Comparative analysis of the synergetic effects of Diwuyanggan prescription on high fat diet-induced non-alcoholic fatty liver disease using untargeted metabolomics.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders worldwide and had no approved pharmacological treatments. Diwuyanggan prescription (DWYG) is a traditional Chinese medicine preparation composed of 5 kinds of herbs, which has been used for treating chronic liver diseases in clinic. Whereas, the synergistic mechanism of this prescription for anti-NAFLD remains unclear. In this study, we aimed to demonstrate the synergetic effect of DWYG by using the disassembled prescriptions and untargeted metabolomics research strategies. The therapeutic effects of the whole prescription of DWYG and the individual herb were divided into six groups according to the strategy of disassembled prescriptions, including DWYG, Artemisia capillaris Thunb. (AC), Curcuma longa L. (CL), Schisandra chinensis Baill. (SC), Rehmannia glutinosa Libosch. (RG) and Glycyrrhiza uralensis Fisch. (GU) groups. The high fat diets-induced NAFLD mice model was constructed to evaluate the efficacy effects of DWYG. An untargeted metabolomics based on the UPLC-QTOF-MS/MS approach was carried out to make clear the synergetic effect on the regulation of metabolites dissecting the united mechanisms. Experimental results on animals revealed that the anti-NAFLD effect of DWYG prescription was better than the individual herb group in reducing liver lipid deposition and restoring the abnormality of lipidemia. In addition, further metabolomics analysis indicated that 23 differential metabolites associated with the progression of NAFLD were identified and 19 of them could be improved by DWYG. Compared with five single herbs, DWYG showed the most extensive regulatory effects on metabolites and their related pathways, which were related to lipid and amino acid metabolisms. Besides, each individual herb in DWYG was found to show different degrees of regulatory effects on NAFLD and metabolic pathways. SC and CL possessed the highest relationship in the regulation of NAFLD. Altogether, these results provided an insight into the synergetic mechanisms of DWYG from the metabolic perspective, and also supported a scientific basis for the rationality of clinical use of this prescription.

Elucidating the mechanisms underlying the anti-hyperlipidemic effects of Laportea bulbifera using integrated serum metabolomics and network pharmacology.

Hyperlipidemia is a chronic metabolic disorder characterized by alterations in lipid metabolism as well as other pathways. Laportea bulbifera, an indigenous medicinal plant of Chinese herbal medicine, exhibits therapeutic effects on hyperlipidemia, but the mechanisms remain unclear. This study investigated the potential mechanisms underlying the anti-hyperlipidemic effects of L. bulbifera using an integrated strategy based on metabolomics and network pharmacology methods that were established to investigate the potential mechanism of anti-hyperlipidemia effect of L. bulbifera. First, the therapeutic effects of L. bulbifera on body weight reduction and biochemical indices were assessed. Next, 18 significant metabolites distinguishing the control and model groups were identified based on serum metabolomics and multivariate analyses. Then, a compound-target network was constructed by linking L. bulbifera and hyperlipidemia using network pharmacology. Three metabolic pathways involved in treating hyperlipidemia were identified. Finally, five crucial targets were selected by constructing a bionetwork starting from the compounds and ending in the metabolites. This study established an integrated strategy based on metabolomics coupled with network pharmacology and revealed the mechanism underlying the protective effects of L. bulbifera against hyperlipidemia for the first time.

Dynamical changes of tea metabolites fermented by Aspergillus cristatus, Aspergillus neoniger and mixed fungi: A temporal clustering strategy for untargeted metabolomics.

Dark tea fermentation involves various fungi, but studies focusing on the mixed fermentation in tea remain limited. This study investigated the influences of single and mixed fermentation on the dynamical alterations of tea metabolites. The differential metabolites between unfermented and fermented teas were determined using untargeted metabolomics. Dynamical changes in metabolites were explored by temporal clustering analysis. Results indicated that Aspergillus cristatus (AC) at 15 days, Aspergillus neoniger (AN) at 15 days, and mixed fungi (MF) at 15 days had respectively 68, 128 and 135 differential metabolites, compared with unfermentation (UF) at 15 days. Most of metabolites in the AN or MF group showed a down-regulated trend in cluster 1 and 2, whereas most of metabolites in the AC group showed an up-regulated trend in cluster 3 to 6. The three key metabolic pathways mainly composed of flavonoids and lipids included flavone and flavonol biosynthesis, glycerophospholipid metabolism and flavonoid biosynthesis. Based on the dynamical changes and metabolic pathways of the differential metabolites, AN showed a predominant status in MF compared with AC. Together, this study will advance the understanding of dynamic changes in tea fermentation and provide valuable insights into the processing and quality control of dark tea.

