Identification of a ß-Carboline Alkaloid from Chemoselectively Derived Vanilla Bean Extract and Its Prevention of Lipid Droplet Accumulation in Human Hepatocytes (HepG2).

Targeting bioactive compounds to prevent lipid droplet accumulation in the liver, we explored an antioxidative extract from vanilla bean (Vainilla planifolia) after chemo-selective derivatization through heating and acid modification. The chemical analysis of vanilla bean extract through chemoselective derivatization resulted in the identification of sixteen compounds (34-50) using LC-MS/MS analysis. A ß-carboline alkaloid with a piperidine C-ring and a vanillin moiety at C-1 (34) was identified by molecular networking and diagnostic fragmentation filtering approaches. ß-carboline alkaloid 34 exhibited significant inhibitory activity of lipid droplet accumulation (LDAI) in oleic acid-loaded hepatocellular carcinoma HepG2 cells. The LDAI activity was associated with both activation of lipolysis and suppression of lipogenesis in the cells. The study indicates that crude plant extracts, following chemoselective derivatization, may contain bioactive compounds that could be beneficial in preventing hepatosteatosis and could serve as a source of lead compounds for drug development. This approach may be useful to investigate other mixtures of natural products and food resources.

Food-Derived ß-Carboline Alkaloids Ameliorate Lipid Droplet Accumulation in Human Hepatocytes.

Lipid droplet accumulation (LDA) in hepatocytes is the initial stage of nonalcoholic fatty liver disease (NAFLD). In the search for natural compounds for the prevention of NAFLD, a series of ß-carboline alkaloid derivatives, inspired by flazin and its derivative, newly identified in Crassostrea gigas Thunberg. extracts, were examined for LDA inhibition (LDAI) activity in oleic acid-loaded hepatocytes (HepG2). Eight compounds with a piperidine or pyridine C-ring were chemically synthesized (1-8). Among them, compounds 2 and 4 (flazin) with a carboxy group at C-3 and furfuryl alcohol moiety at C-1 showed low cytotoxicity and they exhibited significant LDAI activity. Compound 2 with piperidine C-ring was identified for the first time in C. gigas extract, and ameliorated the lipid accumulation with the LDAI value of 25.4%. Active compounds 2 and 4 significantly inhibited triacylglycerol species accumulation in cells. These compounds upregulated ATGL and downregulated SREBP1, FASN, and SCD1 genes, suggesting that they activated lipolysis and suppressed lipogenesis, respectively. These results suggest that ß-carboline alkaloids, especially compounds 2 and 4, might be potentially useful for preventing NAFLD.

Oxidative Stress Linked Organ Lipid Hydroperoxidation and Dysregulation in Mouse Model of Nonalcoholic Steatohepatitis: Revealed by Lipidomic Profiling of Liver and Kidney.

Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.

Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs' plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

Novel Fluorescence-Based Method To Characterize the Antioxidative Effects of Food Metabolites on Lipid Droplets in Cultured Hepatocytes.

A fluorescence microscopic method for characterizing size, quantity, and oxidation of lipid droplets (LDs) in HepG2 cells was developed. LDs were induced by palmitic (PA), oleic (OA), or linoleic acids (LA) and stained with two fluorescent probes for neutral lipids and lipid peroxides. Each fatty acid increased the number of LDs and oxidized LDs (oxLDs) and the degree of LD oxidation time dependently, as well as increased intracellular triglyceride hydroperoxides. LDs induced by LA without 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) showed the most significant oxidation degree over PA and OA, especially in large LDs (area ≥ 3 µm2, oxLD/LD = 52.3 ± 21.7%). Under this condition, two food-derived antioxidants were evaluated, and both of them significantly improved the LD characteristics. Moreover, chlorogenic acid reduced the quantity of large LDs by 74.0-87.6% in a dose-dependent manner. The proposed method provides a new approach to evaluate the effect of dietary antioxidants on LD characteristics.

Separating and Profiling Phosphatidylcholines and Triglycerides from Single Cellular Lipid Droplet by In-Tip Solvent Microextraction Mass Spectrometry.

The analysis of lipid droplets (LDs) by mass spectrometry at the single LD level is still an analytical challenge. In this work, we developed a novel technique termed in-tip solvent microextraction mass spectrometry for the separation and profiling of phosphatidylcholines and triglycerides within a single LD. This method has been successfully used to analyze LDs in mammalian cells and to compare the profiles of triglycerides and phosphatidylcholines in LDs induced at different conditions. Our method has the potential to be applied to such fields as fundamental lipid biology to further our understanding on the mechanisms of lipid production, lipid packaging, and their pathophysiological roles.

