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Although serving as the workhorse, MS/MS cannot fully satisfy the analytical requirements of quantitative sub-metabolome characterization. Because more information intrinsically correlates to more structural and concentration clues, here, efforts were devoted to comprehensively tracing and deciphering MS/MS behaviors through constructing triple three-dimensional (3×3D)-MS/MS spectrum. Ginsenosides-targeted metabolomics of notoginseng, one of the most famous edible medicinal plants, was employed as a proof-of-concept. Serial authentic ginsenosides were deployed to build the correlations between 3×3D-MS/MS spectra and structure/concentration features. Through assaying ginsenosides with progressive concentrations using QTOF-MS to configure 1st 3D spectrum, the generations of MS1 spectral signals, particularly multi-charged multimer anions, e.g., [2M–2H]2– and [2M+2HCOO]2– ions, relied on both concentration and the amount of sugar chains. By programming progressive collision energies to the front collision cell of Qtrap-MS device to gain 2nd 3D spectrum, optimal collision energy (OCE) corresponding to the glycosidic bond fission was primarily correlated with the masses of precursor and fragment ions and partially governed by the glycosidation site. The quantitative relationships between OCEs and masses of precursor and fragment ions were utilized to build large-scale quantitative program for ginsenosides. After applying progressive exciting energies to the back collision chamber to build 3rd 3D spectrum, the fragment ion and the decomposition product anion exhibited identical dissociation trajectories when they shared the same molecular geometry. After ginsenosides-focused quantitative metabolomics, significant differences occurred for sub-metabolome amongst different parts of notoginseng. The differential ginsenosides were confirmatively identified by applying the correlations between 3×3D-MS/MS spectra and structures. Together, 3×3D-MS/MS spectrum covers all MS/MS behaviors and dramatically facilitates sub-metabolome characterization from both quantitative program development and structural identification.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA)-caused infections greatly threaten public health. The discovery of natural-product-based anti-MRSA agents for treating infectious diseases has become one of the current research focuses. Objectives: This study aims to identify promising anti-MRSA agents with a clear mechanism based on natural norharmane modified by quaternization or dimerization. Methods: A total of 32 norharmane analogues were prepared and characterized. Their antibacterial activities and resistance development propensity were tested by the broth double-dilution method. Cell counting kit-8 and hemolysis experiments were used to assess their biosafety. The plasma stability, bactericidal mode, and biofilm disruption effects were examined by colony counting and crystal violet staining assays. Fluorescence microscopy, metabolomic analysis, docking simulation and spectra titration revealed its anti-MRSA mechanisms. The mouse skin infection model was used to investigate the in vivo efficacy. Results: Compound 5a was selected as a potential anti-MRSA agent, which exhibited potent anti-MRSA activity in vitro and in vivo, low cytotoxicity and hemolysis under an effective dose. Moreover, compound 5a showed good stability in 50% plasma, a low tendency of resistance development and capabilities to disrupt bacterial biofilms. The mechanism studies revealed that compound 5a could inhibit the biosynthesis of bacteria cell walls, damage the membrane, disturb energy metabolism and amino acid metabolism pathways, and interfere with protein synthesis and nucleic acid function. Conclusions: These results suggested that compound 5a is a promising candidate for combating MRSA infections, providing valuable information for further exploiting a new generation of therapeutic antibiotics.

Abstract The activation of cyclic GMP‐AMP (cGAMP) synthase (cGAS) and its adaptor, stimulator of interferon genes (STING), is known to reprogram the immunosuppressive tumor microenvironment for promoting antitumor immunity. To enhance the efficiency of cGAS‐STING pathway activation, macrophage‐selective uptake, and programmable cytosolic release are crucial for the delivery of STING agonists. However, existing polymer‐ or lipid‐based delivery systems encounter difficulty in integrating multiple functions meanwhile maintaining precise control and simple procedures. Herein, inspired by cGAS being a natural DNA sensor, a modularized DNA nanodevice agonist (DNDA) is designed that enable macrophage‐selective uptake and programmable activation of the cGAS‐STING pathway through precise self‐assembly. The resulting DNA nanodevice acts as both a nanocarrier and agonist. Upon local administration, it demonstrates the ability of macrophage‐selective uptake, endosomal escape, and cytosolic release of the cGAS‐recognizing DNA segment, leading to robust activation of the cGAS‐STING pathway and enhanced antitumor efficacy. Moreover, DNDA elicits a synergistic therapeutic effect when combined with immune checkpoint blockade. The study broadens the application of DNA nanotechnology as an immune stimulator for cGAS‐STING activation.

Abstract Plants usually produce defence metabolites in non-active forms to minimize the risk of harm to themselves and spatiotemporally activate these defence metabolites upon pathogen attack. This so-called two-component system plays a decisive role in the chemical defence of various plants. Here, we discovered that Panax notoginseng, a valuable medicinal plant, has evolved a two-component chemical defence system composed of a chloroplast-localized β-glucosidase, denominated PnGH1, and its substrates 20(S)-protopanaxadiol ginsenosides. The β-glucosidase and its substrates are spatially separated in cells under physiological conditions, and ginsenoside hydrolysis is therefore activated only upon chloroplast disruption, which is caused by the induced exoenzymes of pathogenic fungi upon exposure to plant leaves. This activation of PnGH1-mediated hydrolysis results in the production of a series of less-polar ginsenosides by selective hydrolysis of an outer glucose at the C-3 site, with a broader spectrum and more potent antifungal activity in vitro and in vivo than the precursor molecules. Furthermore, such β-glucosidase-mediated hydrolysis upon fungal infection was also found in the congeneric species P. quinquefolium and P. ginseng. Our findings reveal a two-component chemical defence system in Panax species and offer insights for developing botanical pesticides for disease management in Panax species.

Abstract Ulcerative colitis (UC) is a digestive disease with a high incidence and is difficult to be cured due to its complex etiology. It has evidenced that intestinal barrier dysfunction plays a predominant role in UC. 20(S)‐protopanaxadiol saponins (PDS) isolated from Panax notoginseng possess anti‐inflammatory and antioxidative activities, suggesting its potential of treating UC. Herein, the therapeutic effects of PDS against UC and underlying mechanisms in the aspect of intestinal barrier dysfunction were investigated in vivo and in vitro. The results showed PDS had protective effects against dextran sulfate sodium–induced colitis, including attenuating weight loss, disease activity index score elevation, colon length shortening, and histological lesions. Additionally, PDS reduced the colonic activity of myeloperoxidase and the cytokine levels of TNF‐α, IL‐6, and IL‐1β, decreased MDA production, and elevated colonic activities of SOD and GSH‐Px in the colitis mice. The expressions of proteins related to tight junction (TJ), including ZO‐1, claudin‐5, occludin, caveolin‐1 (Cav‐1), and Nrf2 were downregulated, whereas that of Keap1 was upregulated after colitis induction. These changes were reversed by PDS. Cav‐1 expression was downregulated in lipopolysaccharides (LPS)‐ and H2O2‐induced HCT116 cells, and the expressions of ZO‐1, claudin‐5, and occludin were suppressed in HCT116 cells stimulated by LPS and H2O2 combined with Cav‐1 small interfering RNA transfection, which were ameliorated by PDS, suggesting PDS targeted Cav‐1 against intestinal barrier damage. Collectively, PDS alleviates inflammatory injury and oxidative stress by regulating the Nrf2/Keap1 pathway, contributing to targeting Cav‐1 against intestinal epithelial TJ proteins loss. It suggests PDS might be a promising therapeutic natural product for UC treatment.

