Study On The Substance Benchmark And Preparation Of Famous Classical Formula Huanglian Decoction

Background:Traditional Chinese medicine（TCM）is a great treasure house in our country,and the famous classical formula is the core resplendence representative of TCM.The research of famous classical formulas compound preparations is the key to the inheritance and development of TCM.The publication of Key Information Table of Ancient famous classical formula（7 Formulas）and other relevant documents has solved some core problems in the research of classical famous formula,and effectively promoted the research and development（R&D）process of famous classical formula.Objective:1.To determine the preparation method of substance benchmark of Huanglian Decoction and draw up its quality standard through multiple batches of substance benchmarks.2.The modern preparation technology was determined and the test with the preliminary amplified was carried out of Huanglian Decoction and explore the preparation technology of Huanglian Decoction granules and establish the quality standard of Huanglian Decoction granules,in order to lay the experimental foundation for the in-depth R&D of the famous classical formula Huanglian Decoction.Methods:1.Literature textual research:Through the investigation of ancient books,the prescription origin,historical evolution,the derivation of the prescription and the origin of the herb were confirmed.2.Preparation and quality evaluation of the substance benchmark:（1）To determine the preparation method of substance benchmarks of15 batches of Huanglian Decoction according to literature textual research and relevant documents.The substance benchmark was freeze-dried in vacuum to obtain the substance benchmark freeze-dried powder（hereinafter referred to as“the corresponding material”）,and the determination of water,extractum and other substances were carried out.（2）With reference to the Chinese pharmacopoeia 2020 edition（hereinafter referred to as the“pharmacopoeia”）rules,to explore the TLC method of specific identification for the substance benchmark of the corresponding material,and establish the UPLC fingerprint method of a substance benchmark corresponding material,and evaluate the similarity of 15 batches substance benchmarks,and made the determination of more index component content,analyzed their quantity value transmissibility.3.Preparation process study:The modern decoction process parameters were screened out by orthogonal method with reference to the quality evaluation indexes of substance benchmark,and the decoction process was verified and the test of the decoction process was carried out.After spray drying and dry compaction,the granules of Huanglian Decoction were obtained,and the optimal preparation technology was preliminarily selected.4.Quality standard of Huanglian Decoction granules:TLC and UPLC quality analysis were carried out on Huanglian Decoction granules according to the substance benchmark quality assessment method.Granules were checked according to the relevant requirements of the general rules of Pharmacopoeia-Book 4,and the quality standard of Huanglian Decoction granules was established.5.Preliminary stability study:The stability of Huanglian Decoction granules was studied by accelerated test.Results:1.Literature textual research:The Following were confirmed.Coptidis Rhizoma was used from Coptis chinensis Franch.;Glycyrrhizae Radix Et Rhizoma is from Glycyrrhiza uralensis Fisch.,and processed by plain-frieding（hereinafter referred to as plain-fried Glycyrrhiza）.Cinnamomi Ramulus is from Cinnamomi cortex（hereinafter referred to as Cinnamon cortex）.Pinelliae Rhizoma was used from Pinelliae Rhizoma Praeparatum Cum Alumine（hereinafter referred to as Pinelliae Rhizoma Praeparatum）,and the rest of the herbal medicine-botanical origins were in accordance with the corresponding categories in Pharmacopoeia.The dosage used was one Liang for 13.8 g,half liter Pinelliae Rhizoma was 34.5 g,one Jujubae Fructus with the core removed was equal to 2.5 g,one“Dou”was 10 L,and 1“Sheng”was 200 m L.2.Study on preparation of substance benchmark and quality standard:（1）Take Coptidis Rhizoma 41.4 g,Cinnamomi cortex 41.4 g,Zingiberis Rhizoma 41.4 g,plain-fried Glycyrrhizae Radix Et Rhizoma41.4 g,Pinelliae Rhizoma Praeparatum Cum Alumine 34.5 g,Ginseng Radix Et Rhizoma 27.6 g,Jujubae Fructus（seedless）30 g,add 2 000 m L of water,heat to boiling at 500 watt（W）,simmer at 300 W and keep it slightly boiling for about 90 min,filter through 400-mesh sieve,and 1 200g of decoction was obtained as the substance benchmark of Huanglian Decoction.