Protective effect of hot-water and ethanol-aqueous extracts from Anneslea fragrans against acetaminophen-induced acute liver injury in mice.

Anneslea fragrans Wall. (AF) is an important medicinal and edible plant in China. The principal objectives of this study are to explore the hepatoprotective effect of ethanol-aqueous (AFE) and hot-water (AFW) extracts in vitro and in vivo. UPLC-ESI-MS/MS analysis showed that AFW and AFE are rich in dihydrochalcones. Both AFW and AFE significantly up-regulated the expressions of SOD, CAT and GSH, reduced the MDA content in acetaminophen (APAP)-induced HepG2 cells, and suppressed the expressions of NO, TNF-α, IL-1ß, and IL-6 in LPS-induced RAW246.7 cells. In APAP-induced mice, AFW and AFE administration significantly decreased the plasma levels of AST and ALT, and improved liver tissue damage, the collagen deposition and fibrosis formation. Moreover, AFW and AFE decreased the MDA and ROS accumulations via activating Nrf2 pathway to increase the hepatic GSH contents and activities of SOD, CAT, HO-1, and NQO-1, reduced the levels of NO, TNF-α, IL-1ß, and IL-6 by suppressing the JNK/p38/ERK/NF-κB pathways, and alleviated apoptosis via regulating Bcl-2, Bax, caspase-3/9 protein expressions. This study provides a new sight that AFW and AFE may have a potential natural resource for the treatment of liver injury.

Influence of Ultra-High-Pressure Pretreatment Method on Chemical Constituents, Antioxidant and Cytoprotective Activities of Free, Esterified, and Bound Phenolics from Anneslea Fragrans Wall. Leaves.

Anneslea fragrans Wall., an edible and medicinal plant, is traditionally used to treat liver and gastrointestinal diseases. This paper aimed to investigate the influence of ultra-high pressure (UHP) pretreatment on the phenolics profiling, antioxidant, and cytoprotective activities of free (FP), esterified (EP), and bound (BP) phenolics from A. fragrans leaves. A total of 32 compounds were characterized and quantified. The davidigenin (44.46 and 113.37 mg/g extract) was the highest in A. fragrans leaves. The vitexin (9), afzelin (10), coreopsin (15), and davidigenin (28) were analyzed with MS2 fragment pathways. Results showed that UHP treated A. fragrans leaves had higher total phenolic (TPC) and total flavonoid (TFC) contents of FP, EP, and BP fractions than those in the raw leaves. Moreover, UHP pretreated A. fragrans leaves had higher scavenging activities on DPPH+• and ABTS+•, and inhibitory effects on the intracellular ROS generation in H2O2-induced HepG2 cells. UFP showed the highest inhibition of ROS production among the samples. Therefore, UHP pretreatment method might be used as an effective strategy for elevating the availabilities of A. fragrans leaves to develop functional foods.

Effect of ultra-high pressure pretreatment on the phenolic profiles, antioxidative activity and cytoprotective capacity of different phenolic fractions from Que Zui tea.

This study aims to explore whether ultra-high pressure (UHP) pre-treatment strengthened the bioaccessibility and bioactivities of the free (QF), esterified (QE) and insoluble-bound phenolics (QIB) from Que Zui tea (QT). The results revealed that the extraction yields, the total phenolic (TPC) and total flavonoid contents (TFC) of three phenolic fractions from QT were markedly increased after ultra-high pressure (UHP) processing (p < 0.05). A total of 19 and 20 compounds were characterized and quantified in non- and UHP-treated QT, respectively, including the content of 6'-O-caffeoylarbutin (11775.68 and 13248.87 µg/g of dry extract) was highest in QF, the content of caffeic acid was highest in QE (2131.58 and 7362.99 µg/g of dry extract) and QIB (9151.89 and 10930.82 µg/g of dry extract). QF, QE and QIB from QT after UHP processing had better antioxidant, ROS scavenging, and anti-apoptosis effects. The possible mechanism of cytoprotective effect was related to Keap1-Nrf2 pathway.

