Analysis Of The Chemical Components And Multidimentional Fingerprint Of Acanthopanax Giraldii Harms

Acanthopanax giraldii Harms is a kind of herbal plant that belongs to the family of Araliaceae, and it is fully covered with the reddish brown thorn, so it is given the name of "Hongmaowujia". In this thesis, the analysis of the components of A. giraldii Harms by LC-DAD-ESI-MS/MS technique, compounds preparation and identification, contents determination, multidimensional fingerprint analysis of A. giraldii Harms and anti-inflammation pharmacological experiments were investigated.1. Analysis of the components of A. giraldii Harms by LC-DAD-ESI-MS/MS techniqueThe LC-DAD-ESI-MS/MS technique was used for analysis of the components in the stem bark, root bark, leaf and fruit of A. giraldii Harms, and those of the stem bark, root bark, leaf and fruit of the of A. senticosus, A. gracilistylus W.W. Smith and A. sessiliflorus were also investigated and compared. 10 compounds were identified in the A. giraldii Harms by the comparison of the LC retention time, UV spectra and the mass spectra with corresponding authentic samples. There were uridine, adenosine, guanosine, liriodendrin, protocatechuic acid, syringin, chlorogenic acid, caffeic acid, hyperoside and rutin, and the last 6 compounds in the A. giraldii Harms have not been reported to date. Caffeoylquinic acid, dicaffeoylquinic acid and feruloylquinic acid were also inferred by their UV spectra, mass spectra and fragments of their MS/MS spectra. The components analysis provided scientific basis for the quality control study of A. giraldii Harms.2. Preparation and identification of liriodendrin and protocatechuic acid from A. giraldiiHarmsThe water extract of A. giraldii Harms was pre-isolated by the macroporous adsorption resin (D-101) and C₁₈ solid phase extraction (SPE) cartridge, and the enriched extract was purified by semi-preparative RP-HPLC to give two compounds. Structure identification was performed by the analysis of UV, IR, ¹H-NMR, ¹³C-NMR and MS data, and they were confirmed as liriodendrin and protocatechuic acid. The developed method was simple, fast, reproducible and easy to operate.3. Determination of 9 compounds in the A. giraldii HarmsBased on the LC-DAD-ESI-MS/MS analysis, syringin, liriodendrin, chlorogenic acid, caffeic acid, rutin, hyperoside, adenosine, ganosine and protocatechuic acid in the A. giraldii Harms were determined, and the corresponding components in the A. senticosus, A. gracilistylus W. W. Smith and A. sessiliflorus were also detected and compared. Only uridine was not determined because of its low content in the A. giraldii Harms.As was shown in the determination results, the content of syringin in the stem bark of A. giraldii Harms was lower than that of liriodendrin, while the content of syringin in the A.senticosus was very high. The content of liriodendrin in the root bark of A. giraldii Harms was higher than that in the stem bark. Cutting the stem of A. giraldii Harms into stem bark, xylem and center three parts and determining, it was found that there was a great difference in the contents of liriodendrin in the three parts. The content of liriodendrin in the xylem part was 3 times of that in the stem bark, and that in the center part was the lowest. Traditionally the stem bark of A. giraldii Harms was peeled to be used for the treatment, and the xylem was then discarded. While the determination results suggested that the content of liriodendrin was greatly decreased.It was found in the determination results of chlorogenic acid and caffeic acid that the contents of chlorogenic acid and caffeic acid in the various batches of A. giraldii Harms were significantly different. However, there was a trend that as the storage time increasing, the content of chlorogenic acid decreased, and the content of caffeic acid rose. The content of caffeic acid in the A. senticosus was lower than that in the A. giraldii Harms. The content of chlorogenic acid in the root bark of A. giraldii Harms was 14.2 mg.g^-1 which was the highest in the all determined samples.By the LC-DAD-MS/MS analysis, it was found that the leaves and fruits of Araliaceae contained some flavone components, and rutin and hyperoside were the main flavone compounds in the leaf and fruit of A. giraldii Harms. By the determination results, the content of rutin in the leaf and fruit of A. giraldii Harms was higher than that of hyperoside, while the content of rutin in the fruit of A. senticosus, A. gracilistylus W. W. Smith and A. sessiliflorus was lower than that of hyperoside.In