Simultaneous determination of 78 pesticide residues and 16 mycotoxins in tsampa by an improved QuEChERS method coupled with ultra performance liquid chromatography-tandem mass spectrometry.

Tsampa may contain pesticide residues and mycotoxins, which may pose a risk to human health. Currently, pesticide detection and mycotoxin detection are two independent experiments. To improve the efficiency of the analysis, a method based on QuEChERS combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of 78 pesticides and 16 mycotoxins in tsampa was developed. All the target compounds showed good linear correlation with correlation coefficients (R2) greater than 0.9990. The limits of detection (LODs) and limits of quantification (LOQs) were in the ranges of 0.10-3.00 µg kg-1 and 0.40-10.00 µg kg-1, respectively. The average recoveries of the pesticides and mycotoxins spiked at the 1, 2, and 10-fold LOQ were in the range of 73.0-115.2%, and the relative standard deviations (RSDs) were lower than 11.7%. This method was applied to 19 batches of real samples in which 32% of samples exceeded the maximum residue limits of the European Union involving aflatoxin G2, ochratoxin A, and hexaconazole. It proved to be excellent, efficient, greatly simplified, and highly applicable, which could reduce the workload and time significantly for the daily monitoring of the pesticides and mycotoxins in tsampa.

[Rapid determination of 15 N-nitrosamines in air-dried yak meat using one-step QuEChERS-gas chromatography-triple quadrupole mass spectrometry].

A method based on gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) coupled with one-step QuEChERS technique was developed for the simultaneous determination of 15 N-nitrosamines in air-dried yak meat. The hydration volume, extraction solvent, extracting salt, and cleaning material were optimized according to the characteristics of the N-nitrosamines and sample matrix. The optimized conditions were as follows: 10 mL of purified water for sample hydration, acetonitrile as the extraction solvent for the sample after hydration, 4.0 g of anhydrous MgSO4 and 1.0 g of NaCl as extracting salts, 500 mg of MgSO4+25 mg of C18+50 mg of PSA as cleaning materials. Favorable recoveries of the 15 N-nitrosamines were obtained when the extraction solution was incompletely dried. Thus, the final extract was dried to below 0.5 mL under a mild nitrogen stream and then redissolved to 0.5 mL with acetonitrile. After filtration, 200 µL of the sample was transferred to an autosampler vial for GC-MS/MS analysis. The 15 N-nitrosamines were determined using GC-MS/MS on a DB-HeavyWAX column (30 m×0.25 mm×0.25 µm) with an electron impact ion source in multiple-reaction monitoring (MRM) mode, and quantified using an external standard method. Under the optimized experimental conditions, the results showed that the calibration curves exhibited good linearities for the 15 N-nitrosamines, with correlation coefficients (r2) greater than 0.9990. The limits of detection (LODs) and the limits of quantification (LOQs) ranged from 0.05 to 0.20 µg/kg and from 0.10 to 0.50 µg/kg, respectively. At spiked levels of 1LOQ, 2LOQ, and 10LOQ, the average recoveries were 79.4%-102.1%, 80.6%-109.5%, and 83.0%-110.6%, respectively, and the relative standard deviations were in the range of 0.8%-16.0%. The low matrix effects of the 15 N-nitrosamines indicated the high sensitivity of the proposed method. The method was applied to detect representative commercial air-dried yak meat samples obtained using different processing techniques. Seven N-nitrosamines, including N-nitrosodimethylamine, N-nitrosodiisobutylamine, N-nitrosodibutylamine, N-methyl-N-phenylnitrous amide, N-ethyl-N-nitrosoaniline, N-nitrosopyrrolidine, and N-nitrosodiphenylamine were detected in all samples. The average contents of the seven N-nitrosamines was 0.08-20.18 µg/kg. The detection rates and average contents of the N-nitrosamines in cooked air-dried yak meat samples were higher than those in traditional raw air-dried yak meat samples. Compared with the manual QuEChERS method, the one-step QuEChERS method developed integrated the extraction and clean-up procedures into one single run, and the detection efficiency was considerably improved. The developed method is simple, rapid, highly sensitive, and insusceptible to human errors. Thus, it is useful for the determination of N-nitrosamines in air-dried yak meat and can be extended to the qualitative and quantitative analysis of N-nitrosamines in other meat products. It also provides method support and a data reference for the general determination of N-nitrosamines, which is of great significance for food safety.