Fatty acids (FAs), which were initially recognized as energy sources and essential building blocks of biomembranes, serve as the precursors of important signaling molecules. Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events. Inspired by the advances in click chemistry for protein enrichment, we herein established a click chemistry-based enrichment (CCBE) strategy for tracing the cellular metabolism of eicosapentaenoic acid (EPA, 20:5 n-3) in neural cells. Terminal alkyne-labeled EPA (EPAA) used as a surrogate was incubated with N2a, mouse neuroblastoma cells, and alkyne-labeled metabolites (ALMs) were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction for enrichment. After removing unlabeled metabolites, ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatography mass spectrometry (LC-MS) analysis. The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from the complicated matrices. In addition, this method can overcome current detection limits by enhancing MS sensitivity of targets, improving the chromatographic separation of sn-position glycerophospholipid regioisomers, facilitating structural characterization of ALMs by a specific MS/MS fragmentation signature, and providing versatile fluorescence detection of ALMs for cellular distribution. This CCBE strategy might be expanded to trace the metabolism of other FAs, small molecules, or drugs.

Abstract Background Red yeast rice (RYR), a nutraceutical with a profound cholesterol-lowering effect, was found to attenuate non-alcoholic fatty liver disease (NAFLD) in mice. Despite monacolin K in RYR being a specific inhibitor of hydroxymethylglutaryl-coenzyme A reductase (HMCGR), the mechanisms underlying the protective effects of RYR against NAFLD are not fully elucidated. Methods Using a mouse model of high-fat diet (HFD) feeding and a cellular model of HepG2 cells challenged by lipopolysaccharide (LPS) and palmitic acid (PA), the possible molecular mechanisms were exploited in the aspects of NF-κB/NLRP3 inflammasome and mTORC1-SREBPs signaling pathways by examining the relevant gene/protein expressions. Subsequently, the correlation between these two signals was also verified using cellular experiments. Results RYR ameliorated lipid accumulation and hepatic inflammation in vivo and in vitro. RYR improved lipid metabolism through modulating mTORC1-SREBPs and their target genes related to triglyceride and cholesterol synthesis. Furthermore, RYR suppressed hepatic inflammation by inhibiting the NF-κB/NLRP3 inflammasome signaling. Interestingly, the treatment with RYR or MCC950, a specific NLRP3 inhibitor, resulted in the reduced lipid accumulation in HepG2 cells challenged by LPS plus PA, suggesting that the inhibitory effects of RYR on NLRP3 inflammasome-mediated hepatic inflammation may partially, in turn, contribute to the lipid-lowering effect of RYR. Conclusions The modulation of NF-κB/NLRP3 inflammasome and lipid synthesis may contribute to the ameliorative effects of RYR against HFD-induced NAFLD.

Luminescence bioimaging and biosensing in the second near‐infrared (NIR‐II) spectral window (1,000–1,700 nm wavelengths) is a newly emerging technique that is extensively used in fundamental research and clinical practice. Owing to its advantages, including deep tissue penetration and excellent temporal resolution, this technique has shown tremendous promise for target‐specific imaging and noninvasive monitoring. However, developing bioimaging modalities that enable target‐specific imaging with high resolution and sensitivity remains a great challenge today. Compared with NIR‐II fluorescence imaging, NIR‐II luminescence lifetime imaging with advantages such as lower background noises, higher sensitivity, and higher spatial resolution has opened up new avenues for deep tissue imaging and biomedical applications. Herein, various types of long‐lived NIR‐II luminescent probes, including complex dyes, rare‐earth‐doped nanoparticles, organic compound‐loaded nanoparticles, inorganic‐based dots and nanotubes, as well as the main types of time‐resolved luminescence techniques including time‐domain (e.g., time‐correlated single‐photon counting, time‐gated detection, pulse sampling, etc.) and frequency‐domain systems are first summarized. The unique advantages of long‐lived NIR‐II luminescent probes and their critical roles in bioimaging and biosensing are also highlighted. Finally, the present challenges and future directions of long‐lived NIR‐II luminescent probes in biomedical research, translational research, and (pre‐)clinical studies are discussed.

Abstract Background The Chinese medicinal wine made from herbal medicines became prevalent among Chinese people. The Chinese herbal spirit is composed of several herbal extracts, and has the certain health functions, such as anti-fatigue and immune regulation. The quality evaluation of Chinese herbal spirit is greatly challenged by the enormous and complex components with great structural diversity and wide range of concentration distribution. Methods An ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) with multiple reaction monitoring (MRM) method was developed to simultaneously determine forty-three bioactive components in the Chinese herbal spirits produced by year 2014 and 2018. Results Quantitative results showed that 11 components, i.e.., puerarin (5), purpureaside C (7), daidzin (8), echinacoside (9), acteoside (15), epimedin B (22), epimedin C (23), icariin (24), eugenol (27), chikusetsusaponin iva (30) and Z-ligustilide (40), significantly decreased along with the increasing years of storage, while 5 compounds, i.e.., geniposidic acid (1), protocatechuic acid (2), crustecdysone (14), daidzein (18) and icariside I (35), were basically stable in all samples across the years. Concusion The established method allowing to simultaneously determined 43 components with wide structural diversity and trace amounts will facilitate the quality control research of Chinese herbal spirits.

