Herbal Leaves Can Suppress Oxidation of Perilla Oil.

Perilla oil is a valuable food source of α-linolenic acids. However, its high reactivity with oxygen shortens its shelf-life after opening. This study investigated the antioxidative profiles of 15 plant materials, including herbs, and examined methods to suppress the oxidation of perilla oil using these plant materials. These plant materials had wide ranges of phenolic, carotenoid, and chlorophyll contents. They exhibit radical scavenging activities and suppress lipid peroxidation, which show highly positive correlations with the phenolic contents. Dipping most of the plant materials examined in perilla oil suppressed its oxidation, and the peroxide values of the oil mixtures indicated a negative correlation with the carotenoid and chlorophyll contents of the plant materials. The leaves of Angelica, Astragalus, and Thyme herbs exhibited the same effect as that of ascorbyl palmitate, which was used as a positive control after 8 wk of incubation in the dark. The suppression of lipid peroxidation was found to be related to the herbal contents of carotenoids and chlorophylls, rather than phenols. Hence, herbal leaves can suppress the oxidation of perilla oil in the dark. The oxidation of n-3 polyunsaturated fatty acids could be suppressed effectively by utilizing plant materials with abundant carotenoids and chlorophylls.

Antioxidative Activities of Plants and Fungi Used as Herbal Medicines.

Plants and fungi classified as herbs are utilized for the maintenance of health in humans. In this study, to evaluate the beneficial effects of herbs, we investigated the phenolic content and antioxidative activity of 20 samples. Some herbs, including Cistanche herb, had high phenol levels and exhibited high activities for radical-scavenging and suppression of lipid peroxidation. Phenolic contents and antioxidative activities showed a high positive correlation. In contrast, some herbal medicines with low phenolic content exhibited high suppressive effects on lipid peroxidation, and it was thought that carotenoids contributed to their suppression. The results of this study are expected to support the clarification of the mechanism of herbal medicines in promoting wellness.

Characterization of Glycolipids in the Strain Chlorella pyrenoidosa.

Plant-derived polar lipids have been reported to exhibit various beneficial effects on human health. The green alga Chlorella is known to be abundant in nutrients, including lipophilic components, and has varying nutrient content depending on the strain. In this study, to assess the nutritional functions of the strain Chlorella pyrenoidosa, we comprehensively analyzed the composition of fatty acids, polar glycerolipids, and sphingolipids. We found that n-3 polyunsaturated fatty acids (PUFAs) comprised 45.6 mol% of fatty acids in the total lipids and 62.2 mol% of n-3 PUFAs in the total lipids occurred in the glycolipids. Monogalactosyldiacylglycerol was the primary glycolipid class, and n-3 PUFA constituted 73.5 mol% of the fatty acids. Although glucosylceramide was observed in trace amounts, highly polar sphingolipids (HPSs), including glycosyl inositol phosphoryl ceramide, were found in much higher amounts compared to rice bran, which is a common source of sphingolipids. These results suggest that the examined Chlorella strain, which is abundant in glycolipids bearing n-3 PUFAs and HPS, is potentially useful as a dietary supplement for improving human health.

Protective Mechanism of Rice-Derived Lipids and Glucosylceramide in an In Vitro Intestinal Tract Model.

We previously reported that the ethanol extract from polished rice suppresses inflammation and the formation of aberrant crypt foci in the mouse colon and particularly focused on the plant sphingolipid glucosylceramide (GlcCer). Here, we investigated the effects of rice lipid fractions and GlcCer on differentiated Caco-2 cells treated with lipopolysaccharide (LPS), in particular, we evaluated the mechanism of action of GlcCer using related substances and metabolic enzyme inhibitors. Rice-derived polar lipids suppressed the LPS-induced reduction in the number of cells. The polar lipids with higher GlcCer content exerted a better effect than the other fractions. GlcCer-related substances reversed the LPS-induced reduction in the number of cells, and GlcCer-metabolic inhibitors, including a sphingosine kinase inhibitor, suppressed the beneficial effects of GlcCer-related substances. These results suggest that GlcCer is a rice component with intestinal protection. Secondly, GlcCer is metabolized during inflammation and protects intestinal cells by maintaining the sphingolipid levels in cells and producing sphingoid base-1-phosphate.

Effects of Dietary Ethanol Extracts from Sake Rice and Sake Lees on Intestinal Impairment in Mice.