Antioxidant effects of pH-regulating agents on the thermal deterioration of vegetable oils.

pH-Regulating agents, such as sodium tartrate, disodium succinate, and trisodium citrate, were investigated for their antioxidant activities during the thermal deterioration of vegetable oils. Refined rapeseed and rice bran oils, supplemented with pH-regulating agents and α-tocopherol (0.1%) were heated at 180℃. After heating, acid values (AVs), carbonyl values (CVs), polar material contents, and color (absorbance at 420 nm) of each sample were measured. All pH-regulating agents gave rise to reduced AVs, CVs, and polar material contents of vegetable oils during heating relative to samples not containing a pHregulating agent. Rapeseed and rice bran oils supplemented with sodium tartrate showed the lowest AVs, CVs, polar material contents and absorbances at 420 nm after heating. Sodium tartrate not only retarded the hydrolysis, thermal oxidation, polymerization, and coloration of both oils while heating at high temperatures, but it also showed antioxidant activity at the supplementation level of 0.01%. The antioxidant activity of sodium tartrate was higher than that of α-tocopherol during the deterioration of vegetable oils. Sodium tartrate was particularly effective retarding hydrolysis while heating at high temperatures, resulting in increase of AVs of vegetable oils. Sodium tartrate is therefore expected to be an effective antioxidant for the thermal deterioration of fats and oils during deep-fat frying.

Effect of caffeine and capsaicin on the blood glucose levels of obese/diabetic KK-A(y) mice.

The present study is aimed to measure the effect of caffeine and capsaicin on the blood glucose level of obese/diabetic model mice. The blood glucose level of KK-A(y) obese/diabetic mice decreased significantly after dietary supplementations with less than 0.031% caffeine and less than 0.0042% capsaicin, while both food ingredients and the combination had little effect on body weight gain and abdominal white adipose tissue (WAT) weight at this dose. The blood glucose level of the control mice was 234.4 mg/dL at 10 days after feeding and it increased to 500.6 mg/dL after 28 days, while levels after intake of caffeine, capsaicin, and both together increased from 180, 131.6, and 140 mg/dL to 183.7, 255.1, and 151.6 mg/dL, respectively. The anti-diabetic activity of caffeine and capsaicin was confirmed by a decrease in total drinking water and in the blood insulin level.

The allenic carotenoid fucoxanthin, a novel marine nutraceutical from brown seaweeds.

Obesity and type 2 diabetes are pathologies with rapidly growing prevalence throughout the world. A few molecular targets offer the most hope for anti-obesity and anti-diabetic therapeutics. One of the keys to success will be the induction of uncoupling protein 1 (UCP1) in abdominal white adipose tissue (WAT) and the regulation of cytokine secretions from both abdominal adipose cells and macrophage cells infiltrated into adipose tissue. Anti-obesity and anti-diabetic effects of fucoxanthin, a characteristic carotenoid found in brown seaweeds, have been reported. Nutrigenomic studies reveal that fucoxanthin induces UCP1 in abdominal WAT mitochondria, leading to the oxidation of fatty acids and heat production in WAT. Fucoxanthin improves insulin resistance and decreases blood glucose levels through the regulation of cytokine secretions from WAT. The key structure of carotenoids for the expression of anti-obesity effect is suggested to be the carotenoid end of the polyene chromophore, which contains an allenic bond and two hydroxyl groups.

Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-Ay mice.

Fucoxanthin, a marine carotenoid found in edible brown seaweeds, attenuates white adipose tissue (WAT) weight gain and hyperglycemia in diabetic/obese KK-A(y) mice, although it does not affect these parameters in lean C57BL/6J mice. In perigonadal and mesenteric WATs of KK-A(y) mice fed fucoxanthin, mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), which are considered to induce insulin resistance, were markedly reduced compared to control mice. In contrast to KK-A(y) mice, fucoxanthin did not alter MCP-1 and TNF-α mRNA expression levels in the WAT of lean C57BL/6J mice. Interleukin-6 (IL-6) and plasminogen activator inhibitor-1 mRNA expression levels in WAT were also decreased by fucoxanthin in KK-A(y) mice. In differentiating 3T3-F442A adipocytes, fucoxanthinol, which is a fucoxanthin metabolite found in WAT, attenuated TNF-α-induced MCP-1 and IL-6 mRNA overexpression and protein secretion into the culture medium. In addition, fucoxanthinol decreased TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression in RAW264.7 macrophage-like cells stimulated by palmitic acid. These findings indicate that fucoxanthin regulates mRNA expression of inflammatory adipocytokines involved in insulin resistance, iNOS, and COX-2 in WAT and has specific effects on diabetic/obese KK-A(y) mice, but not on lean C57BL/6J mice.