The effect of ultra-high pretreatment on free, esterified and insoluble-bound phenolics from mango leaves and their antioxidant and cytoprotective activities.

Ultra-high pressure (UHP) is a novel non-thermal pretreatment method in food processing for improving the extraction yield of polyphenols and functional properties. The present work investigated the phenolic profiles, antioxidant activities, and cytoprotective effects of the free, esterified, and insoluble-bound phenolic fractions from mango leaves before and after ultra-high pressure (UHP) treatment. UHPLC-Q-Orbitrap-MS/MS analysis resulted in the identification of 42 phenolic compounds in the different phenolic forms. UHP pretreatment could significantly influence the contents of total phenols, total flavonoids and individual compounds in the different phenolic fractions (p < 0.05). After UHP pretreatment, these phenolic fractions exhibited greater antioxidant activity, and inhibited reactive oxygen species production and cell apoptosis (p < 0.05). Meanwhile, IBP were the most potential antioxidative and cytoprotective ingredients. Therefore, UHP pretreated mango leaves with enhanced bioactivity could be used as biological agents in the health food industry to improve its application and economic values.

Chemical constituents and anti-inflammatory activity of the total alkaloid extract from Melodinus cochinchinensis (Lour.) Merr. and its inhibition of the NF-κB and MAPK signaling pathways.

BACKGROUND: Melodinus cochinchinensis (Lour.) Merr. is a medicinal plant, which is used as a folk medicine for treating meningitis and fractures. However, the anti-inflammatory activity of total alkaloid extract from M. cochinchinensis (MCTA) and its molecular mechanism are still not studied. PURPOSE: The aim of this study is to investigate the main chemical constituents of MCTA and explore its anti-inflammatory potential in both in vitro and in vivo assessments. METHODS: UHPLC-ESI-HRMS/MS was applied to analyze the chemical profiling. The anti-inflammatory efficacy of MCTA was evaluated on lipopolysaccharide (LPS) induced RAW 264.7 cells and two common inflammation models in mice. The production of pro-inflammatory mediator and cytokine was tested using the ELISA method. The pathological change was analyzed by histological assessment. The expression of NF-κB, MAPKs and PPAR-γ proteins was evaluated using western blot analysis. RESULTS: A total of 21 monoterpenoid indole alkaloids (MIAs) were characterized by UHPLC-ESI-HRMS/MS. Aspidospermine- and quinolone-type alkaloids were found to be the major compounds. MCTA significantly decreased the production of NO, IL-1ß, IL-6 and TNF-α in LPS-induced RAW 264.7 macrophages. MCTA significantly inhibited the phosphorylation of ERK1/2, JNK and p38 MAPK, suppressed the NF-κB transcriptional activation and improved the PPAR-γ expression. Moreover, the in vivo experiment exhibited that MCTA pretreatment markedly alleviated the xylene-induced ear edema and carrageenan-induced paw edema in mice and decreased the IL-1ß, IL-6 and TNF-α expressions. CONCLUSION: MCTA is rich in MIAs and exhibited a significant inhibitory effect on the production proinflammatory cytokines. The mechanism might be related to the inhibition of activation of NF-κB and MAPK pathways.

Safety evaluation and hypolipidemic effect of aqueous-ethanol and hot-water extracts from E Se tea in rats.