Glucosylceramide (GlcCer), a major sphingolipid in plants and fungi, is known to have food functions, such as preventing intestinal impairment and enhancing the moisture content of skin. This study investigated the influence of fermentation on the composition and function of lipophilic components containing GlcCer in plant-based foods; we compared the effects of ethanol extracts from sake rice (SR) and sake lees (SL) on colon impairment in mice. GlcCer and ceramide (Cer) levels in SL were much higher than those in SR, and GlcCer in SL contained 9-methyl-trans-4,trans-8-sphingadienine as a fungi-specific sphingoid base. 1,2-dimethylhydrazine (DMH) treatment markedly increased the formation of aberrant crypt foci (ACF) and the levels of TNF-α and lipid oxidation in mice colons. However, dietary SR or SL significantly suppressed these DMH-induced changes, and SR demonstrated stronger effects than SL. In addition, dietary SR or SL suppressed the expression of apoptotic and anti-apoptotic proteins induced by DMH treatment. This study suggests that SR or SL intake could reduce colon ACF formation via the suppression of inflammation and oxidation-induced cell cycle disturbances. When compared to SR, the weaked effects of SL rich in GlcCer may be the result of the changes in sphingolipid composition (sphingoid base and Cer) and differences in the concentration of other bioactive compounds produced or digested during fermentation.

Effects of Sphingolipid Fractions from Golden Oyster Mushroom (Pleurotus citrinopileatus) on Apoptosis Induced by Inflammatory Stress in an Intestinal Tract in vitro Model.

Previously, we reported that the polar lipid fraction from the golden oyster mushroom, Pleurotus citrinopileatus, suppresses colon injuries which result from apoptosis induced by inflammatory stresses in vivo and in vitro (Yamashita et al., J. Oleo Sci., 69, 751-757 (2020)). Here, we investigated the use of lipid classes in mushroom polar lipid fraction in alleviating colon injury using differentiated Caco-2 cells as an intestinal tract model. The mushroom polar lipid fraction was separated into four fractions using silica thin layer chromatography. Each mushroom polar lipid fraction suppressed lipopolysaccharide (LPS)-induced decreases in the viability of intestinal cells, and the effects of sphingolipid fractions were significantly stronger than those of fraction that did not contain sphingolipids. Addition of sphingolipid fractions suppressed the expression of apoptosis-related proteins (e.g., death receptors and caspases) in the LPS-treated cells. Mushroom polar lipids, especially sphingolipids suppress intestinal apoptosis induced by inflammatory stress, and highly polar sphingolipids may exert stronger suppressive effects.

Polar Lipid Fraction from Golden Oyster Mushrooms (Pleurotus citrinopileatus) Suppresses Colon Injuries from Inflammatory Stresses in vivo and in vitro.

The rising incidence of inflammatory bowel disease (IBD) in East Asian countries has necessitated the implementation of preventive methods in the form of dietary supplementation and changes in dietary habits. We have previously reported that dietary golden oyster mushroom (Pleurotus citrinopileatus) ethanol extract (GOMEE) suppresses intestinal inflammation in mouse models of IBD induced by dextran sulfate sodium salt (DSS). Here, we investigated the components of GOMEE that exert suppressive effects on colon inflammation in vivo and in vitro. The total lipid fraction was extracted from GOMEE, and the polar and neutral lipid fractions were subsequently separated via solvent fractionation. Mice were assigned to dietary groups-control, 1% total lipid, 1% polar lipid, or 1% neutral lipid diet-and fed the respective diets for one week; mice were administered 1.5% DSS in drinking water ad libitum for 20 days. Dietary supplementation with the total or polar lipid fraction alleviated DSS-induced chorionic crypt injury as determined by morphological observation, while dietary supplementation with the neutral lipid fraction did not produce such effects. In the in vitro study, using differentiated Caco-2 cells as the colon model, treatment with the total or polar lipid fraction suppressed cell decrease by lipopolysaccharide (LPS)-induced apoptosis whereas treatment with the neutral lipid fraction did not. Moreover, accumulation of glucosylceramide (GlcCer), a fungal sphingolipid, was observed in the intestinal cells after treatment with polar lipid fraction. These results suggest that the active components of GOMEE that suppress colon inflammation are polar lipids, especially GlcCer. The structure of mushroom GlcCer differs from that of the plant counterpart and is therefore expected to exert different food functions.

Chemical Properties and Nutritional Value of Plant-Origin Glucosylceramide.

Glucosylceramide (GlcCer), a representative sphingolipid in cell membranes of plants and fungi, is known to have certain benefits, such as prevention of intestinal impairment and improved skin moisturizing, when consumed. Recently, incidence rates of intestinal impairments have increased in East Asian countries due to changes of people's diet and life style. Therefore, the occurrence of these impairments needs to be prevented through dietary improvement and supplements containing GlcCer. The in vitro and in vivo effects of GlcCer on colon impairment were explored in our previous studies, with focus on sphingolipid structure. Conversely, plant cell membrane contents such as GlcCer are known to be difficult to extract due to the thick cell wall. Therefore, human and other mammals may not be able to utilize GlcCer when digesting food of plant origin. To confirm this hypothesis, we investigated the effects of polished rice and the extract on intestinal impairment. In addition, we discuss the intestinal function of GlcCer contained in polished rice and the relationship between GlcCer and other lipophilic functional components.