Abstract Background The dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a blood-activating and stasis-removing traditional Chinese medicine for 1000 years. Our previous studies have shown the inhibitory activity on platelet and thrombin (THR) of Chuanxiong. THR and factor Xa (FXa) play significant roles in the coagulation cascade and their inhibitors are of valuable in the treatment of thromboembolic diseases. The aim of the present study is to screen THR and FXa inhibitors from Chuanxiong. Methods Four extracts [ethyl acetate (EA), butanol (BA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Chuanxiong were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Following silica-gel column chromatography (SC), the active EA extract and BA extract was further partitioned, respectively. Their active fractions (EA-SC1 to EA-SC5; BA-SC1 to BA-SC5) were obtained and analyzed by LC–MS. After modeling by the principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA), the specific marker compounds were predicted and identified. Their enzyme inhibitory was assessed in vitro and interactions with THR/FXa were investigated by molecular docking analysis. Results Chuanxiong EA extract showed strong activity against THR and BA extract was more effective in inhibiting FXa activity, and their fractions exhibited obvious difference in enzyme inhibitory activity. Furthermore, marker compounds a–h were predicted by PCA and OPLS-DA, and their chemical structures were identified. Among them, senkyunolide A, Z-ligustilide, ferulic acid and senkyunolide I (IC50 was determined as 0.77 mM) with potential THR inhibitory activity, as well as isochlorogenic acid A with FXa inhibitory activity were screened out. It was found that the four components could interact with the active site of THR, and the binding energy was lower than − 5 kcal/mol. Isochlorogenic acid A were bound to the active site of FXa, and the binding energy was − 9.39 kcal/mol. The IC50 was determined as 0.56 mM. Conclusions THR/FXa inhibitory components in different extracts of Chuanxiong were successfully characterized by the method of enzyme inhibition activity assays with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry-based multivariate statistical analysis.

Due to poor specificity, the current pharmacotherapy of atherosclerosis has limited therapeutic efficacy. Here, the authors show that a macrophage-biomimetic nanomedicine effectively alleviates atherosclerosis via targeted pharmacotherapy and sequestration of proinflammatory cytokines and chemokines.

Abstract Background The dry root and rhizome of Salvia miltiorrhiza Bunge, or Danshen, is a well-known traditional Chinese medicine with anticoagulant activity. Taking into account that thrombin (THR) and factor Xa (FXa) play crucial roles in the coagulation cascade, it is reasonable and meaningful to screening THR and/or FXa inhibitors from Danshen. Methods Four extracts [butanol (BA), ethyl acetate (EA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Danshen were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Then, the active EA extract was further separated by silica-gel column chromatography (SC), and its fractions (SC1–SC5) were analyzed by LC–MS. The principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA) were employed for predicting the specific marker compounds. The chemical structures of targeted compounds were identified by LC–MS/MS and their interactions with THR/FXa were analyzed by the molecular docking analysis. Results Danshen EA extract showed strong activity against THR and FXa, and its fractions (SC1–SC5) exhibited obvious difference in inhibitory activity against these two enzymes. Furthermore, four marker compounds with potential THR/FXa inhibitory activity were screened by PCA and OPLS-DA, and were identified as cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA. The molecular docking study showed that all these four tanshinones can interact with some key amino acid residues of the THR/FXa active cavities, such as HIS57 and SER195, which were considered to be promising candidates targeting THR and/or FXa with low binding energy (

Abstract Background Alzheimer’s disease (AD) is ranked as the most prevalent neurodegenerative disease. However, the exact molecular mechanisms underlying pathophysiological alterations in AD remain unclear, especially at the prodromal stage. The decreased proteolytic degradation of Aβ, blood–brain barrier (BBB) disruption, and neuroinflammation are considered to play key roles in the course of AD. Methods Male APPswe/PS1dE9 C57BL/6 J double-transgenic (APP/PS1) mice in the age range from 1 month to 6 months and age-matched wild type mice were used in this study, intending to investigate the expression profiles of Aβ-degrading enzymes for Aβ degradation activities and zonula occludens-1 (zo-1) for BBB integrity at the prodromal stage. Results Our results showed that there were no significant genotype-related alterations in mRNA expression levels of 4 well-characterized Aβ-degrading enzymes in APP/PS1 mice within the ages of 6 months. Interestingly, a significant decrease in zo-1 expression was observed in APP/PS1 mice starting from the age of 5 months, suggesting that BBB disrupt occurs at an early stage. Moreover, treatment of fish oil (FO) for 4 weeks remarkably increased zo-1 expression and significantly inhibited the glial activation and NF-κB activation in APP/PS1 mice. Conclusion The results of our study suggest that FO supplement could be a potential therapeutic early intervention for AD through protecting the BBB integrity and suppressing glial and NF-κB activation.

As a prevalent medicinal liquor among Chinese people, a type of Chinese herbal spirit from Jing Brand Co., Ltd (CHS-J) is a newly developed health beverage with the health functions of anti-fatigue and immune enhancement. The researchers from the enterprise found that the contents of several components in CHS-J samples have been significantly decreasing during the stated storage period, as detected by the HPLC-UV method, which would make a great challenge for quality control of CHS-J. Furthermore, the chemical stability of CHS-J during the storage period is greatly challenged affected, especially in the environment of high temperature and light exposure. To systematically reveal the unstable components and promote the quality control of CHS-J, the chemical stability of CHS-J during the shelving storage period was characterized by the UPLC/Q-TOFMS-based metabolomics approach. First, the targeted and untargeted metabolomics approaches discovered the significantly changed components in CHS-J samples produced in different years. Furthermore, the accelerated tests of newly produced CHS samples and several authorized standards were conducted to validate the above results and elucidate the possible mechanisms underlying these chemical changes. Moreover, these chemical changes during the storage period had little influence on the anti-fatigue effect of CHS-J samples. These findings will offer new insight into the understanding of the chemical stability of CHS-J and will facilitate the quality control of CHS-J.

Hepatotoxicity caused by an overdose of acetaminophen (APAP) is the leading reason for acute drug-related liver failure. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a protein that helps to regulate redox homeostasis and coordinate stress responses via binding to the Kelch-like ECH-associated protein 1 (Keap1). Targeting the Keap1-Nrf2 interaction has recently emerged as a potential strategy to alleviate liver injury caused by APAP. Here, we designed and synthesized a number of iridium (III) and rhodium (III) complexes bearing ligands with reported activity against oxidative stress, which is associated with Nrf2 transcriptional activation. The iridium (III) complex 1 bearing a bioactive ligand 2,9-dimethyl-1,10-phenanthroline and 4-chloro-2-phenylquinoline, a derivative of the bioactive ligand 2-phenylquinoline, was identified as a direct small-molecule inhibitor of the Keap1–Nrf2 protein-protein interaction. 1 could stabilize Keap1 protein, upregulate HO-1 and NQO1, and promote Nrf2 nuclear translocation in normal liver cells. Moreover, 1 reversed APAP-induced liver damage by disrupting Keap1–Nrf2 interaction and without inducing organ damage and immunotoxicity in mice. Our study demonstrates the identification of a selective and efficacious antagonist of Keap1–Nrf2 interaction possessed good cellular permeability in cellulo and ideal pharmacokinetic parameters in vivo, and, more importantly, validates the feasibility of conjugating metal complexes with bioactive ligands to generate metal-based drug leads as non-toxic Keap1–Nrf2 interaction inhibitors for treating APAP-induced acute liver injury.