E Se tea, processed by the fresh leaves of Malus toringoides (Rehd.) Hughes, is a traditional herbal tea with various human benefits. The present study was aimed to evaluate the toxicity and hypolipidemic effect of aqueous-ethanol extract (EE) and hot-water extract (WE) from E Se tea. Eight main chemical constituents in EE and WE were respectively identified and quantified by UHPLC-HRMS/MS. EE is rich in TPC and TFC, while WE had higher TPS content. Both EE and WE exhibited strong antioxidant activity with no significant difference. The acute toxicity study revealed that the LD50 values were higher than 5000 mg/kg, while both WE and EE had no significant adverse effect in rats by subacute toxicity assay. However, the triglyceride (TG) content in experiment groups (male) and highest doses groups (female) significantly decreased. Furthermore, the hypolipidemic effect of WE and EE were performed on high fat diet induced hyperlipidemic rats. The result exhibited that either WE or EE could effectively regulate lipid droplet accumulation in liver, and reduce the adipocyte size. These results demonstrated that these two extracts from E Se tea could be regarded as a potential functional dietary supplement in preventing and treating diet induced metabolic diseases.

Liquid-liquid interfacial self-assembled triangular Ag nanoplate-based high-density and ordered SERS-active arrays for the sensitive detection of dibutyl phthalate (DBP) in edible oils.

DBP, one of the phthalic acid esters (PAEs), is known as an endocrine disruptor and is toxic to humans in abnormal concentrations. Here, a high-density and ordered SERS substrate based on the self-assembly of triangular Ag nanoplate (TAgNP) arrays is developed for DBP detection. Benefiting from the ordered arrangement and sharp corners of TAgNPS, the arrays can provide sufficient and uniform hotspots for reproducible and highly active SERS effects. Using Rhodamine 6G (R6G) as a reporter molecule, the SERS enhancement factor (EF) of the TAgNP arrays was found to be as high as 1.2 × 107 and the relative standard deviation was 6.56%. As a trial for practical applications, the TAgNP array substrates were used for the detection of dibutyl phthalate (DBP) in edible oils. In this assay, edible oil samples were added to hexane as an organic phase for the formation of the TAgNP arrays, which caused DBP to be loaded at hotspots. DBP in edible oils could be identified at concentrations as low as 10-7 M. This SERS substrate based on the TAgNP arrays has great potential applications in the high sensitivity and reproducible detection of contaminants in food.

Bioactivity-Guided Isolation of Phytochemicals from Vaccinium dunalianum Wight and Their Antioxidant and Enzyme Inhibitory Activities.

Vaccinium dunalianum Wight, usually processed as a traditional folk tea beverage, is widely distributed in the southwest of China. The present study aimed to investigate the antioxidant, α-glucosidase and pancreatic lipase inhibitory activities of V.dunalianum extract and isolate the bioactive components. In this study, the crude extract (CE) from the buds of V. dunalianum was prepared by the ultrasound-assisted extraction method in 70% methanol and then purified with macroporous resin D101 to obtain the purified extract (PM). Five fractions (Fr. A-E) were further obtained by MPLC column (RP-C18). Bioactivity assays revealed that Fr. B with 40% methanol and Fr. D with 80% methanol had better antioxidant with 0.48 ± 0.03 and 0.62 ± 0.01 nM Trolox equivalent (TE)/mg extract for DPPH, 0.87 ± 0.02 and 1.58 ± 0.02 nM TE/mg extract for FRAP, 14.42 ± 0.41 and 19.25 ± 0.23 nM TE/mg extract for ABTS, and enzyme inhibitory effects with IC50 values of 95.21 ± 2.21 and 74.55 ± 3.85 for α-glucosidase, and 142.53 ± 11.45 and 128.76 ± 13.85 µg/mL for pancreatic lipase. Multivariate analysis indicated that the TPC and TFC were positively related to the antioxidant activities. Further phytochemical purification led to the isolation of ten compounds (1-10). 6-O-Caffeoylarbutin (7) showed significant inhibitory effects on α-glucosidase and pancreatic lipase enzymes with values of 38.38 ± 1.84 and 97.56 ± 7.53 µg/mL, and had the highest antioxidant capacity compared to the other compounds.

Acute and Sub-Acute Toxicological Evaluations of Bioactive Alkaloidal Extract from Melodinus henryi and Their Main Chemical Constituents.

