Xiao Liu1, Xiao-li Wang2, Li Wu3, Huan Li2, Kun-ming Qin2, Hao Cai3, Ke Pei3, Ting Liu4, Bao-chang Cai5. 1. College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, PR China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, PR China; Nanjing Haichang Chinese Medicine Group Corporation, Nanjing 210061, PR China; College of Science, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA. 2. College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, PR China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, PR China; Nanjing Haichang Chinese Medicine Group Corporation, Nanjing 210061, PR China. 3. College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, PR China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, PR China. 4. Shanghai AB Sciex Analytical Instrument Trading Co., Ltd, Shanghai 200233, PR China. 5. College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, PR China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, PR China; Nanjing Haichang Chinese Medicine Group Corporation, Nanjing 210061, PR China. Electronic address: bccai@126.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Da-Huang-Fu-Zi-Tang (DHFZT) is a crucial TCM formula commonly used for the treatment of acute pancreatitis in Chinese clinical application. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP). The goal of this paper was to study the underlying correlations between the chemical spectra and the protective effect of DHFZT on pancreatic acinar cell to reveal the real bioactive compounds in DHFZT. MATERIALS AND METHODS: The fingerprint chromatograms of rat serum after oral administration of DHFZT were established by UHPLC-ESI-Q-TOF-MS technique. At the same time, the model of anti-acute pancreatitis on cells was established by adding 10(-7) mol/L cerulein to AR42J cell line, and the protective effects of the serum on pancreatic acinar cell from injury was evaluated by detecting the efficacy of amylase. Then, the spectrum-effect relationships between UHPLC fingerprints and anti-acute pancreatitis activities were evaluated using canonical correlation analysis (CCA) statistical method. The chromatogram separation was performed on a C18 reversed phase UHPLC column (2.1 mm × 100 mm, 3.5 μm, Agilent), the column temperature was set at 35°C. The mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution. The serum samples were analyzed both in negative and positive ion mode. The mother and productive ions were scanned within the mass range of m/z 100-1200 and 50-1200, respectively. A thorough analysis of a great deal of information of the constituents in the rat serum was undertaken. The structure identification of the detected compounds was achieved by using high resolution MS values as well as the MS/MS fragments. RESULTS: Eighteen peaks in rat serum after oral administration of DHFZT were detected within only 30 min recorded chromatograms. The structure of the 18 compounds were then given out, of which 10 were the original form of compounds absorbed from DHFZT, 8 were the metabolites of the compounds existed in rat serum. According to the CCA results, talatisamine, rhein glucoside, rhein isomer methylation, hypaconine, hydroxyl-chrysophanol, emodin glucuronide conjugation, and chrysophanol glucuronide conjugation were finally found to be the main anti-acute pancreatitis components in DHFZT. CONCLUSIONS: The model presented in this paper successfully discovered the spectrum-effect relationships of DHFZT, which showed a representative way to discover the primary active ingredients from the complicated herbal drugs.
ETHNOPHARMACOLOGICAL RELEVANCE: Da-Huang-Fu-Zi-Tang (DHFZT) is a crucial TCM formula commonly used for the treatment of acute pancreatitis in Chinese clinical application. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP). The goal of this paper was to study the underlying correlations between the chemical spectra and the protective effect of DHFZT on pancreatic acinar cell to reveal the real bioactive compounds in DHFZT. MATERIALS AND METHODS: The fingerprint chromatograms of rat serum after oral administration of DHFZT were established by UHPLC-ESI-Q-TOF-MS technique. At the same time, the model of anti-acute pancreatitis on cells was established by adding 10(-7) mol/L cerulein to AR42J cell line, and the protective effects of the serum on pancreatic acinar cell from injury was evaluated by detecting the efficacy of amylase. Then, the spectrum-effect relationships between UHPLC fingerprints and anti-acute pancreatitis activities were evaluated using canonical correlation analysis (CCA) statistical method. The chromatogram separation was performed on a C18 reversed phase UHPLC column (2.1 mm × 100 mm, 3.5 μm, Agilent), the column temperature was set at 35°C. The mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution. The serum samples were analyzed both in negative and positive ion mode. The mother and productive ions were scanned within the mass range of m/z 100-1200 and 50-1200, respectively. A thorough analysis of a great deal of information of the constituents in the rat serum was undertaken. The structure identification of the detected compounds was achieved by using high resolution MS values as well as the MS/MS fragments. RESULTS: Eighteen peaks in rat serum after oral administration of DHFZT were detected within only 30 min recorded chromatograms. The structure of the 18 compounds were then given out, of which 10 were the original form of compounds absorbed from DHFZT, 8 were the metabolites of the compounds existed in rat serum. According to the CCA results, talatisamine, rhein glucoside, rhein isomer methylation, hypaconine, hydroxyl-chrysophanol, emodin glucuronide conjugation, and chrysophanol glucuronide conjugation were finally found to be the main anti-acute pancreatitis components in DHFZT. CONCLUSIONS: The model presented in this paper successfully discovered the spectrum-effect relationships of DHFZT, which showed a representative way to discover the primary active ingredients from the complicated herbal drugs.