Metabolic profile of Phellodendron amurense Rupr. in vivo of rat and its metabolomic study on intervention in rheumatoid arthritis.

To analyze the metabolites (blood, urine and feces) in normal rats after intragastric administration of the decoction of Phellodendri Amurensis Cortex (PAC) and to map the metabolic profile of PAC in vivo of rat; meanwhile, to evaluate the anti-rheumatoid arthritis (RA) effect of PAC by blood metabolomics technique and to explore its mechanism. Performing on UPLC-Q-TOF-MS technology with a Waters ACQUITY UPLC BEH-C18 column (100 mm × 2.1 mm, 1.7 µm), the mobile phase was acetonitrile-0.1% formic acid aqueous solution (gradient elution). Prior to and following the administration of the decoction of PAC, the samples of blood, urine, and fecal were collected from the rats, in the positive ion mode, pharmacogenic metabolites in each biological sample were identified according to the accurate mass, fragment ions, retention time, metabolic reaction type, comparison of reference substance and retrieval of Pub Med database; The adjuvant-type arthritis (AA) rat model was established, and blood metabonomics method was used to study the improvement effect of rheumatoid arthritis after drug intervention with PAC, and its mechanism was preliminarily explored through analysis of metabolic pathway. A total of 72 exogenous components were identified, including 17 prototype components and 55 metabolites; 14 biomarkers were screened by blood metabolomics techniques combined with multivariate statistical analysis, and PAC significantly improved symptoms of rheumatoid arthritis in rats, and the metabolic pathway analysis mainly involves 5 metabolic pathways. The components in the aqueous decoction of PAC mainly undergo phase I metabolic reactions in rats, such as oxidation, reduction, dehydrogenation, demethylation, and phase II metabolic reactions, such as acetylation, glucuronidation, methylation; PAC has anti-rheumatoid arthritis effects, and its mechanism of action may be related to biosynthesis of aminoacyl-tRNA, metabolism of phenylalanine, metabolism of tryptophan, degradation of valine, leucine and isoleucine and biosynthesis of pantothenic acid and coenzyme A, providing a scientific basis for the study of the pharmacodynamic substances and the action mechanism of PAC against RA.

Evaluation of the key ingredient from the main production areas of Phellodendri Amurensis Cortex using ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry and ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry.

Phellodendri Amurensis Cortex (PAC) is a medicinal herb that has been generally used to treat diarrhea and jaundice. In order to comprehensively evaluate the PAC in the main production areas quality, a qualitative and quantitative method with highly effective, sensitive, and reliable was developed. The chemical compositions of PAC were analyzed, and fingerprints were established by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS). Then, the determination of berberine, canthin-6-one, dictamnine, γ-fagarine, and magnoflorine from PAC samples was simultaneously performed using UPLC-QQQ-MS. Furthermore, the chemical components of PAC from different regions were compared and analyzed by combining hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. A total of 58 compounds were identified, including 36 alkaloids, four phenylpropanoids, seven terpenoids, four flavonoids and their glycosides, an organic acid compound, and six other components. The fingerprint results show that samples have good similarity. Meanwhile, the content of the five ingredients in different habitats is quite different. By multivariate statistical analysis, 18 batches of PAC could be divided into three categories, and 20 components were identified as differential markers of various origins. A comprehensive method of PAC quality evaluation and chemical composition difference analysis was established, which provided the scientific basis for quality evaluation and further pharmacological mechanism research.

[Characterization and identification of alkaloids in Phellodendri Chinensis Cortex and Phellodendri Amurensis Cortex based on UHPLC-IM-Q-TOF-MS].

A strategy combining collision cross section(CCS) prediction and quantitative structure-retention relationship(QSRR) model for quinoline and isoquinoline alkaloids was established based on UHPLC-IM-Q-TOF-MS and applied to Phellodendri Chinensis Cortex and Phellodendri Amurensis Cortex. The strategy included the following three steps.(1) The molecular features were extracted by the "find features" algorithm.(2) The potential quinoline and isoquinoline alkaloids were screened by filtering the original characteristic ions extracted from Phellodendri Chinensis Cortex and Phellodendri Amurensis Cortex by the established CCS vs m/z prediction interval.(3) According to the retention time of candidate compounds predicted by QSRR model, the chemical constituents were identified in combination with the characteristic fragment ions and pyrolysis law of secondary mass spectrometry. With the strategy, a total of 80 compounds were predicted, and 15 were identified accurately. The strategy is effective for the identification of small analogs of traditional Chinese medicine.

