| Acanthopanax sessiliflorus (Rupr. et Maxim) Seem, belongs to Acanthopanax genus of the family Araliaceae. It is a shrub which mainly presents in China, Korea and Japan. Its root bark is considered as one of the sources of Wujiapi and widely used for dispelling rheumatism, reinforcing kidney, strengthening bones and promoting blood circulation based on the Chinese Materia Medica. In this thesis, the analysis of the constituents of A. sessiliflorus roots by isolation and identification as well as UPLC-MS/MS technique, fingerprint technique, content determination and pharmacokenetics were investigated.1. Constituents isolation and identification of A. sessiliflorus rootsThe chemical constituents of 70% ethanol extract of A sessiliflorus roots were isolated by using multiple column chromatographic techniques. The structures of 29 compounds were fully elucidated by chemical and spectroscopic methods. The identified compounds include twelve lignans:(+)-Asarinin (2), (-)-Sesamin (3), Helioxanthin (4), Savinin (5), Taiwanin C (6), Taiwanin E (7),7,7’-Dihydrotaiwanin C (8), (+)-Medioresinol (9), (+)-Pinoresinol-4,4’-di-O-β-D-glucopyranoside (10), (+)-Syringaresinol (11), Acanthoside B (12), Acanthoside D (13); fourteen organic acids: 3-ethyl-5-hydroxy-3-(hydroxymethyl)pentyl-3-(3,4-dihydroxyphenyl)acrylate (1), Caffeic acid methyl ester (14), Protocatechuric acid (15), Protocatechuic acid methyl ester (16), Hydroxycinnamic acid (17), Caffeic acid (18), Tricosanoic acid (21),1-Caffeoylquinic acid (23),3-Caffeoylquinic acid (24),5-Caffeoylquinic acid (25),1,3-Dicaffeoylquinic acid (26), 3,4-Dicaffeoylquinic acid (27),3,5-Dicaffeoylquinic acid (28),4,5-Dicaffeoylquinic acid (29); two sterides:Stignasterol (19), Daucosterol (20); one alkaloid:2,2’,2"-Nitrilotriethanol (22). Among them, compound (1) is a new compound; (22) is a new natural product; (8) and (21) are isolated from the family of Araliaceae for the first time; (7), (23), (24), (25), (26), (27), (28), (29) are isolated from this plant for the first time.2. Analysis of the constituents of A. sessiliflorus by UPLC-MS/MS technique and comparison with A. gracilistylus and A. senticosusThe constituents of the roots, stems, leaves, fruits of A. sessiliflorus were analyzed by using the UPLC-MS/MS technique and compared with those of the roots, stems, leaves of A. gracilistylus and A. senticosus. Twenty-five compouds were identified by the comparison of the LC retention time and mass spectra with standard compounds. Thirty-three compounds were also inferred in this study. The fragmentation patterns of the quininic acids, lignans, flavonoids, coumarins, phenolic glycosides and triterpenoids were elaborated. The study is focused on the fragmentation regularities of mass spectra of lignan compounds, which provided the basis for accurate identification of lignan constituents. The constituents of A. sessiliflorus, A. gracilistylus and A. senticosus were also compared with each other in this study. Quininic acids and furofurano lignans are common constituents of all the three Acanthopanax species. A. senticosus roots contained many phenolic glycosides which were not detected in A. sessiliflorus and A. gracilistylus roots. The roots of A. sessiliflorus and A. gracilistylus contained similar constituents. Aside from different number and types of triterpenoids detected in A. sessiliflorus and A. gracilistylus stems, the constituents of the stems were similar to those in the roots of the three Acanthopanax species. In comparison with the roots and stems, the leaves contained more coumarins and triterpenoids and fewer lignans. This research has great significance in the developing new medicinal resources and expanding product categories of Acanthopanax species.3. Fingerprint analysis of the roots of A. sessiliflorusThe chromatographic fingerprint of A. sessiliflorus roots from different resources was established by UPLC with PDA detection (UPLC-PDA). Eighteen common peaks were achieved after analyzing nineteen batches of A. sessiliflorus roots samples. Among them, thirteen common peaks were identified based on their fragmentation patterns in mass spectra and/or by comparison with standard compounds. The research made some beneficial exploration in using multiple pattern recognition