Discovery of Unusual Ajmaline-Macroline Type Bisindole Alkaloids from Alstonia macrophylla by Building Blocks-Based Molecular Networking.

Three unusual ajmaline-macroline type bisindole alkaloids, alsmaphylines A-C, together with their postulated biogenetic precursors, were isolated from the stem barks and leaves of Alstonia macrophylla via the building blocks-based molecular network (BBMN) strategy. Alsmaphyline A represents a rare ajmaline-macroline type bisindole alkaloid with an S-shape polycyclic ring system. Alsmaphylines B and C are two novel ajmaline-macroline type bisindole alkaloids with N-1-C-21' linkages, and the former possesses an unconventional stacked conformation due to the presence of intramolecular noncovalent interactions. The chemical structures including absolute configurations of alsmaphylines A-C were established by comprehensive spectroscopic analyses, electronic circular dichroism (ECD) calculations, and single-crystal X-ray crystallography. In addition, a plausible biosynthetic pathway of these bisindole alkaloids as well as their ability to promote the protein synthesis on HT22 cells were discussed.

Undescribed matrine-type alkaloids from Sophora alopecuroides with anti-inflammatory activity.

A phytochemical investigation on the alkaloid fractions of Sophora alopecuroides L. led to the production of 11 undescribed matrine-type alkaloids, sophaloseedlines I-S (1-11), 12 known analogs (12-23), and an unexpected artificial matrine-derived Al(III) complex (24). The corresponding structures were elucidated by the interpretation of spectroscopic analyses, quantum chemical calculation, and six instances (1-4, 18, and 24), verified by X-ray crystallography. The biological activities screening demonstrated that none of the isolates exhibited cytotoxicity against four human cancer cell lines (HepG2, A549, THP-1, and MCF-7) and respiratory syncytial virus (RSV) at 50 µM, while moderate anti-inflammatory activity with IC50 value from 15.6 to 47.8 µM was observed. The key structure-activity relationships of those matrine-type alkaloids for anti-inflammatory effects have been summarized. In addition, the most potent 7-epi-sophoramine (19) and aluminum sophaloseedline T (24) could effectively inhibit the release of pro-inflammatory factors (TNF-α, IL-6, and IL-1ß), as well as the expression of iNOS and COX-2 proteins.

Simultaneous Screening and Quantification of 479 Pesticides in Green Tea by LC-QTOF-MS.

A high-throughput screening and quantification method for 479 pesticides in green tea was established based on solid-phase extraction combined with liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Pesticides were extracted from samples using an optimized SPE (TPT cartridges) procedure. LC-QTOF-MS in All Ions MS/MS scan mode acquired full MS data for quantification and product ion spectra for identification. LC-QTOF-MS quantification was achieved using matrix-matched standard calibration curves to achieve the optimal method accuracy. The method performance characteristics included the linearity, overall recovery, precision, and measurement uncertainty being evaluated. The validation results exhibited a good sensitivity with the LOQs of 5-55 µg/kg, which was satisfactory for their MRLs in China or the EU. The recoveries of more than 92.7% of the 479 pesticides in green tea were 70-120% at the three spiked levels with a precision of ≤20%. Finally, this method was employed to analyze 479 pesticides in 95 tea samples from markets in China. The test results of the tea samples showed that tolfenpyrad, buprofezin, and pyridaben were found with lower concentrations. The method has effectively improved the determination efficiency of pesticide residue screening by high-resolution mass spectrometry in green tea.

Promoted iron corrosion and enhanced phosphate removal by micro-electric field driven zero-valent iron.