Extraction of Lipophilic Fraction from Polished Rice Improves Its Ameliorative Effect on Intestinal Impairment.

Glucosylceramide (GlcCer), a major sphingolipid in plants and fungi, is known to have food functions such as preventing intestinal impairment and enhancing the moisture content of skin. However, there is little information about functions of GlcCer in food sources as most of the studies on GlcCer functions are done using purified GlcCer. This study was performed to investigate the effects of GlcCer contained in food on intestinal impairment; polished rice flour (RF) and this ethanol extract (RE) were used as sources of GlcCer, and these were evaluated by studying the formation of aberrant crypt foci (ACF) in 1,2-dimethylhydrazine (DMH)-treated mice, which is a model of colon cancer. Mice were fed with either a control diet, a RF diet where RF replaces cornstarch (150 g/kg), or a plus RE diet (0.5 g/kg; RE was extracted from the same amount of RF present in the RF diet). The amount of GlcCer was similar in both the RF and RE diets (3.0 and 2.7 mg/kg, respectively). DMH treatment induced the formation of ACF and the production of inflammation-related cytokines. Both dietary RF and RE suppressed ACF formation and RE, in particular, showed a significant suppressive effect. Dietary RE inhibited the production of almost all of the inflammation-related cytokines studied, while RF suppressed only a few of these cytokines. The present study suggests that the lipophilic fraction including GlcCer, present in polished rice has protective effects against intestinal impairment, but it requires extraction since digestion alone is not enough to elicit its complete protective action.

Analysis of Plasmalogen Species in Foodstuffs.

Ethanolamine plasmalogen (PlsEtn), which is present at high levels in brains, is believed to be involved in neuronal protection. The present study was performed to search for PlsEtn resources in foodstuffs. The foodstuffs examined showed a wide range of PlsEtn contents from 5 to 549 µmol/100 g wet wt. The marine invertebrates, blue mussel, and ascidian had high PlsEtn contents (over 200 µmol/100 g wet wt). Profiling of the molecular species showed that the predominant fatty acids of PlsEtn species were 20:5 (EPA) and 22:6 (DHA) at the sn-2 position of the glycerol moiety in marine foodstuffs, whereas major PlsEtn species in land foodstuffs were 20:4. Following quantitative analysis by multiple reaction monitoring, the ascidian viscera were shown to contain the highest levels of 18:0/20:5-PlsEtn and 18:0/22:6-PlsEtn (86 and 68 µmol/100 g wet wt, respectively). In order to evaluate a neuronal antiapoptotic effect of these PlsEtn species, human neuroblastoma SH-SY5Y cells were treated with ethanolamine glycerophospholipid (EtnGpl), purified from the ascidian viscera, under serum starvation conditions. Extrinsic EtnGpl from ascidian viscera showed stronger suppression of cell death induced by serum starvation than with bovine brain EtnGpl. The EtnGpl from ascidian viscera strongly suppressed the activation of caspase 3. These results suggest that PlsEtn, especially that containing EPA and DHA, from marine foodstuffs is potentially useful for a therapeutic dietary supplement preventing neurodegenerative diseases, such as Alzheimer's disease (AD).

Effects of Dietary Plant-Origin Glucosylceramide on Bowel Inflammation in DSS-Treated Mice.

The effects of dietary plant-origin glucosylceramide (GlcCer) on symptoms similar to those of inflammatory bowel diseasewere investigated in dextran sulfate sodium salt (DSS)-treated mice. Dietary GlcCer suppressed decreases in body weight due to DSS administration. To determine its effects on the colon, we examined its surface under a microscope following toluidine blue staining. Dietary GlcCer decreased DSS-induced chorionic crypt injury and elevated myeloperoxidase levels. Moreover, dietary GlcCer significantly suppressed the production of cytokines by the intestinal mucosa. These results provide evidence for the suppression of DSS-induced inflammation by dietary GlcCer.

Effect of dietary Maitake (Grifola frondosa) mushrooms on plasma cholesterol and hepatic gene expression in cholesterol-fed mice.