Melodinus henryi is a good source of terpenoid indole alkaloids, and traditionally used as a folk medicine in the treatment of meningitis and fracture. In order to further exploit their potential uses, its anti-inflammatory and immunosuppressive activities, safety evaluations and chemical profiles have been illustrated. Compared to the crude methanol extract from M. henryi and its non-alkaloidal fraction, the total alkaloidal fraction (MHTA) had the strongest anti-inflammatory and immunosuppressive activities. In the acute oral toxicity assay, the half lethal dose (LD50) of MHTA was more than 2000 mg/kg. The sub-acute toxicity assay for consecutive 28 days exhibited MHTA at a lower concentrations of less than 500 mg/kg might be regarded as safe, and might damage spleen, liver, kidney, and heart when the dose is higher than 1000 mg/kg. In addition, a phytochemical investigation on MHTA led to the isolation of 15 monoterpenoid indole alkaloids. Thus, in regard with the potent side effects of MHTA, it should be used with caution in the development of phytomedicine.

In vitro and in vivo anti-inflammatory effects of different extracts from Epigynum auritum through down-regulation of NF-κB and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Epigynum auritum has been historically used as a "dai" or traditional medicine for the treatment of inflammation, swelling and severe pain during injury; these may reduce risk of disease and lead to healthier aging. Apart from this, Epigynum auritum extract was also used in arhritis treatment which is also a type of inflammation. Previous phytochemical studies of E. auritum revealed that steroids are main characteristic components with a number of biological activities (especially immunosuppressive and anti-inflammatory activity) Nevertheless, the underlying mechanism of the E. auritum on inflammatory diseases is still unresolved. AIM OF THE STUDY: This study aimed to comparatively investigate the anti-inflammatory potential of different fractions from the extract of E. auritum (EAE), with their possible active ingredients to reveal the underlying mechanism. MATERIALS AND METHODS: The EAE was fractionated by column chromatography with macroporous resin D101 which yielded six fractions. The potential anti-inflammatory properties of different fractions of EAE were evaluated in in vitro and in vivo model. The lipopolysaccharide (LPS)-induced RAW264.7 macrophages cells were used for in vitro studies however two typical acute inflammation murine models (xylene-induced ear edema and carrageenan-induced paw edema) were used for anti-inflammatory studies. The important molecular mechanisms related to inflammation were also analyzed by ELISA, western blotting and immunofluorescence. UHPLC-MS/MS was used to analyze the chemical composition of 100% EAE fraction. RESULTS: Different EAE fractions (especially the Fr. 100% of MeOH:H2O) significantly reduced the productions of NO, ROS, TNF-α, and IL-6 by LPS-induced RAW264.7 macrophages and increased the expression of IL-10. The expression levels of iNOS and COX-2 enzymes were significantly down-regulated by 100% EAE fraction. Furthermore, 100% EAE fraction inhibited the phosphorylation of the ERK1/2, JNK, and p38 MAPK, and reduced the nuclear translocation of NF-κB which prevents its activation by blocking the phosphorylation and degradation of inhibitor protein of IκBα. In addition two inflammatory animal models; xylene-induced ear edema and carrageenan-stimulated paw edema were also developed with significantly ameliorated inflammatory cytokines. The treatment of these inflammatory models with 100% EAE fraction (Fr. 100%) suppressed the expressions of elevated inflammatory cytokines. Besides the UHPLC-HRMS/MS analysis was also carried out in which the androstane analogues were found to be as a main chemical components. CONCLUSION: Different fractions (especially Fr. 100%) exert inhibitory effect on inflammation by regulating the release of inflammatory mediators through the NF-κB and MAPK signaling pathways. The androstane and its derivatives might be performing an important role in the observed anti-inflammatory activity. Therefore, Fr. 100% of EAE could be applied as a potential drug candidate for the prevention and treatment of inflammatory diseases.

A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay.

