Novel Guanidinium-Functionalized Stigmasterol for Bile Salt Binding and Serum Cholesterol Reduction: Synthesis, Interaction Mechanisms, and In Vivo Function.

A novel amphiphilic guanidyl-functionalized stigmasterol hydrochloride (GFSH) was designed and synthesized as bile salt sequestrants for cholesterol reduction. GFSH exhibited a considerable in vitro capacity for bile salt binding in gastrointestinal digestion and alleviated hypercholesterolemia in vivo. GFSH spontaneously interacted with sodium cholate via synergistic electrostatic, hydrophobic, and hydrogen-bonding interactions. The effects of GFSH on serum cholesterol reduction in mice fed a high-fat-high-cholesterol diet were explored by measuring the expression of key transcription factors related to bile acid metabolism. GFSH produced a dose-dependent reduction in weight gain, hepatic fat accumulation, and fecal and blood markers. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analyses demonstrated GFSH-induced expression of hepatic CYP7A, LXRα, and LDL-R. GFSH exerts the cholesterol-lowering activity by inducing the bile acid metabolism.

Long-term hybrid stability and matrix metalloproteinase inhibition by fucosterol in resin-dentin bonding biomechanics.

Fucosterols have been widely studied for their antioxidant, anticancer, and anti-inflammatory properties. However, they have not yet been studied in the field of dentistry. This study aimed to determine whether pretreatment of dentin with fucosterol before resin restoration enhances bond stability in resin-dentin hybrid layers. After applying 0.1, 0.5, and 1.0 wt% fucosterol to demineralized dentin, microtensile bond strength (MTBS) and nanoleakage tests were performed before and after collagenase aging, and the surface was observed using scanning electron microscope (SEM). The fucosterol-treated group showed better bond strength and less nanoleakage both before and after collagenase aging, and the corresponding structures were confirmed using SEM. MMP zymography confirmed that the activity of MMPs was relatively low along the concentration gradient of fucosterol, and the FTIR analysis confirmed the production of collagen crosslinks. In addition, fucosterol exhibits cytotoxicity against Streptococcus mutans, the main cause of dental decay. The results of this study suggest that fucosterol pretreatment improves bond strength and reduces nanoleakage at the resin-dentin interface, possibly through a mechanism involving collagen cross-link formation via the inhibition of endogenous and exogenous MMP activity. This study demonstrates the potential of fucosterol as an MMP inhibitor in dentin, which contributes to long-term resin-dentin bond stability and can be used as a restorative material.

Improvement and prediction of the extraction parameters of lupeol and stigmasterol metabolites of Melia azedarach with response surface methodology.

BACKGROUND: Melia azedarach is known as a medicinal plant that has wide biological activities such as analgesic, antibacterial, and antifungal effects and is used to treat a wide range of diseases such as diarrhea, malaria, and various skin diseases. However, optimizing the extraction of valuable secondary metabolites of M. azedarach using alternative extraction methods has not been investigated. This research aims to develop an effective, fast, and environmentally friendly extraction method using Ultrasound-assisted extraction, methanol and temperature to optimize the extraction of two secondary metabolites, lupeol and stigmasterol, from young roots of M. azedarach using the response surface methodology. METHODS: Box-behnken design was applied to optimize different factors (solvent, temperature, and ultrasonication time). The amounts of lupeol and stigmasterol in the root of M. azedarach were detected by the HPLC-DAD. The required time for the analysis of each sample by the HPLC-DAD system was considered to be 8 min. RESULTS: The results indicated that the highest amount of lupeol (7.82 mg/g DW) and stigmasterol (6.76 mg/g DW) was obtained using 50% methanol at 45 °C and ultrasonication for 30 min, and 50% methanol in 35 °C, and ultrasonication for 30 min, respectively. Using the response surface methodology, the predicted conditions for lupeol and stigmasterol from root of M. azedarach were as follows; lupeol: 100% methanol, temperature 45 °C and ultrasonication time 40 min (14.540 mg/g DW) and stigmasterol 43.75% methanol, temperature 34.4 °C and ultrasonication time 25.3 min (5.832 mg/g DW). CONCLUSIONS: The results showed that the amount of secondary metabolites lupeol and stigmasterol in the root of M. azedarach could be improved by optimizing the extraction process utilizing response surface methodology.

