Prospects of Marine Sterols against Pathobiology of Alzheimer's Disease: Pharmacological Insights and Technological Advances.

Alzheimer's disease (AD) is a degenerative brain disorder characterized by a progressive decline in memory and cognition, mostly affecting the elderly. Numerous functional bioactives have been reported in marine organisms, and anti-Alzheimer's agents derived from marine resources have gained attention as a promising approach to treat AD pathogenesis. Marine sterols have been investigated for several health benefits, including anti-cancer, anti-obesity, anti-diabetes, anti-aging, and anti-Alzheimer's activities, owing to their anti-inflammatory and antioxidant properties. Marine sterols interact with various proteins and enzymes participating via diverse cellular systems such as apoptosis, the antioxidant defense system, immune response, and cholesterol homeostasis. Here, we briefly overview the potential of marine sterols against the pathology of AD and provide an insight into their pharmacological mechanisms. We also highlight technological advances that may lead to the potential application of marine sterols in the prevention and therapy of AD.

Recent advances in ABCG5 and ABCG8 variants.

PURPOSE OF REVIEW: In this review, we summarize the genetics and mechanisms of sitosterolemia and sterol trafficking, and provide an update on the understanding of the prevalence of ABCG5 and ABCG8 variants and their role in human disease. RECENT FINDINGS: Defects in ABCG5/G8 result in the accumulation of xenosterols. It had been previously thought that near total LoF of one of the proteins was required to cause pathology. However, recently there was the first report of a patient with Sitosterolemia who was heterozygous for mutations in both genes. Moreover, large population studies have demonstrated the even simple heterozygous carriers are associated with altered lipid profiles and cardiovascular risk. Broader screening has added to the rapidly growing list of gene variants indicating that the prevalence of ABCG5/G8 variants is higher than previous thought, especially in patients with hypercholesterolemia. SUMMARY: These findings support a strategy of measuring xenosterol levels in patients with hypercholesterolemia to screen for ABCG5/G8 variants, and then tailoring treatment with a sterol absorption inhibitor, like ezetimibe, where indicated. Xenosterol trafficking affects remnant clearance and maybe pathogenically linked to the increased risk of atherosclerosis.

Dietary Supplementation of Black Rice Anthocyanin Extract Regulates Cholesterol Metabolism and Improves Gut Microbiota Dysbiosis in C57BL/6J Mice Fed a High-Fat and Cholesterol Diet.

SCOPE: This study explores the beneficial effects of dietary supplementation of black rice anthocyanin extract (BRAE) on cholesterol metabolism and gut dysbiosis. METHODS AND RESULTS: C57BL/6J mice are grouped into the normal chow diet group (NCD), the high-fat and the cholesterol diet group (HCD), and three treatment groups feeding HCD supplemented with various dosage of BRAE for 12 weeks. Results reveal that BRAE alleviates the increased body weight, serum triglyceride (TG), total cholesterol (TC), non-high-density lipoprotein cholesterol levels (non-HDL-C), and increased fecal sterols excretion and caecal short-chain fatty acids (SCFAs) concentration in HCD-induced hypercholesterolemic mice. Moreover, BRAE decreases hepatic TC content through the fundamental regulation of body energy balance gene, adenosine 5'-monophosphate activated protein kinase α (AMPKα). Meanwhile, BRAE improves the genes expression involved in cholesterol uptake and efflux, and preserves CYP7A1, ATP-binding cassette subfamily G member 5/8 mRNA expression, and the relative abundance of gut microbiota. Additionally, the antibiotic treatment experiment indicates that the beneficial effects of BRAE in reducing hypocholesterolemia risk largely depends on the gut microbiota homeostasis. CONCLUSION: BRAE supplement could be a beneficial treatment option for preventing HCD-induced hypocholesterolemia and related metabolic syndromes.

Anti-Inflammatory Activities of Marine Algae in Neurodegenerative Diseases.