Characterization and identification of alkaloids in Phellodendri Chinensis Cortex and Phellodendri Amurensis Cortex based on UHPLC-IM-Q-TOF-MS / 中国中药杂志

A strategy combining collision cross section(CCS) prediction and quantitative structure-retention relationship(QSRR) model for quinoline and isoquinoline alkaloids was established based on UHPLC-IM-Q-TOF-MS and applied to Phellodendri Chinensis Cortex and Phellodendri Amurensis Cortex. The strategy included the following three steps.(1) The molecular features were extracted by the "find features" algorithm.(2) The potential quinoline and isoquinoline alkaloids were screened by filtering the original characteristic ions extracted from Phellodendri Chinensis Cortex and Phellodendri Amurensis Cortex by the established CCS vs m/z prediction interval.(3) According to the retention time of candidate compounds predicted by QSRR model, the chemical constituents were identified in combination with the characteristic fragment ions and pyrolysis law of secondary mass spectrometry. With the strategy, a total of 80 compounds were predicted, and 15 were identified accurately. The strategy is effective for the identification of small analogs of traditional Chinese medicine.

Identification of Prototype Components and Their Metabolites in Rat Serum, Urine and Feces After Oral Administration of Phellodendri Amurensis Cortex Aqueous Extract by UPLC-Orbitrap Fusion Lumos Tribrid-MS / 中国实验方剂学杂志

Objective:An ultra-performance liquid chromatography coupled with Orbitrap Fusion Lumos Tribrid mass spectrometry （UPLC-Orbitrap Fusion Lumos Tribrid-MS） was applied to analyze the prototypes and their metabolites of Phellodendri Amurensis Cortex aqueous extract in the serum， urine and feces of normal rats， and to investigate the pharmacodynamic material basis of Phellodendri Amurensis<italic> </italic>Cortex in rats. Method:Chromatographic separation was performed on the ACQUITY UPLC^® CSH^TM C₁₈ column （2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-15 min， 2%-25%B； 15-25 min， 25%-50%B； 25-28 min， 50%-98%B）， flow rate was 0.3 mL·min^-1， the injection volume was 10 μL and the column temperature was 40 ℃. Heated electrospray ionization （HESI） was used to collect data in the positive ion modes with the scanning range of <italic>m</italic>/<italic>z</italic> 100-1 000. By comparing chromatogram differences between the blank samples and the samples after administration， prototypes and their metabolites of biological samples after oral administration of Phellodendri Amurensis Cortex aqueous extract were identified. Result:After oral administration of Phellodendri Amurensis Cortex aqueous extract， a total of 70 compounds including 15 prototypes and 55 metabolites in rat serum， urine and feces were detected. Among them， 15 prototypes included 12 alkaloids and 3 limonoids， and 55 metabolites included 52 alkaloids and 3 limonoids. Desaturation， methylation， oxidation， sulfonation and glucuronide conjugation were observed as the primary metabolic pathways for the chemical constituents of Phellodendri Amurensis Cortex aqueous extract. Conclusion:Alkaloids in Phellodendri Amurensis Cortex aqueous extract undergo phase Ⅰ and phase Ⅱ metabolism in rats， and limonoids mainly undergo phase Ⅰ metabolism in rats. This paper can provide experimental basis for further analyzing the process <italic>in vivo</italic> of Phellodendri Amurensis Cortex and elucidating its pharmacodynamic substance basis<italic>.</italic>

Characterizing metabolites and potential metabolic pathways changes to understanding the mechanism of medicinal plant Phellodendri Amurensis cortex against doxorubicin-induced nephritis rats using UPLC-Q/TOF-MS metabolomics.

