Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry analysis of icariside II metabolites in rats

<div><p><b>:</b> The possible metabolic pathways of icariside II were proposed. An ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry method was used for analysing the faecal, bile, plasma and urine samples of rats administrated with icariside II. In all, 27 metabolites were identified in the biosamples. Of these, 20, including F1–F12, D3, D4, D6, D7–D9 and M3, M4, were, to our knowledge, reported for the first time. The results indicated that icariside II was metabolised via desugarisation, dehydrogenation, hydrogenation, hydroxylation, demethylation, glucuronidation, dehydration and glycosylation pathways <i>in vivo</i>. Specific hydrolysis of 7-<i>O</i> glucosides in the gut lumen and glucuronic acid conjugation in the liver were considered as the main physiologic processes of icariside II. This study revealed the possible metabolite profiles of icariside II in rats.</p></div

DataSheet1_Zengshengping improves lung cancer by regulating the intestinal barrier and intestinal microbiota.docx

Lung cancer is a common malignant tumor in clinical practice, and its morbidity and mortality are in the forefront of malignant tumors. Radiotherapy, chemotherapy, and surgical treatment play an important role in the treatment of lung cancer, however, radiotherapy has many complications and even causes partial loss of function, the recurrence rate after surgical resection is high, and the toxic and side effects of chemotherapy drugs are strong. Traditional Chinese medicine has played a huge role in the prognosis and improvement of lung cancer, among them, Zengshengping (ZSP) has the effect of preventing and treating lung cancer. Based on the “gut-lung axis” and from the perspective of “treating the lung from the intestine”, the purpose of this study was to research the effect of Zengshengping on the intestinal physical, biological, and immune barriers, and explore its role in the prevention and treatment of lung cancer. The Lewis lung cancer and urethane-induced lung cancer models were established in C57BL/6 mice. The tumor, spleen, and thymus were weighed, and the inhibition rate, splenic and thymus indexes analyzed. Inflammatory factors and immunological indexes were detected by enzyme-linked immunosorbent assay. Collecting lung and colon tissues, hematoxylin and eosin staining was performed on lung, colon tissues to observe histopathological damage. Immunohistochemistry and Western blotting were carried out to detect tight junction protein expression in colon tissues and expression of Ki67 and p53 proteins in tumor tissues. Finally, the feces of mice were collected to investigate the changes in intestinal microbiota using 16SrDNA high-throughput sequencing technology. ZSP significantly reduced tumor weight and increased the splenic and thymus indexes. It decreased expression of Ki67 protein and increased expression of p53 protein. Compared with Model group, ZSP group reduced the serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and ZSP group increased the concentration of secretory immunoglobulin A (sIgA) in the colon and the bronchoalveolar lavage fluid (BALF). ZSPH significantly increased the level of tight junction proteins such as ZO-1, Occludin and Claudin-1. Model group significantly reduced the relative abundance of Akkermansia (p < 0.05) and significantly promoted the amount of norank_f_Muribaculaceae, norank_f_Lachnospiraceae (p < 0.05) compared with that in the Normal group. However, ZSP groups increased in probiotic strains (Akkermansia) and decreased in pathogens (norank_f_Muribaculaceae, norank_f_Lachnospiraceae). Compared with the urethane-induced lung cancer mice, the results showed that ZSP significantly increased the diversity and richness of the intestinal microbiota in the Lewis lung cancer mice. ZSP played an important role in the prevention and treatment of lung cancer by enhancing immunity, protecting the intestinal mucosa and regulating the intestinal microbiota.</p

DataSheet3_Zengshengping improves lung cancer by regulating the intestinal barrier and intestinal microbiota.docx

Lung cancer is a common malignant tumor in clinical practice, and its morbidity and mortality are in the forefront of malignant tumors. Radiotherapy, chemotherapy, and surgical treatment play an important role in the treatment of lung cancer, however, radiotherapy has many complications and even causes partial loss of function, the recurrence rate after surgical resection is high, and the toxic and side effects of chemotherapy drugs are strong. Traditional Chinese medicine has played a huge role in the prognosis and improvement of lung cancer, among them, Zengshengping (ZSP) has the effect of preventing and treating lung cancer. Based on the “gut-lung axis” and from the perspective of “treating the lung from the intestine”, the purpose of this study was to research the effect of Zengshengping on the intestinal physical, biological, and immune barriers, and explore its role in the prevention and treatment of lung cancer. The Lewis lung cancer and urethane-induced lung cancer models were established in C57BL/6 mice. The tumor, spleen, and thymus were weighed, and the inhibition rate, splenic and thymus indexes analyzed. Inflammatory factors and immunological indexes were detected by enzyme-linked immunosorbent assay. Collecting lung and colon tissues, hematoxylin and eosin staining was performed on lung, colon tissues to observe histopathological damage. Immunohistochemistry and Western blotting were carried out to detect tight junction protein expression in colon tissues and expression of Ki67 and p53 proteins in tumor tissues. Finally, the feces of mice were collected to investigate the changes in intestinal microbiota using 16SrDNA high-throughput sequencing technology. ZSP significantly reduced tumor weight and increased the splenic and thymus indexes. It decreased expression of Ki67 protein and increased expression of p53 protein. Compared with Model group, ZSP group reduced the serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and ZSP group increased the concentration of secretory immunoglobulin A (sIgA) in the colon and the bronchoalveolar lavage fluid (BALF). ZSPH significantly increased the level of tight junction proteins such as ZO-1, Occludin and Claudin-1. Model group significantly reduced the relative abundance of Akkermansia (p < 0.05) and significantly promoted the amount of norank_f_Muribaculaceae, norank_f_Lachnospiraceae (p < 0.05) compared with that in the Normal group. However, ZSP groups increased in probiotic strains (Akkermansia) and decreased in pathogens (norank_f_Muribaculaceae, norank_f_Lachnospiraceae). Compared with the urethane-induced lung cancer mice, the results showed that ZSP significantly increased the diversity and richness of the intestinal microbiota in the Lewis lung cancer mice. ZSP played an important role in the prevention and treatment of lung cancer by enhancing immunity, protecting the intestinal mucosa and regulating the intestinal microbiota.</p

