Pharmacokinetic and tissue distribution analysis of bioactive compounds from Fuke Qianjin capsules in rats by a validated UPLCMS/MS method.

Fuke Qianjin capsules (FKQJ) exhibit obvious advantages and characteristics in the treatment of pelvic inflammatory disease. At present, information regarding the in vivo process of FKQJ is lacking, which has become a bottleneck in further determining the therapeutic effect of this traditional Chinese medicine. In the present study, a sensitive, simple and reliable method was developed and validated for the simultaneous quantification of 12 main components (4 flavonoids, 4 alkaloids, 2 phthalides and 2 diterpene lactones) in plasma and seven tissues of rats to study the pharmacokinetic and distribution characteristics of these components in vivo by using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the first time. Plasma and tissue were prepared by protein precipitation with acetonitrile and methanol, followed by its separation on a Waters Acquity UPLC BEH C18 column. The quantification was performed via multiple reaction monitoring (MRM) by a triple quadrupole mass spectrometer under positive electrospray ionization (ESI) mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect and stability. For 12 analytes, the low limit of quantification (LLOQs) reached 0.005-2.44 ng/mL, and all calibration curves showed good linearity (r2 ≥ 0.990) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.96%, and the accuracies were in the range of 85.29%-114.97%. Extraction recoveries and matrix effects of analytes were acceptable. The pharmacokinetic results showed that the main components could be absorbed quickly, had a short residence time, and were eliminated quickly in vivo. At different time points, the 12 components were widely distributed with uneven characteristics in the body, which tended to be distributed in the liver, kidney and lung and to a lesser extent in the uterus, brain and heart. The pharmacokinetic process and tissue distribution characteristics of FKQJ were expounded in this study, which can provide a scientific theory for in-depth development of FKQJ and guide FKQJ use in the clinic.

[Mechanism of Fuke Qianjin Capsules in treating intrauterine adhesion in rats through TGF-ß1-PI3K/Akt signaling pathway].

To explore the effect of Fuke Qianjin Capsules on anti-endometrial fibrosis in intrauterine adhesion(IUA) rats through TGF-ß1-PI3 K/Akt signaling pathway. With female SD rats as the object, IUA rat models were established through mechanical injury and infection, and they were randomly divided into normal group, sham operation group, Bujiale group(0.63 mg·kg~(-1)·d~(-1)), and high-dose Fuke Qianjin Capsules group(1.008 g·kg~(-1)·d~(-1)), medium-dose Fuke Qianjin Capsules group(0.504 g·kg~(-1)·d~(-1)), low-dose Fuke Qianjin Capsules group(0.252 g·kg~(-1)·d~(-1)). The rats were sacrificed 21 days after drug administration, and the uterus and liver were removed after blood collection from the abdominal aorta. The morphology of the uterus was observed with the naked eyes; the pathological and morphological changes of the uterine tissue and liver were observed by HE staining; the degree of fibrosis of the uterine tissue was observed by Masson staining; the expressions of TGF-ß1, TNF-α and IL-6 in serum were detected; the expressions of TGF-ß1, PI3 K, Akt, p-Akt protein in uterine tissue were detected by Western blot. The results showed that Fuke Qianjin Capsules could improve the pathological changes of uterine tissues in IUA rats, without damage to liver tissues, and reduce the expressions of TGF-ß1, TNF-α and IL-6 in serum(P<0.01); significantly reduce TGF-ß1, PI3 K, p-Akt protein expression in uterine tissues(P<0.05, P<0.01). It is indicated that Fuke Qianjin Capsules could exert the anti-endometrial fibrosis effect by regulating the TGF-ß1-PI3 K/Akt signal pathway, so as to achieve the effect in treating IUA rats, especially with the best effect in medium-dose Fuke Qianjin Capsules group.

[Mechanisms of Fuke Qianjin Capsules in treatment of pelvic inflammatory disease based on GC-MS metabolomics].

Pelvic inflammatory disease( PID) rat model was induced by the mixture of Escherichia coli,Staphylococcus aureus,and Streptococcus hemolytic-ß． Gas chromatography-mass spectrometry( GC-MS) based metabolic profiling method was combined with multivariate statistical analysis,such as PCA,PLS-DA and OPLS-DA to analyze endogenous small molecule metabolites in serum of rats after treatment of Fuke Qianjin Capsules. The results showed that Fuke Qianjin Capsules could significantly improve the inflammatory pathological characteristics and tissue damages in model rats. Based on the principle of VIP>1 and P<0. 05,a total of 6 different metabolic biomarkers were identified,including L-valine,L-isoleucine,L-threonine,butanedioic acid,serine and D-glucose,respectively.The contents of these six different metabolites were significantly reversed after administration. Further analysis of the metabolite pathways through KEGG database showed that Fuke Qianjin Capsules achieved the effect possibly through glycine,serine and threonine metabolism,aminoacyl-tRNA biosynthesis and valine,leucine and isoleucine biosynthesis. Therefore,this study came to the conclusion that Fuke Qianjin Capsules can be used in the treatment of mixed bacteria induced pelvic inflammatory disease possibly by regulating amino acid and its derivative metabolism.

