Acanthopanax senticosus ameliorates steatohepatitis through HNF4 alpha pathway activation in mice.

Non-alcoholic fatty liver disease is a common liver disease worldwide, and is associated with dysregulation of lipid metabolism, leading to inflammation and fibrosis. Acanthopanax senticosus Harms (ASH) is widely used in traditional medicine as an adaptogen food. We examined the effect of ASH on steatohepatitis using a high-fat diet mouse model. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet with ASH extract (ASHE). After 6 weeks, liver RNA transcriptome sequencing (RNA-Seq) was performed, followed by Ingenuity Pathway Analysis (IPA). Our findings revealed that mice fed a high-fat diet with 5% ASHE exhibited significantly reduced liver steatosis. These mice also demonstrated alleviated inflammation and reduced fibrosis in the liver. IPA of RNA-Seq indicated that hepatocyte nuclear factor 4 alpha (HNF4 alpha), a transcription factor, was the activated upstream regulator (P-value 0.00155, z score = 2.413) in the liver of ASHE-fed mice. Adenosine triphosphate binding cassette transporter 8 and carboxylesterase 2, downstream targets of HNF4 alpha pathway, were upregulated. Finally, ASHE-treated HepG2 cells exposed to palmitate exhibited significantly decreased lipid droplet contents. Our study provides that ASHE can activate HNF4 alpha pathway and promote fat secretion from hepatocytes, thereby serving as a prophylactic treatment for steatohepatitis in mice.

Simultaneous Intake of Chlorella and Ascidian Ethanolamine Plasmalogen Accelerates Activation of BDNF-TrkB-CREB Signaling in Rats.

Brain-derived neurotrophic factor (BDNF) plays an important role in neurogenesis, synaptic plasticity, and cognition. BDNF is a neurotrophin that binds to tropomyosin receptor kinase B (TrkB), a specific receptor on target cell surfaces; it acts on neuronal formation, development, growth, and repair via transcription factors, such as cAMP response element-binding protein (CREB), and it is involved in learning and memory. BDNF expression is decreased in patients with Alzheimer's disease (AD). Exercise and the intake of several different foods or ingredients can increase BDNF expression, as confirmed with lutein, xanthophylls (polar carotenoids), and ethanolamine plasmalogen (PlsEtn), which are present at high levels in the brain. This study examined the effects of combining lutein and PlsEtn using lutein-rich Chlorella and ascidian extracts containing high levels of PlsEtn bearing docosahexaenoic acid, which is abundant in the human brain, on the activation of the BDNF-TrkB-CREB signaling pathway in the hippocampus of Sprague-Dawley rats. Although activation of the BDNF-TrkB-CREB signaling pathway in the hippocampus was not observed in Chlorella or ascidian PlsEtn monotherapy, activation was observed with combination therapy at an equal dose. The results of this study suggest that the combination of Chlorella and ascidian PlsEtn may have a preventive effect against dementia, including AD.

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.

Removal of chlorophyll and pheophorbide from Chlorella pyrenoidosa by supercritical fluid extraction: potential of protein resource.

This study tried to quantitatively clarify the usefulness of supercritical fluid extraction for removal of chlorophyll and pheophorbide from Chlorella pyrenoidosa. C. pyrenoidosa powder was subjected to supercritical fluid extraction, and chlorophyll a and pheophorbide a in its extracted fractions were measured by HPLC-UV. Chlorophyll a and pheophorbide a in residue after supercritical fluid extraction became below of detection limit.

Acanthopanax senticosus Harms extract causes G0/G1 cell cycle arrest and autophagy via inhibition of Rubicon in human liver cancer cells.

Acanthopanax senticosus (Rupr. et Maxim) Harms (ASH), also known as Siberian ginseng or eleuthero, is a hardy shrub native to China, Korea, Russia and the northern region of Japan. ASH is used for the treatment of several diseases such as heart disease, hypertension, rheumatoid arthritis, allergies, chronic bronchitis, diabetes and cancer. In the present study, the inhibitory effect of the root extract of ASH (ASHE) on HuH­7 and HepG2 liver cancer cells was examined. ASHE suppressed liver cancer cell proliferation by inducing cell cycle arrest at the G0/G1 phase, as well as apoptosis, as indicated by the increased number of Annexin V and 7­AAD­positive cells. Furthermore, the expression of LC3­II, an autophagy marker, in these cells also increased post treatment with ASHE. LC3­II induction was further enhanced by co­treatment with chloroquine. Fluorescence and transmission electron micrographs of ASHE­treated liver cancer cells showed the presence of an increased number of autophagic vesicles. A decreased protein expression level of run domain Beclin­1­interacting and cysteine­rich domain­containing, an autophagy inhibitor, with no change in RUBCN mRNA expression was observed, indicating activation of the autophagosome­lysosome fusion step of autophagy. In conclusion, ASHE exerts cytostatic activity on liver cancer cells via both apoptosis and autophagy, and may serve as a potential therapeutic agent for management of liver cancer and autophagy­related diseases.