DataSheet2_Zengshengping improves lung cancer by regulating the intestinal barrier and intestinal microbiota.xlsx

Lung cancer is a common malignant tumor in clinical practice, and its morbidity and mortality are in the forefront of malignant tumors. Radiotherapy, chemotherapy, and surgical treatment play an important role in the treatment of lung cancer, however, radiotherapy has many complications and even causes partial loss of function, the recurrence rate after surgical resection is high, and the toxic and side effects of chemotherapy drugs are strong. Traditional Chinese medicine has played a huge role in the prognosis and improvement of lung cancer, among them, Zengshengping (ZSP) has the effect of preventing and treating lung cancer. Based on the “gut-lung axis” and from the perspective of “treating the lung from the intestine”, the purpose of this study was to research the effect of Zengshengping on the intestinal physical, biological, and immune barriers, and explore its role in the prevention and treatment of lung cancer. The Lewis lung cancer and urethane-induced lung cancer models were established in C57BL/6 mice. The tumor, spleen, and thymus were weighed, and the inhibition rate, splenic and thymus indexes analyzed. Inflammatory factors and immunological indexes were detected by enzyme-linked immunosorbent assay. Collecting lung and colon tissues, hematoxylin and eosin staining was performed on lung, colon tissues to observe histopathological damage. Immunohistochemistry and Western blotting were carried out to detect tight junction protein expression in colon tissues and expression of Ki67 and p53 proteins in tumor tissues. Finally, the feces of mice were collected to investigate the changes in intestinal microbiota using 16SrDNA high-throughput sequencing technology. ZSP significantly reduced tumor weight and increased the splenic and thymus indexes. It decreased expression of Ki67 protein and increased expression of p53 protein. Compared with Model group, ZSP group reduced the serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and ZSP group increased the concentration of secretory immunoglobulin A (sIgA) in the colon and the bronchoalveolar lavage fluid (BALF). ZSPH significantly increased the level of tight junction proteins such as ZO-1, Occludin and Claudin-1. Model group significantly reduced the relative abundance of Akkermansia (p < 0.05) and significantly promoted the amount of norank_f_Muribaculaceae, norank_f_Lachnospiraceae (p < 0.05) compared with that in the Normal group. However, ZSP groups increased in probiotic strains (Akkermansia) and decreased in pathogens (norank_f_Muribaculaceae, norank_f_Lachnospiraceae). Compared with the urethane-induced lung cancer mice, the results showed that ZSP significantly increased the diversity and richness of the intestinal microbiota in the Lewis lung cancer mice. ZSP played an important role in the prevention and treatment of lung cancer by enhancing immunity, protecting the intestinal mucosa and regulating the intestinal microbiota.</p

DataSheet1_Integrating strategies of metabolomics, network pharmacology, and experiment validation to investigate the processing mechanism of Epimedium fried with suet oil to warm kidney and enhance yang.docx

Introduction: Epimedium, a traditional Chinese medicine (TCM) commonly used in ancient and modern China, is one of the traditional Chinese medicines clinically used to treat kidney yang deficiency syndrome (KYDS). There are differences in the efficacy of Epimedium before and after processing, and the effect of warming the kidney and enhancing yang is significantly enhanced after heating with suet oil. However, the active compounds, corresponding targets, metabolic pathways, and synergistic mechanism of frying Epimedium in suet oil to promote yang, remain unclear.Methods: Herein, a strategy based on comprehensive GC-TOF/MS metabolomics and network pharmacology analysis was used to construct an “active compounds-targets-metabolic pathways” network to identify the active compounds, targets and metabolic pathways involved. Subsequently, the targets in kidney tissue were further validated by real-time quantitative polymerase chain reaction (RT-qPCR). Histopathological analysis with physical and biochemical parameters were performed.Results: Fifteen biomarkers from urine and plasma, involving five known metabolic pathways related to kidney yang deficiency were screened. The network pharmacology results showed 37 active compounds (13 from Epimedium and 24 from suet oil), 159 targets, and 267 pathways with significant correlation. Importantly, integrated metabolomics and network pharmacologic analysis revealed 13 active compounds (nine from Epimedium and four from suet oil), 7 corresponding targets (ALDH2, ARG2, GSTA3, GSTM1, GSTM2, HPGDS, and NOS2), two metabolic pathways (glutathione metabolism, arginine and proline metabolism), and two biomarkers (Ornithine and 5-Oxoproline) associated with improved kidney yang deficiency by Epimedium fried with suet oil.Discussion: These finds may elucidate the underlying mechanism of yang enhancement via kidney warming effects. Our study indicated that the mechanism of action mainly involved oxidative stress and amino acid metabolism. Here, we demonstrated the novel strategies of integrating metabolomics and network pharmacology in exploring of the mechanisms of traditional Chinese medicines.</p