Anti-Inflammatory Activity and Acute Toxicity Of Pereskia aculeata, In Zophobas morio Larvae.

Pereskia aculeata has been widely investigated due to its anti-inflammatory potential. Among the metabolites found in this species are the phytosterols beta-sitosterol (ß-SIT) and stigmasterol (STIG). The objective of the study was to evaluate the anti-inflammatory and toxicity activities of the hexane partition of P. aculeata (PHEX), as well as ß-SIT and STIG. PHEX was prepared and the phytosterols were quantified. In terms of toxicity against L929 fibroblast cells, PHEX showed toxicity up to 200 µg/mL; STIG and ß-SIT showed toxicity up to 25 µg/mL. PHEX inhibited 66 % of nitric oxide radicals, while STIG and ß-SIT inhibited 33.73 % and 34.94 %, respectively. In an anti-inflammatory test against Zophobas morio larvae, all samples significantly reduced hemocyte levels. Additionally, the LD50 values were calculated: 229.6 mg/kg for PHEX, 101.5 mg/kg for STIG, and 103.8 mg/kg for ß-SIT. In conclusion, the study indicates that the phytosterols present in PHEX may contribute to its anti-inflammatory activity.

In silico and network pharmacology analysis of fucosterol: a potent anticancer bioactive compound against HCC.

The Fucaceae family of marine brown algae includes Ascophyllum nodosum. Fucosterol (FSL) is a unique bioactive component that was identified through GC-MS analysis of the hydroalcoholic extract of A. nodosum. Fucosterol's mechanism of action towards hepatocellular cancer was clarified using network pharmacology and docking study techniques. The probable target gene of FSL has been predicted using the TargetNet and SwissTargetPred databases. GeneCards and the DisGNet database were used to check the targeted genes of FSL. By using the web programme Venny 2.1, the overlaps of FSL and HCC disease demonstrated that 18 genes (1.3%) were obtained as targeted genes Via the STRING database, a protein-protein interaction (PPI) network with 18 common target genes was constructed. With the aid of CytoNCA, hub genes were screened using the Cytoscape software, and the targets' hub genes were exported into the ShinyGo online tool for study of KEGG and gene ontology enrichment. Using the software AutoDock, a hub gene molecular docking study was performed. Ten genes, including AR, CYP19A1, ESR1, ESR2, TNF, PPARA, PPARG, HMGCR, SRC, and IGF1R, were obtained. The 10 targeted hubs docked with FSL successfully. The active components FSL of ASD, the FSL, are engaged in fatty liver disease, cancer pathways, and other signalling pathways, which could prove beneficial for the management of HCC.

Multi-targeted therapeutic potential of stigmasterol from the Euphorbia ammak plant in treating lung and breast cancer.

Cancer is the most prevalent disease globally, which presents a significant challenge to the healthcare industry, with breast and lung cancer being predominant malignancies. This study used RNA-seq data from the TCGA database to identify potential biomarkers for lung and breast cancer. Tumor Necrosis Factor (TNFAIP8) and Sulfite Oxidase (SUOX) showed significant expression variation and were selected for further study using structure-based drug discovery (SBDD). Compounds derived from the Euphorbia ammak plant were selected for in-silico study with both TNFAIP8 and SUOX. Stigmasterol had the greatest binding scores (normalized scores of -8.53 kcal/mol and -9.69 kcal/mol) with both proteins, indicating strong stability in their binding pockets throughout the molecular dynamics' simulation. Although Stigmasterol first changed its initial conformation (RMSD = 0.5 nm with the starting conformation) in SUOX, it eventually reached a stable conformation (RMSD of 1.5 nm). The compound on TNFAIP8 showed a persistent shape (RMSD of 0.35 nm), indicating strong protein stability. The binding free energy of the complex was calculated using the MM/GBSA technique; TNFAIP8 had a ΔGTOTAL of -24.98 kcal/mol, with TYR160 being the most significant residue, contributing -2.52 kcal/mol. On the other hand, the SUOX complex had a binding free energy of -16.87 kcal/mol, with LEU151 being the primary contributor (-1.17 kcal/mol). Analysis of the complexes' free energy landscape unveiled several states with minimum free energy, indicating robust interactions between the protein and ligand. In its conclusion, this work emphasises the favourable ability of Stigmasterol to bind with prospective targets for lung and breast cancer, indicating the need for more experimental study.

