Comparative pharmacokinetic study of the five anti-inflammatory active ingredients of Inula cappa in a normal and an LPS-induced inflammatory cell model.

Inula cappa is a commonly used medicine in the Miao area of Guizhou Province in China. We established an in vitro inflammatory model of mouse macrophage RAW264.7 cells to study the different pharmacokinetics of five anti-inflammatory active ingredients in the I. cappa extract namely luteolin (LUT), chlorogenic acid (CA), cryptochlorogenic acid (CCA), 3,4-dicaffeoylquinic acid (3,4-DCQA) and 4,5-dicaffeoylquinic acid (4,5-DCQA), in a normal and an inflammatory cell model. First, RAW264.7 cells were treated in vitro with l µg/mL lipopolysaccharide (LPS) for 24 h to establish an inflammatory cell model. Then, the pharmacokinetic characteristics of the five ingredients were compared in normal and inflammatory cells after treatment with 200 µg/ml and 800 µg/ml of I. cappa extracts. After treatment with 1 µg/ml LPS for 24 h, the volume of RAW264.7 cells was increased, the morphology was changed, the antennae were obvious, and the secretion of inflammatory factors nitric oxide and TNF-α was increased. The pharmacokinetics results showed that the five ingredients in normal and inflammatory cells exhibited an increase in Cmax and AUC values with increasing doses, and the Cmax and AUC values of five ingredients were positively correlated with the extract concentration. Each of these five ingredients presented nonlinear pharmacokinetic characteristics. After treatment with 200 µg/ml of I. cappa extract, the uptake of five ingredients increased in inflammatory cells, Tmax was prolonged, MRT and t1/2 were prolonged, and CL_F and Vz_F were decreased, while after treatment with 800 µg/ml of I. cappa extract, the uptake of five ingredients decreased, Tmax was prolonged, absorption was faster, and MRT and t1/2 were prolonged. The five analyzed components in I. cappa extract exerted different effects on normal cells and LPS-induced inflammatory cells. Compared to normal cells, the uptake of five ingredients in inflammatory cells was faster and the AUC and Cmax values increased with increasing doses, showing a dose-dependent nonlinear pharmacokinetic profile. These results indicate that the pharmacokinetic effects of the five analyzed ingredients in I. cappa extract are changed in the inflammatory state.

[Establishment of PK-PD model in anti-inflammatory active components in Inula cappa extract based on lipopolysaccharide-induced in vitro inflammation model].

In the present study, a pharmacokinetics(PK)-pharmacodynamics(PD) model in the anti-inflammatory active components in Inula cappa extract was established based on the lipopolysaccharide(LPS)-induced in vitro inflammation model in order to clarify the relationship between the dynamic changes of anti-inflammatory active components in inflammatory cells and their efficacy. Firstly, the inflammation model in vitro was induced by 1 µg·mL~(-1) LPS in RAW264.7 cells for 24 h. After treatment with 400 µg·mL~(-1) I. cappa extract, the pharmacokinetics(PK) of five anti-inflammatory active components, including luteolin(LUT), chlorogenic acid(CA), cryptochlorogenic acid(CCA), 3,4-dicaffeoylquinic acid(3,4-DCQA), and 4,5-dicaffeoylquinic acid(4,5-DCQA), in normal cells and inflammatory cells was compared. Meanwhile, the PD study was carried out by measuring the inflammatory factors NO and TNF-α in the cell supernatant at each time point, which was fitted with PK by the Phoenix Model in the WinNonlin 8.2 to establish the PK-PD model for five components including LUT, CA, CCA, 3,4-DCQA, and 4,5-DCQA. The results showed that compared with normal cells, the model cells showed increased or decreased uptake of five components, advanced T_(max), faster absorption, prolonged MRT and t_(1/2), and increasing or decreasing trend of CL_(z/F) and V_(z/F). When NO was used as the efficacy index, the PK-PD model after the integration of the multi-effect components in I. cappa was E=7.45×\[1-Ce~(5.74)/(78.24~(5.74)+Ce~(5.74))\], while with TNF-α as the efficacy index, the PK-PD model after the integration of the multi-effect components in I. cappa was E=79.28×[1-Ce~(6.45)/(85.10~(6.45)+Ce~(6.45))]. The results of the study suggested that the inflammatory state could change the cellular PK of I. cappa. The anti-inflammatory effect of active components in I. cappa might be related to the down-regulation of the secretion of NO and TNF-α in inflammatory cells, and NO and TNF-α might serve as the anti-inflammatory targets of active components of I. cappa.

[Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis].

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

Effect of Inula cappa on CYP450 Enzyme in Rats by Cocktail Probe Drugs / 中国实验方剂学杂志

Objective:To study on the effect of Inula cappa extract on the activities of six cytochrome P450(CYP450) enzymes in rats by Cocktail probe method. Method:Rats in the I. cappa high and low dose groups were given oral administration of active fractions of I. cappa at a dose of 8.127,2.709 g·kg-1·d-1 of the material for 7,14 d,repectively.Probe drugs(caffeine,midazolam,tolbutamide,omeprazole,metoprolol,chlorzoxazone) were simultaneously injected to rats after administration.Plasma was collected at different time after the administration of probe drugs.The plasma concentrations of these six probes were measured by UPLC-MS and their corresponding pharmacokinetic parameters were calculated with DAS 2.0. Result:Compared with the control group,only the apparent volume of distribution(V) of midazolam was increased;area under the curve(AUC0-t and AUC0-∞)and half-life period(T1/2) of caffeine,midazolam,tolbutamide and omeprazole were increased and the clearance rate(CL) of them was decreased in rats of I. cappa groups.But there were no differences in pharmacokinetic parameters of chlorzoxazone and metoprolol. Conclusion:I. cappa can reduce the enzymatic activities of CYP3A,CYP1A2,CYP2C9 and CYP2C19 in rats at different degree,among which CYP3A is the strongest.

Investigation of the Excretion of Seven Constituents of Inula cappaort in Rat Urine by UPLC-MS/MS / 中国药学杂志

OBJECTIVE: To establish an UPLC-MS/MS method for the analysis of seven compounds of Inula cappaort in rat urine to study their excretion. METHODS: The urine samples in 0-2, 2-6, 6-12, 12-24, and 24-36 h were collected. Acquity UPLC BEH C18 column (2.1 mm×50 mm, 1.7 μm) was used and the column temperature was set at 45 ℃, the mobile phase was 0.1% formic acid acetonitrile -0.1% formic acid aqueous solution in a gradient elution mode and flow rate was 0.25 mL•min-1. The detection was carried out by a triple quadrupole linear ion trap mass spectrometer in positive and negative ion mode with an electrospray source. Multiple reactions monitoring (MRM) mode was employed. RESULTS: The calibration curves showed good linearity, with correlation coefficients of greater than 0.991 0 for all of the analytes within the concentration ranges. The intra-day and inter-day precisions (RSD) were all less than 15%. The extraction recoveries of the seven components were more than 84.41%, without obvious matrix effect, which met the requirements for analysis. CONCLUSION: The established method is simple, rapid, and sensitive. It can be applied in the excretion study of the seven components of Inula cappaort extract in rat urine. The urine excretion test showed that the prototype excretion rates are low in rats, and the cumulative excretion rates are all less than 5%.

Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis / 中国中药杂志

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

[Absorption of Inula cappa extract based on everted intestinal sac method].

To investigate the absorptive characteristics of Inula cappa extract based on the rat everted intestinal sac method in vitro. Nine representative ingredients in I. cappa extract were selected as the study objects. An UPLC-MS/MS method was established to determine and detect their cumulative absorption amount for expounding the absorptive characteristics of ingredients in different intestinal sections. According to the results， the transport mechanism of 8 compounds showed passive diffusion by the reverted gut sac method. And scopolin was actively transported in the intestine. The best absorption site of chlorogenic acid was duodenum. The best absorption site of cryptochlorogenic acid， 1，3-O-dicaffeoylquinic acid， luteolin-7-glucoside and 3，4-O-dicaffeoylquinic acid were jejunum. The best absorption site of neochlorogenic acid， scopolin， 4，5-O-dicaffeoylquinic acid and 3，5-O-dicaffeoylquinic acid was ileum. The absorption of all the compounds was affected by pH and bile. All of the nine ingredients in I. cappa extract could be absorbed in intestines， but with differences in the absorption rate， the best absorptive site and mechanism， indicating that the intestinal absorption of I. cappa extract was selective.