Neuroinflammation is one of the main contributors to the onset and progression of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Microglial and astrocyte activation is a brain defense mechanism to counteract harmful pathogens and damaged tissues, while their prolonged activation induces neuroinflammation that can trigger or exacerbate neurodegeneration. Unfortunately, to date there are no pharmacological therapies able to slow down or stop the progression of neurodegeneration. For this reason, research is turning to the identification of natural compounds with protective action against these diseases. Considering the important role of neuroinflammation in the onset and development of neurodegenerative pathologies, natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Marine organisms represent a huge source of natural compounds, and among them, algae are appreciated sources of important bioactive components such as antioxidants, proteins, vitamins, minerals, soluble dietary fibers, polyunsaturated fatty acids, polysaccharides, sterols, carotenoids, tocopherols, terpenes, phycobilins, phycocolloids, and phycocyanins. Recently, numerous anti-inflammatory compounds have been isolated from marine algae with potential protective efficacy against neuroinflammation. This review highlights the key inflammatory processes involved in neurodegeneration and the potential of specific compounds from marine algae to counteract neuroinflammation in the CNS.

Simultaneous determination of four furostanol glycosides in rat plasma by UPLC-MS/MS and its application to PK study after oral administration of Dioscorea nipponica extracts.

A novel, sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method for simultaneous quantification of four furostanol glycosides in rat plasma was established and validated. Ginsenoside Rb1 was used as an internal standard. Plasma samples were pretreated by liquid-liquid extraction with n-butanol and chromatographed on a C18 column (2.1×50 mm i.d., 2.6 µm) using a gradient elution program consisting of acetonitrile and water (containing 0.03% formic acid and 0.1 mM lithium acetate) at a flow rate of 0.4 mL/min. Lithium adduct ions were employed to enhance the response of the analytes in electrospray positive ionization mode and multiple reaction monitoring transitions were performed for detection. All calibration curves exhibited good linearity (r>0.999) over the range of 10-20,000 ng/mL for protodioscin and 2-4000 ng/mL for protogracillin, pseudoprotodioscin and pseudoprotogracillin. The recoveries of the whole analytes were more than 80.3% and exhibited no severe matrix effect. Meanwhile, the intra- and inter-day precisions were all less than 10.7% and accuracies were within the range of -8.1-12.9%. The four saponins showed rapid excretion and relative high plasma concentrations when the validated method was applied to the PK study of Dioscorea nipponica extracts by intragastric administration at low, medium and high dose to rats. Moreover, the T(1/2) and AUC(0-t) of each compound turned out to behave in a dose-dependent pattern by comparing them at different dose levels.

Pharmacokinetics, tissue distribution and excretion study of a furostanol glycoside-based standardized fenugreek seed extract in rats.

The furostanol glycoside isolated from the seed of fenugreek (SFSE-G) has an array of pharmacological activities. To date, no validated high-performance liquid chromatography (HPLC) method has been reported for quantification of SFSE-G in biological samples. Hence, the aim of the present study was to study the pharmacokinetics, tissue distribution and excretion profiles of SFSE-G after oral administration in rats. A rapid, sensitive, selective, robust and reproducible HPLC method has been developed for determination of SFSE-G in the rat biological samples. The chromatographic separation was accomplished on a reversed-phase C18 column using formic acid and acetonitrile (80:20) as mobile phase at a flow rate of 1.0 mL/min and 274 nm as a detection wavelength. The assay was linear for SFSE-G with the correlation coefficients (R(2)) >0.996. The analytes were stable during samples storage and handling, and no matrix effects were observed. After oral dosing of SFSE-G at a dose of 200 mg/kg, the elimination half-life was app. 40.10 h. It showed relatively slowly distribution and eliminated in urine and feces after 24 h, and could be detected until 108 h post-dosing. Following oral single dose (200 mg/kg), SFSE-G was detected in lung and brain which indicated that it could cross the blood-brain barrier. It is a major route of elimination is excretion through urine and feces. In conclusion, oral administration of SFSE-G showed slow distribution to tissues, such as lung and brain, but showed fast renal elimination.

Quantification of taraxasterol in rat plasma by LC/MS/MS: application to a pharmacokinetic study.