Conjugates of 1,3- and 1,2-Acylglycerols with Stigmasterol: Synthesis, NMR Characterization, and Impact on Lipid Bilayers.

The main aim of research was synthesis and spectroscopic characterization of new conjugates in which stigmasterol was linked via carbonate or succinyl linker with 1,3- and 1,2-acylglycerols of palmitic and oleic acid. Acylglycerols containing stigmasterol residue at internal position have been synthesized from 2-benzyloxypropane-1,3-diol or dihydroxyacetone. Their asymmetric counterparts containing stigmasterol residue attached to sn-3 position have been obtained from (S)-solketal. Eight synthesized conjugates were used to create the liposomes as nanocarriers of phytosterols to increase their stability and protect them from degradation during thermal-oxidative treatments. Fluorimetric and ATR-FTIR methods were used to determine the impact of synthesized conjugates on the physicochemical properties of the lipid bilayer. The results indicate that conjugates with palmitic acid are better candidates for use as the potential stigmasterol nanocarriers compared to those with oleic acid because they increase the stiffness of the lipid bilayer and temperature of the main phase transition. The obtained results are the first step in designing of stigmasterol-enriched liposomal carriers with higher thermo-oxidative stability for their potential use in the food industry.

Unraveling the Role of Scutellaria baicalensis for the Treatment of Breast Cancer Using Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation.

Scutellaria baicalensis is often used to treat breast cancer, but the molecular mechanism behind the action is unclear. In this study, network pharmacology, molecular docking, and molecular dynamics simulation are combined to reveal the most active compound in Scutellaria baicalensis and to explore the interaction between the compound molecule and the target protein in the treatment of breast cancer. In total, 25 active compounds and 91 targets were screened out, mainly enriched in lipids in atherosclerosis, the AGE-RAGE signal pathway of diabetes complications, human cytomegalovirus infection, Kaposi-sarcoma-associated herpesvirus infection, the IL-17 signaling pathway, small-cell lung cancer, measles, proteoglycans in cancer, human immunodeficiency virus 1 infection, and hepatitis B. Molecular docking shows that the two most active compounds, i.e., stigmasterol and coptisine, could bind well to the target AKT1. According to the MD simulations, the coptisine-AKT1 complex shows higher conformational stability and lower interaction energy than the stigmasterol-AKT1 complex. On the one hand, our study demonstrates that Scutellaria baicalensis has the characteristics of multicomponent and multitarget synergistic effects in the treatment of breast cancer. On the other hand, we suggest that the best effective compound is coptisine targeting AKT1, which can provide a theoretical basis for the further study of the drug-like active compounds and offer molecular mechanisms behind their roles in the treatment of breast cancer.

Acylglycerols of Myristic Acid as New Candidates for Effective Stigmasterol Delivery-Design, Synthesis, and the Influence on Physicochemical Properties of Liposomes.

New carriers of phytosterols; acylglycerols containing natural myristic acid at sn-1 and sn-3 positions and stigmasterol residue linked to sn-2 position by carbonate and succinate linker have been designed and synthesized in three-step synthesis from dihydroxyacetone (DHA). The synthetic pathway involved Steglich esterification of DHA with myristic acid; reduction of carbonyl group of 1,3-dimyristoylpropanone and esterification of 1,3-dimyristoylglicerol with stigmasterol chloroformate or stigmasterol hemisuccinate. The structure of the obtained hybrids was established by the spectroscopic methods (NMR; IR; HRMS). Obtained hybrid molecules were used to form new liposomes in the mixture with model phospholipid and their effect on their physicochemical properties was determined, including the polarity, fluidity, and main phase transition of liposomes using differential scanning calorimetry and fluorimetric methods. The results confirm the significant effect of both stigmasterol-containing acylglycerols on the hydrophilic and hydrophobic region of liposome membranes. They significantly increase the order in the polar heads of the lipid bilayer and increase the rigidity in the hydrophobic region. Moreover, the presence of both acylglycerols in the membranes shifts the temperature of the main phase transition towards higher temperatures. Our results indicate stabilization of the bilayer over a wide temperature range (above and below the phase transition temperature), which in addition to the beneficial effects of phytosterols on human health makes them more attractive components of novel lipid nanocarriers compared to cholesterol.