Absorption of Inula cappa extract based on everted intestinal sac method / 中国中药杂志

To investigate the absorptive characteristics of Inula cappa extract based on the rat everted intestinal sac method . Nine representative ingredients in I. cappa extract were selected as the study objects. An UPLC-MS/MS method was established to determine and detect their cumulative absorption amount for expounding the absorptive characteristics of ingredients in different intestinal sections. According to the results， the transport mechanism of 8 compounds showed passive diffusion by the reverted gut sac method. And scopolin was actively transported in the intestine. The best absorption site of chlorogenic acid was duodenum. The best absorption site of cryptochlorogenic acid， 1，3--dicaffeoylquinic acid， luteolin-7-glucoside and 3，4--dicaffeoylquinic acid were jejunum. The best absorption site of neochlorogenic acid， scopolin， 4，5--dicaffeoylquinic acid and 3，5--dicaffeoylquinic acid was ileum. The absorption of all the compounds was affected by pH and bile. All of the nine ingredients in I. cappa extract could be absorbed in intestines， but with differences in the absorption rate， the best absorptive site and mechanism， indicating that the intestinal absorption of I. cappa extract was selective.

[Metabolism of Inula cappa extract by rat intestinal bacteria in vitro].

To investigate the metabolism of major components in Inula cappa by rat intestinal bacteria in vitro. I. cappa extract was incubated for 24 h with rat intestinal bacteria under anaerobic environment. After the samples were precipitated by n-butanol, UPLC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites, combined with data software such as Metabolite Tools, Data Analysis and so on. The potential metabolites in rat intestinal bacteria were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results injected that fourteen metabolites were detected in rat intestinal flora. Various types of metabolic reactions happen to caffeoylquinic acid in intestinal flora, including isomerization, hydrolyzation, there were also methylation, hydrogenation and acetylation of caffeic acid. At the same time, a methylate of dicaffeoylquinic acid was also detected. Presumably, caffeoylquinic acids were gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.

Anti-inflammatory and immunomodulatory activities of Inula cappa roots (Compositae).

Background The medicinal properties of Inula cappa, a perennial shrub, are ascribed to its roots. In this article, we study the anti-inflammatory and immunomodulatory activities of I. cappa root extracts based on its utilization in traditional medicine-treatment of rheumatoid arthritis, menoxenia, fever, jaundice and many others. Methods Anti-inflammatory and immunomodulatory activities of I. cappa extracts were studied. The anti-inflammatory activity was determined by carrageenan-induced rat paw edema and cotton pellet-induced granulation method while the immunomodulatory activity was estimated by phagocytic assay method, hemagglutinating antibody (HA) titer assay, delayed-type hypersensitivity assay method, plaque forming cell assay and determination of immunoprophylatic activity. Results The methanolic extract showed maximum reduction in the rat paw edema and showed significant inhibition of the cotton pellet-induced granulomas in rats. The methanolic extract also showed potential immunomodulatory activity in all the assays performed. Two sesquiterpenes, isoalantolactone and germacranolide were also isolated from the methanolic extract. Conclusions The present study supports the evidence that the roots of I. cappa can be used as a potent anti-inflammatory and immunomodulatory agent.