We report in this study the identification of a natural product-like antagonist (1a) of Vps34 as a potent autophagy modulator via structure-based virtual screening. Aurone derivative 1a strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, 1a prevents autophagy in human cells induced either by starvation or by an mTOR inhibitor. In silico modeling and kinetic data revealed that 1a could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy in vivo and without inducing heart or liver damage in mice. 1a could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases. KEY WORDS: Autophagy, Natural products, Vps34, Inhibitor, Structure-based virtual screening, Vesicle trafficking, Heart or liver damage, Aurone derivative

Background: Ginsenosides with less sugar moieties may exhibit the better adsorptive capacity and more pharmacological activities. Methods: An efficient method for the separation of four minor saponins, including gypenoside XVII, notoginsenoside Fe, ginsenoside Rd2, and notoginsenoside Fd, from Panax notoginseng leaves (PNL) was established using biotransformation, macroporous resins, and subsequent preparative high-performance liquid chromatography. Results: The dried PNL powder was immersed in the distilled water at 50°C for 30 min for converting the major saponins, ginsenosides Rb1, Rc, Rb2, and Rb3, to minor saponins, gypenoside XVII, notoginsenoside Fe, ginsenoside Rd2, and notoginsenoside Fd, respectively, by the enzymes present in PNL. The adsorption characteristics of these minor saponins on five types of macroporous resins, D-101, DA-201, DM-301, X-5, and S-8, were evaluated and compared. Among them, D-101 was selected due to the best adsorption and desorption properties. Under the optimized conditions, the fraction containing the four target saponins was separated by D-101 resin. Subsequently, the target minor saponins were individually separated and purified by preparative high-performance liquid chromatography with a reversed-phase column. Conclusion: Our study provides a simple and efficient method for the preparation of these four minor saponins from PNL, which will be potential for industrial applications. Keywords: ginsenoside Rd2, gypenoside XVII, notoginsenoside Fd, notoginsenoside Fe, Panax notoginseng leaves

Although n-3 polyunsaturated fatty acids (PUFAs) exhibit biological activity in many inflammatory diseases, their renoprotective effects against diabetic nephropathy (DN) have been controversial. Here, fat-1 transgenic mice were used to investigate the effects of n-3 PUFAs on streptozocin (STZ)-induced DN. Fat-1 mice exhibited increased renal n-3 PUFAs and n-3 PUFA-derived lipid mediators, compared to wild type (WT) mice. Endogenous n-3 PUFAs protected STZ-treated fat-1 mice against hyperglycemia-induced renal dysfunction, as reflected by significant decreases in kidney/body weight ratio, urinary protein, blood urea nitrogen levels and creatinine clearance. In addition, endogenous n-3 PUFAs significantly ameliorated hyperglycemia-induced renal damage by modulating E-cadhein expression. It was also found that DN-induced NLRP3 inflammasome activation was attenuated by endogenous n-3 PUFAs in STZ-treated fat-1 mice. Our findings suggest that endogenous n-3 PUFAs ameliorate DN potentially through the: (i) formation of n-3 lipid mediators, (ii) induction of E-cadherin expression and (iii) inhibition of NLRP3 inflammasome.

Cerebral microinfarcts have significant effects on the development of geriatric neurological disorders, including vascular dementia and Alzheimer's disease. However, little is known about the pathophysiological mechanisms involved in the evolution of microinfarcts and potential treatment and prevention against these microvascular ischemic lesions. In the present study, the “single cortical microinfarct model” generated via occluding a penetrating arteriole by femtosecond laser ablation and the “multiple diffuse microinfarcts model” induced by unilateral injection of cholesterol crystals through the internal carotid artery were established to investigate the pathophysiological mechanisms underlying the evolution of microinfarcts and the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on alleviating microinfarct burdens and functional deficits. The occlusion of a single penetrating arteriole led to a distinct cortical microinfarct, which manifested as neuronal loss and occupation of activated glial cells in the ischemic core. Using Fat-1 transgenic mice and fish oil supplements, we demonstrated that both endogenously-generated and exogenously-delivered ω-3 PUFAs significantly inhibited the activation of receptor-interacting serine/threonine protein kinases 1 (RIPK1) and its downstream apoptosis-associated proteins, mitigated cell apoptosis, and anatomically reduced the microinfarct size. The protective effects of ω-3 PUFAs against microinfarcts were further verified in a multiple diffuse microinfarcts model, where ω-3 PUFAs significantly attenuated cell apoptosis as revealed by TUNEL staining, alleviated the diffuse microinfarct burdens and remarkably improved the functional deficits as evidenced by reduced spontaneous anxiety, increased preference for the novel object, and improved hippocampal-based learning and short-term memory. Together, these findings demonstrate that enriched brain ω-3 PUFAs are effective for reducing microinfarct burdens and improving the function deficits, which support the clinical research and application of ω-3 PUFAs in the treatment or prophylaxis in vascular dementia. Keywords: Fish oil, Cognitive decline, Vascular dementia, Neuropsychiatric disorders

Background: Panax notoginseng leaves (PNL) exhibit extensive activities, but few analytical methods have been established to exclusively determine the dammarane triterpene saponins in PNL. Methods: Ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC/Q-TOF MS) and HPLC-UV methods were developed for the qualitative and quantitative analysis of ginsenosides in PNL, respectively. Results: Extraction conditions, including solvents and extraction methods, were optimized, which showed that ginsenosides Rc and Rb3, the main components of PNL, are transformed to notoginsenosides Fe and Fd, respectively, in the presence of water, by removing a glucose residue from position C-3 via possible enzymatic hydrolysis. A total of 57 saponins were identified in the methanolic extract of PNL by UPLC/Q-TOF MS. Among them, 19 components were unambiguously characterized by their reference substances. Additionally, seven saponins of PNL—ginsenosides Rb1, Rc, Rb2, and Rb3, and notoginsenosides Fc, Fe, and Fd—were quantified using the HPLC-UV method after extraction with methanol. The separation of analytes, particularly the separation of notoginsenoside Fc and ginsenoside Rc, was achieved on a Zorbax ODS C8 column at a temperature of 35°C. This developed HPLC-UV method provides an adequate linearity (r2>0.999), repeatability (relative standard deviation, RSD < 2.98%), and inter- and intraday variations (RSD < 4.40%) with recovery (98.7–106.1%) of seven saponins concerned. This validated method was also conducted to determine seven components in 10 batches of PNL. Conclusion: These findings are beneficial to the quality control of PNL and its relevant products. Keywords: ginsenoside transformation, notoginsenoside Fd, notoginsenoside Fe, Panax notoginseng leaves, UPLC/Q-TOF MS