Taraxasterol, a pentacyclic triterpene from Taraxacum officinale, is one of the main active constituents of the herb. This study developed and validated a highly selective and sensitive liquid chromatography/tandem mass spectrometry for the determination of taraxasterol in rat plasma over the range of 9.0-5000 ng/mL. Chromatographic separation was achieved on a C18 (4.6 × 50 mm, 5.0 µm) column with methanol-isopropanol-water-formic acid (80:10:10:0.1, v/v/v/v) as mobile phase with an isocratic elution. The flow rate was 0.7 mL/min. After adding cucurbitacin IIa as an internal standard (IS), liquid-liquid extraction was used for sample preparation using ethyl acetate. The atmospheric pressure chemical ionization source was applied and operated in positive ion mode. Selected reaction monitoring mode was used for the quantification of transition ions m/z 409.4 → 137.1 for taraxasterol and m/z 503.4 → 113.1 for IS. The mean recoveries of taraxasterol in rat plasma ranged from 85.3 to 87.2%. The matrix effects for taraxasterol were between 98.5 and 104.0%. Intra- and inter-day precision were both <11.8%, and the accuracy of the method ranged from -7.0 to 12.9%. The method was successfully applied to a pharmacokinetic study of taraxasterol after oral administration of 7.75, 15.5 and 31.0 mg/kg in rats.

Mung bean decreases plasma cholesterol by up-regulation of CYP7A1.

Our results affirmed that supplementation of 1 or 2% mung bean could decrease plasma total cholesterol and triacylglycerol level. Mung bean increased mRNA 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. Most importantly, mung bean increased not only the protein level of cholesterol-7α-hydroxylase (CYP7A1) but also mRNA CYP7A1. It was concluded that the hypocholesterolemic activity of mung bean was most probable mediated by enhancement of bile acid excretion and up-regulation of CYP7A1.

Enhanced oral bioavailability and tissue distribution of a new potential anticancer agent, Flammulina velutipes sterols, through liposomal encapsulation.

This study innovatively investigated the anticancer effect of Flammulina velutipes sterols (FVSs), the in vivo pharmacokinetics, and the tissue distribution of FVS-loaded liposomes. The FVS consisting of mainly 54.8% ergosterol and 27.9% 22,23-dihydroergosterol exhibited evident in vitro antiproliferative activity (liver HepG-2, IC50 = 9.3 µg mL(-1); lung A549, IC50 = 20.4 µg mL(-1)). To improve the poor solubility of FVS, F. velutipes sterol liposome (FVSL) was originally prepared. The encapsulation efficiency of ergosterol was 71.3 ± 0.1% in FVSL, and the encapsulation efficiency of 22,23-dihydroergosterol was 69.0 ± 0.02% in FVSL. In comparison to its two free sterol counterparts, the relative bioavailability of ergosterol and 22,23-dihydroergosterol in FVSL was 162.9 and 244.2%, respectively. After oral administration in Kunming mice, the results of tissue distribution demonstrated that the liposomal FVS was distributed mostly in liver and spleen. The drug was eliminated rapidly within 4 h. These findings support the fact that FVS, a potential nutraceutical and an effective drug for the treatment of liver cancer, could be encapsulated in liposomes for improved solubility and bioavailability.

Cytotoxic effect of novel Flammulina velutipes sterols and its oral bioavailability via mixed micellar nanoformulation.

The aim of this study was to investigate the anti-tumor effect of sterols initially separated from Flammulina velutipes and the pharmacokinetics and tissue distribution after oral administration of F. velutipes sterol nanomicelles (FVSNs). F. velutipes sterol (FVS) consisted of mainly ergosterol (54.78%), 22,23-dihydroergosterol (27.94%) and ergost-8(14)-ene-3ß-ol (discovered for the first time in F. velutipes). In vitro cytotoxicity assay of FVS against U251 cells and HeLa cells showed that at 72h treatment, the FVS (IC50=23.42µg/mL) exhibited strong inhibitory effect against U251 cells, even overwhelmed the standard anti-tumor drug (5-fluorouracil) to an extent, while the HeLa cells were not significantly susceptible to the FVS. To improve the solubility and bioavailability of FVS, a model for insoluble anti-tumor drugs, FVSNs were prepared. In vitro characterization of FVSNs revealed satisfactory size distribution, loading capacity and encapsulation efficiency. Pharmacokinetic study in SD rats demonstrated that the mixed micellar nanoformulation significantly enhanced the bioavailability of FVS than free drug. Additionly, tissue distribution in mice manifested that the biodistribution of FVSNs as compared to the free FVS suspension were significantly improved. In conclusion, the nanomicelles developed in our study provided a promising delivery system for enhancing the oral bioavailability and selective biodistribution of FVS, a potential anti-tumor agent.