Simultaneous detection of different types of cancer biomarkers (nucleic acids and proteins) could facilitate early diagnosis of cancer and clinical treatment. Herein, a simultaneous detection platform of proteins and nucleic acids has been developed using a single substrate probe combining a label-free and background-eliminated fluorescence assay. Telomerase and telomerase RNA (TR) were chosen as the models. The molecular beacon (dU-BIO-HP) that contains deoxyuridine/biotin in its side arm, a TR recognition sequence in the loop and a telomerase substrate primer at the stem end was ingeniously designed. In the presence of telomerase, the stem of dU-BIO-HP is elongated by the addition of telomere repeats complementary to the assistant DNA. Furthermore, the formed dsDNA performed as engaging primers to initiate a SDA reaction, generating abundant G-quadruplex monomers. Similarly, on TR, the hybridization between TR and dU-BIO-HP can open its stem, triggering another SDA reaction, producing abundant short ssDNAs. With the G-quadruplex binding with ZnPPIX and ssDNA binding with SG for specific fluorescence responses, the label-free multiple detection can be achieved. In our strategy, the deoxyuridine of dU-BIO-HP acts as a barrier to block the DNA extension due to its strong inhibitory effects on DNA polymerase activity and to make sure that the two SDA reactions occurred independently. The biotin of dU-BIO-HP enables the reduction of the background from the binding between SG, ZnPPIX and dU-BIO-HP through streptavidin-biotin interaction. This method showed an excellent sensitivity with telomerase and TR detection limit of 2.18 HeLa cells per mL and 0.16 × 10-12 M, respectively. Furthermore, the telomerase and TR in different cell lines have been evaluated as powerful tools for biomedical research and clinical diagnosis.

Magnetic bead-liposome hybrids enable sensitive and portable detection of DNA methyltransferase activity using personal glucose meter.

DNA methyltransferase (MTase) plays a critical role in maintaining genome methylation patterns, which has a close relationship to cancer and bacterial diseases. This encouraged the need to develop highly sensitive, simple, and robust assays for DNA MTase detection and inhibitor screening. Herein, a simple, sensitive, and specific DNA MTase activity assay was developed based on magnetic beads-liposome hybrids combined with personal glucose meter (PGM) for quantitative detection of DNA MTase and inhibitor screening. First, a magnetic beads-liposome hybrid probe is designed by the hybridization of p1DNA-functionalized magnetic bead with p2DNA-functionalized glucoamylase-encapsulated liposome (GEL). It integrates target recognition, magnetic separation and signal amplification within one multifunctional design. Then, in the presence of Dam MTase, the hybrids probe was methylated, and cleaved by methylation-sensitive restriction endonuclease Dpn I, making liposome separated from magnetic bead by magnetic separation. Finally, the separated liposome was decomposed, liberating the encapsulated glucoamylase to catalyze the hydrolysis of the signal substrate amylose with multiple turnovers, producing a large amount of glucose for quantitative readout by the PGM. In the proposed assay, the magnetic beads-liposome hybrids offered excellent sensitivity due to primary amplification via releasing numerous glucoamylase from a liposome followed by a secondary enzymatic amplification. The use of portable quantitative device PGM bypasses the requirement of complicated instruments and sophisticated operations, making the method simple and feasible for on-site detection. Moreover, the proposed assay was successfully applied in complex biological matrix and screen suitable inhibitor drugs for DAM for disease(s) treatment. The results reveal that the approach provides a simple, sensitive, and robust platform for DNA MTases detection and screening potential drugs in medical research and early clinical diagnostics.

Target-controlled gating liposome "off-on" cascade amplification for sensitive and accurate detection of phospholipase D in breast cancer cells with a low-background signal.

Here we developed a simple, sensitive and accurate PLD detection method based on a target-controlled gating liposome (TCGL) "off-on" cascade amplified strategy and personal glucose meters (PGMs). It showed excellent sensitivity with a detection limit of 0.005 U L(-1) and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.