Abstract Background Penthorum chinense Pursh (Penthoraceae, PCP), a well-known Miao ethnomedicine, has been traditionally used to treat several liver-related diseases, such as jaundice and viral hepatitis. The aims of the present study were to evaluate the probable properties of the aqueous extract of PCP on carbon tetrachloride (CCl4)—induced acute liver injury in mice. Methods C57BL/6 mice were orally administered an aqueous extract of PCP (5.15 and 10.3 g/kg BW) or silymarin (100 mg/kg) once daily for 1 week prior to CCl4 exposure. Silymarin serves as a positive drug to validate the effectivenes of PCP. Results A single dose of CCl4 exposure caused severe acute liver injury in mice, as evidenced by the elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alanine phosphatase (ALP), and the increased TUNEL-positive cells in liver, which were remarkably ameliorated by the pretreatment of PCP. PCP was also found to decrease the levels of malondialdehyde (MDA), restore the glutathione (GSH) and enhance the activities of superoxide dismutase (SOD) and catalase (CAT) in the liver. In addition, the pretreatment of PCP inhibited the degradation of hepatic cytochrome P450 2E1 (CYP2E1), up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target proteins in CCl4-treated mice. Conclusion Results indicated that the pretreatment of PCP (10.3 g/kg BW) effectively protected against CCl4-induced acute liver injury, which was comparable to efficacy of silymarin (100 mg/kg). This hepatoprotective effects might be attributed to amelioration of CCl4-induced oxidative stress via activating Nrf2 signaling pathway.

Berberine (BBR) is a renowned natural compound that exhibits potent neuroprotective activities. However, the cellular and molecular mechanisms are still unclear. Hormesis is an adaptive mechanism generally activated by mild oxidative stress to protect the cells from further damage. Many phytochemicals have been shown to induce hormesis. This study aims to investigate whether the neuroprotective activity of BBR is mediated by hormesis and the related signaling pathways in 6-OHDA-induced PC12 cells and zebrafish neurotoxic models. Our results demonstrated that BBR induced a typical hormetic response in PC12 cells, i.e. low dose BBR significantly increased the cell viability, while high dose BBR inhibited the cell viability. Moreover, low dose BBR protected the PC12 cells from 6-OHDA-induced cytotoxicity and apoptosis, whereas relatively high dose BBR did not show neuroprotective activity. The hormetic and neuroprotective effects of BBR were confirmed to be mediated by up-regulated PI3K/AKT/Bcl-2 cell survival and Nrf2/HO-1 antioxidative signaling pathways. In addition, low dose BBR markedly mitigated the 6-OHDA-induced dopaminergic neuron loss and behavior movement deficiency in zebrafish, while high dose BBR only slightly exhibited neuroprotective activities. These results strongly suggested that the neuroprotection of BBR were attributable to the hormetic mechanisms via activating cell survival and antioxidative signaling pathways. Keywords: Berberine, Hormesis, Neuroprotection, PC12 cells, Zebrafish

Context: Corydalis yanhusuo W.T. Wang (Papaveraceae) (Rhizoma Corydalis) showed inhibitory effects on rabbit platelet aggregation induced by ADP, thrombin (THR) or arachidonic acid (AA). Objective: This study separates and identifies the possible target-related platelet proteins and suggests possible signal cascades of RC antiplatelet aggregation. Materials and methods: Based on comparative proteomics, the differentially expressed platelet proteins treated before and after with 50 mg/mL RC 90% ethanol extract (for 15 min at 37 °C) were analyzed and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS. To further verify the possible signalling pathways of RC antiplatelet aggregation function, the concentration of calcium (Ca2+) was measured by Fura-2/AM fluorescence (Ex 340/380 nm, Em 500 nm) (RC final concentrations of 0.0156–0.1563 mg/mL), the levels of P-selectin and cyclic guanosine monophosphate (cGMP) were quantified by ELISA (OD. 450 nm) (RC final concentrations of 0.0156–1.5625 mg/mL), and the 5-hydroxytryptamine (5-HT) level was measured using ortho-phthalaldehyde (OPT) fluorescence (Ex 340 nm, Em 470 nm) (RC final concentrations of 0.3125–1.5625 mg/mL). Results: The expression of 52 proteins were altered in rabbit platelets after the treatment and the MALDI-TOF-MS analysis indicated that those proteins include 12 cytoskeleton proteins, 7 cell signalling proteins, 3 molecular chaperone proteins, 6 proteins related to platelet function, 16 enzymes and 7 other related proteins. Furthermore, RC extract could decrease the levels of 5-HT [inhibition rate of 96.80% (p

Glycine tabacina (Labill.) Benth, a renowned Chinese medicinal herb, has been used for treatment of rheumatoid arthritis (RA) and joint infection. This study aimed to investigate inhibitory effect of its ethanol extract (GTE) on RA-associated inflammation. Nine isoflavonoids of twelve ones which were found in G. tabacina roots were identified by UHPLC-DAD-MS/MS. GTE treatment suppressed lipopolysaccharide-stimulated overproduction of nitric oxide, TNF-α and IL-6, and iNOS expression in macrophages. GTE blocked the activation of NF-κB and MAPK pathways, as indicated by reduced levels of phosphorylation of IKKα/β, IκBα, p38, ERK and JNK, and the suppressed nuclear translocation of NF-κB p65. Additionally, GTE administration decreased swelling of arthritic paw and serum TNF-α and IL-1β levels in collagen-induced arthritis (CIA) mice. These results demonstrated that GTE exhibited strong anti-inflammatory and anti-arthritic activities through suppressing NF-κB and MAPK pathways activation in activated macrophages and CIA mice, suggesting the potential applications of this species in RA prevention and treatment as functional food or therapeutic agent.

Malignant melanoma is an aggressive cancer of neuroectodermal origin. The mechanisms of n-3 polyunsaturated fatty acids (PUFAs) against melanoma metastasis are not clear. Here fat-1 and wild type (WT) mice were injected with B16F10 melanoma cells via the tail vein to establish lung metastasis model. Endogenous n-3 PUFAs were associated with a reduction in grossly visible pulmonary metastases and outgrowth through inhibition of mesenchymal to epithelial transition (MET), which was characterized by decreased expression of epithelial markers E-cadherin and ZO-1, and increased expression of mesenchymal marker vimentin in tumour tissues from fat-1 mice compared with WT controls. In addition, n-3 PUFA-mediated inhibition of MET may have been associated, in part, with the: (i) formation of n-3 PUFA-derived lipid mediators and (ii) decreased expression of mature IL-1β and p-NF-κB, and enhanced expression of superoxide dismutase-1. These results suggest that n-3 PUFAs exert their antitumourigenic activities, in part, via their anti-inflammatory properties.

