Combination of tenuigenin-based Polygala tenuifolia willd. root extract and forsythoside a improved Alzheimer's disease.

Tenuigenin is a kind of the main active ingredients in roots of Polygala tenuifolia Willd. (a species in the genus Polygala, family Polygalaceae) and forsythoside A (FA) is one of the main active ingredients of Forsythia suspensa (Thunb.) Vahl. (a species in the genus Forsythia, family Meliaceae). The studies have shown that tenuigenin-based Polygala tenuifolia Willd. extract (YZ) and FA have protective effects on nervous damage. In the study the combination (YF) of YZ and FA showed synergistic neuro-protective effects on PC12 cell model of Alzheimer's disease (AD). YF (2:1) which was made up of 2/3 YZ and 1/3 FA increased cell viability, inhibited AChE expression and activity, alleviated apoptosis and slowed Aß aggregation, while YF (1:2) which consisted of 1/3 YZ and 2/3 FA depressed inflammation and oxidative stress. There was no obvious synergistic effect of YF on Tau phosphorylation.

A specific dual-locked fluorescence probe to visualize the dynamic changes of lipid droplets and hypochlorous acid in inflammation.

Inflammation is a key factor leading to the occurrence and development of many diseases, both lipid droplets (LDs) and hypochlorous acid (HClO/ClO-) are regarded as the important biomarkers of inflammation. Therefore, it is of great significance to develop an efficient single chemical sensor that can simultaneously detect these two biomarkers. To achieve the goal, we developed a dual-locked fluorescence probe (TPA-DNP) by fusing two targets activated reporting system, its implementation was achieved by turning-on the fluorescence of TPA-DNP through LDs and HClO/ClO- simultaneously. In simulated LDs environment, TPA-DNP displayed excellent selectivity to HClO/ClO-, high sensitivity (LOD = 0.527 µM) and strong anti-interference ability. In addition, cell and zebrafish imaging experiments showed that TPA-DNP could be utilized to visualize exogenous/endogenous HClO/ClO- in LDs environment, and could also be used to observe the impact of LDs changes on the HClO/ClO- detection. On the basis, TPA-DNP served as a favorable tool to achieve visualization of inflammatory dynamic changes.

Interactions between forsythoside E and two cholinesterases at the different conditions: fluorescence sections.

Forsythoside E is one secondary metabolite ofForsythia suspensa(Thunb.) Vahl. In the study, the interactions between forsythoside E and two types of cholinesterases, acetylcholinesterase and butyrylcholinesterase were investigated in the different conditions. Forsythoside E increased the fluorescence intensity of acetylcholinesterase but quenched the fluorescence of butyrylcholinesterase. Aß25-35used in the study may not form complexes with cholinesterases, and did not affect the interaction between forsythoside E and cholinesterases. The charged quaternary group of AsCh interacted with the 'anionic' subsite in acetylcholinesterase, which did not affect the interaction between forsythoside E and acetylcholinesterase. The enhancement rate of forsythoside E to acetylcholinesterase fluorescence from high to low was acid solution (pH 6.4), neutral solution (pH 7.4) and alkaline solution (pH 8.0), while the reduction rate of forsythoside E to butyrylcholinesterase fluorescence was in reverse order. Metal ions may interact with cholinesterases, and increased the effects of forsythoside E to cholinesterases fluorescence, in order that Fe3+was the highest, followed by Cu2+, and Mg2+. A forsythoside E-butyrylcholinesterase complex at stoichiometric ratio of 1:1 was spontaneously formed, and the static quenching was the main quenching mode in the process of forsythoside E binding with butyrylcholinesterase. TheKvalues of two complexes were pretty much the same, suggesting that the interaction between cholinesterases and forsythoside E was almost unaffected by acid-base environment and metal ions. Thennumbers of two cholinesterases approximately equaled to one, indicating that there was only one site on each cholinesterase applicable for forsythoside E to bind to.

The Interaction Between Two Metabolites of Polygala tenuifolia and Cholinesterases.

