Establishment of a UPLC-MS/MS Method for Studying the Effect of Salt-Processing on Tissue Distribution of Twelve Major Bioactive Components of Qing’e Pills in Rats

Qing’e pills is clinically used for treating osteoporosis in postmenopausal women in China. Eucommiae Cortex and Psoraleae Fructus are the main herbs of Qing’e pills and are both required to be salt-processed. In order to study the influence of salt-processing on the tissue distribution of Qing’e pills, a UPLC-MS/MS method was established for studying the tissue distribution of 12 main bioactive ingredients of Qing’e pills in rats. The linear relationships of the 12 compounds in each tissue were good. The method was fully validated for its selectivity, accuracy, precision, stability, matrix effect, and extraction recovery. Then, the validated method was successfully applied for simultaneous determination of the 12 chemical components in Qing’e pills in tissues for the first time. Areas under the curve (AUC) results showed that, except for pinoresinol diglucoside, psoralen, and isopsoralen, the distribution of the other components was increased in the kidney, uterus, ovary, and testes. Relative targeting efficiency (RTE) results showed that all 12 chemical components targeted the kidney and sexual organs. The results indicated that the Eucommiae Cortex and Psoraleae Fructus after salt-processing could significantly increase the distribution of components to the kidney and generative organs.

Pinoresinol Diglucoside Relieves Osteoporosis Through Enhancing Osteogenic Differentiation via Activating Phosphatidylinositol-3-Kinase/Protein Kinase B Signaling Pathway

Background: The purpose of the current study was to explore whether Pinoresinol diglucoside (PD) could relieve osteoporosis through promoting osteogenic differentiation by activating phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Methods: Firstly, human bone marrow mesenchymal stem cells (hBMMSCs) and mouse embryo steoblast recursor cells (MC3T3-E1) were induced for osteogenic differentiation, and then the cells were subjected to 1 mol/l PD. Then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot assay were used to detect the expression of osteogenic marker genes/proteins such as bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), and Osterix. The activity of alkaline phosphatase (ALP) in cells was also detected. Cell viability was further detected by Cell Count Kit-8 (CCK-8), and cell apoptosis was detected by flow cytometer. Finally, the protein expression of p-AKT and AKT was detected by Western blot assay. Results: The results showed that osteogenic differentiation of hBMMSCs and MC3T3-E1 cells were successfully induced, evidenced by increased BMP2, Runx2, Osterix mRNA expression and ALP activity enhancement. The osteogenic differentiation of MC3T3-E1 cells and hBMMSCs was enhanced by PD administration. At the same time, PD promoted the viability of MC3T3-E1 cells and reduced the MC3T3-E1 cell apoptosis. In addition, PD increased the protein expression of p-AKT and the ratio of p-AKT/AKT in MC3T3-E1 cells, suggesting PI3K/AKT pathway activation. Conclusion: PD promoted osteogenic differentiation of hBMMSCs and MC3T3-E1 cells, and it could promoted osteoblast proliferation and inhibit apoptosis, thereby playing a protective role in osteoporosis.

Tracing the mass flow from glucose and phenylalanine to pinoresinol and its glycosides in Phomopsis sp. XP-8 using stable isotope assisted TOF-MS

Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (Eucommia ulmoides Oliv) showed capability to biosynthesize pinoresinol (Pin) and pinoresinol diglucoside (PDG) from glucose (glu) and phenylalanine (Phe). To verify the mass flow in the biosynthesis pathway, [13C6]-labeled glu and [13C6]-labeled Phe were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry. As results, [13C6]-labeled Phe was incorporated into [13C6]-cinnamylic acid (Ca) and p-coumaric acid (p-Co), and [13C12]-labeled Pin, which revealed that the Pin benzene ring came from Phe via the phenylpropane pathway. [13C6]-Labeled Ca and p-Co, [13C12]-labeled Pin, [13C18]-labeled pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glu was used, demonstrating that the benzene ring and glucoside of PDG originated from glu. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study identified the occurrence of phenylalanine- lignan biosynthesis pathway in fungi at the level of mass flow.