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OBJECTIVE To investigate the improvement effects of Arisaema Cum Bile on Parkinson’s disease （PD） model mice and its potential mechanism. METHODS Sixty male C57BL/6J mice were randomly divided into normal group， model group， Arisaema Cum Bile low-dose group [0.39 g/（kg·d）]， Arisaema Cum Bile high-dose group [1.56 g/（kg·d）] and positive control drug Levodopa tablet group [80 mg/（kg·d）]， with 12 mice in each group. Except that normal group was given constant volume of normal saline， other groups were given 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine [MPTP，35 mg/（kg·d）] intraperitoneally for 5 consecutive days to induce subacute PD model； after modeling， they were given relevant medicine continuously for 7 d； rod climbing test and line suspension test were performed 1 d before modeling， on the 5th day of modeling and after the last medication. The number of tyrosine hydroxylase （TH）-positive neurons in the substantia nigra of mice were measured by immunofluorescence； the levels of interleukin 1β （IL-1β） and tumor necrosis factor α（ TNF-α） in serum and the levels of IL- E-mail：qhwang668@sina.com 1β， TNF-α， cyclooxygenase-2 （COX-2） and inducible nitric oxide synthase （iNOS） in the substantia nigra of mice were measured by enzyme-linked immunosorbent assay. The expression levels of cAMP-dependent protein kinase catalytic subunit α （PKA C-α）， glutathione peroxidase 4 （GPX4） and ferritin heavy chain polypeptide 1 （FTH1） proteins in the substantia nigra of mice was measured by Western blot. RESULTS After last medicine， compared with the normal group， mice in the model group had significantly longer pole-climbing time （P＜0.01）， significantly lower line suspension scores （P＜0.01）， significantly fewer TH-positive neurons in the substantia nigra （P＜0.01）， significantly higher serum concentrations of IL-1β and TNF-α and nigrostriatal concentrations of IL-1β， TNF-α， COX-2 and iNOS （P＜0.01）， while lower protein expression levels of GPX4， PKA C-α and FTH1 in the substantia nigra （P＜0.05 or P＜0.01）. Compared with the model group， the above indexes of mice were significantly returned in Arisaema Cum Bile high-dose group （P＜0.05 or P＜ 0.01）. CONCLUSIONS Arisaema Cum Bile can improve motor impairment and reduce apoptosis of nigrostriatal TH neurons in MPTP-induced PD mice， and has neuroprotective effects on model mice； this may be related to its inhibition of neuroinflammation and the inhibition of ferroptosis by up-regulating PKA signaling pathway.
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ObjectiveIn order to solve the problem that the quality and stability of Arisaema Cum Bile in the fermentation process with hybrid bacteria were not easy to control， the microorganism in the fermentation process of Arisaema Cum Bile was isolated and identified， the dominant strains were screened and the fermentation process of Arisaema Cum Bile with compound bacteria was investigated. MethodThe submerged culture during the fermentation process of Arisaema Cum Bile was taken out for strain separation and purification. Bacteria and fungi multiphase identification and detection methods and automatic microbial analysis system were used to analyze and compare DNA sequences and identify microorganisms. The isolated and identified strains were respectively inoculated and fermented. After screening the dominant strains， a preliminary exploration of compound strain fermentation were carried out. The contents of index components in Arisaema Cum Bile fermented by compound strain and traditional Arisaema Cum Bile were compared by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry （UPLC-QqQ-MS/MS）. Mmobile phase was 0.1% formic acid acetonitrile solution （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-2 min， 35%-45%A； 2-10 min， 45%-48%A； 10-12 min， 48%-100%A； 12-12.01 min， 100%-35%A； 12.01-15 min， 35%-65%A）， the flow rate was set at 0.35 mL·min-1. The mass spectrographic analysis employed electrospray ionization （ESI）， negative ion acquisition mode and multiple reaction monitoring （MRM） scanning mode were adopted to collect information， the collection range was m/z 50-1 000. ResultEight microorganisms were isolated and identified from the submerged culture of Arisaema Cum Bile. Among them， Enterococcus sp. （anaerobic） and E. casseliflavus were selected as the dominant strains in the fermentation process. Compared with the traditional fermentation method， the contents of chenodeoxycholic acid， hyodeoxycholic acid and hyocholic acid in free cholic acid increased by 1.76， 0.06， 0.19 mg·g-1， respectively. In bound cholic acid， glycochenodeoxycholic acid， taurochenodeoxycholic acid， glycohyodeoxycholic acid， taurohyodeoxycholic acid， glycohyocholic acid， taurine porcine cholic acid decreased by 0.63， 0.23， 0.26， 0.16， 0.03， 0.04 mg·g-1， respectively. ConclusionArisaema Cum Bile with compound strain fermentation （Enterococcus sp. and E. casseliflavus） can be fermented more completely， the fermentation cycle can be shortened， and the quality and stability of products can be improved.