Metabolism of Inula cappa extract by rat intestinal bacteria in vitro / 中国中药杂志

To investigate the metabolism of major components in Inula cappa by rat intestinal bacteria in vitro. I. cappa extract was incubated for 24 h with rat intestinal bacteria under anaerobic environment. After the samples were precipitated by n-butanol, UPLC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites, combined with data software such as Metabolite Tools, Data Analysis and so on. The potential metabolites in rat intestinal bacteria were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results injected that fourteen metabolites were detected in rat intestinal flora. Various types of metabolic reactions happen to caffeoylquinic acid in intestinal flora, including isomerization, hydrolyzation, there were also methylation, hydrogenation and acetylation of caffeic acid. At the same time, a methylate of dicaffeoylquinic acid was also detected. Presumably, caffeoylquinic acids were gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.

Chemical Constituents of Inula cappa / 中国药学杂志

OBJECTIVE: To study the chemical constituents of Inula cappa. METHODS: Chromatographic techniques were employed for isolation and purification of the constituents and their structures were determined by spectral analysis and chemical evidence. RESULTS: Seventeen compounds were obtained and identified as friedelin(1), epifriedelanol(2), α-amyrin(3), β-amyrin(4), benzyl 2-O-β-D-glucopyranosy-2, 6-dihydroxybenzoate(5), scopoletin(6), luteolin-7-O-β-D-glucuronide ethyl ester(7), benzyl alcohol glucoside(8), ophiopogonoside A(9), apigenin-7-O-β-D-glucoside(10), luteolin-7-O-β-D-rutinoside(11), hydnocarpin-D(12), luteolin(13), luteolin-7-O-β-D-glucoside (14), luteolin-4'-O-β-D-glucoside (15), quercetin-3-O-β-D-glucoside (16), and juglans cerebroside A(17). CONCLUSION: Compounds 5, 7, 9, 11, 12, and 17 are for the first time obtained from the genus Inula and compounds 8, 10, 14, and 16 are isolated from Inula cappa for the first time.

Analysis of components in plasma from effective fractions of Inula cappa based on UHPLC/Q-TOF/MS / 中国药理学通报

Aim To analyze the main components of effective fractions of Inula cappa in rat plasma with UHPLC/Q-TOF/MS technology and serum pharmacochemistry theory.Methods After gavage administration with Inula cappa extracts,blood was collected from abdominal aorta,and the protein in plasma sample was precipitated by methanol.Eclipse Plus C18 RRHD (2.1 mm × 100 mm,1.8 μm) was used,with 0.1％ formic acid agueous solution (A)-0.1％ formic acid and acetonitrile (B) as the mobile phase for gradient elution,and the flow rate was 0.3 mL ·min-1 Detected at negative ion mode,and with the help of Metabolite Tools software from Bruker Corporation,components in plasma were defined.Results A total of 16 compounds were identified,including 9 prototypes and 7 metabolites.Main metabolites were isomerization,methylation,glucuronidation and recombination of caffeoylquinic acid.Conclusions Therefore,our study has comprehensively expounded Inula cappa extracts' constituents migrating to rat plasma,and provided a reference for further studies for in vivo metabolic process and effective material base of Inula cappa.

Quantitative determination of seven constituents and fingerprint research on Inula cappa by UPLC / 中草药

Objective: This study is aimed at developing an UPLC method for simultaneous determination of 1, 3-O-dicaffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid and fingerprint analysis of Inula cappa. Methods: WATERS ACQUITY UPLC BEH C18 column with gradient elution of 0.1% acetic acid-acetonitrile at a flow rate of 0.4 mL/min was employed for analysis of 20 batches of samples from three provinces or autonomous region. The detected wavelength was set at 329 nm. The column temperature was maintained at 30℃ and the sample solutions were maintained at 4℃ before analysis. Results: Twenty peaks were selected as the common peaks in fingerprint and the similarity of samples was above 0.900 except S18. The variance analysis, hierarchical clustering analysis, and principle component analysis were employed for the quality evaluation based on the contents of seven components and fingerprint similarity. The results indicated that the contents of 1,3- and 1,5-O-dicaffeoylquinic acid were significantly different among three provinces or autonomous region. The clustering analysis results showed that 20 batches of samples were divided into two classes and the quality of samples from Guangxi provinves were more stable than those from Guizhou and Yunnan. Conclusion: The established method could be rapid and accurate to evaluate the quality of I. cappa.