To investigate the effects of dietary Grifola frondosa on cholesterol, normal mice were fed a diet containing 1% cholesterol (HC group) or 1% cholesterol and 10% freeze-dried G. frondosa powder (HC+G group) for 4 weeks and hepatic and plasma lipid levels were compared with those of a cholesterol-free diet-fed mice (N group). Hepatic total cholesterol (TC), triacylglycerol contents were considerably increased and plasma TC / phospholipid (PL) was also increased significantly in the HC group compared with the N group. However, plasma TC content decreased in the HC+G group compared with the HC group. To characterize the mechanisms responsible for lowered plasma cholesterol in G. frondosa-supplemented mice, hepatic gene expression was profiled using DNA microarray and gene ontology. Genome analyses revealed that de novo cholesterol synthesis genes were suppressed following cholesterol intake. However, expression of bile acid biosynthesis and low-density lipoprotein receptor genes showed little change. Scarb1, Abcg5, and Abcg8, involved in cholesterol transport and excretion, were slightly upregulated in the HC+G group compared with the HC group. These data indicate the plasma cholesterol-lowering effect of G. frondosa. Moreover, fatty acid (FA) ß-oxidation was promoted via adipocytokine signaling pathways, and Saa, encodes serum amyloid A related to arteriosclerosis, was suppressed in the HC+G group.

Functional expression of a humanized gene for an omega-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells.

Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of alpha-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.

Anti-inflammatory effect of buckwheat sprouts in lipopolysaccharide-activated human colon cancer cells and mice.

In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.

Effect of dietary wine pomace extract and oleanolic acid on plasma lipids in rats fed high-fat diet and its DNA microarray analysis.

The aim of this study was to evaluate the concentration of oleanolic acids (OA) in pomace, a winemaking byproduct, and its influence on the levels of plasma lipids in rats fed a high-fat diet and on hepatic gene expression using DNA microarray analysis in vivo. HPLC analyses of pomace ethanol extract (PEE) revealed a high amount of OA ranging from 4 to 11 g/100 g. Male Sprague-Dawley rats were fed a normal-fat diet (NF group), a high-fat diet with 21% lard (HF group), a high-fat diet with 0.05% OA (OA group, 50 mg/kg/day), or a high-fat diet with 0.45% PEE (PEE group, 450 mg/kg/day). Plasma triacylglycerol and phospholipid concentrations were significantly lower in the OA and PEE groups than in the HF group. The microarray analysis of hepatic mRNA revealed reduced expression levels of lipogenic genes including acetyl-CoA carboxylase and glycerol-3-phosphate acyltransferase, probably resulting from the suppression of transcription factor Srebf1 expression. Gene expression of gluconeogensis and inflammatory cytokines was also down-regulated in the OA and PEE groups, suggesting that administration of OA or PEE could ameliorate obesity-induced insulin resistance, as well as prevent hyperlipidemia.

Danshen inhibits oxysterol-induced endothelial cell apoptosis in vivo.

Oxysterols induce apoptosis in vascular endothelial cells in vitro, but it is not clear whether they do so in vivo. We intravenously injected an oxysterol, cholestan-3beta, 5alpha, 6beta-triol, into rats and quantitatively analyzed endothelial cell apoptosis in the aorta. Oxysterol significantly promoted apoptosis in a time- and dose-dependent fashion. The apoptosis had increased 4.5-fold 6 hrs after injection, and returned to the background level at 48 hrs. An extract of the Chinese herb Danshen as well as probucol abolished triol-induced endothelial cell apoptosis in vitro and in vivo. Since apoptotic cells are quickly cleared, oxysterol-induced apoptosis could significantly affect endothelial integrity over a long period of time. Radical scavengers may be useful for the prevention of endothelial damage.

Presence of higher alcohols as ferulates in potato pulp and its radical-scavenging activity.

Higher alcohols with a carbon length ranging from 16 to 30 found in the lipophilic fraction from potato pulp were shown to be present as ferulate and in a free form, but not as wax. Thin-layer chromatography of the neutral lipids in potato pulp indicated a few spots with scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical, the major active component being characterized as alkyl ferulate which showed almost the same level of activity as gamma-oryzanol.

Increased levels of policosanol and very long-chain fatty acids in potato pulp fermented with Rhizopus oryzae.

Significant amounts of policosanol and very long-chain fatty acids (VLFAs) ranging in carbon length from 22 to 30 were found in the lipophilic fraction obtained from potato pulp fermented with Rhizopus oryzae. It is believed that these compounds would have originally been present as suberin-related compounds, but not as wax, in the periderm of potato tubers and concentrated into potato pulp during the process of starch production. Moreover, the policosanol and VLFAs extracted from potato pulp with organic solvents were found to have increased after fermentation.

Functional expression of a Delta12 fatty acid desaturase gene from spinach in transgenic pigs.

Linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Delta12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were approximately 10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained approximately 20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.