Zero-valent iron (Fe0) is restricted in phosphate removal due to the formation of a passive P-Fe layer on its surface. A micro-electric field (0.20 mA cm-2) was employed in Fe0 column to facilitate iron corrosion for enhanced phosphate removal with a Fe0 column as the control. The performance of two columns was compared by batch experiment at a Fe0 filling rate of 10 vol% with quartz sand as dispersing media. The stability and reusability of micro-electric field driven Fe0 (MFD-Fe0) column was estimated by cyclic test. Solid phase analysis showed promoted iron corrosion, iron ion generation, and secondary mineral production such as lepidocrocite and magnetite in the MFD-Fe0 column. Since iron ions tended to precipitate with phosphate, and iron minerals provided reaction sites for phosphate adsorption, the MFD-Fe0 column achieved an enhanced phosphate removal of 94.1%, 2.8 times higher than that of the Fe0 column. The increase of current density from 0 to 0.20 mA cm-2 significantly improved phosphate removal from 24.5% to 94.1%, further demonstrating the promoting effect of micro-electric field on iron corrosion. The MFD-Fe0 column also possessed excellent stability and reusability. It only showed a slight decrease of phosphate removal from 94.1% to 89.7% in eight cycles. It restored a phosphate removal capacity of 97.4% as compared to the initial MFD-Fe0 column by eluting iron (hydro)oxides on Fe0 and quartz sand surfaces with sulfuric acid. This study indicated that MFD-Fe0 is a promising method to remove phosphate from water and an alternative strategy for overcoming Fe0 passivation.

Sophflarine A, a novel matrine-derived alkaloid from Sophora flavescens with therapeutic potential for non-small cell lung cancer through ROS-mediated pyroptosis and autophagy.

BACKGROUND: Novel compounds and more efficient treatment options are urgently needed for the treatment of non-small cell lung cancer (NSCLC). The decoction of Sophora flavescens has been used to treat NSCLC in the clinic, and matrine-type alkaloids are generally considered to be the key pharmacodynamic material basis. But the previous study showed that common matrine-type alkaloids exhibit significant cytotoxicity only when at concentrations close to the millimolar (mM) level. The key antitumor alkaloids in S. flavescens seem to have not yet been revealed. PURPOSE: The aim of this study was to screen water-soluble matrine alkaloid with novel skeleton and enhanced activity from S. flavescens, and to reveal the pharmacological mechanism of its therapeutic effect on NSCLC. METHODS: Alkaloid was obtained from S. flavescens by chromatographic separation methods. The structure of alkaloid was determined by spectroscopic methods, and single-crystal X-ray diffraction. The mechanism of anti-NSCLC in vitro with cellular models was evaluated by MTT assay, western blotting, cell migration and invasion assay, plate colony-formation assay, tube formation assay, immunohistochemistry assay, hematoxylin and eosin staining. The antitumor efficacy in vivo was test in NSCLC xenograft models. RESULTS: A novel water-soluble matrine-derived alkaloid incorporating 6/8/6/6 tetracyclic ring system, named sophflarine A (SFA), was isolated from the roots of S. flavescens. SFA had significantly enhanced cytotoxicity compared with the common matrine-type alkaloids, having an IC50 value of 11.3 µM in A549 and 11.5 µM in H820 cells at 48 h. Mechanistically, SFA promoted NSCLC cell death by inducing pyroptosis via activating the NLRP3/caspase-1/GSDMD signaling pathway, and inhibited cancer cell proliferation by increasing the ROS production to activate autophagy via blocking the PI3K/AKT/mTOR signaling pathway. Additionally, SFA also inhibited NSCLC cell migration and invasion by suppressing EMT pathway, and inhibited cancer cell colony formation and human umbilical vein endothelial cell angiogenesis. In concordance with the above results, SFA treatment blocked tumor growth in an A549 cell-bearing orthotopic mouse model. CONCLUSION: This study revealed a potential therapeutic mechanism of a novel matrine-derived alkaloid, which not only described a rational explanation for the clinical utilization of S. flavescens, but also provided a potential candidate compound for NSCLC treatment.

Migration behavior and dietary exposure risk assessment of pesticides residues in honeysuckle (Lonicera japonica Thunb.) based on modified QuEChERS method coupled with tandem mass spectrometry.

The honeysuckle was widely appreciated as tea beverage owing to the biological activities and the unique aroma and flavor. It is in urgent requirement to explore the migration behavior and dietary exposure as the pesticide residues would bring about potential risks through honeysuckle intake. The optimized QuEChERS procedure coupled with the HPLC-MS/MS and GC-MS/MS methods were employed to determine 93 pesticide residues of seven classifications including carbamates, pyrethroid, triazoles, neonicotinoids, organophosphorus, organochlorine, and others for 93 honeysuckle samples from four primary production bases. As a result, 86.02% of the samples were contaminated by at least one pesticide. Unexpectedly, the banned pesticide of carbofuran was also identified. The migration behavior of metolcarb was the highest, whereas thiabendazole contributed less risk to the infusion with relative lower transfer rate. Both the chronic and acute exposure yielded low risk for human health with five high risk pesticides of dichlorvos, cyhalothrin, carbofuran, ethomyl, and pyridaben. Besides, this study provides foundation of dietary exposure risk assessment for honeysuckle and other likewise products.