[Characteristic fingerprint and multi-components quantitative determination for Fuke Qianjin Capsules by HPLC].

To establish an HPLC characteristic fingerprint method of Fuke Qianjin Capsules,and determine the contents of its main components. The analysis was carried out on a Kromasil 100-5-C18 analytical column(4. 6 mm ×250 mm,5 µm) with gradient elution by acetonitrile(A)-0. 1% phosphoric acid aqueous solution(B),a flow rate at 1. 0 m L·min-1 and the detection wavelength of 254 nm.The column temperature was 30 ℃,and the injection volume was 10 µL. The determination method of genistin,jatrorrhizine,andrographolide and 14-deoxy-11,12-didehydroandrographolide index components were studied methodologically. The common mode of the characteristic fingerprint of Fuke Qianjin Capsules was set up with 8 common peaks,which were identified as genistin,jatrorrhizine,palmatine,berberine,andrographolide,14-deoxy-11,12-didehydroandrographolide,Z-ligustilide,and Z-3-butylidenephthalide,respectively,in comparison with the references. The similarities of 20 batches of Fuke Qianjin Capsules samples were above 0. 95. All of the above-mentioned 4 analytes could be well separated under the optimized chromatographic conditions. RSD of precision and repeatability experiment were both less than 1. 5%,and the sample solution was stable during 72 h. All of the compounds had a good linearity and linear range. The contents of genistin,jatrorrhizine,andrographolide,and 14-deoxy-11,12-didehydroandrographolide in 20 batches of Fuke Qianjin Capsules samples were 28. 66-56. 04,94. 77-197. 92,1 705. 33-4 148. 93 and 462. 16-1 225. 96 µg in each capsule,respectively. The developed HPLC characteristic fingerprint and quantitative analysis methods were reliable,accurate and sensitive,and could be used effectively evaluate the quality of Fuke Qianjin Capsules samples.

Mechanisms of Fuke Qianjin Capsules in treatment of pelvic inflammatory disease based on GC-MS metabolomics / 中国中药杂志

Pelvic inflammatory disease( PID) rat model was induced by the mixture of Escherichia coli,Staphylococcus aureus,and Streptococcus hemolytic-β． Gas chromatography-mass spectrometry( GC-MS) based metabolic profiling method was combined with multivariate statistical analysis,such as PCA,PLS-DA and OPLS-DA to analyze endogenous small molecule metabolites in serum of rats after treatment of Fuke Qianjin Capsules. The results showed that Fuke Qianjin Capsules could significantly improve the inflammatory pathological characteristics and tissue damages in model rats. Based on the principle of VIP>1 and P<0. 05,a total of 6 different metabolic biomarkers were identified,including L-valine,L-isoleucine,L-threonine,butanedioic acid,serine and D-glucose,respectively.The contents of these six different metabolites were significantly reversed after administration. Further analysis of the metabolite pathways through KEGG database showed that Fuke Qianjin Capsules achieved the effect possibly through glycine,serine and threonine metabolism,aminoacyl-tRNA biosynthesis and valine,leucine and isoleucine biosynthesis. Therefore,this study came to the conclusion that Fuke Qianjin Capsules can be used in the treatment of mixed bacteria induced pelvic inflammatory disease possibly by regulating amino acid and its derivative metabolism.

Characteristic fingerprint and multi-components quantitative determination for Fuke Qianjin Capsules by HPLC / 中国中药杂志

To establish an HPLC characteristic fingerprint method of Fuke Qianjin Capsules,and determine the contents of its main components. The analysis was carried out on a Kromasil 100-5-C18 analytical column(4. 6 mm ×250 mm,5 μm) with gradient elution by acetonitrile(A)-0. 1% phosphoric acid aqueous solution(B),a flow rate at 1. 0 m L·min-1 and the detection wavelength of 254 nm.The column temperature was 30 ℃,and the injection volume was 10 μL. The determination method of genistin,jatrorrhizine,andrographolide and 14-deoxy-11,12-didehydroandrographolide index components were studied methodologically. The common mode of the characteristic fingerprint of Fuke Qianjin Capsules was set up with 8 common peaks,which were identified as genistin,jatrorrhizine,palmatine,berberine,andrographolide,14-deoxy-11,12-didehydroandrographolide,Z-ligustilide,and Z-3-butylidenephthalide,respectively,in comparison with the references. The similarities of 20 batches of Fuke Qianjin Capsules samples were above 0. 95. All of the above-mentioned 4 analytes could be well separated under the optimized chromatographic conditions. RSD of precision and repeatability experiment were both less than 1. 5%,and the sample solution was stable during 72 h. All of the compounds had a good linearity and linear range. The contents of genistin,jatrorrhizine,andrographolide,and 14-deoxy-11,12-didehydroandrographolide in 20 batches of Fuke Qianjin Capsules samples were 28. 66-56. 04,94. 77-197. 92,1 705. 33-4 148. 93 and 462. 16-1 225. 96 μg in each capsule,respectively. The developed HPLC characteristic fingerprint and quantitative analysis methods were reliable,accurate and sensitive,and could be used effectively evaluate the quality of Fuke Qianjin Capsules samples.