qualitative and quantitative analysis indicators and methods of JXP,for providing a scientific method for comprehensively evaluation the quality of JXP.1.The pretreatment method and TLC inspection condition for qualitative identification of Pseudostellariae Radix by TLC were optimized.The single factor experiment was used to investigate the extraction solvent,extraction method,extraction time and solvent volume of the sample pretreatment.The optimal pretreatment method for thin layer identification of Pseudostellariae Radix in JXP was determined as follows: 1g of sample fine powder was added with 10 m L of absolute ethanol were extracted for ultrasonic treatment for 30 min,and the filtrate was concentrated to 1m L as the test solution.Through the comparison of the unfold effects of four development systems,the identification method of toluene:ethyl acetate:2-butanone:formic acid:water(5:15:2:4:2)as the development solvent and 365 nm with fluorescent lamp was determined.By comparing the unfold effection of thin plates from different manufacturers and at different temperatures and humidities,it showed that this method has good applicability.The identification of 34 batches of market samples verified that the pre-treatment method of the new method was simpler than the preparation of Pseudostellariae Radix in the Pharmacopoeia and the new method had better results in identification.2.The identification method of Dioscoreae Rhizoma by TLC was established.The single factor experiment was used to investigate the extraction solvent,extraction method,extraction time and solvent volume of the sample pretreatment.The optimal pretreatment method for the identification of Dioscoreae Rhizoma thin layer in JXP was determined as follows: 1g of sample fine powder was added with 10 m L of methanol for ultrasonic treatment for 30 min.After the filtrate was evaporated,it was dissolved with 1m L of chloroform: methanol(1:1)mixed solution as the test solution.Through the comparison of the unfold effection of three development systems,the identification method using trichloromethane:methanol(100:1)and 10%phosphomolybdic acid ethanol solution as color developing agent was determined.This method had good durability by comparing the effections of thin plates from different manufacturers and at different temperatures and humidities.The 34 batches of market samples were verified,and the results showed that the method was stable and reliable.3.A dual wavelength HPLC method was established for the simultaneous determination of cyclopeptide B in Pseudostellariae Radix,nobiletin and tangeretin in Citri Reticulatae Pericarpium,and the pretreatment method of samples was optimized.Through the optimization of mobile phase,elution conditions,column types and column temperature conditions,the applicability of the system method was investigated,and the chromatographic conditions were determined as follows: Shiseido C18 column TYPE MG(4.6×250 mm,5μm),acetonitrile(A)-water(B)as the mobile phase,gradient elution:0～10min(20% A→55%A),10～30min(55%A),30～35min(20%A),the flow rate was 1.0 m L/min,the detection wavelength was 203 nm and330nm,the column temperature was 40℃,injection volume was 20 μL.The number of theoretical plates shall not be less than 1000 according to the calculation of Heterophyllin B,nobiletin and tangeretin.The single factor experiment was used to investigate the extraction method,extraction solvent,extraction time and material to liquid ratio of sample pretreatment.The preparation method of sample solution was determined as follows: 2g of sample fine powder,added 50 m L of 60% methanol,weighted,heated and refluxed for 60 min,weighted again,replenished the weight with 60% methanol,filtered,took 25 m L of subsequent filtrate and evaporated,dissolved the residue with methanol to a constant volume of 10 m L.The content range of HB in 34 batches of samples was 4.9262 μg/g～89.5418 μg/g,the total content range of nobiletin and tangeretin was 2.0777 μg/g～91.8618 μg/g.According to the comprehensive analysis,we suggested that the content limit of HB in JXP shall not be less than 9.4938 μg/g,the total content of nobiletin and tangeretin shall not be less than 5.3 μg/g.4.The HPLC method for the determination of allantoin in Dioscoreae Rhizoma was determined and the pretreatment method was optimized.The applicability of the method system was investigated by optimizating mobile phase proportion and flow rate,column type and column temperature,and the chromatographic condition was determined as follows: Agilent column Polaris 5 C18 Ether(4.6 × 250 mm,5 μ m),the mobile phase was water:acetonitrile(98:2),flow rate was 0.5 m L/min,the detection wavelength was 220 nm,column temperature was20℃ and injection volume was 10μL.The number of theoretical plates shall not be less than1000 according to the allantoin peak.The single factor experiment was used to investigate the extraction method,extraction solvent,extraction time and material to liquid ratio of sample pretreatment.The preparation method of sample solution was determined as follows: 1g of sample fine powder,added 10 m L of 30% methanol,weighted the weight,ultrasonic extractied for 30 min,supplement the weight,filtered,and took the subsequent filtrate.The content range of allantoin in 34 batches of samples was 0.0469 mg/g～0.6810 mg/g.According to the comprehensive analysis,we suggested that the content limit of allantoin in JXP shall not be less than 0.17 mg/g.5.It was planned to draw up the draft of the quality detection standard for JXP.In the draft,the TLC identification method of Dioscoreae Rhizoma was added under the identification items,and the TLC identification method of Pseudostellariae Radix was replaced.Under the content determination items,the HPLC method was added to determine the content of HB,nobiletin and tangeretin,and HPLC method to determine the content of allantoin.