Steroid toxicity and detoxification in ascomycetous fungi.

In the last couple of decades fungal infections have become a significant clinical problem. A major interest into fungal steroid action has been provoked since research has proven that steroid hormones are toxic to fungi and affect the host/fungus relationship. Steroid hormones were found to differ in their antifungal activity in ascomycetous fungi Hortaea werneckii, Saccharomyces cerevisiae and Aspergillus oryzae. Dehydroepiandrosterone was shown to be the strongest inhibitor of growth in all three varieties of fungi followed by androstenedione and testosterone. For their protection, fungi use several mechanisms to lower the toxic effects of steroids. The efficiency of biotransformation in detoxification depended on the microorganism and steroid substrate used. Biotransformation was a relatively slow process as it also depended on the growth phase of the fungus. In addition to biotransformation, steroid extrusion out of the cells contributed to the lowering of the active intracellular steroid concentration. Plasma membrane Pdr5 transporter was found to be the most effective, followed by Snq2 transporter and vacuolar transporters Ybt1 and Ycf1. Proteins Aus1 and Dan1 were not found to be involved in steroid import. The research of possible targets of steroid hormone action in fungi suggests that steroid hormones inhibit ergosterol biosynthesis in S. cerevisiae and H. werneckii. Results of this inhibition caused changes in the sterol content of the cellular membrane. The presence of steroid hormones most probably causes the degradation of the Tat2 permease and impairment of tryptophan import.

Enhanced oral bioavailability of a sterol-loaded microemulsion formulation of Flammulina velutipes, a potential antitumor drug.

PURPOSE: To investigate the growth inhibition activity of Flammulina velutipes sterol (FVS) against certain human cancer cell lines (gastric SGC and colon LoVo) and to evaluate the optimum microemulsion prescription, as well as the pharmacokinetics of encapsulated FVS. METHODS: Molecules present in the FVS isolate were identified by gas chromatography/mass spectrometry analysis. The cell viability of FVS was assessed with methyl thiazolyl tetrazolium (MTT) bioassay. Based on the solubility study, phase diagram and stability tests, the optimum prescription of F. velutipes sterol microemulsions (FVSMs) were determined, followed by FVSMs characterization, and its in vivo pharmacokinetic study in rats. RESULTS: The chemical composition of FVS was mainly ergosterol (54.8%) and 22,23-dihydroergosterol (27.9%). After 72 hours of treatment, both the FVS (half-maximal inhibitory concentration [IC(50)] = 11.99 µg · mL(-1)) and the standard anticancer drug, 5-fluorouracil (IC(50) = 0.88 µg · mL(-1)) exhibited strong in vitro antiproliferative activity against SGC cells, with IC(50) > 30.0 µg · mL(-1); but the FVS performed poorly against LoVo cells (IC(50) > 40.0 µg · mL(-1)). The optimal FVSMs prescription consisted of 3.0% medium chain triglycerides, 5.0% ethanol, 21.0% Cremophor EL and 71.0% water (w/w) with associated solubility of FVS being 0.680 mg · mL(-1) as compared to free FVS (0.67 µg · mL(-1)). The relative oral bioavailability (area-under-the-curve values of ergosterol and 22,23-dihydroergosterol showed a 2.56-fold and 4.50-fold increase, respectively) of FVSMs (mean diameter ~ 22.9 nm) as against free FVS were greatly enhanced. CONCLUSION: These results indicate that the FVS could be a potential candidate for the development of an anticancer drug and it is readily bioavailable via microemulsion formulations.

Reversal of fluconazole resistance by sulfated sterols from the marine sponge Topsentia sp.