methods in combination for the fingerprint analysis of A. sessiliflorus root. Principle component analysis (PCA) and hierarchy clustering analysis (HCA) were applied in this study. The consistant results proved the objectivity and reliability. Both SPSS software and Simca-P software were used to appraise the PCA, and the differences between them were investigated, which provided new thoughts for the application of fingerprint analysis software.GC technique was applied to develop the fingerprint of the essential oil of A. sessiliflorus roots. Twenty-nine common peaks were identified by GC/MS with NIST database. Nineteen batches of samples were classified by HCA, computer aided similarity evaluation system was used to appraise the similarity and set up the common mode. The results of HCA of UPLC-PDA fingerprint and GC fingerprint were not consistant, which showed that multiple methods of sample preparation and different detection techniques should be used in combination to reveal whole chemical information of a medicinal plant.4. Determination of seven organic acids in A. sessiliflorus and comparison with A. gracilistylus and A. senticosusA high performance liquid chromatography (HPLC) method was developed for simultaneous quantitative determination of 3-caffeoylquinic acid,5-caffeoylquinic acid, caffeic acid,1,3-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid. The analysis was performed on a Hypersil ODS2 C18 column using gradient elution with the mobile phase consisting of 0.1% fomic acid in acetonitrile and 0.1% fomic acid in water. The method has been applied to quantitative determination of seven organic acids in the A. sessiliflorus roots of different growing period. The result showed the total content of the seven organic acids increased with age. The contents of seven organic acids in the bark and xylem parts of the root and stem of A. sessiliflorus were determined, the result showed that the total contents of seven organic acids in the barks of root and stem were both much higher than those in the xylems of root and stem. The contents of seven organic acids in different parts of A. sessiliflorus were determined, the composition of organic acids in fruits was quite different from that in other parts of A. sessiliflorus. The main organic acids in fruits were 5-caffeoylquinic acid and 4,5-dicaffeoylquinic acid. The contents of the seven organic acids in different parts of A. gracilistylus and A. senticosus were also determined and compared. The contents of the seven organic acid in the roots of the three Acanthopanax species were basically the same, while there were certain differences in the stems and leaves among the three Acanthopanax species. The method showed good accuracy and good precision, which provided a foundation for the quality control and comparison with other Acanthopanax species.5. LC-MS/MS determination and pharmacokinetic study of four lignan components of A. sessiliflorus roots in rat plasmaA selective and sensitive UPLC-MS/MS method was developed and validated for the determination and pharmacokinetic study of asarinin, sesamin, helioxanthin and savinin in rat plasma. Sample preparation involved a liquid-liquid extraction of the analytes with methyl tert-butyl ether (MTBE). LC separation was achieved on a UPLC C18 column with a mobile phase consisting of 2 mmol/L ammonium acetate-methanol (68:32, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning with electrospray ionization (ESI) source operating in the positive ionization mode. The current UPLC-MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability and was applied to a pharmacokinetic study of the four lignans after oral administration of Acanthopanax sessiliflorus extract to rats. The maximum concentration (Cmax) was 392.81±51.2 ng/mL for asarinin,1285.6±117.4 ng/mL for sesamin,39.1±4.1 ng/mL for helioxanthin and 21.2±21.2 ng/mL for savinin. The time to reach the maximum plasma concentration (Tmax) was 2.50±0.15 h for asarinin,1.94±0.28 h for sesamin,2.22± 0.48 h for helioxanthin and 2.83±0.29 h for savinin. The elimination half-time (t1/2) of asarinin, sesamin, helioxanthin and savinin was 6.08±1.10,11.69±0.50,7.16± 0.52 and 6.26±0.57 h, respectively. |
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