Two new aryltetralin-type lignans from Camellia oleifera husk.

Two new aryltetralin-type lignans (1-2) were isolated from the dichloromethane fraction of 95% ethanol extract of Camellia oleifera fruit husk. Their structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations of compounds 1-2 were determined by the comparison of measured ECD curves with the quantum chemical calculated ones. The new compounds were tested for their antioxidant activities and cytotoxicity against three human cancer cell lines (Huh-7, H460 and MCF-7). While compounds 1 and 2 only showed slight DPPH radical scavenging activities with the IC50 values of 38.68 ± 5.02 and 56.62 ± 1.49 µM, respectively.

Two new chemical constituents from the stem and branch of Tripterygium wilfordii.

A chemical investigation of 95% ethanol extract from the stem and branch of Tripterygium wilfordii has resulted in the isolation and characterization of two new compounds, one neolignan (1) and one phenylalanine derivative (2), as well as four known compounds (3-6). The structures of the new compounds were determined based on extensive spectroscopic analyses. The absolute configuration of compound 1 was defined by X-ray crystallographic analyses and electronic circular dichroism calculation. In addition, compounds 2 and 4-6 exhibited inhibitory effects against NO production in LPS-induced RAW 264.7 macrophages with the IC50 value ranging from 3.51 µM to 30.40 µM.

Quassinoids from Eurycoma longifolia with antiviral activities by inhibiting dengue virus replication.

BACKGROUND: Dengue caused by dengue virus (DENV) spreads rapidly around the world. However, there are no worldwide licensed vaccines or specific antivirals to combat DENV infection. Quassinoids are the most characteristic components of Eurycoma longifolia, which have been reported to display a variety of biological activities. However, whether quassinoids exert anti-DENV activities remains unknown. PURPOSE: To test the quassinoids of E. longifolia for their activity against DENV and to clarify the potential mechanisms. METHODS: The quassinoids from E. longifolia were isolated by chromatography techniques, and their chemical structures were elucidated by spectroscopic analysis. The anti-DENV activities of quassinoids on baby hamster kidney cells BHK-21 were determined by lactate dehydrogenase (LDH) assay. The synthesis of progeny virus was measured by plaque assay. The expression levels of envelope protein (E) and non-structural protein 1 (NS1) were evaluated by qRT-PCR, Western blot and immunofluorescence assays. Molecular docking was used to screen the potential targets of the most active quassinoid against DENV-2, and surface plasmon resonance analysis was employed to confirm the direct binding between the most active quassinoid and potential target. RESULTS: Twenty-four quassinoids, including three new quassinoids (1 - 3), were isolated from the ethanol extract of E. longifolia. Quassinoids 4, 5, 9, 11, 12, 15, 16, 17, 19 and 20 significantly reduced the LDH release at the stages of viral binding and entry or intracellular replication. Among them, 19 (6α-hydroxyeurycomalactone, 6α-HEL) exhibited the best anti-DENV-2 activities with an EC50 value of 0.39 ± 0.02 µM. Further experiments suggested that 6α-HEL remarkably inhibited progeny virus synthesis and mRNA and protein expression levels of E and NS1 of DENV-2. Time-of-drug-addition assay suggested that 6α-HEL inhibited intracellular replication of DENV-2 at an early stage. Moreover, 6α-HEL was shown to interact with NS5-RdRp domain at a binding affinity of -8.15 kcal/mol. SPR assay further verified 6α-HEL bound to RdRp protein with an equilibrium dissociation constant of 1.49 × 10-7 M. CONCLUSION: Ten quassinoids from E. longifolia showed anti-DENV activities at processes of virus binding and entry or intracellular replication. The most active quassinoid 6α-HEL exerts the anti-DENV-2 activities at intracellular replication stage by directly targeting the NS5-RdRp protein. These results suggest that 6α-HEL could be a promising candidate for the treatment of DENV-2 infection.

Bixasteroid, a new compound from the fruits of Bixa orellana and its anti-inflammatory activity.