Thermo-oxidative stability of asymmetric distigmasterol-modified acylglycerols as novel derivatives of plant sterols.

The study investigated the thermo-oxidative stability of distigmasterol-modified acylglycerols as a new structured acylglycerols. Samples were heated at 60 and 180 °C for 8 h. Their percentage degradation and products formed during heating were compared with free stigmasterol and stigmasteryl esters. The remaining of stigmasterol and fatty acid parts, the formation of stigmasterol oxidation products and the composition of polar and non-polar fractions were analysed using chromatographic methods. The cytotoxicity and genotoxicity were determined with the use of an MTT test and a comet assay, respectively. The highest stability during heating was observed for 2,3-distigmasterylsuccinoyl-1-oleoyl-sn-glycerol (dStigS-OA) and the lowest for 2,3-distigmasterylcarbonoyl-1-oleoyl-sn-glycerol (dStigC-OA). Data showed that the formation of thermo-oxidative degradation products is affected by the temperature and chemical structure of lipids present in the molecule. The dStigMAs bonded by a succinate linker and products formed during their thermo-oxidation showed no cytotoxic or genotoxic activity to normal human cells.

The potential mechanism of Bupleurum against anxiety was predicted by network pharmacology study and molecular docking.

Bupleurum chinense DC. (Chaihu) is a traditional Chinese medicine (TCM) used in the treatment of anxiety. But the anxiolytic mechanisms of bupleurum are still unclear. Therefore, this unknown is predicted by network pharmacology study with molecular docking in the present study. The components of bupleurum were obtained from the databases. Genes associated with components and disease were also provided by databases. Overlapping genes between components and disease were analyzed. The network of medicine-components-targets-disease was constructed, visualized, and analyzed. Protein-protein interaction (PPI), gene ontology (GO), pathway enrichment (KEGG) and molecular docking were conducted to predict the potential mechanisms of bupleurum on anxiety. A total of 9 bioactive components derived from bupleurum with 80 target genes were involved in anxiety. Neurotransmitter receptor activity, G protein-coupled amine receptor activity, regulation of blood circulation, neuroactive ligand-receptor interaction, calcium signaling pathway and salivary secretion may play significant roles in the anxiolytic of bupleurum. Molecular docking implicated that ACHE and MAOA showed high affinity for stigmasterol. Based on network pharmacology study with molecular docking, multi-component-multi-target-multi-pathway action mode of bupleurum on anxiety was elaborated. Stigmasterol might be the core bioactive component, while ACHE and MAOA might be the core target genes in the pharmacological profile of bupleurum on anxiety.

The edible seaweed Laminaria japonica contains cholesterol analogues that inhibit lipid peroxidation and cyclooxygenase enzymes.

In this study, 5 sterols were isolated and purified from Laminaria japonica, commonly known as edible brown seaweed, and their structures were identified based on detailed chemical methods and spectroscopic analyses. Spectroscopic analyses characterized 5 sterols as 29-Hydroperoxy-stigmasta-5,24(28)-dien-3ß-ol, saringosterol (24-vinyl-cholest-5-ene-3ß,24-diol), 24-methylenecholesterol, fucosterol (stigmasta-5,24-diene-3ß-ol), and 24-Hydroperoxy-24-vinyl-cholesterol. The bioactivities of these sterols were tested using lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Fucosterol exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. Saringosterol, 24-methylenecholesterol and fucosterol showed higher LPO inhibitory activity at >50% than the other compounds. In addition, the results of molecular docking revealed that the 5 sterols were located in different pocket of COX-1 and -2 and fucosterol with tetracyclic skeletons and olefin methine achieved the highest binding energy (-7.85 and -9.02 kcal/mol) through hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica and its significant anti-inflammatory and antioxidant activity.

Distigmasterol-Modified Acylglycerols as New Structured Lipids-Synthesis, Identification and Cytotoxicity.

