Metabolomics and serum pharmacochemistry revealed the preventive mechanism of Gushudan in kidney-yang-deficiency-syndrome rats.

Kidney-yang-deficiency-syndrome (KYDS) is a metabolic disease caused by neuroendocrine disorder. Gushudan (GSD) is a traditional Chinese medicine prescription with the effect of nourishing kidney and strengthening bones. In this study, the mechanism of preventive effect of GSD on KYDS was explored by integrating metabolomics and serum pharmacochemistry. Reversed-phase/hydrophilic interaction chromatography-ultra-high-performance liquid chromatography-Quadrupole-Orbitrap high-resolution mass spectrometry (RP/HILIC-UHPLC-Q-Orbitrap HRMS)-based serum metabolomics indicated metabolic disturbances of KYDS rats, and 50 potential biomarkers including l-threonine, succinic acid and phytosphingosine were obtained, which were mainly involved in alanine, aspartate and glutamate metabolism, citrate cycle (tricarboxylic acid cycle) and glycerophospholipid metabolism, among others. Serum pharmacochemistry identified 29 prototypical ingredients and 9 metabolites of GSD after administration, such as icaritin and xanthotoxol. The combination of 10 serum migration ingredients in GSD, including icaritin and osthole, with 7 important targets, including AKT serine/threonine kinase 1 (AKT1) and MAPK14, was found to be key for GSD to prevent KYDS in the network pharmacology study. This study provided a new idea for the research of pathogenesis of diseases and the pharmacodynamic mechanism of traditional Chinese medicine.

Combined 1H NMR fecal metabolomics and 16S rRNA gene sequencing to reveal the protective effects of Gushudan on kidney-yang-deficiency-syndrome rats via gut-kidney axis.

Based on traditional Chinese medicine (TCM) theory, kidney is regarded as governing the bones and dominating the storage of essence ('jing' in Chinese). Gushudan (GSD) is a traditional Chinese medicine prescription with the effects of strengthening bone and nourishing kidney, which has been used to treat osteoporosis for years. Several anti-osteoporosis effects of GSD have been investigated based on metabolomics in previous studies. However, the specific mechanism of GSD on kidney tonifying and its alterations in gut microbiota are still unclear. In this study, 1H NMR fecal metabolomics and 16 S rRNA gene sequencing technology were integrated to comprehensively explore the microbiota and metabolic changes in kidney-yang-deficiency-syndrome (KYDS) rats and to elucidate the protective mechanism of GSD through the gut-kidney axis. GSD significantly regulated the levels of 12 out of 31 potential metabolites and the abundance of 11 out of 16 potential microbial biomarkers related to KYDS, respectively. Fecal metabolomics showed that GSD could reserve the abnormal levels of gut microbial-mediated metabolites of KYDS rats, such as tryptophan, lysine, dimethylamine, creatinine, acetate and butyrate, which mainly involved in amino acid metabolism, methylamine metabolism, energy metabolism and short-chain fatty acid metabolism. Specifically, GSD could promote butyrate-producing bacteria g_Lachnospiraceae_NK4A136_group and lactate-producing bacteria g_Lactobacillus. Interestingly, there was a strong relationship between altered fecal metabolites and perturbed intestinal microflora in genus. For example,lysine was negatively correlated with g_Lactobacillus, while acetate was positively correlated with g_Barnesiella. In conclusion, the study showed that the gut-kidney axis had scientific implications, which not only offered new insights into the in-depth understanding of the pathogenesis of KYDS, but also provided further evidence for the efficacy evaluation of GSD.

1H NMR serum metabolomics and its endogenous network pharmacological analysis of Gushudan on kidney-yang-deficiency-syndrome rats.

The pharmacodynamics, 1H NMR metabolomics and endogenous network pharmacology strategy approaches were integrated to investigate the preventive mechanism of Gushudan (GSD) on kidney-yang-deficiency-syndrome (KYDS) rats in this study. Firstly, the KYDS rat model was achieved by hydrocortisone induction, and the efficacy of GSD on KYDS model rats was assessed by the pharmacodynamic indicators. Next, the comprehensive untargeted serum metabolic profile of rats was obtained in 1H NMR metabolomics study, 29 potential biomarkers closely associated with KYDS were identified, which were mainly involved in carbohydrate metabolism, amino acid metabolism and intestinal flora metabolism. In addition, the potential biomarkers-targets-pathways-disease metabolic network was further investigated for deeper understanding the preventive effects of GSD on KYDS rats and its mechanism, which was further obtained for the important targets related to biomarkers and diseases such as NOS3, PTGS2 and CXCL8, and important metabolic pathways such as glyoxylate and dicarboxylate metabolism, arginine and proline metabolism, and microbial metabolism in diverse environments. Finally, compared with our previous anti-osteoporosis study of GSD, it suggested that some similar metabolic pathways, which would provide some scientific reference of the existence of the kidney-bone axis under the traditional Chinese medicine (TCM) theory of "kidney dominates bone".