Combination of syringaresinol-di-O-ß-D-glucoside and chlorogenic acid shows behavioral pharmacological anxiolytic activity and activation of hippocampal BDNF-TrkB signaling.

Mental stress, such as anxiety and conflict, causes physiological changes such as dysregulation of autonomic nervous activity, depression, and gastric ulcers. It also induces glucocorticoid production and changes in hippocampal brain-derived neurotrophic factor (BDNF) levels. We previously reported that Acanthopanax senticosus HARMS (ASH) exhibited anxiolytic activity. Thus, we attempted to identify the anxiolytic constituents of ASH and investigated its influence on hippocampal BDNF protein expression in male Sprague Dawley rats administered chlorogenic acid (CHA), ( +)-syringaresinol-di-O-ß-D-glucoside (SYG), or a mixture of both (Mix) for 1 week using the open field test (OFT) and improved elevated beam walking (IEBW) test. As with ASH and the benzodiazepine anxiolytic cloxazolam (CLO), Mix treatment significantly increased locomotor activity in the OFT. CHA and Mix increased the time spent in the open arm in the IEBW test. SYG and Mix treatment inhibited the significant increase in normalized low-frequency power, indicative of sympathetic nervous activity, and significant decrease in normalized high-frequency power, indicative of parasympathetic nervous activity, as observed in the IEBW test. SYG and Mix treatment significantly increased hippocampal BDNF protein expression. The combination of CHA and SYG possibly induces anxiolytic behavior and modulates autonomic regulation, activates hippocampal BDNF signaling as with ASH.

Anxiolytic Effects of Acanthopanax senticosus HARMS Occur via Regulation of Autonomic Function and Activate Hippocampal BDNF⁻TrkB Signaling.

Mental stress, such as anxiety and conflict, causes physiological changes, such as changes in autonomic nervous activity and gastric ulcers. In addition, stress induces glucocorticoids and changes the hippocampal brain-derived neurotrophic factor (BDNF) expression levels. We previously reported that Acanthopanax senticosus HARM (ASH) prevents stress-induced gastric ulcers. Thus, we investigated the potential anxiolytic effect and influence of ASH on the hippocampus BDNF-related protein in male Sprague-Dawley rats fed 1% and 5% ASH extract-containing food for one week using novelty suppressed feeding (NSF) and improved elevated beam walking (IEBW) tests. ASH treatment significantly decreased latency to eat in the NSF test and increased the time spent on the open arm in the IEBW test. ASH5% treatment showed a significant decrease in LFnu, indicative of sympathetic nervous activity, and a significant increase in HFnu, indicative of parasympathetic nervous activity, in the NSF test. In addition, ASH1% and ASH5% treatments significantly decreased LFnu and significantly increased HFnu in the IEBW test. ASH5% treatment significantly increased hippocampal BDNF protein expression in both Western blotting and immunohistochemistry experiments. Our findings suggest that anxiolytic effects of ASH occur via the regulation of autonomic function and increased hippocampal BDNF signaling.

Ingestion of Chlorella reduced the oxidation of erythrocyte membrane lipids in senior Japanese subjects.

Accumulation of phospholipid hydroperoxide (PLOOH) in erythrocyte membranes is an abnormality found in patients with senile dementia, including those with Alzheimer's disease. In our previous studies, dietary xanthophylls (polar carotenoids such as lutein) were hypothesized to inhibit lipid peroxidation. In the present study, we conducted a randomized, double-blind, placebo-controlled human trial to assess the impact for a total of 2 months Chlorella supplementation (8 g Chlorella/day/person; equivalent to 22.9 mg lutein/day/person) on PLOOH and carotenoid concentrations in erythrocytes as well as plasma of 12 normal senior subjects. After 1 or 2 months of treatment, erythrocytes and plasma lutein concentrations increased in the Chlorella group but not in the placebo group. In the Chlorella-supplemented group, erythrocyte PLOOH concentrations after a total of 2 months of treatment were lower than the concentrations before supplementation. These results suggest that Chlorella ingestion improved erythrocyte antioxidant status and lowered PLOOH concentrations. These reductions might contribute to maintaining the normal function of erythrocytes and prevent the development of senile dementia.