Plant sterols, also referred as phytosterols, have been known as bioactive compounds which have cholesterol-lowering properties in human blood. It has been established that a diet rich in plant sterols or their esters alleviates cardiovascular diseases (CVD), and also may inhibit breast, colon and lung carcinogenesis. Phytosterols, in their free and esterified forms, are prone to thermo-oxidative degradation, where time and temperature affect the level of degradation. Looking for new derivatives of phytosterols with high thermo-oxidative stability for application in foods, our idea was to obtain novel structured acylglycerols in which two fatty acid parts are replaced by stigmasterol residues. In this work, asymmetric (1,2- and 2,3-) distigmasterol-modified acylglycerols (dStigMAs) were synthesized by the covalent attachment of stigmasterol residues to sn-1 and sn-2 or sn-2 and sn-3 positions of 3-palmitoyl-sn-glycerol or 1-oleoyl-sn-glycerol, respectively, using a succinate or carbonate linker. The chemical structures of the synthesized compounds were identified by NMR, HR-MS, and IR data. Moreover, the cytotoxicity of the obtained compounds was determined. The dStigMAs possessing a carbonate linker showed potent cytotoxicity to cells isolated from the small intestine and colon epithelium and liver, whereas the opposite results were obtained for compounds containing a succinate linker.

Stigmasterol alleviates interleukin-1beta-induced chondrocyte injury by down-regulatingsterol regulatory element binding transcription factor 2 to regulateferroptosis.

Stigmasterol (STM), one of the main active components of Achyranthes bidentata, has been shown to effectively inhibit proinflammatory factors and matrix degradation in chondrocytes. However, the effect of STM on interleukin (IL)-1ß-induced chondrocytes and its specific mechanism remain unclear. The purpose of the present study was to explore the effect and mechanism of sterol regulatory element binding transcription factor 2 (SREBF2) on IL-1ß induced chondrocytes in the presence of STM. CCK-8 was used to detect the effect of STM on the cell viability of mouse chondrogenic cells (ATDC5). After ATDC5 cells were induced by IL-1ß, the expression of SREBF2 in osteoarthritis cells was detected by RT-qPCR. The content of iron ion in the cells was detected by using an iron colorimetric assay kit. After further transfection of a SREBF2 overexpressing vector (Oe-SREBF2) or addition of a ferroptosis inhibitor, the expression levels of inflammation and matrix degradation-related proteins were detected via Western blotting. The levels of oxidative stress in cells were determined by using an ELISA kit. The results revealed that STM had no significant effect on the viability of ATDC5 cells. STM reduced IL-1ß-induced ATDC5 cell damage and ferroptosis through SREBF2 and enhanced the inhibitory effect of ferroptosis inhibitors on IL-1ß-induced ATDC5 cell injury. The present data suggest that STM attenuated chondrocyte injury induced by IL-1ß by regulating ferroptosis via down-regulation of SREBF2, and may have potential as a novel therapeutic method for knee osteoarthritis.

Pectin-zein based stigmasterol nanodispersions ameliorate dextran sulfate sodium-induced colitis in mice.

Due to the insolubility of phytosterols in both water and oil, their application in the medicine and health and food industries is limited. In this study, zein and pectin were selected as wall materials of phytosterol nanoparticles to enhance the solubility and bioactivity of phytosterols. The colitis-inhibitory effects of zein-based stigmasterol nanodispersions (ZNs) and zein/pectin-based stigmasterol nanodispersions (ZPNs) were investigated in the sodium dextran sulfate (DSS)-induced colitis mouse model. The results showed that ZPNs' therapeutic effect was better than that of ZNs. According to electron microscopy observation, pectin adsorbed on the surface of zein appeared to form an elastic network structure, which increased the stability of stigmasterol nanodispersions. ZPNs not only relieved the adverse physiological symptoms of colitis in mice, but additionally prevented colonic length shortening and reduced fecal hemoglobin content. Immunohistochemical analysis showed that ZPNs could alleviate colitis by inhibiting the NF-κB signaling pathway involved in the expression of inflammatory factors TNF-α, IL-6, IL-1ß, CSF-1 and coenzyme COX-2. This study suggests that supplement of nano-embedded stigmasterol based on zein and pectin has a positive therapeutic effect on alleviating colitis in mice. Such activities of nano-embedded stigmasterol in humans remain to be investigated.

Phytosterols: Potential Metabolic Modulators in Neurodegenerative Diseases.