Characterization of metabolites of sagittatoside B in rats using UPLC-QTOF-MS spectrometry

Sagittatoside B is one of the principal diglucosides in Herba Epimedii. In this work, an ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was applied to the rapid analysis of sagittatoside B metabolites in rats after oral administration. A total number of 17 metabolites were detected or tentatively identified from rat plasma, bile, urine and feces. The major metabolic pathways of sagittatoside B in rats were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, decarbonylation and conjugation with glucuronic acid and different sugars. This work revealed the metabolism of sagittatoside B in vivo, and reported the characteristic metabolic reactions of sagittatoside B for the first time. This provided the basis for the further research and development of sagittatoside B, and also provided reference for the metabolism of active flavonoid compounds.</p

Revolving Door Action of Breast Cancer Resistance Protein (BCRP) Facilitates or Controls the Efflux of Flavone Glucuronides from UGT1A9-Overexpressing HeLa Cells

Cellular production of flavonoid glucuronides requires the action of both UDP-glucuronosyltransferases (UGT) and efflux transporters since glucuronides are too hydrophilic to diffuse across the cellular membrane. We determined the kinetics of efflux of 13 flavonoid glucuronides using the newly developed HeLa-UGT1A9 cells and correlated them with kinetic parameters derived using expressed UGT1A9. The results indicated that, among the seven monohydroxylflavones (HFs), there was moderately good correlation (<i>r</i>² ≥ 0.65) between the fraction metabolized (<i>f</i>_met) derived from HeLa-UGT1A9 cells and CL_int derived from the UGT1A9-mediated metabolism. However, there was weak or no correlation between these two parameters for six dihydroxylflavones (DHFs). Furthermore, there was weak or no correlation between various kinetic parameters (<i>K</i>_m, <i>V</i>_max, or CL_int) for the efflux and the metabolism regardless of whether we were using seven HFs, six DHFs, or a combination thereof. Instead, the cellular excretion of many flavonoid glucuronides appears to be controlled by the efflux transporter, and the poor affinity of glucuronide to the efflux transporter resulted in major intracellular accumulation of glucuronides to a level that is above the dosing concentration of its aglycone. Hence, the efflux transporters appear to act as the “Revolving Door” to control the cellular excretion of glucuronides. In conclusion, the determination of a flavonoid’s susceptibility to glucuronidation must be based on both its susceptibility to glucuronidation by the enzyme and resulting glucuronide’s affinity to the relevant efflux transporters, which act as the “Revolving Door(s)” to facilitate or control its removal from the cells

Efficient preparation of rare Sagittatoside A from epimedin A, by recyclable aqueous organic two-phase enzymatic hydrolysis

The rare secondary flavonol glycoside Sagittatoside A has much better in vivo bioactivities than epimedin A in Epimedii Folium. However, its current preparation methods are of low efficiency, with byproducts generated. The aim of this study was to establish a novel catalysis system for effective and convenient preparation of Sagittatoside A from epimedin A based on recyclable and integrated aqueous organic two-phase enzymatic hydrolysis. The system was consisted of propyl acetate and HAc-NaAc buffer (pH4.5) containing β-dextranase/epimedin A, and the hydrolysis was performed at 60 °C for 1 h. Consequently, epimedin A was completely hydrolyzed to sagittatoside A, and 95.02% of the product was transferred into the organic phase. Moreover, 90% of its initial activity was retained after seven cycles of hydrolysis. Additionally, the procedure was simpler than conventional enzymatic hydrolysis. Collectively, the newly proposed strategy is an efficient and promising approach for the preparation of sagittatoside A in industrial application.</p

A novel PCR-based technology for rapid and non-sequencing authentication of <i>Bombyx batryticatus</i> using species-specific primers

<p>A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of <i>Bombyx batryticatus</i> based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for <i>Beauveria bassiana</i> and 100 pg for <i>Bombyx mori</i> and <i>Metarhizium anisopliae</i>. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes.</p

Additional file 1 of Characterization of a novel polysaccharide from red ginseng and its ameliorative effect on oxidative stress injury in myocardial ischemia

Additional file 1: Figure S1. GC–MS spectra of methylation of RGP 1-1