Supplementation with Ginseng, Lilii Bulbus, and Poria induces alterations in the serum metabolic profile of healthy adults.

The preventive and therapeutic effects of herbal supplementation containing Ginseng, Lilii Bulbus, and Poria (GLP) on inflammation and oxidative stress in healthy adults have been demonstrated in our previous studies. However, the underlying mechanisms of organism protection by GLP remain unclarified, and few studies have used metabolomics to investigate comprehensive changes before and after GLP supplementation. Based on previous research, we conducted a placebo-controlled trial among 82 healthy adults in Wuhan, China, using a metabolomics approach with ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) and multivariate statistical methods to analyze serum metabolite alterations in participants before and after GLP supplementation. Furthermore, 14 discriminant metabolites related to lipid metabolism, inflammation and oxidative stress, energy metabolism, and coenzyme A metabolism were significantly different between the before- and after-GLP groups (P < 0.0001). Nine metabolites were significantly decreased in the serum samples from the after-GLP group compared with the before-GLP group, while five metabolites were significantly increased. These metabolites could be critical components associated with the anti-inflammatory, antioxidant, and hypolipidemic activities of GLP, indicating the potential complementary role of GLP supplements in the primary prevention of dysfunctional metabolism caused by potential diseases such as cardiovascular disease. This study provides a valuable reference for cardiovascular health protection and disease prevention.

Potential hypoglycemic metabolites in dark tea fermented by Eurotium cristatum based on UPLC-QTOF-MS/MS combining global metabolomic and spectrum-effect relationship analyses.

The preventive and therapeutic effects of dark tea fermented by Eurotium cristatum (DTE) in glucose metabolism have been demonstrated. However, few studies have investigated comprehensive changes in the chemical composition and activity in DTE before and after fermentation. In this study, the metabolic profiling of raw samples and fermented samples was determined by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). Furthermore, a systematic analytical strategy combining global metabolomics and the spectrum-effect relationship based on α-glucosidase inhibition was employed for screening discriminant metabolites. As a result, 15 discriminant metabolites were identified in DTE samples. Among them, 10 metabolites (4 fatty acids, 1 dyphylline derivative, 3 lysophosphatidylcholines, and 2 triterpenes) increased in relative contents and the contents of the other 5 polyphenol metabolites decreased after fermentation. These metabolites were critical constituents possibly associated with DTE's hypoglycemic activity, which also might be suitable as quality evaluation indicators. This study provided a worthy insight into the exploration of representative active constituents or quality indicators of DTE.

Changes in Triterpenes in Alismatis rhizoma after Processing Based on Targeted Metabolomics Using UHPLC-QTOF-MS/MS.

Alismatis rhizoma (AR) has been used as an herbal medicine in China for over a thousand years. Crude AR, salt-processed AR (SAR), and bran-processed AR (BAR) are recorded in the Pharmacopoeia of the People's Republic of China. However, the differences of chemical composition between crude AR and its processing products remain limited. In this study, triterpenes were identified from crude AR, SAR, and BAR by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometer (UHPLC-QTOF-MS/MS). Subsequently, the differences of triterpenes between the crude AR and processed ARs were compared via a targeted metabolomics approach. Finally, a total of 114 triterpenes were identified, of which 83, 100, and 103 triterpenes were found in crude AR, SAR, and BAR, respectively. After salt-processing, there were 17 triterpenes newly generated, 7 triterpenes with trends of increasing, and 37 triterpenes decreased. Meanwhile, 56 triterpenes including 21 newly generated and 35 with significant increases were observed in BAR. This study could be benefit to investigate the processing mechanism of AR, as well as support their clinical applications.

The hypoglycemic effect of extract/fractions from Fuzhuan Brick-Tea in streptozotocin-induced diabetic mice and their active components characterized by LC-QTOF-MS/MS.