（2）The average extraction rate of 15 batches of substance benchmarks decoction was 18.23%,the average water content of the corresponding material was 6.52%,and the average extract rate was45.74%.（3）The thin-layer Chromotograph（TLC）identification method of substance benchmark corresponding material showed good specificity for the Coptidis Rhizoma,Cinnamomi cortex,Zingiberis Rhizoma,plain-fried Glycyrrhizae Radix Et Rhizoma,Ginseng Radix Et Rhizoma,Pinelliae Rhizoma Praeparatum Cum Alumine,Jujubae Fructus.Two sets of UPLC fingerprinting methods were established,and multi-index component content determination was carried out at the same time.The methodological verification was good.（4）A total of 26 common peaks and 13 exclusive peaks were identified for UPLC-PDA Fingerprint of the substance benchmarks of Huanglian Decoction.The fingerprint similarity of 15 batches of substance benchmarks was>0.92.The adding standard recoveries of 11 components were in the range of 95%～105%.The corresponding material contents of the 15 batches of substance benchmarks are as follows:magnolitin1.65～2.477 mg·g^-1,coptidine 11.48～16.77 mg·g^-1,epiberberine 2.21～3.19mg·g^-1,palmatine 1.20～1.65 mg·g^-1,jatrorrhizine 1.03～2.18 mg·g^-1,berberine 13.58～23.29 mg·g^-1,palmatin 3.46～4.69 mg·g^-1,cinnaldehydum1.61～6.67 mg·g^-1,6-ginger capingol 0.83～1.46 mg·g^-1,glycyrrhizin1.61～4.52 mg·g^-1,glycyrrhizic acid 2.14～6.74 mg·g^-1.The transfer rates from decoction pieces to substance benchmarks were as follows:coptidine55.00%～94.56%,epiberberine20.74%～35.13%,berberine19.44%～40.76%,palmatin21.22%～30.97%,cinnaldehydum3.91%～19.44%,6-ginger 9.93%～18.06%,glycyrrhizin 20.81%～53.28%,and glycyrrhizic acid 10.01%～32.42%.（5）A total of 12 common peaks were calibrated for UPLC-ELSD Fingerprint of the substance benchmarks of Huanglian Decoction and 6components were identified.The fingerprint similarity of 15 batches of substance benchmarks>was 0.897.The adding standard recoveries of three components were 90%～110%.The corresponding material contents of the 15 batches of substance benchmarks are ginsenoside Rg₁0.61～0.99mg·g^-1,ginsenoside Re 0.47～1.00 mg·g^-1and ginsenoside Rb₁0.53～0.80mg·g^-1.The transfer rates of the sum of ginsenoside Rg₁and ginsenoside Re ranged 36.86%～76.82%,and ginsenoside Rb₁32.49%～60.03%.3.Preparation technology study:By modern decoction orthogonal screened method of Huanglian Decoction,namely“10 multiple water,soaking 0.5 h,decocting 2 times,each time boiling for 0.5 h”the decoction was prepared,and atmospherically concentrated till the Fried fluid amount to about a quarter[ρ=1.026 g·cm^-3（20℃）],After directly spray drying,spray powder for dry crushing to made the granules,the yield rate of the granules was not less than 65%assayed by double sieve method,the result of related assays of the granules was good.4.Quality standard of granules:（1）According to the substance benchmark assay method,the identification of Huanglian Decoction granules was carried out by the TLC identification method of each herbal slice.Except for the volatilization loss of cinnamaldehyde in the granulation process,the exclusive identification results of the other components were the same as the substance benchmark.（2）The Huanglian Decoction granules（10 g）for each bag contained each no less than magnolitin 13.63 mg,coptidine 91.75 mg,epiberberine19.55 mg,palmatine 9.75 mg,jatrorrhizine 9.97 mg,berberine 132.29 mg,glycyrrhizin 17.85 mg,glycyrrhizic acid 29.05 mg,palmatin 29.97 mg,6-ginger capingo 7.73 mg,the sum of ginsenoside Rg₁and ginsenoside Re10.42 mg,ginsenoside Rb₁4.61 mg.5.Preliminary stability study:After the stability test for 6 months,all the indexes of Huanglian Decoction granules meet the requirements of the initial quality standard.It was determined that the storage temperature should not exceed 40℃,the humidity should not exceed 75%,and the period of validity was at least 6months when the granules was stored at room temperature.Conclusion:The preparation process of substance benchmark of Huanglian Decoction was made up and stable.TLC was used to identify Coptidis Rhizoma,Cinnamomi cortex,Zingiberis Rhizoma,plain-fried Glycyrrhizae Radix Et Rhizoma,Ginseng Radix Et Rhizoma,Pinelliae Rhizoma Praeparatum Cum Alumine,Jujubae Fructus in multiple batches of substance benchmarks,with good specificity.The established fingerprint can better characterize the characteristic components of Huanglian Decoction and effectively control the quality of each processing check piont.Huanglian Decoction granules was prepared by modern technology.The results of each inspection item were good,The above have laid the experimental foundation for the further R&D study of Huanglian Decoction compound preparation.