Bioassay-guided fractionation of the extract of Topsentia sp. led to the identification of two new sulfated sterols, geodisterol-3-O-sulfite (1) and 29-demethylgeodisterol-3-O-sulfite (2), the active constituents reversing efflux pump-mediated fluconazole resistance. Both compounds enhanced the activity of fluconazole in a Saccharomyces cerevisiae strain overexpressing the Candida albicans efflux pump MDR1, as well as in a fluconazole-resistant Candida albicans clinical isolate known to overexpress MDR1. These results provide insight into the clinical utility of combining efflux pump inhibitors with current antifungals to combat the resistance associated with opportunistic fungal infections caused by C. albicans.

Niemann-Pick C proteins in sterol transport and absorption: flies in the ointment.

The Niemann-Pick C proteins have slowly emerged as regulators of subcellular lipid transport and sterol absorption at the small intestine. A recent article in Cell Metabolism suggests that in addition to their significant structural and sequence homology, these proteins may orchestrate their functions in a previously unappreciated fashion (Voght et al., 2007).

Uptake and trafficking of exogenous sterols in Saccharomyces cerevisiae.

The proper distribution of sterols among organelles is critical for numerous cellular functions. How sterols are sorted and moved among membranes remains poorly understood, but they are transported not only in vesicles but also by non-vesicular pathways. One of these pathways moves exogenous sterols from the plasma membrane to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae. We have found that two classes of proteins play critical roles in this transport, ABC transporters (ATP-binding-cassette transporters) and oxysterol-binding protein-related proteins. Transport is also regulated by phosphoinositides and the interactions of sterols with other lipids. Here, we summarize these findings and speculate on the role of non-vesicular sterol transfer in determining intracellular sterol distribution and membrane function.

Multiple rare variants in NPC1L1 associated with reduced sterol absorption and plasma low-density lipoprotein levels.

An approach to understand quantitative traits was recently proposed based on the finding that nonsynonymous (NS) sequence variants in certain genes are preferentially enriched at one extreme of the population distribution. The NS variants, although individually rare, are cumulatively frequent and influence quantitative traits, such as plasma lipoprotein levels. Here, we use the NS variant technique to demonstrate that genetic variation in NPC1L1 contributes to variability in cholesterol absorption and plasma levels of low-density lipoproteins (LDLs). The ratio of plasma campesterol (a plant sterol) to lathosterol (a cholesterol precursor) was used to estimate relative cholesterol absorption in a population-based study. Nonsynonymous sequence variations in NPC1L1 were five times more common in low absorbers (n = 26 of 256) than in high absorbers (n = 5 of 256) (P < 0.001). The rare variants identified in low absorbers were found in 6% of 1,832 African-Americans and were associated with lower plasma levels of LDL cholesterol (LDL-C) (96 +/- 36 mg/dl vs. 105 +/- 36 mg/dl; P = 0.005). These data, together with prior findings, reveal a genetic architecture for LDL-C levels that does not conform to current models for quantitative traits and indicate that a significant fraction of genetic variance in LDL-C is due to multiple alleles with modest effects that are present at low frequencies in the population.

ABC transporters and sterol absorption.

Recent molecular studies, in particular investigations of subjects with monogenic disorders of lipoprotein metabolism and studies of induced-mutant mice, have increased the understanding of intestinal sterol absorption. Some of these genes encode adenosine triphosphate [ATP] binding cassette (ABC) transporters that transport dietary cholesterol from enterocytes back out to the intestinal lumen, thereby limiting the amount of cholesterol absorbed. ABC transporters also provide an effective barrier against the absorption of plant sterols, which are normally not absorbed in significant quantities by humans. This mechanism was clarified by the discovery that defects in two adjacent genes encoding ABC transporters are the molecular basis of sitosterolemia, a rare autosomal recessive disease in which plant sterols are absorbed due to failure of intestinal barrier to their absorption. Furthermore, recent experiments performed in induced-mutant mice have solidified the importance of these transporters in intestinal sterol absorption. Together with new developments in the biology of bile acids, sterol absorption is providing interesting directions for metabolism research. In addition to elucidating some of the molecular mechanisms of sterol absorption, these recent findings may lead to new therapeutic options to treat hypercholesterolemia and to help patients at risk of vascular disease reach ever-more stringent target levels.