Phytosterols constitute a class of natural products that are an important component of diet and have vast applications in foods, cosmetics, and herbal medicines. With many and diverse isolated structures in nature, they exhibit a broad range of biological and pharmacological activities. Among over 200 types of phytosterols, stigmasterol and ß-sitosterol were ubiquitous in many plant species, exhibiting important aspects of activities related to neurodegenerative diseases. Hence, this mini-review presented an overview of the reported studies on selected phytosterols related to neurodegenerative diseases. It covered the major phytosterols based on biosynthetic considerations, including other phytosterols with significant in vitro and in vivo biological activities.

24(R, S)-Saringosterol - From artefact to a biological medical agent.

Enhancing the cholesterol turnover in the brain via activation of liver x receptors can restore memory in a mouse model for Alzheimer's disease. The edible Asian brown alga Sargassum fusiforme (Hijiki) contains high amounts of oxysterols such as (3ß, 24ξ)-stigmasta-5, 28-dien-3, 24-diol (24[R, S]-saringosterol) that are a potent liver x receptor agonists. We aimed to find native European seaweed species with contents of 24(R, S)-saringosterol that are comparable to those found in Sargassum fusiforme. Additionally, we hypothesize that seasonal variations modify the amount of 24(R, S)-saringosterol in seaweeds. Sterols and oxysterols were extracted with chloroform/methanol from various seaweed species harvested in the Eastern Scheldt in different seasons between October 2016 and September 2017. Identification and quantification of the lipids was performed by gas chromatography- mass spectrometry and gas chromatography- flame ionization detection. We confirmed that brown algae Undaria pinnatifida harvested in February and Sargassum muticum harvested in October contained the highest amounts of 24(R, S)-saringosterol (32.4 ± 15.25 µg/g, mean ± S.D. and 32.95 ± 2.91 µg/g, respectively) and its precursor fucosterol (1.48 ± 0.11 mg/g), higher than Sargassum fusiforme (20.94 ± 3.00 µg/g, mean ± S.D.), while Ascophyllum nodosum and Fucus vesiculosus and Fucus serratus contained amounts of 24(R, S)-saringosterol (22.09 ± 3.45 µg/g, 18.04 ± 0.52 µg/g and 19.47 ± 9.01 µg/g, mean ± S.D., respectively) comparable to Sargassum fusiforme. In other algae only minor amounts of these sterols were observed. The green algae Ulva lactuca contained only 0.29 mg/g fucosterol and 10.3 µg/g 24 (R, S)-saringosterol, while all investigated red algae did not contain any 24(R, S)-saringosterol or fucosterol. In the Eastern Scheldt algae harvested in September/October delivered the highest yield for 24(R, S)-saringosterol, with the exception of Undaria pinnatifida that showed the highest levels in February. We showed that exposure of lipid extracts of Ulva lactuca to sunlight at room temperature or in the presence of oxygen to UV-C light lead to the quantitative conversion of fucosterol into 24(R, S)-saringosterol. Exposing pure fucosterol to UV-light did not convert any fucosterol into 24(R, S)-saringosterol underscoring the requirement of seaweed constituents in the conversion of fucosterol into 24(R, S)-saringosterol. In conclusion, we showed that brown seaweeds harvested from the Eastern Scheldt contain amounts of 24(R, S)-saringosterol comparable to Sargassum fusiforme, varying per season and showing the highest amounts in spring. In accordance with these observations the amount of 24(R, S)-saringosterol in the brown seaweeds can be modulated by light.

Eustigmatophyte strains with potential interest in cancer prevention and treatment: partial chemical characterization and evaluation of cytotoxic and antioxidant activity.