Fuzhuan Brick-Tea is a postfermented product with the hypoglycemic effect, which is prepared from the leaves of Camellia sinensis var. sinensis. However, the material basis associated with the hypoglycemic effect was not clear. The present research was designed to explore the hypoglycemic effect of extract/fractions from Fuzhuan Brick-Tea in streptozotocin-induced type II diabetic mice. Then an ultra-high pressure liquid chromatography along with quadrupole time of flight mass spectrometry was used to analyze the phytochemicals in Fuzhuan Brick-Tea fractions. As a result, the hypoglycemic and hypolipidemic effects were evidently observed from the serum biochemical indexes and liver pathological examination in type II diabetic mice. In addition, there were total of 20 major components including eight lysophosphatidylcholines (Lyso-PCs), five fatty acids, and seven novel theophylline derivatives tentatively identified in the active fraction from water extract. Therefore, these components were assumed to contribute partly to the hypoglycemic effect of Fuzhuan Brick-Tea. These findings also give the evidence that the Lyso-PCs, fatty acids, and novel theophylline derivatives in Fuzhuan Brick-Tea may provide benefits in ameliorating disorders of glucose and lipid metabolism. PRACTICAL APPLICATION: This study suggests that the Lyso-PCs, fatty acids, and novel theophylline derivatives in Fuzhuan Brick-Tea may provide benefits in ameliorating disorders of glucose and lipid metabolism. It can be taken as a beneficial diet additive or nutraceutical.

A novel predict-verify strategy for targeted metabolomics: Comparison of the curcuminoids between crude and fermented turmeric.

Curcuminoids are the major bioactive constituents of turmeric, the application of which are limited by the poor bioavailability. In this study, turmeric was fermented by the Monascus purpureus and Eurotium cristatum fungi to enhance its bioavailability. To explore the variations in the curcuminoids contents in fermented turmeric, a targeted predict-verify strategy was established. For targeted analysis of curcuminoids, a compound library containing all possible curcuminoids based on their structural skeleton was predicted and built for targeted scanning. Then, the MS data were automatically matched with the predicted library to verify the corresponding curcuminoids. As a result, 115 curcuminoids (48 novel compounds and 14 compounds reported in turmeric for the first time) were fully characterized in crude and fermented turmeric. Among these curcuminoids, 31 were newly generated in fermented turmeric. The established predict-verify strategy allows for an efficient and automatic metabolomic analysis to screen for curcuminoids with potentially better bioavailability.

The metabolic change in serum lysoglycerophospholipids intervened by triterpenoid saponins from Kuding tea on hyperlipidemic mice.

Triterpenoid saponins from Kuding tea have demonstrated preventive effects on hyperlipidaemia induced by a high-fat diet. Lysoglycerophospholipids (Lyso-GPLs) are known to be associated with proatherogenic conditions such as hyperlipidaemia. In this study, a target profiling strategy based on a multiple reaction monitoring mode was applied for the analysis of Lyso-GPLs. The metabolic changes were evaluated by the qualitative and relative quantitative distribution of six classes of Lyso-GPLs in mouse serum. A total of 153 Lyso-GPL regioisomers, consisting of 85 lysophosphatidylcholines, 15 lysophosphatidic acids, 23 lysophosphatidylethanolamines, 5 lysophosphatidylserines, 19 lysophosphatidylinositols and 6 lysophosphatidylglycerols, were detected and quantified. The results showed decreased trends in the content of total Lyso-GPLs in the serum of hyperlipidemic mice compared with that in normal controls. The content of total Lyso-GPLs significantly increased after treatment with triterpenoid saponins from Kuding tea. Among them, the proportions of most Lyso-GPLs with a higher degree of unsaturation or a longer carbon chain in fatty acyl chains dramatically decreased in hyperlipidemic mice. However, this tendency reversed after the treatment of triterpenoid saponins from Kuding tea. This is the first study regarding a target profiling strategy for the quantitative analysis of six different types of Lyso-GPLs on high-fat diet-induced hyperlipidemic mice intervened by Kuding tea. Those Lyso-GPLs changed significantly may be potential biomarkers for hyperlipidaemia, and involved in the mechanism of the preventive intervention of Kuding tea on Lipid metabolic diseases.

Identification of the lipid-lowering component of triterpenes from Alismatis rhizoma based on the MRM-based characteristic chemical profiles and support vector machine model.

