Comparative metabolic profiling of wild Cordyceps species and their substituents by liquid chromatography-tandem mass spectrometry.

Cordyceps is a genus of ascomycete fungi and used widely in fungal drugs. However, in-depth studies of the metabolites of wild Cordyceps species and their substituents are lacking. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics analysis was carried out to comprehensively profile the metabolites in wild Chinese Cordyceps species (Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora 2007) from Naqu (NCs) and Yushu (YCs) and their substituents including artificially cultivated Cordyceps species (CCs) and mycelia. A total of 901 metabolites were identified in these samples, including lipids, amino acids, nucleosides, carbohydrates, organic acids, coenzymes, vitamins, alkaloids and their derivatives. Univariate and multivariate statistical analyses revealed remarkable differences and significantly different metabolites among them. Seventy amino acid-relevant metabolites were analyzed quantitatively in four samples for the first time. The four samples contained abundant L-glutamic acid and oxidized glutathione as well as multiple unique amino acid-relevant metabolites (e.g., 3-chloro-L-tyrosine, 6-aminocaproic acid, L-theanine, anserine, γ-glutamyl-cysteine). Collectively, our study provides rich metabolic information of wild Cordyceps species and their substituents, which could facilitate their quality control and optimal utilization.

Serum anion gap at admission predicts all-cause mortality in critically ill patients with cerebral infarction: evidence from the MIMIC-III database.

BACKGROUND: Recent studies reported that serum anion gap could be regarded as a prognostic biomarker for patients admitted to intensive care units. However, the association between AG and mortality in cerebral infarction patients remained largely unknown. METHODS: Relevant clinical data were collected from Medical Information Mart for Intensive Care III. Patients were divided into three groups according to tertiles of AG. Kaplan-Meier curve and Cox proportional hazards models were used to evaluate the association between AG levels and all-cause mortality. Subgroup analyses were performed to verify the predictive role of AG on mortality. RESULTS: Kaplan-Meier analysis showed that patients with higher AG had shorter survival time. Cox regression model indicated high AG as an independent risk factor of 30-day, 60-day and 180-day all-cause mortality (30-day: HR = 2.45, 95% CI = 1.21-4.97, 60-day: HR = 2.04, 95% CI = 1.07-3.89, and 180-day: HR = 1.85, 95% CI = 1.02-3.36). However, no significance was observed between AG and 365-day all-cause mortality (HR = 1.56, 95% CI = 0.87-2.78). CONCLUSIONS: High AG was associated with increased risk of all-cause mortality, and AG could be an independent short-term prognostic factor for cerebral infarction.

Fast quantification of free amino acids in food by microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry.

A method based on microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry (VAL-DESI-MS/MS) has been developed for fast quantification of free amino acids in food. Food extracts were transferred to the microfluidic platform and analyzed by liquid desorption ESI-MS/MS. Deuterated aspartic acid (i.e., 2,2,3-d3-Asp) was used as internal standard for analysis. The method had linear calibration curves with r2 values > 0.998. Limits of detection were at the level of sub µM for the amino acids tested, i.e., glutamic acid (Glu), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). To validate the proposed method in food analysis, extracts of Cordyceps fungi were analyzed. Amino acid contents were found in the range from 0.63 mg/g (Tyr in Cordyceps sinensis) to 4.44 mg/g (Glu in Cordyceps militaris). Assay repeatability (RSD) was ≤ 5.2% for all the five amino acids measured in all the samples analyzed. Recovery was found in the range from 95.8 to 105.1% at two spiking concentrations of 0.250 mg/g and 1.00 mg/g. These results prove that the proposed microfluidic VAL-DESI-MS/MS method offers a quick and convenient means of quantifying free amino acids with accuracy and repeatability. Therefore, it may have potential in food analysis for nutritional and quality assessment purposes. Graphical abstract.

Simultaneous Quantification of Adenosine and Deoxyadenosine Isomers in Foods with High Sensitivity.

Simultaneous quantification of adenosine and deoxyadenosine isomers, including 2'-deoxyadenosine (dA) and 3'-deoxyadenosine (cordycepin, COR) is a challenge because they are very similar in chemical structure. In some previous studies on food ingredients, adenine and dA might be mistakenly detected as COR that has been shown to have multiple health benefits. In this work, we developed a novel HPLC method with fluorescence detction (HPLC-FD) to simultaneously quantify COR, adenosine and dA. Pre-column derivatization with chloroacetaldehyde (CAA) was deployed. The proposed method has a limit of detection at the nM level for COR and adenosine, and is far more sensitive than the methods previously deveopled for COR determination. Using the present method, caterpillar fungi were analyzed as model food samples. The analysis revealed that COR was present in cordyceps militaris and cordyceps flowers in a concentration range from 0.314 to 0.735 mg/g, but not in cordyceps sinensis (C. sinensis), a natural and the priciest caterpillar fungus. These results suggest that the profile of active ingredients in C. sinensis has been wrongly claimed for many years. This finding was also supported by the results from further HPLC-MS/MS analyses.