OBJECTIVE: The work aimed to compare the binding between the two main components of Polygala tenuifolia Willd. and two cholinesterases (ChEs) by using a variety of spectral techniques. METHODS: Two main components of Polygala tenuifolia Willd. included Tenuifolin (Ten) and Onjisaponin B (Onj B), and two ChEs included acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). RESULTS: The UV-visible absorption spectra results showed that Ten had no effect on the structure of ChEs, and the combination of Onj B with ChEs changed its structure. Onj B statically quenched the endogenous fluorescence of both of ChEs, Ten dynamically quenched the endogenous fluorescence of AChE with no effect on BChE. The fluorescence quenching rate of ChEs by Onj B was much higher than that of AChE by Ten, and only one binding site of each protein spontaneously interacted with the compound to bind to or collide. Synchronous fluorescence results showed that Ten and Onj B quenched the fluorescence intensity by affecting tryptophan and tyrosine residues in cholinesterases, respectively. Hydrophobic force played an important role in the interaction between Ten and AChE, and van der Waals force and hydrogen bond were the main driving forces for the binding of Onj B to ChEs. The Enzyme activity test showed that Onj B inhibited ChE activity, and Ten never inhibited ChE activity. CONCLUSION: Onj B has the potential to inhibit ChE activity and increase the neurotransmitter acetylcholine content in the nerve system, improving the Alzheimer's disease (AD).

Humanin protects cortical neurons from calyculin A-induced neurotoxicities by increasing PP2A activity and SOD.

BACKGROUND: Humanin (HN) is an extensive neuroprotective peptide. This study aims to investigate the neuroprotective effects of HN on Calyculin A (CA)-induced neurotoxicities in cortical neurons and the underlying mechanism. METHODS: CA was added into the cultured cortical neurons to induce neurotoxicity. Cortical neurons were preincubated with HN which plays a protective role. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and Calcein-AM were applied to evaluate the neural insults. Caspase 3 signal and Tunnel were performed to test neural apoptosis. Western blot analysis was used to detect the expressions of phosphorylated tau. The corresponding kits were used to measure the contents of malondialdehyde (MDA) and superoxide dismutase (SOD), and the activity of PP2A, respectively. RESULTS: HN preincubation preserved cell viability, protected the neurons, alleviated oxidative stress, and reserved PP2A activity. It also blocked tau overphosphorylation at Ser199/202, Ser396, and Thr231 sites and protected neurons against CA-induced insults. CONCLUSION: These results suggest that HN may serve as a potential therapeutic agent to prevent the pathological changes induced by CA via modulating the activity of PP2A and oxidative stress in neurodegenerative diseases.

Metabolic profile and underlying antioxidant improvement of Ziziphi Spinosae Folium by human intestinal bacteria.

Ziziphi Spinosae Folium, the leaf of Ziziphus jujuba Mill. Var. spinosa (Bunge) Hu ex H. F. Chou (LZJS), is currently used as a healthy tea in China. This study evaluated the chemical components and antioxidant activities of LZJS flavonoid (LZJSF) and fermented LZJSF (FLZJSF) using human intestinal bacteria (HIB) through dynamic fermentation. Eighteen flavonoids were simultaneously identified in LZJSF using UHPLC-Q-Orbitrap-MS method, nine of which were targeted for a HIB metabolism study. Seven small phenolic acids were identified in FLZJSF. Not only at chemical level but also at PC12 cell level, FLZJSF samples fermented for 4 and 6 h showed significant positive correlation between their activities and flavonoid aglycones, which were transformed from LZJSF. However, FLZJSF samples (8 h and longer time) mainly contained phenolic acids and indicated weak activities. Thus, LZJSF was found to result in increased antioxidant activity and could be commercially utilized as a novel functional food.

Determination of five neurotransmitters in the rat brain for the study of the hypnotic effects of Ziziphi Spinosae Semen aqueous extract on insomnia rat model by UPLC-MS/MS.

Ziziphi Spinosae Semen (ZSS) has been used for treatment of insomnia in China for centuries. To reveal the influence of insomnia on the levels of the neurotransmitters including serotonin (5-HT), glutamic acid (Glu), γ-aminobutyric acid (GABA), noradrenaline (NE) and dopamine (DA), and to study the role of ZSS aqueous extract in the treatment of insomnia, an UPLC-ESI- MS/MS method was developed and validated for simultaneous determination of five neurotransmitters in the rat brain. The brain samples were pretreated by one-step direct protein precipitation with acetonitrile. The analytes were detected in positive mode with multiple reaction monitoring (MRM) and the procedure was completed in less than 10 min. The method showed a good linearity (R2 > 0.9967) with the other validation parameters were within acceptance range. The results indicated that the concentration of 5-HT, GABA and DA is significantly lower (P < 0.01) in para-chlorophenylalanine (PCPA)-induced insomnia rat model group, while Glu and NE significantly higher than those in control group (P < 0.01). Treatment with ZSS aqueous extract (4 or 8 g·kg-1·d-1 for seven days) could ameliorate the symptoms of insomnia by significantly changing the levels of the neurotransmitter parameters mentioned above. The data obtained in this study demonstrate that ZSS aqueous extract could ameliorate the symptoms of insomnia by modulating the levels of monoamines and amino acid neurotransmitters in the brain.