It has been demonstrated that triterpenes in Alismatis rhizoma (Zexie in Chinese, ZX) contributed to the lipid-lowering effect on high-fat diet-induced hyperlipidemia. Alisol B 23-acetate, one of the abundant triterpenes in ZX, was used as the marker of quality control for ZX in Chinese Pharmacopoeia, while it could not reflect the lipid-lowering effect because other triterpenes in ZX also had prominent medicinal efficacy. To identify the significantly bioactive triterpenes in ZX, a multiple reaction monitoring (MRM)-based characteristic chemical profile (CCP)-support vector machine (SVM) model was used to explore the relationship between triterpenes and lipid-lowering effect of ZX. Firstly, the content of 87 targeted triterpenes was quantified by the MRM-based CCP using UHPLC-QTRAP-MS/MS. Secondly, the lipid-lowering effect of 30 ZX samples was assessed by 3T3-L1 preadipocytes. Thirdly, 9 of the 87 triterpenes possessing high mean impact value were identified to have significant lipid-lowering effect via the particle swarm-optimized SVM model. The new SVM model constructed by the 9 triterpenes showed good prediction performance and the overall prediction accuracy reached 81.94%. Finally, the real activity of these triterpenes was partly confirmed and was consistent with the prediction of SVM. These results showed that the method for discovery of triterpenes with prominent lipid-lowering activity in ZX was reliable. The proposed method is expected to provide an efficient and rapid approach for screening of active component and drug discovery in traditional herbs. Graphical abstract.

Association between prenatal nickel exposure and preterm low birth weight: possible effect of selenium.

There is a proposed link between prenatal nickel (Ni) exposure and preterm low birth weight (PLBW); however, this association remains unclear. Selenium (Se) may modify this relationship by protecting against Ni toxicity. Concentrations of Ni and Se were measured in urine samples collected from 408 pregnant women (102 PLBW cases and 306 matched controls) in China. Conditional logistic regression was utilized to explore the association between Ni levels and PLBW, as well as the effect modification by Se on this association. A significant association was observed between higher maternal urinary Ni levels and risk of PLBW [adjusted odds ratio (OR) = 2.80 (95% confidence interval (CI): 1.44, 5.44) for the highest tertile], and this association was more apparent among female infants than that among male infants. Further analyses showed that mothers with high urinary Ni and low urinary Se levels had a significantly increased risk for PLBW [adjusted OR = 2.87 (95% CI: 1.09, 7.56)] compared with the mothers with low urinary Ni and high urinary Se levels. Our study indicates that prenatal exposure to Ni was a risk factor for PLBW. Se might provide protection against the toxicity of Ni.

Predictors of thallium exposure and its relation with preterm birth.

Thallium (Tl) is a well-recognized hazardous toxic heavy metal that has been reported to have embryotoxicity and fetotoxicity. However, little is known about its association with preterm birth (PTB) in humans. We aimed to evaluate the predictors of Tl exposure and assessed its relation with PTB. The study population included 7173 mother-infant pairs from a birth cohort in Wuhan, China. Predictors of Tl concentrations were explored using linear regression analyses, and associations of Tl exposure with risk of PTB or gestational age at birth were estimated using logistic regression or generalized linear models. The geometric mean and median values of urinary Tl concentrations were 0.28 µg/L (0.55 µg/g creatinine) and 0.29 µg/L (0.53 µg/g creatinine). We found that maternal urinary Tl concentrations varied by gestational weight gain, educational attainment, multivitamin and iron supplementations. Women with Tl concentrations higher than 0.80 µg/g creatinine were at higher risk of giving birth prematurely versus those with Tl concentrations lower than 0.36 µg/g creatinine [adjusted odds ratio (95% confidence interval (CI)): 1.55 (1.05, 2.27)], and the association was more pronounced in PTB with premature rupture of membranes (PROM) rather than in PTB without PROM. About 3-fold increase in creatinine-corrected Tl concentrations were associated with 0.99-day decrease in gestational length (95% CI: -1.36, -0.63). This is the first report on the associations between maternal Tl exposure and the risk of PTB.

Enrichment and Cytotoxic Activity of Curcuminoids from Turmeric Using Macroporous Resins.