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ObjectiveTo predict the possible quality markers （Q-markers） of Arisaema Cum Bile in the prevention and treatment of stroke based on ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spetrometry （UPLC-QTOF-MS/MS） and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine （TCMIP） v2.0. MethodUPLC-QTOF-MS/MS was employed with the mobile phase of 0.1% formic acid aqueous solution （A）-0.1% formic acid acetonitrile solution （B） for gradient elution （0-3 min， 0.2%-5%B； 3-5 min， 5%-8%B； 5-8 min， 8%-10%B； 8-14 min， 10%-25%B； 14-18 min， 25%-50%B； 18-20 min， 50%-70%B； 20-21 min， 70%-98%B； 21-23 min， 98%B； 23-24 min， 98%-0.2%B； 24-26 min， 0.2%B）， the flow rate of 0.5 mL·min-1 and electrospray ionization （ESI）. High quality MS/MS data were scanned in positive and negative ion modes with scanning range of m/z 50-1 500. A local database of the chemical constituents in Arisaema Cum Bile was established by UNIFI 1.8. Then the chemical constituents in Arisaema Cum Bile were characterized by matching with the local database and comparing with the reference substances and literature information. TCMIP v2.0 was used to obtain the targets corresponding to the identified components of Arisaema Cum Bile and stroke， and the "disease-formula" correlation analysis was carried out to screen the core targets by topological eigenvalues. DAVID 6.8 was used for enrichment analysis of Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway of core targets. According to the "five principles" of Q-markers and combined with literature reports， the Q-markers of Arisaema Cum Bile in the prevention and treatment of stroke were predicted， and the core components acting on these target genes were obtained. Cytoscape 3.8.0 was employed to draw the network diagram of "medicinal materials-active ingredients-target genes-pathways". Finally， AutoDock Vina 1.2.2 was used to calculate and verify the molecular docking between the candidate components and the key targets. ResultA total of 76 chemical components was identified in positive and negative ion modes， 85 core targets were collected for Arisaema Cum Bile in the prevention and treatment of stroke. A total of 31 stroke-related pathways， 23 target genes and 9 main active components of Arisaema Cum Bile acting on these genes were screened， and then we determined 4 possible Q-markers for Arisaema Cum Bile in the prevention and treatment of stroke according to the "five principles". ConclusionThe possible Q-markers of Arisaema Cum Bile for stroke are gallic acid， apigenin-6，8-di-C-glucoside， apigenin and cholic acid， and the target of these four components may be estrogen receptor alpha （ESR1）.
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<b>Objective::To compare the protective effect of different bile (porcine bile, oxgall and sheep bile) and their Arisaema cum Bile on rats with acute lung injury, so as to provide reference for the selection of bile and the classification of decoction pieces of Arisaema cum Bile. <b>Method::Wistar male rats were randomly divided into 8 groups (<italic>n</italic>=12), including blank group, model group, porcine bile group, oxgall group, sheep bile group, Arisaema cum Bile with porcine bile group, Arisaema cum Bile with oxgall group and Arisaema cum Bile with sheep bile group. Rats in each treatment group were given corresponding drug solution by gavage at 2.52 g·kg<sup>-1</sup> every day, and rats in the model group and the blank group were given the same volume of normal saline by gavage every day for a total of 8 days. On the 8<sup>th</sup> day, after 1 h of administration, rats in the model group and each treatment group were intraperitoneally injected lipopolysaccharide (LPS, 2 mg·kg<sup>-1</sup>) to prepare rat lung injury model. Blood and lung tissues were collected from every four rats at 3, 6, 24 h after LPS injection, respectively. Lung coefficient, lung water content and wet weight/dry weight ratio of lung tissue (<italic>W</italic>/<italic>D</italic>) were measured, and the levels of tumor necrosis factor (TNF)-<italic>α</italic>, interleukin (IL)-6 and thromboxane B<sub>2</sub> (TXB<sub>2</sub>) in serum and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the contents of malondialdehyde (MDA) and matrix metalloproteinase-9 (MMP-9) in lung tissue were determined by enzyme linked immunosorbent assay (ELISA). The pathological morphology of rat lung tissue was observed and the score of lung tissue injury was calculated. <b>Result::Compared with the model group at the same time point, the lung coefficient, <italic>W</italic>/<italic>D,</italic> lung water content, contents of TNF-<italic>α</italic>, IL-6 and TXB<sub>2</sub> in serum, contents of MDA and MMP-9 in lung tissue of rats in each treatment group were all decreased, and most of them had significant differences (<italic>P</italic><0.05, <italic>P</italic><0.01), but the activities of GSH-Px and SOD were all increased, and most of them had significant differences (<italic>P</italic><0.05, <italic>P</italic><0.01). The oxgall group and the sheep bile group were superior to the porcine bile group in most of the indexes, the Arisaema cum Bile with oxgall group and the Arisaema cum Bile with sheep bile group were superior to the Arisaema cum Bile with porcine bile group, and some of them had significant differences(<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion::Each bile group and each Arisaema cum Bile group all have protective effects on rats with acute lung injury induced by LPS, and the therapeutic effect of oxgall and sheep bile is better than that of porcine bile, the therapeutic effect of Arisaema cum Bile prepared by oxgall and sheep bile is better than that of Arisaema cum Bile prepared by porcine bile.