Effects of Chinese medicinal herbs on expression of brain-derived Neurotrophic factor (BDNF) and its interaction with human breast cancer MDA-MB-231 cells and endothelial HUVECs.

BACKGROUND: Our previous study demonstrated that an up-regulation of the Brain-Derived Neurotrophic Factor (BDNF) signaling pathway is involved the mechanism causing the recurrence of triple negative breast cancer. The aim of this study is to investigate the effects of commonly used Chinese medicinal herbs on MDA-MB-231 and HUVEC cells and how they interact with BDNF. METHODS: Human TNBC MDA-MB-231 cells and human endothelial HUVEC cells were used to explore the effect of commonly used Chinese herbal medicines on cancer cells alone, on endothelial cells alone and on cancer cell/endothelial cell interactions; this was done via functional studies, including migration and invasion assays. Furthermore, Western blot analysis and real-time PCR investigations were also used to investigate migration signal transduction, invasion signal transduction, and angiogenic signal transduction in these systems. Finally, the effect of the Chinese medicinal herbs on cancer cell/endothelial cell interactions was assessed using co-culture and ELISA. RESULTS: In terms of autoregulation, BDNF up-regulated TrkB gene expression in both MDA-MB-231 and HUVEC cells. Furthermore, BDNF enhanced migration by MDA-MB-231 cells via Rac, Cdc42 and MMP, while also increasing the migration of HUVEC cells via MMP and COX-2 expression. As measured by ELISA, the Chinese herbal medicinal herbs A. membranaceus, P. lactiflora, L. chuanxiong, P. suffruticosa and L. lucidum increased BDNF secretion by MDA-MB-231 cells. Similarly, using a co-culture system with MDA-MB-231 cells, A. membranaceus and L. lucidum modulated BDNF-TrkB signaling by HUVEC cells. CONCLUSION: We conclude that BDNF plays an important role in the metastatic interaction between MDA-MB-231 and HUVEC cells. Some Chinese medicinal herbs are able to enhance the BDNF-related metastatic potential of the interaction between cancer cells and endothelial cells. These findings provide important information that should help with the development of integrated medical therapies for breast cancer patients.

[Efficacy of Zaozhu Yinchen Recipe for Treating Non-alcoholic Steatohepatitis and its Effect on Free Fatty Acid and TNF-alpha].

OBJECTIVE: To observe the efficacy of Zaozhu Yinchen Recipe (ZZYCR) on non-alcoholic steatohepatitis (NASH) patients, and to explore its effect on serum free fatty acid (FFA) and tumor necrosis factor alpha (TNF-alpha). METHODS: Totally 120 patients with NASH were randomly assigned to the treatment group (60 cases, treated with ZZYCR, one dose per day) and the control group (60 cases, treated with Silibin Meglumine Tablets, 20 mg each time, thrice per day). The therapeutic course for all was 24 weeks. Serum levels of ALT and AST activities, TC and TG levels were detected before and after treatment. Peritoneal CT was performed in all patients, and CT ratios of liver and spleen calculated. NAFLD activity score (NAS) and degree of hepatic fibrosis were assessed using pathological examinations of liver tissue, and efficacy also evaluated. Serum contents of FFA and TNF-alpha were also detected. RESULTS: Compared with before treatment in the same group, activities of ALT and AST, serum levels of TC, TG, FFA, and TNF-alpha, NAS, scores of symptoms and signs all obviously decreased, degree of hepatic fibrosis was obviously improved in the two groups (P < 0.05, P < 0.01). These changes were more obviously seen in the treatment group (P < 0.05). After 24-week treatment, the total effective rate and total clinical efficacy were 80.00% (48/60 cases) and 85.00% (51/60 cases) in the treatment group, obviously higher than those in the control group [60.00% (36/60 cases) and 73.33% (44/60 cases) respectively], with significant difference (P < 0.05, P < 0.01). CONCLUSION: ZZYCR could improve the clinical efficacy of NASH patients, and its mechanism might be associated with inhibiting serum levels of FFA and TNF-alpha.

[Mechanism of geniposide in improving free fatty acid metabolism in rats with non-alcoholic fatty liver disease].