The effects of α-linolenic acid (ALA)-rich flaxseed oil (FDO) against ethanol-induced liver injury and the probable molecular mechanisms in a mouse model of chronic-plus-single-binge ethanol feeding were evaluated. Mice were fed Lieber-DeCarli ethanol or control liquid diets with corn oil (CNO) or flaxseed oil for 10 days. On day 11, mice are gavaged with a single dose of ethanol or maltose dextrin. Ethanol exposure with CNO caused severe liver injury, inflammation and oxidative stress in liver, which were remarkably ameliorated by FDO. FDO supplementation decreased the elevation of plasma endotoxin level, which might be attributed to ameliorating ethanol-induced intestinal barrier dysfunction via upregulating the expressions of tight junction proteins. Additionally, FDO supplementation suppressed endotoxin-triggered inflammation via blocking TLR4/MyD88/NF-κB cascades in liver. These findings suggest that ALA-rich flaxseed oil may have potential to be developed as an effective agent for ethanol-induced liver injury.

Effects of saponins from leaves of Panax notoginseng (LPNS) against alcoholic liver injury were evaluated, and their probable molecular mechanisms were further elucidated in a mouse model of chronic-plus-single-binge ethanol feeding. Alcohol exposure dramatically increased plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triacylglycerol (TG) and endotoxin; these elevations were attenuated by treatment with LPNS at a dose of 100 or 300 mg/kg. LPNS was also found to suppress thiobarbituric acid reactive substances (TBARS) production, enhance glutathione (GSH) level and superoxide dismutase (SOD) activity in liver. The protective of LPNS against oxidative stress was associated with down-regulation of hepatic cytochrome P450 2E1 (CYP2E1), and upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and haem oxygenase-1 (HO-1). Additionally, LPNS ameliorated gut leakiness via upregulating the expressions of tight junction proteins, and suppressed endotoxin-mediated inflammation via down-regulating toll-like receptor-4 (TLR-4), CD14 and p-NF-κB p65. These results demonstrate that treatment with LPNS effectively protects against alcoholic liver injury through reducing ethanol-induced oxidative stress and ameliorating gut-derived endotoxin-mediated inflammation.

Hepatic ischemia/reperfusion (I/R), characterized by severe inflammation and cell death, causes significant liver damage, and there are no effective approaches to prevent this pathological condition. We evaluated the potentially protective functions of ginsenoside Rg1 in hepatic I/R injury and the underlying mechanisms. A mouse hepatic I/R model was established by 60 min of induced ischemia followed by 0, 6, 12 or 24 h of reperfusion. Ginsenoside Rg1 (20 mg/kg/day) significantly promoted hepatic function and suppressed liver necrosis and inflammatory responses. Mechanistically, the JNK/MAPK signaling was dramatically diminished by ginsenoside Rg1. Furthermore, we unexpectedly found that only the cytotoxicity induced by inflammation, rather than oxidative stress, was significantly inhibited by ginsenoside Rg1 in vitro, and this inhibition was almost completely abolished by treatment with a specific JNK activator, anisomycin. Thus, ginsenoside Rg1 exerts protective effects against hepatic I/R in a JNK signaling-dependent manner.

By comparing the target proteins (TPs) of classic traditional Chinese medicine (TCM) herbal formulas and modern drugs used for treating coronary artery disease (CAD), this study aimed to identify potential therapeutic TPs for treating CAD. Based on the theory of TCM, the Xuefu-Zhuyu decoction (XZD) and Gualou-Xiebai-Banxia decoction (GXBD), both of which are classic herbal formulas, were selected for treating CAD. Data on the chemical ingredients and corresponding TPs of the herbs in these two formulas and data on modern drugs approved for treating CAD and related TPs were retrieved from professional TCM and bioinformatics databases. Based on the associations between the drugs or ingredients and their TPs, the TP networks of XZD, GXBD, and modern drugs approved for treating CAD were constructed separately and then integrated to create a complex master network in which the vertices represent the TPs and the edges, the ingredients or drugs that are linked to the TPs. The reliability of this master network was validated through statistical tests. The common TPs of the two herbal formulas have a higher possibility of being targeted by modern drugs in comparison with the formula-specific TPs. A total of 114 common XZD and GXBD TPs that are not yet the target of modern drugs used for treating CAD should be experimentally investigated as potential therapeutic targets for treating CAD. Among these TPs, the top 10 are NOS3, PTPN1, GABRA1, PRKACA, CDK2, MAOB, ESR1, ADH1C, ADH1B, and AKR1B1. The results of this study provide a valuable reference for further experimental investigations of therapeutic targets for CAD. The established method shows promise for searching for potential therapeutic TPs based on herbal formulas. It is crucial for this work to select beneficial therapeutic targets of TCM, typical TCM syndromes, and corresponding classic formulas.

Transient global cerebral ischemia, one of the consequences of cardiac arrest and cardiovascular surgery, usually leads to delayed death of hippocampal cornu Ammonis1 (CA1) neurons and cognitive deficits. Currently, there are no effective preventions or treatments for this condition. Omega-3 (ω-3) PUFAs have been shown to have therapeutic potential in a variety of neurological disorders. Here, we report that the transgenic mice that express the fat-1 gene encoding for ω-3 fatty acid desaturase, which leads to an increase in endogenous ω-3 PUFAs and a concomitant decrease in ω-6 PUFAs, were protected from global cerebral ischemia injury. The results of the study show that the hippocampal CA1 neuronal loss and cognitive deficits induced by global ischemia insult were significantly less severe in fat-1 mice than in WT mice controls. The protection against global cerebral ischemia injury was closely correlated with increased production of resolvin D1, suppressed nuclear factor-kappa B activation, and reduced generation of pro-inflammatory mediators in the hippocampus of fat-1 mice compared with WT mice controls. Our study demonstrates that fat-1 mice with high endogenous ω-3 PUFAs exhibit protective effects on hippocampal CA1 neurons and cognitive functions in a global ischemia injury model.

The omega-3 polyunsaturated fatty acids (ω-3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), derived mainly from fish oil, play important roles in brain development and neuroplasticity. Here, we reported that application of ω-3 PUFAs significantly protected mouse neural progenitor cells (NPCs) against H2O2-induced oxidative injury. We also isolated NPCs from transgenic mice expressing the Caenorhabditis elegans fat-1 gene. The fat-1 gene, which is absent in mammals, can add a double bond into an unsaturated fatty acid hydrocarbon chain and convert ω-6 to ω-3 fatty acids. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that a marked decrease in apoptotic cells was found in fat-1 NPCs after oxidative injury with H2O2 as compared with wild-type NPCs. Quantitative RT-PCR and Western blot analysis demonstrated a much higher expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor for antioxidant genes, in fat-1 NPCs. The results of the study provide evidence that ω-3 PUFAs resist oxidative injury to NPCs.