Comparison of the Quenching Effects of Two Main Components of Ziziphi Spinosae Semen on Serum Albumin Fluorescence.

Swertisin (6-glucosyl flavonoid) and spinosin (2″-ß-O-glucopyranosyl swertisin) are two main components from Ziziphi Spinosae Semen, with anti-anxiety and hypnosis effects. The paper aims to compare the differences between the two compounds binding with serum albumins (BSA and HSA). Swertisin and spinosin statically quench intrinsic fluorescence of serum proteins by binding to proteins to form complexes. The fluorescence quenching rates of BSA induced by swertisin or spinosin are faster than those of HSA resulted by swertisin or spinosin, respectively. Each serum protein has only one binding site respectively accessible to the two compounds. Hydrophobic force and hydrogen bond play the important roles during the biding process of swertisin with proteins, but van der Waals force and hydrogen bond are major driving forces for spinosin binding to proteins. Synchronous fluorescence data show that spinosin binds to BSA and HSA and thus changes Tyr and Trp residue microenvironments, and has a greater effect on the latter. Compared with swertisin, spinosin has a stronger effect on the α-helix of proteins. But the distance between swertisin and proteins is slightly closer than spinosin. These findings will contribute to further understand the reaction of Ziziphi Spinosae Semen in the liver phase I oxidation, intestinal hydrolysis and deparaffin metabolism.

Different effects of Forsythia suspensa metabolites on bovine serum albumin (BSA).

Forsythia suspensa metabolites have many bioactivities, such as selective immuno suppression, antioxidation, anti-hepatic injury, etc. In the present study, the interactions of the three metabolites with BSA have been investigated in a buffer (pH 7.40) using multi-spectroscopic techniques in combination with molecular docking methods. Two isoformers, forsythoside A and forsythoside I can statically quench BSA intrinsic fluorescence by forming the complexes with BSA at stoichiometric ratio of 1:1 that is again proved by UV-visible absorption. During the binding, the proportion of α-helix in BSA increases, the microenvironment around Tryptophan 213 changes and FRET is one of the major factors to quench fluorescence. Forsythoside E forms BSA-forsythoside E complex (1:1) and thus enhances the intrinsic fluorescence of BSA. During the process, forsythoside E affects not only Tryptophan residues but also Tyrosine residues so that the conformation of BSA is consequently changed. All above binding processes are spontaneous mainly through hydrogen bonding and the hydrophobic force interaction, which is supported by docking analysis and thermodynamic parameters. In addition, three compounds do not induce BSA aggregation. These findings are beneficial to understand the detailed information of the interactions of Forsythia suspensa metabolites with BSA.

Forsythiaside prevents ß-amyloid-induced hippocampal slice injury by upregulating 2-arachidonoylglycerol via cannabinoid receptor 1-dependent NF-κB pathway.

In the study, the neuroprotectivities of forsythiaside, a main constituent of Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese), were investigated in the hippocampal slices. Forsythiaside suppressed the overexpression of cyclooxygenase-2 (COX-2) and monoacylglycerol lipase (MAGL) proteins induced by ß-amyloid (Aß25-35) to upregulate the levels of 2-arachidonoylglycerol (2-AG), an endogenous endocannabinoids. Then the inhibition of forsythiaside on COX-2 was deeply studied by the molecular docking. Forsythiaside prevented neuroinflammation and apoptosis from Aß25-35 insults, and this action appeared to be mediated via cannabinoid receptor 1 (CB1R)-dependent nuclear factor-κB (NF-κB) signaling pathways. More importantly, forsythiaside functionally improved Aß25-35-induced learning and memory deficits, which was indicated by long term potentiation (LTP). Taken together, forsythiaside may have therapeutic potential for Alzheimer's diseases (AD) by increasing the levels of 2-AG.

Study of the interactions of forsythiaside and rutin with acetylcholinesterase (AChE).