Phellodendri Amurensis cortex (PAC), a famous traditional Chinese herb with good anti-inflammatory efficacy, is used to treat various liver and kidney sickness in clinical practice. However, the potential mechanisms protecting against nephritis of PAC have not been comprehensively elucidated. The aim of this research was to explore the mechanism of PAC against doxorubicin- induced nephritis in rats by characterizing metabolites and potential metabolic pathways changes. The rat models of nephritis were established using 6.5 mg/kg doxorubicin injection from caudal vein for five weeks. The rats in the treatment group were respectively received PAC extract at the dose of 216, 432, and 864 mg/kg once a day during the experiment. Then, urine metabolomics strategy based on ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-Q/TOF-MS) has been employed to discover the possible significant metabolites and metabolic pathway of nephritis rats. At the end of the experiment, serum, urine and kidney tissue were collected for biochemical and pathological examination. The results showed that PAC treatment notably decreased urinary protein, serum Cr content and renal tissue lesions, and increased serum TP and ALB content. A total of potential twenty- eight metabolites such as 5'-methylthioadenosine, cGMP, dehydroepiandrosterone sulfate, salbuta, 2-phenylaminoadenosine contributing to nephritis rat model were selected and identified in the urine samples. Compared with the model group, the high-dose PAC group can recall 18 metabolites level, the medium-dose group can recall 13 metabolites level, and the low-dose PAC group can recall 8 metabolites level, which were involved in nine primary metabolic pathways such as steroid hormone biosynthesis, alanine,aspartate and glutamate metabolism, cysteine and methionine metabolism as well as glyoxylate and dicarboxylate metabolism. The protein expressions of key enzymes involving methylthioadenosine phosphorylase (Mtap), cytidine deaminase (Cda), thymidine kinase (Tk), argininosuccinate synthase (Ass) in metabolic pathways were further verified by Western blot. The results showed that Phellodendron chinense up-regulated the protein expressions of Cda and Tk and down-regulated the protein expressions of Mtap and Ass. In conclusion, PAC possesses renoprotective effect against doxorubicin-induced nephritis, which may be mediated via regulating differential metabolites, reducing oxidative stress response, improving renal function, enhancing the ability of the immune system, regulating the role of key enzymes.

A New Method for Simultaneous Determination of Phenolic Acids, Alkaloids and Limonoids in Phellodendri Amurensis Cortex.

Phellodendri Amurensis Cortex (PAC) is a well-known herbal medicine in China with complex components, but the previous research has mostly focused on its alkaloids analysis. For the first time, a simpler and more efficient method was proposed in this paper to simultaneously determine the content of three different kinds of compounds-phenolic acids, alkaloids and limonoids-in PAC. The phenolic acids included 3-O-feruloylquinic acid, 4-O-feruloylquinic acid and syringin. The alkaloids include magnoflorine, phellodendrine, jatrorrhizine, palmatine and berberine, while the limonoids include obaculactone and obacunone. An approach combining multi-wavelength and HPLC-DAD was used in this study due to the great difference in maximum absorption wavelength of the various components. Four wavelengths at 215, 275, 280 and 310 nm, respectively, were chosen for monitoring. It has been indicated through appropriate tests that this approach is of high accuracy, good repeatability and stability and provides a scientific basis for the quality assessment of PAC and associated derivatives. In addition, the chromatographic fingerprints method combined with multivariate statistical analysis chosen in this study was proved to be effective and reasonable for an accurate classification of 33 batches of samples collected from different locations.

Quality Evaluation of Phellodendri Chinensis Cortex by Fingerprint⁻Chemical Pattern Recognition.

Phellodendri Chinensis Cortex (PCC) and Phellodendri Amurensis Cortex (PAC) are increasingly being used as traditional herbal medicines, but they are often mistaken for each other. In this study, the fingerprints of PCC from six different geographical sources were obtained by high-performance liquid chromatography, and multivariate chemometric methods were used for comprehensive analysis. Two unsupervised pattern recognition models (principal component analysis and hierarchical cluster analysis) and a supervised pattern recognition model (partial least squares discriminant analysis) were established on the basis of the chemical composition and physical traits of PCC and PAC. PCC and PAC were found to be distinguishable by these methods. The PCC category was divisible into two categories, one with more crude cork and a maximum thickness of ~1.5 mm, and the other with less net crude cork and a maximum thickness of 0.5 mm. According to the model established by partial least squares discriminant analysis (PLS-DA), the important chemical marker berberine hydrochloride was obtained and analyzed quantitatively. From these results combined with chemometric and content analyses, the preliminary classification standards for phellodendron were established as three grades: superior, first-order and mixed. Compared with the traditional identification methods of thin layer chromatography identification and microscopic identification, our method for quality evaluation is relatively simple. It provides a basis and reference for identification of PCC and enables establishment of grade standards. It also could be applied in quality control for compound preparations containing PCC.

[Regional characteristic analysis of alkaloids and chlorogenic acid in wild Phellodendri Amurensis Cortex].