Ginseng, the root of Panax ginseng C.A. Meyer, has been used as a tonic to enhance bodily functions against various ailments for hundreds of years in Far Eastern countries without apparent adverse effects. Ginsenoside Rb1, one of the most active ingredients of ginseng, has been shown to possess various pharmacological activities. Here we report that Rb1 exhibits potent neuroprotective effects against oxidative injury induced by tert-butylhydroperoxide (t-BHP). Lactate dehydrogenase (LDH) assay demonstrated that incubation with 300 µm t-BHP for 2.5 h led to a significant cell loss of cultured rat embryonic cortex-derived neural progenitor cells (NPCs) and the cell viability was pronouncedly increased by 24 h pretreatment of 10 µm Rb1. TUNEL staining further confirmed that pretreatment of Rb1 significantly reduced the cell apoptosis in t-BHP-induced oxidative injury. Real time PCR revealed that pretreatment with Rb1 activated Nrf2 pathway in cultured NPCs and led to an elevated expression of HO-1. The results of the present study demonstrate that Rb1 shows a potent anti-oxidative effect on cultured NPCs by activating Nrf2 pathway.

Atherosclerosis mainly contributes to cardiovascular disease, a leading cause of global morbidity and mortality. Panax notoginseng saponins (PNS) are proved to therapeutically attenuate the formation of atherosclerotic lesions. According to different sapogenin, PNS are generally classified into 20(S)-protopanaxadiol saponins (PDS) and 20(S)-protopanaxatriol saponins (PTS). It was reported that PDS and PTS might exert diverse or even antagonistic bioactivities. In this study, the probable effects of PTS and PDS on atherosclerotic development were investigated and compared in ApoE-deficient mice (ApoE−/−). Male mice were gavaged daily by PNS (200 mg/kg/d), PTS (100 mg/kg/d), or PDS (100 mg/kg/d), respectively for eight weeks. The treatments of PNS and PDS, but not PTS, showed decreased atherosclerotic lesions in the entire aorta by 45.6% and 41.3%, respectively, as evaluated by an en-face method. Both PNS and PDS can improve the plaque vulnerability, as evidenced by the increased collagen fiber, increased expression of α- smooth muscle actin (α-SMA), and decreased Cluster of differentiation 14 (CD14). Additionally, PDS also inhibit the nuclear factor kappa B (NF-κB)-mediated vascular inflammation in the aorta. In conclusion, PDS, but not PTS, might mainly contribute to the anti-atherosclerosis of P. notoginseng.

Multi-origin Chinese herbal medicines, with herbs originating from more than one species of plants, is a common phenomenon but an important issue in Traditional Chinese Medicines (TCMs). In the present study, a gas chromatography-mass spectrometry (GC-MS)—based fatty acid profiling approach to rapidly discriminate multi-origin Chinese medicines in terms of species and medicinal parts was proposed and validated using tuberous roots (Curcumae Radix) and rhizomes (Curcumae Rhizoma and Curcumae Longae Rhizoma) derived from four Curcuma species (e.g., C. wenyujin, C. kwangsiensis, C. phaeocaulis and C. longa) as models. Both type and content of fatty acids varied among different species of either tuberous roots or rhizomes, indicating each species has its own fatty acid pattern. Orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical clustering analysis (HCA) based on dataset of global fatty acid profiling showed that either tuberous roots or rhizomes samples could be clearly classified into four clusters according to their species. Furthermore, those tested samples could also be discriminated in terms of their medicinal parts (e.g., tuberous root and rhizome). Our findings suggest that the proposed GC-MS-based fatty acid profiling followed by multivariate statistical analysis provides a reliable platform to discriminate multi-origin Chinese herbal medicines according to species and medicinal parts, which will be helpful for ensuring their quality, safety and efficacy.

Panax notoginseng saponins (PNS) are major active constituents of P. notoginseng. This study investigated the mechanisms of the hypocholesterolaemic and atheroscleroprotective effects of PNS. It was found that treatment of PNS (30 or 100 mg/kg/day, p.o.) could significantly reduce elevated serum total cholesterol, triacylglycerol, low-density lipoprotein cholesterol and atherogenic index induced by feeding rats with high cholesterol diet for 28 days. High-density lipoprotein cholesterol could also be increased by PNS treatment. Additionally, PNS significantly enhanced the hypercholesterolaemia-induced reduction of superoxide dismutase activity in the liver. In hypercholesterolaemic rats, PNS significantly improved the endothelium-dependent vasodilatation, acetylcholine-induced nitric oxide production and endothelial nitric oxide synthase mRNA expression in a dose-dependent manner. It also significantly reversed the hypercholesterolaemia-induced changes in mRNA expressions of cholesterol 7α-hydroxylase and peroxisome proliferator-activated receptor α but not HMG-CoA reductase. The findings provided evidence for using PNS to prevent the development of hypercholesterolaemia and atherosclerosis. The hypocholesterolaemic activity of PNS is likely to be resulted from inducing the biosynthesis of bile acids from cholesterol and promoting the β-oxidation of fatty acids in the liver. The vasoprotective function of PNS may be brought about by increasing the gene expression of eNOS in endothelial cells, its antioxidative activity, or both.

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.

Despite the existence of available therapies, the Hepatitis B virus infection continues to be one of the most serious threats to human health, especially in developing countries such as China and India. To shed light on the improvement of current therapies and development of novel anti-HBV drugs, we thoroughly investigated 212 US patents of anti-HBV drugs and analyzed the technology flow in research and development of anti-HBV drugs based on data from IMS LifeCycle databases. Moreover, utilizing the patent citation method, which is an effective indicator of technology flow, we constructed patent citation network models and performed network analysis in order to reveal the features of different technology clusters. As a result, we identified the stagnant status of anti-HBV drug development and pointed the way for development of domestic pharmaceuticals in developing countries. We also discussed about therapeutic vaccines as the potential next generation therapy for HBV infection. Lastly, we depicted the cooperation between entities and found that novel forms of cooperation added diversity to the conventional form of cooperation within the pharmaceutical industry. In summary, our study provides inspiring insights for investors, policy makers, researchers, and other readers interested in anti-HBV drug development.