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Objective: To establish and analyze the HPLC characteristic chromatogram for water extracts of Arisaematis Rhizoma (AR) and its processed products, Arisaematis Rhizoma Preparatum (ARP) and Arisaema Cum Bile (ACB). The research provided reliable method and scientific basis for their quality control. Methods: The separation was performed on an Agilent C18 (200 mm × 4.6 mm, 5 μm) column with gradient elution of 0.2% acetic acid water and 0.2% acetic acid acetonitrile. The similarity was analyzed with software “Similarity Evaluation System for Chromatographic Fingerprint of TCMs (Version 2012)”. The cluster analysis was performed by SPSS 23.0. The principal component analysis was performed by SIMCA 14.1. Results: HPLC characteristic chromatogram for water extracts of AR, ARP, and ACB were established. There were 19, 20 and 13 common peaks in AR, ARP and ACB, respectively. A total of eight characteristic peaks were identified as F1 (xanthine), F3 (uracil), F4 (hypoxanthine), F5 (uridine), F6 (guanosine), F7 (adenosine), F14 (schaftoside), and F16 (isoschaftoside), respectively. The intragroup similarities of AR, ARP, and ACB were all above 0.8 and each was clustered into one type, and the intergroup similarities among AR and its processed products were all below 0.4. The main components of AR were F16 (isoschaftoside), F14 (schaftoside), F17and F15. The main components of ARP were F16 (isoschaftoside) and F1 (xanthine). The main components of ACB were F3 (uracil), F2, F5 (uridine) and F1 (xanthine). Conclusion: The method can effectively identify AR, ARP and ACB, and provide a scientific basis for their quality control.
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Objective To find delayed luminescence parameters that could characterize the cold and hot properties of Arisaematis Rhizoma Preparatum and Arisaema Cum Bile. Methods Delayed luminescence of Arisaematis Rhizoma Preparatum with addition of Scenedesmus sp. within 26 d after decoction was measured in unequal interval, with aim to verify the stability of the natural delayed luminescence average strength and the linear fitting slope value (k) of excitation delayed luminescence. The delayed luminescence of six batches of Arisaematis Rhizoma Preparatum and Arisaema Cum Bile was measured using biological indicator method, and the content of β-sitosterol in Arisaematis Rhizoma Preparatum and β-sitosterol, bilirubin, and cholic acid of Arisaema Cum Bile was determined using high performance liquid chromatograph (HPLC) to analyze the correlation of k value and the above components content of Arisaematis Rhizoma Preparatum and Arisaema Cum Bile. Results K value of excitation delayed luminescence within 14 d after decoction was steadier than natural delayed luminescence average strength, and k values of six batches of Arisaematis Rhizoma Preparatum were all higher than that of Arisaema Cum Bile. A significant negative correlation between β-sitosterol contents and k values of six batches of Arisaematis Rhizoma Preparatum and Arisaema Cum Bile was found, and no significant negative correlation between bilirubin and cholic acid contents and k values of Arisaema Cum Bile was found. Conclusion K value of excitation delayed luminescence could indicate the differences of medicinal properties of Arisaematis Rhizoma Preparatum and Arisaema Cum Bile, which provides a new method for the study of medicinal properties of Chinese materia medica.
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The aim of this study is to analyze the compositions of main bile acids in fermented and mixed processing products of arisame cum bile from pig bile, and to establish a method for content determination of bile acids in fermented Arisaema Cum Bile. Fermented and mixed processing products were prepared from arisaematis rhizome and arisaematis rhizoma preparatum with pig bile respectively. Then the differences in bile acids compositions between such two kinds of products were compared by high performance liquid chromatography and evaporative light-scattering detector (HPLC-ELSD). With three kinds of free bile acid compositions as the indicators, HPLC-ELSD method was adopted to determine the content of bile acid compositions in fermented product,on Agilent Eclipse XDB C₁₈(4.6 mm×250 mm, 5 μm) chromatographic column, with acetonitrile and 0.1% glacial acetic acid solution (55:45) as mobile phase, at a flow rate of 1 mL·min⁻¹, column temperature of 30 °C, drift tube temperature of 90 °C, and a nitrogen flow rate of 2.2 mL·min⁻¹. The results showed that the bile acids in fermented bile Arisaema were mainly in a free form, while in mixed processing product, the compositions were mainly in a conjugated form. Three kinds of free bile acids, namely porcine cholic acid (HCA), porcine deoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) in fermented product, showed a good linear relationship in the range of quantification. The average recovery rate was 95.99%-104.3%, complying with the requirements. The results showed that the conjugated bile acids could be transformed into free bile acids during the fermentation of arisaema cum bile. This established method can effectively control the content of bile acids compositions in fermenting arisaema cum bile.