The interest in bioactive compounds from microalgae is increasing since they have medicinal and nutritional areas. The present work aims to evaluate the potential pharmaceutical interest of extracts from three eustigmatophyte strains from the Coimbra Collection of Algae (ACOI): Chlorobotrys gloeothece, Chlorobotrys regularis and Characiopsis aquilonaris. Antioxidant and antiproliferative activities were determined as well as chlorophyll a, carotenoid and phenolic total contents. In addition, major pigments and sterols were identified and quantified. The three strains were grown until the stationary phase and then the biomass was extracted. Antioxidant activity was measured by TEAC, DPPH and FRAP assays and antiproliferative effect was assessed by the MTT method on MCF-7, PC-3 and NHDF cells. The pigment and phenolic total contents were determined by spectrophotometry. Of these strains, C. aquilonaris showed the highest antioxidant activity measured by TEAC and FRAP assays (23.98 ± 0.01 µmol TE eq g-1 DW and 42.57 ± 0.04 µmol TE eq g-1 DW, respectively), a selective effect in reduting MCF-7 cells proliferation and a larger amount of chlorophyll a, carotenoids and phenolic content (18.40 ± 0.00 µg chlorophyll a mg-1 DW, 2.27 ± 0.00 mg carotenoids g-1 DW and 6.23 ± 0.01 mg GAE g-1 DW, respectively). A positive correlation between chlorophyll a and TEAC assay was observed, as well as between carotenoids and TEAC and FRAP assays, suggesting these compounds as important contributors to significant antioxidant activity. Violaxanthin, cholesterol and stigmasterol were present in larger amount in C. aquilonaris while C. regularis showed a higher amount of ß-carotene. These results suggest that these three ACOI eustigmatophytes are promising for applications in the improvement of human health, particularly in cancer prevention and treatment.

Inhibitors of BRD4 protein from the roots of Astilbe grandis stapf ex E.H. Wilson.

A new monoterpene (1) along with eight known compounds were isolated from the roots of Astilbe grandis Stapf ex E.H. Wilson. Their structures were determined by extensive spectroscopic analysis and ECD experiments as (S)-3-(2-hydroxyethyl)-5-(2-methylprop-1-en-1-yl)furan-2(5H)-one (1), caffeic acid (2), mandelic acid (3), sonchifolinin B (4), α-viniferin (5), euscaphic acid (6), cianidanol (7), ß-sitosterol (8), and stigmasterol (9), respectively. Compounds 5 and 6 exhibited inhibitory effects against BRD4 protein with IC50 values of 13.20 and 17.39 µM, respectively. In vitro, compounds 5 and 6 showed moderate cytotoxicity to A549 cells, HCC827 cells and Hela cells with IC50 values ranging from 31.98 to 154.90 µM.

The potential LXRß agonist stigmasterol protects against hypoxia/reoxygenation injury by modulating mitophagy in primary hippocampal neurons.

BACKGROUND: Neuronal excitotoxicity induces a plethora of downstream signaling pathways, resulting in the calcium overload-induced excitotoxic cell death, a well-known phenomenon in cerebrovascular and neurodegenerative disorders. The naturally occurring phytosterol, stigmasterol (ST) is known for its potential role in cholesterol homeostasis and neuronal development. However, the ability of ST to protect against the induced excitotoxicity in hippocampal neurons has not been investigated yet. PURPOSE: The present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons. METHODS: After H/R, neurons were initially subjected to trypan blue exclusion assay for the assessment of cell viability. Live staining using fluorescence dyes namely JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide), DCFDA (2',7'-dichlorofluorescein diacetate) and FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) were used to measure MMP, ROS and synaptic vesicle pool size. Immunostaining was performed to analyze the expression levels of vesicular glutamate transporter 1 (VGLUT1), N-methyl-D-acetate receptor subunit 2B (GluN2B), LC3BII, p62, and PTEN induced protein kinase 1 (PINK1) in neuron after H/R. Western blotting was carried out to measure the protein expression of GluN2B. The molecular dynamics simulation was employed to elucidate the LXRß agonistic conformation of ST. RESULT: Pre-incubation of neuronal cultures with ST (20 µM) protected against excitotoxicity, and attenuated reactive oxygen species (ROS) generation, double-stranded DNA break, and mitochondrial membrane potential (MMP) loss. ST treatment also resulted in the downregulation of the expressions of VGLUT1 and GluN2B and the reduction of the size of recyclable synaptic vesicle (SV) pool. Like LXRß agonist GW3695, ST suppressed the expression of GluN2B. Furthermore, ST induced mitophagy through upregulating the expressions of LC3BII, p62, and PINK1. The molecular simulation study showed that ST interacted with the ligand binding domain of liver X receptor ß (LXRß), a known binding receptor of ST, through multiple hydrogen bonding. CONCLUSION: Collectively, these findings revealed that ST exhibited a promising neuroprotective effect by regulating both pre- and post-synaptic events following H/R, particularly, attenuation of GluN2B-mediated excitotoxicity and oxidative stress, and induction of mitophagy, and suggested that ST might be a therapeutic promise against ischemic stroke and its associated neurological disorders.