A pressurized liquid extraction (PLE) and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for the qualitative determination of saponins in different parts of P. notoginseng, including rhizome, root, fibre root, seed, stem, leaf and flower. The samples were extracted using PLE. The analysis was achieved on a Zorbax SB-C18 column with gradient elution of acetonitrile and 8 mM aqueous ammonium acetate as mobile phase. The mass spectrometer was operated in the negative ion mode using the electrospray ionization, and a collision induced dissociation (CID) experiment was also carried out to aid the identification of compounds. Forty one saponins were identified in different parts of P. notoginseng according to the fragmentation patterns and literature reports, among them, 21 saponins were confirmed by comparing the retention time and ESI-MS data with those of standard compounds. The results showed that the chemical characteristics were obviously diverse in different parts of P. notoginseng, which is helpful for pharmacological evaluation and quality control of P. notoginseng.

Two minor novel dammarane-type saponins—5,6-didehydroginsenoside Rd (1) and 5,6-didehydroginsenoside Rb1 (2)—were isolated from the dried roots of Panax notoginseng along with sixteen known saponins. The structures of the new compounds were elucidated on the basis of spectroscopic and chemical methods.

Hormetic response is an adaptive mechanism for a cell or organism surviving in an unfavorable environment. It has been an intriguing subject of researches covering a broad range of biological and medical disciplines, in which the underlying significance and molecular mechanisms are under intensive investigation. In the present study, we demonstrated that topoisomerase I inhibitor camptothecin (CPT), a potent anticancer agent, induced an obvious hormetic response in rat pheochromocytoma PC12 cells. Camptothecin inhibited PC12 cell growth at relative high doses as generally acknowledged while stimulated the cell growth by as much as 39% at low doses. Moreover, low doses of CPT protected the cells from hydrogen peroxide (H 2 O 2 )-induced cell death. Phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathways were reported playing pivotal roles in protecting cells from oxidative stress. We observed that these 2 pathways were upregulated by low doses of CPT, as evidenced by increased levels of phosphorylated PI3K, phosphorylated Akt, phosphorylated mammalian target of rapamycin, Nrf2, and HO-1; and abolishment of the growth-promoting and neuroprotective effects of CPT by LY294002, a PI3K inhibitor. These results suggest that the hormetic and neuroprotective effects of CPT at low doses on PC12 cells were attributable, at least partially, to upregulated PI3K/Akt and Nrf2/HO-1 pathways.

Hormesis is a phenomenon of biphasic dose response characterized by exhibiting stimulatory or beneficial effects at low doses and inhibitory or toxic effects at high doses. Increasing numbers of chemicals of various types have been shown to induce apparent hormetic effect on cancer cells. However, the underlying significance and mechanisms remain to be elucidated. Berberine, one of the major active components of Rhizoma coptidis, has been manifested with notable anticancer activities. This study aims to investigate the hormetic effect of berberine and its influence on the anticancer activities of chemotherapeutic agents. Our results demonstrated that berberine at low dose range (1.25 ~ 5 μM) promoted cell proliferation to 112% ~170% of the untreated control in various cancer cells, while berberine at high dose rage (10 ~ 80 μM) inhibited cell proliferation. Further, we observed that co-treatment with low dose berberine could significantly attenuate the anticancer activity of chemotherapeutic agents, including fluorouracil (5-FU), camptothecin (CPT), and paclitaxel (TAX). The hormetic effect and thereby the attenuated anticancer activity of chemotherapeutic drugs by berberine may attributable to the activated protective stress response in cancer cells triggered by berberine, as evidenced by up-regulated MAPK/ERK1/2 and PI3K/AKT signaling pathways. These results provided important information to understand the potential side effects of hormesis, and suggested cautious application of natural compounds and relevant herbs in adjuvant treatment of cancer.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder mainly affecting motor neurons. Mutations in superoxide dismutase-1 (SOD-1) account for about 20% of familial ALS patients. A robust supply of motoneurons carrying the mutated gene would help understand the causes of motoneuron death and develop new therapeutics for the disease. Here, we established induced pluripotent stem (iPS) cell lines from SOD1G93A mice and compared their potency in motoneuron generation with normal iPS cells and mouse embryonic stem cells (E14). Our results showed that iPS cells derived from SOD1G93A mice possessed the similar potency in neuronal differentiation to normal iPS cells and E14 cells and can be efficiently driven to motoneuron-like phenotype. These cells exhibited typical neuronal morphology, expressed key motoneuron markers, including ChAT and HB9, and generated repetitive trains of action potentials. Furthermore, these neurons highly expressed human SOD-1 and exhibited shorter neurites compared to controls. The present study provides evidence that ALS-iPS cells can be used as disease models in high-throughput screening and mechanistic studies due to their ability to efficiently differentiate into specific neuronal subtypes.

Dopaminergic differentiation of embryonic stem cells (ESCs) gains more and more attention worldwide owing to its potential use for neurorestorative therapy for the treatment of Parkinson's disease. The conventional 2D cell culture on petri dishes with various animal derived substrata such as collagen gels, laminin, and Matrigel is widely used to induce dopaminergic differentiation and it may limit the efficiency in the generation of dopaminergic neurons from ESCs and prevent their application for human therapies. Here, we reported that a self-assembling peptide made from natural amino acids has a property to generate a true 3D environment for dopaminergic differentiation. Mouse ESCs (R1) and mouse iPSCs (TTF-1) embedded in RADA16-I peptide-derived nanofiber scaffolds led to a marked increase in dopaminergic differentiation compared to the laminin-coated 2D culture or Matrigel-encapsulated 3D culture. These differentiated neurons expressed specific dopaminergic markers and produced appropriate patterns of action potential firing. Consistent with the increase in the number of dopaminergic neurons differentiated from R1 or TTF-1 in the self-assembling peptide nanofiber scaffold (SAPNS), both the expression levels of genes that involve in dopaminergic differentiation and maturation and the dopamine release in SAPNS culture were significantly elevated. The results of the study suggest that SAPNS provides a promising 3D culture system for dopaminergic differentiation.

Abstract Despite the broad potential applications of C-glycosides, facile synthetic methods remain scarce. Transforming glycosyltransferases with promiscuous or natural O-specific chemoselectivity to C-glycosyltransferases is challenging. Here, we employ rational directed evolution of the glycosyltransferase MiCGT to generate MiCGT-QDP and MiCGT-ATD mutants which either enhance C-glycosylation or switch to O-glycosylation, respectively. Structural analysis and computational simulations reveal that substrate binding mode govern C-/O-glycosylation selectivity. Notably, directed evolution and mechanism analysis pinpoint the crucial residues dictating the binding mode, enabling the rational design of four enzymes with superior non-inherent chemoselectivity, despite limited sequence homology. Moreover, our best mutants undergo testing with 34 substrates, demonstrating superb chemoselectivities, regioselectivities, and activities. Remarkably, three C-glycosides and an O-glycoside are produced on a gram scale, demonstrating practical utility. This work establishes a highly selective platform for diverse glycosides, and offers a practical strategy for creating various types of glycosylation platforms to access pharmaceutically and medicinally interesting products.