Nuclear receptor signaling in the control of cholesterol homeostasis: have the orphans found a home?

Cholesterol is essential for all mammalian cells. Cellular cholesterol requirements are met through de novo synthesis and uptake of plasma lipoproteins, homeostatic responses that are transcriptionally regulated by the sterol regulatory element-binding proteins (SREBPs). To prevent cytotoxicity attributable to accumulation of excess cholesterol, liver X receptors (LXRs) and the farnesoid X receptor (FXR), together with other members of the nuclear receptor superfamily, promote the storage, transport, and catabolism of sterols and their metabolites. Members of this metabolic nuclear receptor family include receptors for oxysterols (LXRs), bile acids (CAR, FXR, and PXR), and fatty acids (PPARs). Through coordinated regulation of transcriptional programs, these nuclear receptors regulate key aspects of cellular and whole-body sterol homeostasis, including cholesterol absorption, lipoprotein synthesis and remodeling, lipoprotein uptake by peripheral tissues, reverse cholesterol transport, and bile acid synthesis and absorption. This review focuses on the nuclear receptors that are central to the lipid metabolic signaling cascades, communication between lipid metabolites and their receptors, and the role of nuclear receptors in orchestrating the complex transcriptional programs that govern cholesterol and bile acid metabolism.

Well-characterized garlic-derived materials are not hypolipidemic in APOE*3-Leiden transgenic mice.

Garlic is reported to have beneficial effects on risk factors associated with cardiovascular disease, including normalization of plasma lipid levels. However, numerous studies do not support this beneficial effect of garlic on plasma lipids. This contradiction may result from the use of different garlic-derived materials, experimental designs, and/or animal models. The present study investigated the hypolipidemic effect of garlic-derived materials in APOE*3-Leiden mice, a model well suited for drug and dietary intervention studies of hyperlipidemia. APOE*3-Leiden mice were fed a garlic-derived sulfur-rich compound, either allicin (0.29 g.L drinking water(-1)) or diallyldisulfide (0.27 g.kg diet(-1)), or powdered garlic, of either the kwai (42 g.kg diet(-1)) or morado (42 g.kg diet(-1)) variety. The amounts of garlic-derived materials supplied allowed free intake of allicin or allicin equivalents (diallyldisulfide, kwai, or morado) at 44 mg.kg body wt(-1).d(-1). Mice were fed a nonpurified diet for 4 wk, followed by a Western diet for 8 wk, both supplemented with the garlic-derived materials. These diets had no consistent effect on plasma lipids and did not affect lipoprotein profiles, which are markers for whole-body cholesterol synthesis and intestinal sterol absorption. The current data indicate that the postulated effects of garlic on cardiovascular disease are not caused via modulation of plasma lipid levels.

Lipid lowering activity of drugs affecting cholesterol absorption.

AIM: Dietary cholesterol absorption, endogenous cholesterol synthesis and biliary cholesterol excretion regulate whole body cholesterol balance as a result of biotransformation into bile acids or direct cholesterol excretion. Recent studies have significantly advanced our understanding of intestinal sterol absorption at molecular level. This review concentrates on two major issues: the mechanisms of sterol absorption, and the currently available or experimental drugs that affect this pathway. DATA SYNTHESIS: Nuclear hormone receptors, such as the liver X, farnesoid X and retinoid X receptors, regulate the absorption of dietary sterols by modulating the transcription of several genes involved in cholesterol metabolism, The ABC proteins transport dietary cholesterol from enterocytes back to the intestinal lumen, thus limiting the amount of absorbed cholesterol. By means of the same mechanism, ABC transporters also provide an efficient barrier against the absorption of plant sterols. Phytosterols, bile acid sequestrants, ezetimibe and ACAT inhibitors are possible means of affecting these pathways. CONCLUSION: In addition to providing an insight into the molecular mechanisms of sterol absorption, these recent findings may lead to new therapeutic options for the treatment of hypercholesterolemia. This is particularly true in the case of patients at high risk of coronary artery disease requiring aggressive lipid-lowering therapy combining a statin with drugs affecting cholesterol absorption in order to ensure the optimal management of dyslipidemia.