this experiment, the main components in the stem bark, root bark, leaf and fruit of A. giraldii Harms were determined, which provided a foundation for the quality control and comparison with other Wujia Pie.4. LC-DAD-ESI-MS/MS multidimensional fingerprint analysis of A. giraldii HarmsA study on the multidimensional fingerprint of the stem bark of A. giraldii Harms was performed. Both the LC-UV and LC-MS fingerprints were developed, and it was found that they were in a complementary manner for the fingerprint analysis of A. giraldii Harms, and the combination of them could reflect the quality of A. giraldii Harms overall.In the LC-UV fingerprint analysis, 22 samples were classified by Hierarchical Cluster and Step Discrimination analysis, and two computer aided similarity evaluation system were used to appraise the Similarity and set up the Common Mode. The differences between Pearson correlation and Cosine of the angle, and differences of similarity calculated by two evaluation software were compared. Quantification coefficient was used to judge the whole amount of the fingerprint, and both the correlation and quantification coefficient were needed to appraise the quality of the material. Based on the results of Hierarchical Cluster and computer aided evaluation system, the samples with similarities more than 0.9 and quantification coefficient more than 0.9 were defined as the valid samples.10 samples were involved to set up the LC-MS fingerprint of the stem bark of A. giraldii Harms, and the extracted ion chromatogram of chlorogenic acid was chosen as the reference peak. The similarities of the LC-UV and LC-MS fingerprints of the stem bark of A. giraldii Harms were compared, and the evaluation results of the two method were identical for most of the samples.5. Pharmacological experiment of anti-inflammation of A. giraldii HarmsMice ear swelling model, rats hind paw edema model and the model of adjuvant arthritis in rats by injection of Freund's complete adjuvant were used to observe the protective and therapeutic action of three different doses of the extract of the stem bark of A. giraldii Harms. Moreover, the effect of the isolated phenolic acids from the A. giraldii Harms on the acute inflammation was also investigated. As was shown in the results, it was possible that the phenolic acid compounds would be the active part of anti-inflammation action of the A. giraldii Harms.Two batches of the stem bark of A. giraldii Harms were used in the pharmacological experiment. It was found that there was no significant difference between the test group and the model control group while using the No.2 batch of A. giraldii Harms. As was shown in the fingerprints of the two batches material, the total contents of the components in the No.2 batch were significantly lower than that in the No.1 batch, and the content of chlorogenic acid was especially low in the No.2 batch. The pharmacological results validated our conclusion that No. 1 layer samples in the Hierarchical Cluster analysis were in good quality, and those of in the No. 2 layer were bad. It also suggested that the fingerprint analysis of the traditional Chinese medicine(TCM) played a very important role in the process of the modernization of TCM.The innovation points of this study(1) The LC-DAD-ESI-MS/MS technique was used for analysis of the components in the stem bark, root bark, leaf and fruit of A. giraldii Harms. 10 compounds were identified from the A. giraldii Harms, and 6 in the l0 compounds in the A. giraldii Harms have not been reported to date. 9 compounds in the A. giraldii Harms were determined except uridine because of its low content.(2) Based on the UV spectra, mass spectra and fragments of the MS/MS spectra, caffeoylquinic acid, dicaffeoylquinic acid and feruloylquinic acid in the A. giraldii Harms were also inferred.(3) The components in the stem bark, root bark, leaf and fruit of A. giraldii Harms were compared, and the components in the A. giraldii Harms, A. senticosus, A. gracilistylus W. W. Smith and A. sessiliflorus were also investigated and compared. Moreover, a deduction was put forward that the content of syringin should be as the standard to distinguish A. giraldii Harms from A. senticosus.(4) A study on the multidimensional fingerprint of the stem bark of A. giraldii Harms was performed. Both the LC-UV and LC-MS fingerprints were developed, and it was found that they were in a complementary manner for the fingerprint analysis of A. giraldii Harms, and the combination of them could reflect the quality of A. giraldii Harms overall.