Acetylcholinesterase (AChE) inhibitors have been considered as candidates for the treatment of Alzheimer's disease (AD) and have been utilized in clinical trials. In the present study, the interactions of forsythiaside and rutin with AChE have been investigated, after discovering the inhibitory AChE activity of the two compounds. Forsythiaside and rutin both can bind to AChE to form forsythiaside-AChE and rutin-AChE complex, and thus quench the intrinsic fluorescence of AChE. The quenching mechanism, the binding sites, the binding forces, the binding constants and the energy transfer involved were studied in details. Forsythiaside and rutin show some properties in common, including the stoichiometric binding ratio of 1:1 with AChE and the full quenching of AChE fluorescence. At the same time, the two compounds distinctly present some different characters, for example, the binding constant of rutin is less than that of forsythiaside, and the interaction force and the affinity between forsythiaside and AChE are much bigger than that of rutin. Spectroscopy data and docking analysis powerfully support the findings that forsythiaside inhibit AChE activity more strongly than rutin. The current study will provide the better understanding on the nature of the possible interactions between forsythiaside and rutin with AChE.

An anthraquinone compound and its protective effects against homocysteine-induced cytotoxicity and oxidative stress.

In this work, we designed an anthraquinone derivative: 1,4-diacrylateanthracene-9,10-dione (DAAD) with antioxidant activity for preventing Alzheimer's disease (AD) through preventing the neurotoxicity of Homocysteine (Hcy). This compound has very low cytotoxicity and protects the cells against Hcy-induced cytotoxicity and oxidative stress. Thus, maybe DAAD can be used as a potential reagent to preventing AD. In addition, we investigated the UV-Vis and fluorescence spectra of DAAD in PBS (pH 7.29)/DMSO (v/v, 1:1) solution for detecting Hcy, and the detection limit of DAAD for Hcy was found to be 0.121 µM. Thus, DAAD also can be used to monitor the Hcy level in plasma and cells.

Comparison of neuroprotective effect of Forsythia suspensa leaf extract and forsythiaside, one of its metabolites.

As one of authentic Chinese medicinal materials in Shanxi Province, leaves of Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese) have been used as alternative for the relief or treatment of inflammation. The study aimed to compare the neuroprotective effects of extract from F. suspensa leaves and forsythiaside against inflammation and apoptosis induced by lipopolysaccharide (LPS) on hippocampal slices. The cultured hippocampal slices were treated with extract (5 and 50 µg/mL) and forsythiaside (5 and 50 µg/mL), respectively, followed by LPS treatment for 24 h. The present study showed that 60% ethanol extract of F. suspensa leaves exhibited better neuroprotective effect than equivalent forsythiaside.

Protective effects of Forsythoside A on amyloid beta-induced apoptosis in PC12 cells by downregulating acetylcholinesterase.

Increasing the acetylcholine level and fighting the neuroinflammation has always been taken as a treatment strategy for Alzheimer's disease (AD). Forsythoside A is a major component in Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese) that has been traditionally used as Chinese herbal medicine to treat the inflammation in China. This study examined the inhibitory acetylcholinesterase activities of Forsythoside A at chemical and biological level. Forsythoside A inhibited acetylcholinesterase in a mixed type of inhibition, with Ki of 47.68µM. Docking analysis strongly supported these findings. In PC12 cells Forsythoside A increased cell viability and suppressed acetylcholinesterase increased by Aß25-35, thus alleviated the corresponding apoptosis. Taken together, these results suggest that Forsythoside A has the protective effects on Aß25-35-induced apoptosis in PC12 cells by downregulating acetylcholinesterase, making it a potential functional food ingredient or drug candidate for the treatment of AD.

Deep Eutectic Solvent-Based Microwave-Assisted Method for Extraction of Hydrophilic and Hydrophobic Components from Radix Salviae miltiorrhizae.

Deep eutectic solvents (DESs) have attracted significant attention as a promising green media. In this work, twenty-five kinds of benign choline chloride-based DESs with microwave-assisted methods were applied to quickly extract active components from Radix Salviae miltiorrhizae. The extraction factors, including temperature, time, power of microwave, and solid/liquid ratio, were investigated systematically by response surface methodology. The hydrophilic and hydrophobic ingredients were extracted simultaneously under the optimized conditions: 20 vol% of water in choline chloride/1,2-propanediol (1:1, molar ratio) as solvent, microwave power of 800 W, temperature at 70 °C, time at 11.11 min, and solid/liquid ratio of 0.007 g·mL-1. The extraction yield was comparable to, or even better than, conventional methods with organic solvents. The microstructure alteration of samples before and after extraction was also investigated. The method validation was tested as the linearity of analytes (r² > 0.9997 over two orders of magnitude), precision (intra-day relative standard deviation (RSD) < 2.49 and inter-day RSD < 2.96), and accuracy (recoveries ranging from 95.04% to 99.93%). The proposed DESs combined with the microwave-assisted method provided a prominent advantage for fast and efficient extraction of active components, and DESs could be extended as solvents to extract and analyze complex environmental and pharmaceutical samples.