Receptor binding activities of Chlorella on cysteinyl leukotriene CysLT, glutamate AMPA, ion channels, purinergic P 2Y, tachykinin NK2 receptors and adenosine transporter.

A Chlorella powder was tested in a total of 129 in vitro receptor binding assay systems. The results showed a potent inhibition of this powder on cysteinyl leukotriene CysLT2, and glutamate AMPA in a dose-concentration manner with IC(50) mean +/- SEM values of 20 +/- 4.5 microg/mL and 44 +/- 14 microg/mL, respectively. Other moderate and weak activities reflected in competitive binding experiments were seen versus adenosine transporter; calcium channel L-type, benzothiazepine; gabapentin; kainate, NMDA-glycine; inositol trisphosphate IP(3); cysteinyl CysLT(1), LTB(4); purinergic P(2Y); tachykinin NK(2); serotonin 5-HT(2B) and prostanoid, thromboxane A(2). Together, the results suggest that the various inhibitory effects of Chlorella powder in these receptor binding assays could reflect its actions in modulating Ca(2+)-dependent signal related targets and might be relevant to the mechanisms of its biological effects. These results reveal important potential biochemical activities that might be exploited for the prevention or treatment of several pathologies. From these results, the possible therapeutic usage of the product is discussed.

Chlorella pyrenoidosa supplementation reduces the risk of anemia, proteinuria and edema in pregnant women.

Pregnancy anemia and pregnancy-induced hypertension (PIH) are common and potentially dangerous disorder in human pregnancy, and nutritional status of pregnant women is one of the leading causes. Chlorella contains large quantities of folate, vitamin B-12 and iron, and can help improve anemia and hypertensive disorder. Our objective was to investigate the preventive effects of Chlorella supplement on pregnancy anemia and PIH in Japanese pregnant women. A total of 70 pregnant women were placed into the control group (n = 38) or the Chlorella group (n = 32). The subjects in the Chlorella group were supplemented daily from 12th-18th wk of gestation until delivery with 6 g of Chlorella supplement. The proportion of anemic (hemoglobin level < 11 g/dL) subjects in the Chlorella group were significantly lower compared with the control group at the second and third trimesters. Additionally, in the Chlorella group, the incidences of proteinuria and edema, signs of PIH, were significantly lower during the third trimester. These results suggest that Chlorella supplementation significantly reduces the risk of pregnancy associated anemia, proteinuria and edema. Chlorella supplement may be useful as a resource of natural folate, vitamin B-12 and iron for pregnant women.

Chlorella powder inhibits the activities of peptidase cathepsin S, PLA2, cyclooxygenase-2, thromboxane synthase, tyrosine phosphatases, tumor necrosis factor-alpha converting enzyme, calpain and kinases.

A Chlorella powder was tested in 118 in vitro enzyme assay systems. The powder showed potent inhibitions of peptidase cathepsin S, thromboxane A(2) synthase and cyclooxygenase-2 in a dose-concentration manner with IC(50)+/-standard error of the mean values of 3.46+/-0.93 microg/ml, 3.23+/-0.69 microg/ml, and 44.26+/-9.98 microg/ml, respectively. Other activities observed were inhibitions of tumor necrosis factor-alpha converting enzyme, protein tyrosine phosphatase (SHP-2), calpain, protein kinases and protein tyrosine phosphatases. Chlorella powder had no significant effect on cyclooxygenase-1. These actions to inhibit cyclooxygenase-2 and thromboxane synthase could contribute to the purported anti-inflammatory and anti-thrombotic effects of Chlorella. These results reveal important potential biochemical activities to be developed that, if confirmed by in vivo studies, might be exploited for the prevention or treatment of several serious pathologies, including inflammatory diseases, immune and cancer.

Chlorella (Chlorella pyrenoidosa) supplementation decreases dioxin and increases immunoglobulin a concentrations in breast milk.

In addition to meeting nutritional requirements, breast milk plays important roles in biodefense for nursing infants. Dioxins have been detected at high concentrations in breast milk, raising concerns about disorders in nursing infants caused by breast milk containing dioxins in Japan. We analyzed dioxin levels in breast milk and maternal blood samples from 35 pregnant women in Japan. We also measured immunoglobulin (Ig) A concentrations in breast milk and investigated correlations with dioxin concentrations. In addition, 18 of the 35 women took Chlorella pyrenoidosa (Chlorella) supplements during pregnancy, and the effects on dioxin and IgA concentrations in breast milk were investigated. Toxic equivalents were significantly lower in the breast milk of women taking Chlorella tablets than in the Control group (P = .003). These results suggest that Chlorella supplementation by the mother may reduce transfer of dioxins to the child through breast milk. No significant correlation was identified between dioxin and IgA concentrations in breast milk in the Control group. It is unlikely that normal levels of dioxin exposure via food have a remarkable influence on IgA in breast milk. IgA concentrations in breast milk in the Chlorella group were significantly higher than in the Control group (P = .03). Increasing IgA levels in breast milk is considered to be effective for reducing the risk of infection in nursing infants. The present results suggest that Chlorella supplementation not only reduces dioxin levels in breast milk, but may also have beneficial effects on nursing infants by increasing IgA levels in breast milk.