To observe the effect of geniposide on non-alcoholic fatty liver disease (NAFLD), and discuss the mechanism of geniposide for NAFLD from the aspect of free fatty acid, forty healthy Wistar male rats were randomly divided into normal group, model group, geniposide and Xuezhikang group. The rats in normal group were fed with normal diets, and the rats in other 3 groups were given with high-fat diet for 8 weeks to induce the NAFLD models. From the week 5 to end of week 8, the rats in geniposide and Xuezhikang group were intervened with corresponding medicines. The body weight, liver wet weight, and fat weight of the rats were recorded. Visual and pathological changes in hepatic tissues were observed with HE staining. The contents of TG, FFA, FAS, AMPK, ACCase and Malonyl-CoA in hepatic tissue, contents of CHO and LDL-C in serum and activities of AST and ALT in serum were detected by using corresponding methods. The results showed that the body weight, liver wet weight, and fat weight of the rats, CHO, LDL-C, ALT and AST levels in serum, TG, FFA, FAS, ACCase and Malonyl-CoA levels in hepatic tissues of the rats in model group were significantly higher than those in normal group (P<0.01), while AMPK activity was significantly lower than that of the normal group (P<0.01), with obvious visual and pathological steatosis in hepatic tissues, and inflammatory injury occurred in model group. Compared with the model group, body weight of the rat, fat weight, levels of FFA in hepatic tissues, ALT and AST activities in serum, liver wet weight, TG, FAS, ACCase and Malonyl-CoA levels were significantly decreased in geniposide group (P<0.01), while the AMPK activity in hepatic tissues was significantly increased (P<0.05),with improvement in visual and pathological performance. Compared with the model group, liver wet weight, fat weight, TG and FFA levels in hepatic tissues, and LDL-C level in serum were significantly decreased in Xuezhikang group (P<0.05). Compared with Xuezhikang group, the body weight of rat, fat weight and FFA level in hepatic tissues were significantly lower in geniposide group (P<0.01), but with no significant difference in other aspects. These findings indicated that geniposide was highly effective in improving the pharmacological effect of NAFLD induced by high-fat diet, and the mechanism was achieved through AMPK-ACCase-Malonyl-CoA-FFA axis.

Decoction vs extracts-mixed solution: effect of yiqihuoxue formula on non-alcoholic fatty liver disease in rats.

OBJECTIVE: To investigate the effects on non-alcoholic fatty liver disease (NAFLD) in rats of the decoction of Yiqihuoxue formula and the solution prepared with the extracts from the individual herbal medicines of the formula. METHODS: The rat models of NAFLD were established with high-fat diet (HFD) for 10 weeks. Thirty-two rats were randomly divided into 4 groups: the control group, the model group, the decoction group and the solution group, 8 for each group. From the 6th week, drinking water, the decoction and the solution were intragastrically administrated accordingly to the rats for 5 weeks. The pathological changes of the liver tissues were observed with Hematoxylin and eosin staining, triglyceride levels in liver tissues measured, serum alanine aminotransferase (ALT) activity measured, and serum gastrin and motilin tested. RESULTS: Fatty degeneration and vacuole-like changes to various degrees occurred in hepatic cells of the model group. Indicators for fat metabolism, serum ALT activity and hepatic triglyceride level significantly increased, while serum gastrin and motilin levels significantly decreased. Serum ALT activity and the fatty deposition in hepatocytes were significantly reduced. In the meantime, the expressions of gastrin and motilin in the serum rose significantly in the treatment groups. CONCLUSION: Both the decoction and the extracts-mixed solution had effect on NAFLD of protecting the liver function and reducing the fatty deposition in liver, which might be achieved by regulating the expression of gastrin and motilin.

Study on effects of zhi zi (fructus gardeniae) on non-alcoholic fatty liver disease in the rat.

OBJECTIVE: To investigate the effects of Zhi Zi (Fructus Gardeniae) on non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet in the rat. METHODS: A rat model of NAFLD was established using a high-fat diet. Twenty one rats were randomly divided into a normal group, a model group and a Zhi Zi treatment group, 7 rats per group. Drinking water and the drug were intragastrically administrated for 5 weeks. Samples were then taken to observe pathological changes of the liver tissue (HE staining); changes in the fat metabolism pathway e. g. triglyceride (TG) and free fatty acid (FFA) content; alterations in liver function, i.e. serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity; and differences in tumor necrosis factor alpha (TNF-alpha) and P-IkB protein expression in the liver tissue. RESULTS: Fatty degeneration and vacuole-like changes of different degrees occurred in hepatic cells of the model group. Markers for fat metabolism, serum ALT and AST activities, and expression of TNF-alpha and P-IkB proteins in liver tissue significantly increased. Fat metabolism in the Zhi Zi group significantly reduced, as shown by a drop in marker levels. Serum ALT and AST activities, and expression of TNF-alpha, P-IkB proteins in liver tissue were also significantly decreased in this group. CONCLUSION: Zhi Zi has a very strong inhibitory action on lipidosis and inflammatory injury in the rat model of NAFLD. This mechanism may possibly be related to the inhibition of the free fatty acid metabolism pathway.