In the present research, 674 wild medicinal material samples of Phellodendri amurensis Cortex were collected from 31 sampling sites in the whole distribution of its original plant Phellodendron amurense. The samples were collected under the premise that the stem diameter of sampling plant, sampling position and time were controlled. And the sampling sites were set at the interval of a latitude. The content of 6 kinds of active ingredients, palmatine chloride, berberine hydrochloride, phellodendrine chloride, jatrorrhizine hydrochloride, magnoflorine, chlorogenic acid, etc in the medicinal material samples were determined, and the results showed that the content of most active ingredients in the medicinal materials showed significant differences due to the difference of sampling sites. Among them, the medicinal materials from Liaoning region had the highest content of active ingredients, followed by Beijing and Jilin regions, and that from Heilongjiang region had the lowest content. The study has important directive significance to the exploration of environmental factors for the formation of active constituent and artificial planting regionalization of high quality Phellodendri amurensis Cortex.

Regional characteristic analysis of alkaloids and chlorogenic acid in wild Phellodendri Amurensis Cortex / 中国中药杂志

In the present research, 674 wild medicinal material samples of Phellodendri amurensis Cortex were collected from 31 sampling sites in the whole distribution of its original plant Phellodendron amurense. The samples were collected under the premise that the stem diameter of sampling plant, sampling position and time were controlled. And the sampling sites were set at the interval of a latitude. The content of 6 kinds of active ingredients, palmatine chloride, berberine hydrochloride, phellodendrine chloride, jatrorrhizine hydrochloride, magnoflorine, chlorogenic acid, etc in the medicinal material samples were determined, and the results showed that the content of most active ingredients in the medicinal materials showed significant differences due to the difference of sampling sites. Among them, the medicinal materials from Liaoning region had the highest content of active ingredients, followed by Beijing and Jilin regions, and that from Heilongjiang region had the lowest content. The study has important directive significance to the exploration of environmental factors for the formation of active constituent and artificial planting regionalization of high quality Phellodendri amurensis Cortex.

Rapid identification and comparative analysis of the chemical constituents and metabolites of Phellodendri amurensis cortex and Zhibai dihuang pill by ultra-performance liquid chromatography with quadrupole TOF-MS.

Zhibai dihuang pill is a famous traditional Chinese medicine formula containing the herbal medicine Phellodendri amurensis cortex. In this work, an integrative pattern recognition approach including principal component analysis and orthogonal partial least squared discriminant analysis based on ultra-performance liquid chromatography coupled to MS was successfully applied for the rapid discovery of natural compounds from herbal medicines. In a 24 min analysis, 93 compounds were identified or tentatively characterized from Zhibai dihuang pill based on their fragmentation behaviors, 23 of which were from Phellodendri amurensis cortex. A total of 26 metabolites in plasma were identified from Phellodendri amurensis cortex in Zhibai dihuang pill formula, among them, 12 prototypes and 14 metabolites through metabolic pathways of demethylation, methylation, hydrolysis, sulfate conjugation, and glucuronide conjugation, which were seven metabolites more than that of the single drug, suggesting the importance of the compatibility of traditional Chinese medicine. The present study provided important structural information on the metabolism of Zhibai dihuang pill. Furthermore, the results of this work have demonstrated the feasibility of ultra-performance liquid chromatography coupled to MS for the rapid and reliable characterization of metabolites from herbal medicines. Based on these results, this method could be a novel approach for identifying potentially bioactive components in other traditional Chinese medicines.

Quantitative analysis of multi-components with single marker for determination of alkaloids in Phellodendri amurensis cortex / 中草药

Objective: To establish an HPLC method for simultaneously measuring the content of berberine, palmatine, and jatrorrhizine in Phellodendri Amurensis Cortex, and on this basis, the multiple components were examined by way of using a single test to validate the method's feasibility and technical suitability of quality control applications for Phellodendri Amurensis Cortex. Methods: Taking berberine, the main component in Phellodendri Amurensis Cortex, as an indicator, a relative correction factor between berberine and palmatine as well as jatrorrhizine was established. Then the correction factor was used to calculate the content of the other two in Phellodendri Amurensis Cortex. In this way, the calculated value was compared with the external standard method. Results: A measuring method of evaluating the three alkaloids in Phellodendri Amurensis Cortex was established via using a single test; There was no significant difference compared with the external standard method. Conclusion: The method for simultaneously measuring the content of berberine, palmatine, and jatrorrhizine in Phellodendri Amurensis Cortex is reliable and accurate, it could be used to control the quality of crude drugs and herbal pieces of Phellodendri Amurensis Cortex.