Design of ratiometric emission probe with visible light excitation for determination of Ca2+ in living cells.

An organic salt as a fluorescent probe based on intramolecular charge transfer for Ca(2+) determination is developed. Ca(2+) can be detected by ratiometric emission at 490 and 594 nm with an excitation wavelength of 405 nm. This probe is highly selective for Ca(2+) over other divalent metal cations and displays a large Stokes shift of 189 nm that can avoid interference of the excitation light beam and autofluorescence of biological samples. The dissociation constant for Ca(2+) is 2.25 ± 0.47 µM and pertinent to Ca(2+) detection in cellular resting and dynamic states. The probe demonstrates its application in monitoring Ca(2+) in living cells under confocal microscopic imaging.

Neuregulin-1 impairs the long-term depression of hippocampal inhibitory synapses by facilitating the degradation of endocannabinoid 2-AG.

Endocannabinoids play essential roles in synaptic plasticity; thus, their dysfunction often causes impairments in memory or cognition. However, it is not well understood whether deficits in the endocannabinoid system account for the cognitive symptoms of schizophrenia. Here, we show that endocannabinoid-mediated synaptic regulation is impaired by the prolonged elevation of neuregulin-1, the abnormality of which is a hallmark in many patients with schizophrenia. When rat hippocampal slices were chronically treated with neuregulin-1, the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids, was enhanced due to the increased expression of its degradative enzyme, monoacylglycerol lipase. As a result, the time course of depolarization-induced 2-AG signaling was shortened, and the magnitude of 2-AG-dependent long-term depression of inhibitory synapses was reduced. Our study reveals that an alteration in the signaling of 2-AG contributes to hippocampal synaptic dysfunction in a hyper-neuregulin-1 condition and thus provides novel insights into potential schizophrenic therapeutics that target the endocannabinoid system.

Inhibition of COX-2 expression by endocannabinoid 2-arachidonoylglycerol is mediated via PPAR-γ.

BACKGROUND AND PURPOSE: Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB(1) receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs). EXPERIMENTAL APPROACH: By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture. KEY RESULTS: Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1ß (IL-1ß) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1ß- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB(1) receptor. CONCLUSIONS AND IMPLICATIONS: Our results suggest that CB(1) receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults.

Enhancement of lanthanum (III) on sodium currents in acutely isolated hippocampal CA1 neurons of rat.

The effects of lanthanum (III) (La(3+)) on voltage-gated sodium channel currents (I (Na)) in freshly dissociated rat hippocampal CA1 neurons were studied using the whole-cell patch clamp techniques. La(3+) reversibly enhanced I (Na) in a concentration- and voltage-dependent manner. The 50% enhancement concentration (EC(50)) of La(3+) on I (Na) was 9.93 microM. In addition, 10 microM La(3+) shifted the steady state activation curve of I (Na) towards positive potential and the steady state inactivation curve towards negative potential without changing the slope factor. These results indicated that La(3+) could increase the amplitudes of I (Na) and change the activation and inactivation courses of I (Na) even in very low concentration.

Effects of 3-benzidino-6-phenylpyridazine, as an acetylcholinesterase inhibitor, on outward potassium current in acutely isolated rat hippocampal pyramidal neurons.

As an acetylcholinesterase (AChE) inhibitor, the effects of 3-benzidino-6-phenylpyridazine (BPP) on outward potassium current including delayed rectifier potassium current (I(K(DR))) and transient outward potassium current (I(K(A))) in acutely isolated rat hippocampal pyramidal neurons were studied, using the whole cell patch-clamp technique. BPP reversibly inhibited electric eel AChE as an inhibitor, with IC(50) of 1.43 microM. BPP (0.10-100 microM) decreased I(K(DR)) and I(K(A)) in a concentration-dependent, voltage-independent and partial reversible manner, with IC(50) of 0.47 and 0.31 microM, respectively. 10 microM BPP did not affect steady-state activation of I(K(DR)) and I(K(A)). In addition, 10 microM BPP shifted the voltage dependence of steady-state inactivation of I(K(A)) towards negative potential. In conclusion, BPP potently inhibits I(K(DR)) and I(K(A)) in rat hippocampal pyramidal neurons, which may contribute to BPP's restoring the damaged central nervous system.