Bixasteroid (1), one new steroid together with five known compounds (2-6), were isolated from the ethyl acetate fraction of ethanol extract of Bixa orellana fruits. All of these known compounds were isolated from the plant for the first time. Their structures were elucidated on the basis of spectroscopic analysis, and the absolute configuration of compound 1 was determined by X-ray crystallographic data analysis as well as by the quantum chemical ECD calculations. All the isolated compounds were tested for their anti-inflammatory activities. Compounds 1 and 2 showed inhibiting NO release activities in LPS-induced RAW 264.7 macrophages with the IC50 values of 4.72 ± 0.28 and 5.48 ± 1.48 µM, respectively.

Development and Validation of a Method for Determination of 43 Antimicrobial Drugs in Western-Style Pork Products by UPLC-MS/MS with the Aid of Experimental Design.

Western-style pork products have attracted many modern urban consumers, and these products have rapidly entered the Chinese market. The current hazard analysis of processed meat products mainly focuses on processing hazards (PAHs, microorganisms, and food additives), with less attention to veterinary drug residues. According to the survey results, the residues of antimicrobial drugs (sulfonamides and quinolones) in pork and its products in China are a severe problem, which may cause metabolic reactions, toxic effects, or enhance drug resistance. This study applied a modified QuEChERS method combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS) to develop a rapid and sensitive method for determining antimicrobial drugs in bacon and ham was successfully evaluated methodologically by EU 2002/657/EC. This study used a three-level, three-factor Box-Behnken design (BBD) to optimize the QuEChERS method by response surface methodology. The excellent linearity of the calibration curve was shown in the corresponding concentration range with a coefficient of determination greater than 0.99. The values of decision limit (CCα) and detection capability (CCß) were in the range of 10.9-31.3 µg/kg and 11.8-52.5 µg/kg, respectively. The method successfully detected two trace levels of antimicrobial drugs in commercially available samples, including sulfadiazine and moxifloxacin.

Multi-residue analysis of 206 pesticides in grass forage by the one-step quick, easy, cheap, effective, rugged, and safe method combined with ultrahigh-performance liquid chromatography quadrupole orbitrap mass spectrometry.

A novel method for detecting pesticide multi-residue in grass forage (alfalfa and oat) was established based on the one-step automatic extraction and purification technology of quick, easy, cheap, effective, rugged, and safe combined with ultrahigh-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry. The crushed sample was extracted with acetonitrile with 1% acetate, followed by a cleanup step with a primary-secondary amine, octadecylsilane, and graphitized carbon black. The extraction and purification were carried out using the one-step automatic pretreatment equipment. The target pesticides were acquired in positive ion electrospray ionization mode and full scan/data dependent secondary scan mode. The calibration curve shows good linearity over the corresponding concentration range, with the coefficient of determination greater than 0.99. The screening detection limits were 0.5-50 µg/kg, and the limit of quantification for the 206 pesticides was set at 1-50 µg/kg. At the spiking levels of one, two, and 10 times of limit of quantification, more than 95% of pesticides had recovery between 70-120%, with a relative standard deviation ≤20%. The method was proved to be simple, rapid, high-sensitivity, and could be routinely used for the high throughput screening and quantitative analysis of pesticide residues in alfalfa and oat.

Anti-inflammatory, anti-angiogenetic and antiviral activities of dammarane-type triterpenoid saponins from the roots of Panax notoginseng.

Panax notoginseng has been used both as a traditional medicine and as a functional food for hundreds of years in Asia. However, the active constituents from P. notoginseng and their pharmacologic properties still need to be further explored. In this study, one new dammarane-type triterpenoid saponin (1), along with fourteen known analogs (2-15) were isolated and identified from the roots of P. notoginseng. The anti-inflammatory, anti-angiogenetic and anti-dengue virus effects of these isolated compounds were further evaluated. Compounds 1, 3, 5-7 and 10-12 exerted anti-inflammatory effects in two different zebrafish inflammatory models. Among them, 11, with the most significant activities, alleviated the inflammatory response by blocking the MyD88/NF-κB and STAT3 pathways. Moreover, compound 15 showed anti-angiogenetic activities in Tg(fli1:EGFP) and Tg(flk1:GFP) zebrafish, while 3 and 5 only inhibited angiogenesis in Tg(fli1:EGFP) zebrafish. Additionally, compounds 1, 3, 6, 8, 9 and 12 suppressed the replication of dengue virus either at the viral adsorption and entry stages or at the intracellular replication step. In conclusion, these findings enrich knowledge of the diversity of saponins in P. notoginseng and suggest that the dammarane-type triterpenoid saponins from P. notoginseng may be developed as potential functional foods to treat inflammation, angiogenesis or dengue-related diseases.