Tailor-made deep eutectic solvents extraction combined with UPLC-MS/MS determination of icarrin and icarisid II in rat plasma and its comparative pharmacokinetic application.

Using green and high efficient solvents to extract and trace active ingredients of traditional Chinese medicine (TCM) in the complex biological samples was still challenging. In this paper, a co-friendly, fast pretreatment method with high extraction efficiency, based on the tailor-made deep eutectic solvent (DES) system, combined with ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination of icarrin and icarisid II in rat plasma samples, which can be further applied to comparative pharmacokinetic studies after oral administration of Herba Epimedii and icarrin monomer in rats, respectively. PrE (l-proline: ethylene glycol = 1:4 mol/mol) and acetonitrile were optimized and combined as the tailor-made DES at the volumetric ratio of 3:7 to extract icarrin and icarisid II, and to precipitate the protein in rat plasma in one step simultaneously. The extraction efficiency of the tailor-made DES was about 1.7 times of DES (PrE). The extraction recovery of icarrin and icarisid II in rat plasma samples by this method were within the range of 90-110 %, and the lower limits of quantification (LLOQ) were 0.32 ng mL-1 (icarrin) and 0.43 ng mL-1 (icarisid II). There was a linear relationship between 0.32-80.16 ng mL-1 (icarrin) and 0.43-107.4 ng mL-1 (icarisid II), which effectively reduced the detection limit. In this comparative pharmacokinetic study, the maximum plasma concentration (Cmax) and the area under plasma concentration-time curve (AUC0-∞) of two analytes in rat plasma of Herba Epimedii group were both much higher than those in the icarrin monomer group, which suggested that other ingredients in Herba Epimedii may contribute to the in vivo absorption of icarrin and icarisid II. This simple, rapid, relatively green and high effeicient method would provide a new approach for the extraction of active ingredients from complex biological samples.

Integrated renal metabolomic and metallomic profiling revealed protective effect and metabolic mechanism of Gushudan on glucocorticoid-induced osteoporotic rat based on GC-MS and ICP-MS.

Based on the traditional Chinese medicine theory, kidney is considered to govern the bones and dominate the store of essence ('jing' in Chinese). Gushudan (GSD) is a traditional Chinese medicine prescription for the treatment osteoporosis in the clinic and is beneficial for improving kidney function and strengthening bone in vivo. This study aims to reveal the renal metabolic profiling of glucocorticoid-induced osteoporosis (GIOP) rats and the potential preventive effect of GSD based on an integrative metabolomic and metallomic approach. Gas chromatography-mass spectrometry (GC-MS) and inductively coupled plasma mass spectrometry (ICP-MS) were combined for the investigation of renal metabolomic and metallomic profiling. In the metabolomic analysis, 17 potential biomarkers were found to be related to GIOP, such as glucose, malate, γ-aminobutyric acid and arachidonic acid. And seven metallic elements, including Zn, Mn, Se, Fe, Mo, As and Ba, were identified in rat kidney tissue in the metallomic analysis. The major metabolic pathways included aerobic glycolysis, and neurotransmitter amino acids metabolism. It was worth mentioning that the levels of trace metal elements (Zn, Mn, Se, Fe, As and Ba) significantly reduced in the model group, while the contents of Zn, Mn, Se, Fe and As were elevated after administration of GSD. Finally, a correlation metabolic regulatory network and the metabolic pathways associated with trace metal elements were further investigated to illuminate the role of potential biomarkers and trace metal elements in GIOP rats. These variations of potential biomarkers and trace metal elements suggested the existence of kidney damage and metabolic disorder in GIOP rats, which indicated a close relationship between bone and kidney in vivo. Moreover, the integrated renal metabolomic and metallomic profiling could be as an effective supplementary measure to the plasma and urine metabolomic research, and it was helpful to further understand the holistic formation process of osetoporosis and the potential preventive effects of GSD on GIOP rats.