In vitro and in vivo evaluation of mutagenicity of fucoxanthin (FX) and its metabolite fucoxanthinol (FXOH).

Mutagenicity of fucoxanthinol (FXOH), the major compound after oral ingestion of fucoxanthin (FX), was evaluated by in vitro Ames test, and of FX by in vivo micronucleus test. In in vitro Ames test, bacterial reverse mutation was examined by using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and Escherichia coli WP2uvrA/pKM101, with or without metabolic activation by S9 mix in the preincubation method, and mutagenicity of FXOH was found to be negative in all cases. In in vivo micronucleus test, mice were orally administered with FX at doses of 500, 1,000 and 2,000 mg/kg, and the bone marrow cells were taken 24 hr after the administration to observe the incidence of micronucleus cells, and mutagenicity of FX was found to be negative at all doses. Based on the data of the present study it can be presumed that orally administered FX is a safe compound in terms of mutagenicity under the experimental conditions employed here.

Single and repeated oral dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice.

Fucoxanthin (FX), a xanthophyll derivative, is an orange-colored pigment present in edible brown algae. As a part of safety evaluation, single and repeated oral dose toxicity study of FX was conducted. In a single dose study, FX purified from seaweed was orally administered to male and female ICR mice at doses of 1,000 and 2,000 mg/kg. In a repeated doses study, FX at doses of 500 and 1,000 mg/kg was orally administered for 30 days. In both studies, no mortality and no abnormalities in gross appearance were observed. In the repeated doses study, histological observation revealed no abnormal changes in liver, kidney, spleen and gonadal tissues of any of the FX-treated groups. However, significantly increased total cholesterol concentrations were shown by plasma biochemical analyses in all FX-treated groups. Although total bilirubin concentrations were increased by FX, it was established that presence of fucoxanthinol, a major metabolite of FX, interfered with bilirubin determination in plasma. To further ascertain the safety of FX, the mechanism by which FX induces hypercholesterolemia in mice and species differences in the induction of hypercholesterolemia should be elucidated.

Seaweed carotenoid, fucoxanthin, as a multi-functional nutrient.

Fucoxanthin has a unique structure including an unusual allenic bond and 5, 6-monoepoxide in its molecule. We found that abdominal white adipose tissue (WAT) weights of rats and mice fed fucoxanthin were significantly lower than those fed a control diet. The daily intake of fucoxanthin in mice also caused a significant reductions of body weight. Clear signals of uncoupling protein 1 (UCP1) and its mRNA were detected by Western and Northern blot analyses in abdominal WAT in mice fed fucoxanthin, although there is little expression of UCP1 in WAT in mice fed a control diet. UCP1 expression in WAT by fucoxanthin intake leads to oxidation of fatty acids and heat production in WAT mitochondria. Substrate oxidation can directly reduce WAT in animals. Fucoxanthin intake also significantly reduced blood glucose and plasma insulin. Furthermore, feeding fucoxanthin significantly increased the level of hepatic docosahexaenoic acid (DHA), a most important n-3 functional polyunsaturated fatty acid in biological systems. These multi-functionalities of fucoxanthin indicate that it is an important bioactive carotenoid that is beneficial for the prevention of the metabolicsyndrome.

Fucoxanthin and fucoxanthinol enhance the amount of docosahexaenoic acid in the liver of KKAy obese/diabetic mice.

This study examined the effect of dietary fucoxanthin or fucoxanthinol on the amount of docosahexaenoic acid (DHA) in the liver of KKAy mice, a model for obese/type II diabetes. In the first experiment, mice were fed diets containing crude fucoxanthin or glyceroglycolipid for 4 weeks. Results showed a significant increase in the level of DHA in mice fed 0.53% crude fucoxanthin, from 2.3% in control mice to 5.1% of fatty acid composition of total liver lipids. On the other hand, in mice fed crude glyceroglycolipid, the level of DHA as a proportion of total liver fatty acids remained unchanged. To clarify the enhancement of hepatic DHA, in the second experiment, KKAy mice were fed a diet containing purified fucoxanthin or its deacetylated derivative, fucoxanthinol. Results from a quantitative analysis using an internal standard showed that in mice fed 0.2% fucoxanthin, the amount of hepatic DHA was 2-fold higher than in control mice, whereas DHA levels in the small intestine remained unchanged. Furthermore, 0.2% fucoxanthinol led to 1.8- and 1.2-fold increases in the amount of hepatic DHA and arachidonic acid compared to control mice, respectively. These results indicate for the first time that dietary fucoxanthin and fucoxanthinol enhance the amount of DHA in the liver of KKAy mice.