Curcuminoids are functional secondary metabolites abundant in turmeric. In the present study, a simple and efficient method for enrichment of curcuminoids from turmeric was developed using macroporous resin. Eight different types of macroporous resins were examined by static adsorption/desorption properties and the type of XDA-7 was selected as the optimum one. Under the optimized conditions, the final contents of refined extract excluded 84.2% of impurities, by comparison with crude extract in a scale-up experiment. Meanwhile, 8 representative curcuminoids including 4 dihydro- and tetrahydro-curcuminoids were enriched, isolated, and identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) data. In addition, the individual curcuminoids were prepared to evaluate their cytotoxic activity toward HeLa tumor cell lines. All compounds, especially the trace amount of curcuminoids, demonstrated notable cytotoxic activity. The results supported that those trace amount of curcuminoids can be good candidates for drug development as anticancer agents. The purification process was simple and efficient, which could afford a potential method to enrich and concentrate not only the major curcuminoids, but also the trace amount of ones from turmeric raw materials for industrial applications.

The Hypolipidemic Effect of Total Saponins from Kuding Tea in High-Fat Diet-Induced Hyperlipidemic Mice and Its Composition Characterized by UPLC-QTOF-MS/MS.

Kuding tea are used as a traditional tea material and widely consumed in China. In this study, total saponins (TS) from water extract of Kuding tea was prepared by D101 macroporous resins and analyzed by UPLC-QTOF-MS/MS. Then the hypolipidemic effect of TS extract was investigated in high-fat diet-induced hyperlipidemic mice. For comprehensive identification or characterization of saponins in TS extract, 3 major saponins of Kudinoside A, Kudinoside F, and Kudinoside D were isolated and used as standards to investigate the MS/MS fragmentation pattern. As a result, 52 saponins were identified or characterized in TS extract from Kuding tea. In addition, the increased levels of mice serum TC, LDL-C, HDL-C, and atherogenic index (AI) were significantly reduced after the treatment of TS extract. Also, the liver protective effect of TS extract was obviously judged from the photographs stained with oil red-O staining. Meanwhile, TS extract significantly upregulated the expression of hepatic scavenger receptors including SR-AI, SR-BI, and CD36. Therefore, it is reasonable to assume that the overexpression of hepatic scavenger receptors was involved in the hypolipidemic effect of Kuding tea on the high-fat diet-induced hyperlipidemic mice. The TS extract could influence these scavenger receptors, and this could be the potential mechanism of TS extract from Kuding tea in the treatment of lipid disorders. These results give the evidence that the saponins in Kuding tea could provide benefits in managing hypercholesterolemia and may be a good candidate for development as a functional food and nutraceutical.

The metabolic change of serum lysophosphatidylcholines involved in the lipid lowering effect of triterpenes from Alismatis rhizoma on high-fat diet induced hyperlipidemia mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Alismatis rhizoma (AR), a Traditional Chinese Medicine with lipid-regulating properties, is usually used to treat hyperlipidemia. Lysophosphatidylcholines (Lyso PCs) play a crucial role in lipid metabolism disorders. In this study, the triterpene fraction purified from boiling water extract of AR was evaluated for its lipid lowering activity using mice with high-fat diet (HFD) induced hyperlipidemia. The metabolic changes of individual Lyso PCs treated with the triterpene fraction were detected by ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometer (UHPLC-QTRAP-MS/MS). MATERIALS AND METHODS: HFD induced hyperlipidemia mice were administrated with triterpene and non-triterpene fractions at doses of 180, 360 and 720 mg/kg body weight/day for 4 weeks, respectively. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and atherogenic Index (AI) in mice serum were measured. The chemical components in the lipid-lowering fraction were characterized by ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). The changes of Lyso PC in the serum of mice treated with the lipid-lowering fraction were quantified by UHPLC-QTRAP-MS/MS. RESULTS: A total of 18 alisol derivatives were identified in the triterpene fraction. The hyperlipidemia mice treated with the triterpene fraction showed a significant decrease in serum TC, LDL-C and AI after continuous consumption of HFD for 4 weeks. The results also showed that 27 serum Lyso PCs in mice fed with HFD were down-regulated, and 19 were up-regulated. The abnormal serum level of Lyso PCs associated with hyperlipidemia was intervened by the alisol derivatives, with increase of unsaturated Lyso PCs and decrease of saturated ones. CONCLUSIONS: The study demonstrated for the first time that triterpenes from the AR extract can lower serum lipid level in HFD induced hyperlipidemia mice. These metabolism changes of Lyso PCs could further improve our understanding of the potential mechanism of lipid lowering effect of AR.