Deep eutectic solvent combined with ultrasound-assisted extraction as high efficient extractive media for extraction and quality evaluation of Herba Epimedii.

Deep eutectic solvent (DES) combined with ultrasound-assisted extraction (UAE) was successfully developed and fully validated to simultaneously determine Icarrin, IcarisidⅡ, Epimcdin A, Epimcdin B and Epimcdin C for the quality evaluation of Herba Epimedii. Twelve kinds of DESs were initially screened, and then the effective extraction was achieved by the tailor-made DES consisting of the mixture of l-proline and ethylene glycol with the molar ratio of 1:4 in this study. The optimal conditions were further optimized by the orthogonal experimental design (OED). 0.2 g sample powder was ultrasonic extracted by using 4.00 mL of aqueous solution containing 70 % (v/v) the above DES for 45 min, resulting to the optimum extraction efficiency. The FT-IR and NMR spectra showed the chemical structural characteristic correlation between l-proline and ethylene glycol, and could infer the formation of hydrogen bonds between the hydroxyl group of ethylene glycol and the nitrogen atom of l-proline. The hierarchical cluster analysis (HCA) was further processed for the quality evaluation of Herba Epimedii. Finally, DES could be used to distinguish different origins and different kinds of Herba Epimedii, and to evaluate the quality of Herba Epimedii. This method provided good linearity, precision and accuracy. The recoveries of the five main bioactive flavonoids in Herba Epimedii were within the range of 88.5-107.7 % (RSD less than 3.4 %). Compared to the traditional extraction method of Icarin in the Chinese Pharmacopoeia (2015 edition), the solvent consumption was decreased by 80 % and the extraction time was shortened by 25 %, leading to more efficient and more convenient of this DES-UAE method. This work indicated that DES would be a promising high effective solvent for the analytical sample preparations of plant herbs, and it might have a broad application in the quality control of traditional Chinese medicines.

The core structure characterization and of ginseng neutral polysaccharide with the immune-enhancing activity.

The study aims to clarify the structural domain required for the immune enhancement of ginseng neutral polysaccharide (GPN). GPN was first obtained through water extraction and ion-exchange chromatography from Panax ginseng. GPN was hydrolyzed by α-amylase for 24 h and fractionated through gel permeation chromatography to give two final fragments GPNE-I and GPNE-II, with molecular weight of 8.03 × 104 Da for GPNE-I and 3.15 × 104 Da for respectively. FT-IR, methylation and 1D/2D NMR analysis demonstrated that GPNE-I was a heteropolysaccharide consisting mainly of a glucan domain and type I and II arabinogalactans (AG-I and AG-II). GPNE II was a glucan consisting of (1 → 4)-α-d-Glcp backbone with a substitution at O-6 on every two residues. (1 → 3)-α-d-Glcp and (1 → 6)-α-d-Glcp were located at the branches. In the two fractions, both α- and ß-t-Glcp as reductive terminals and →4)-α-Glcp as a non-reducing end were detected. The branching degrees of GPNE-I and GPNE-II were 38.17% and 50.78%, respectively. Immunological experiments revealed that GPNE-I exhibited more effectively stimulated lymphocyte proliferation than GPN and GPNE-II, indicating the former showed potential for immunomodulators applications, indicating that GPNE-I might be the core active domain and necessary for GPN to promote lymphocyte proliferation.

Alcohol and tea consumption in relation to the risk of nasopharyngeal carcinoma in Guangdong, China.

To investigate whether alcohol and tea consumption has an etiological association with nasopharyngeal carcinoma (NPC) in a high-incident population, a large scale case-control study was conducted. The study included 2846 individuals in Guangdong Province, China, with 1387 newly diagnosed cases of NPC and 1459 frequency-matched controls. Exposure histories of alcohol and tea consumption were obtained via personal interviews. Information regarding socio-demographic characteristics (age, sex, education, dialect and household type), family history of NPC, Epstein-Barr virus (EBV) infection, dietary habits and other potential confounding factors was also studied. An analysis was performed using unconditional logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI). The risk of NPC was found to be associated with habitual alcohol consumption and tea consumption. Tea consumption has been associated with a decreased occurrence of NPC (OR = 0.62), while consumption of alcohol was associated with a complex effect. Specifically, moderate consumption of alcohol was associated with decreased risk of NPC, while overuse, especially strong distillate spirits, appeared to be a risk factor.