PRIAS: An Intelligent Analysis System for Pesticide Residue Detection Data and Its Application in Food Safety Supervision.

Pesticide residue is a prominent factor that leads to food safety problems. For this reason, many countries sample and detect pesticide residues in food every year, which generates a large amount of pesticide residue data. However, the way to deeply analyze and mine these data to quickly identify food safety risks is still an unresolved issue. In this study, we present an intelligent analysis system that supports the collection, processing, and analysis of detection data of pesticide residues. The system is first based on a number of databases such as maximum residue limit standards for the fusion of pesticide residue detection results; then, it applies a series of statistical methods to analyze pesticide residue data from multiple dimensions for quickly identifying potential risks; it uses the Apriori algorithm to mine the implicit association in the data to form pre-warning rules; finally, it applies Word document automatic generation technology to automatically generate pesticide residue analysis and pre-warning reports. The system was applied to analyze the pesticide residue detection results of 42 cities in mainland China from 2012 to 2015. Application results show that the system proposed in this study can greatly improve the depth, accuracy and efficiency of pesticide residue detection data analysis, and it can provide better decision support for food safety supervision.

Screening of Bufadienolides from Toad Venom Identifies Gammabufotalin as a Potential Anti-inflammatory Agent.

Toad venom (Chansu) is used in the treatment of infectious and inflammatory diseases in China and East/Southeast Asian countries. However, the anti-inflammatory components of toad venom have not yet been systematically evaluated and clearly defined. To investigate the anti-inflammatory effects of toad venom and identify new anti-inflammatory ingredients, we used zebrafish, an alternative drug screening model, to evaluate the anti-inflammatory effects of 14 bufadienolides previously isolated from toad venom. Most of the bufadienolides were found to exert significant anti-inflammatory effects on lipopolysaccharide-, CuSO4-, or tail transection-induced zebrafish inflammatory models. Moreover, gammabufotalin ( 6: ) inhibits lipopolysaccharide-induced inflammation by suppressing the myeloid differentiation primary response 88/nuclear factor-kappa B and STAT3 signal pathways. This study confirms the potential of zebrafish in drug screening, clarifies the anti-inflammatory effects of bufadienolides from toad venom, and indicates that gammabufotalin may be developed as a novel therapeutic agent for inflammatory diseases in the future.

Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage.

OBJECTIVE: To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons. METHODS: The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway. RESULTS: MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01). CONCLUSION: TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.

Quassinoids from the Roots of Eurycoma longifolia and Their Anti-Proliferation Activities.

A phytochemical investigation on the roots of medicinal plant Eurycoma longifolia resulted in the isolation of 10 new highly oxygenated C20 quassinoids longifolactones G‒P (1-10), along with four known ones (11-14). Their chemical structures and absolute configurations were unambiguously elucidated on the basis of comprehensive spectroscopic analysis and X-ray crystallographic data. Notably, compound 1 is a rare pentacyclic C20 quassinoid featuring a densely functionalized 2,5-dioxatricyclo[5.2.2.04,8]undecane core. Compound 4 represents the first example of quassinoids containing a 14,15-epoxy functionality, and 7 features an unusual α-oriented hydroxyl group at C-14. All isolated compounds were evaluated for their anti-proliferation activities on human leukemia cells. Among the isolates, compounds 5, 12, 13, and 14 potently inhibited the in vitro proliferation of K562 and HL-60 cells with IC50 values ranging from 2.90 to 8.20 µM.

Uncommon Bis-Amide Matrine-type Alkaloids From Sophora alopecuroides With Anti-inflammatory Effects.

Four new alkaloids (1-4) belonging to rare examples of bis-amide matrine-type were isolated from the seeds of sophora alopecuroides. Their structures including absolute configuration were determined by extensive spectroscopic analysis, electronic circular dichroism (ECD) interpretation, and X-ray diffraction crystallography. Chemically, bis-amide matrine-type alkaloids can provide new molecular template for structural modification. Compounds 3-4 displayed obvious anti-inflammatory effects based on the inhibition of two key pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in a dose-dependent manner, with IC50 values from 35.6 to 45.8 µm.