Suppression of glutathione S-transferase placental form-positive foci development in rat hepatocarcinogenesis by Chlorella pyrenoidosa.

The modifying effects of dietary administration of dried Chlorella pyrenoidosa powder (C. pyrenoidosa) on the development of glutathione S-transferase placental form-positive foci (GST-P-positive foci), which are putative preneoplastic lesions, in male F344 rats were investigated using a medium-term liver bioassay system. In rats given 10% C. pyrenoidosa in a basal diet, the number and area of GST-P-positive foci in the rat livers, which diethylnitrosamine (DEN) initiated and 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) promoted, were significantly decreased compared with those fed a basal diet not containing C. pyrenoidosa. The inhibition percentage of the number and area of GST-P-positive foci > or =0.2 mm in diameter was 67.6 and 74.2%, respectively (p<0.01). Furthermore, C. pyrenoidosa significantly decreased the number of GST-P-positive foci induced by MeIQx alone. The inhibition percentage of the number of GST-P-positive foci <0.2 mm in diameter was 52% (p<0.01). These results suggest that C. pyrenoidosa has chemopreventive effects against hepatocarcinogenesis in rats. C. pyrenoidosa appears to be a promising chemopreventive agent for human liver neoplasia and carcinogenesis induced by heterocyclic amines such as MeIQx.

Maternal-fetal distribution and transfer of dioxins in pregnant women in Japan, and attempts to reduce maternal transfer with Chlorella (Chlorella pyrenoidosa) supplements.

Dioxins can be transferred from mother to fetus via the placenta, or to nursing infants via breast milk, potentially causing developmental health problems in children. To assess pediatric health risks from dioxins, exposure of mothers and children to dioxins must be clarified. Methods of reducing maternal transfer of dioxins should also be investigated. Concentrations of 28 dioxin (polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and co-planar polychlorinated biphenyls) congeners in blood, adipose tissue, breast milk, cord blood and placenta collected from 44 pregnant Japanese women were measured. In addition, to investigate potential reductions in maternal transfer of dioxins, 23 pregnant women were instructed to take Chlorella pyrenoidosa supplements during pregnancy. Correlations were observed between dioxin total toxic equivalents (total TEQ) in blood and total TEQ in adipose tissue (r=0.913, P<0.0001), breast milk (r=0.695, P=0.0007), and cord blood (r=0.759, P<0.0001). Dioxin levels transferred to fetuses and nursing infants reflect cumulative maternal concentrations of dioxins. A linear regression equation was introduced to predict total TEQ in breast milk and cord blood from dioxin levels in maternal blood, which should prove useful in evaluating fetal and infant risk of dioxin exposure. Total TEQ in cord blood were approximately 26% lower than in maternal blood (P<0.0001). The results of this study suggest that transplacental transfer differs depending on the dioxin congener. Total TEQ in breast milk were approximately 30% lower in the Chlorella group than in controls (P=0.0113). This finding suggests that maternal transfer of dioxins can be reduced using dietary measures such as Chlorella supplements.

Effect of Chlorella pyrenoidosa on fecal excretion and liver accumulation of polychlorinated dibenzo-p-dioxin in mice.

The effect of Chlorella pyrenoidosa on fecal excretion and liver accumulation of polychlorinated dibenzo-p-dioxin in C57BL/6N mice administered dioxin was examined. Mice were administered 2.2 microg of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (H6CDD) dissolved in corn oil once after a period of acclimatization, after which they were fed either a basal diet, a 10% C. pyrenoidosa diet, or a 10% Spinach diet, for five weeks. Among mice fed the 10% C. pyrenoidosa diet, cumulative fecal excretion of H6CDD over the first week following administration was significantly greater (9.2-fold) than that observed among mice fed the basal diet. Moreover, excretion during the fifth week following administration of H6CDD was still significantly greater (3.1-fold) among mice fed the 10% C. pyrenoidosa diet than among mice fed the basal diet. Five weeks after administration of H6CDD, liver accumulation of H6CDD in mice fed the 10% C. pyrenoidosa diet was significantly less than that observed among mice fed either the basal diet and the Spinach diet (by 27.9% and 34.8%, respectively). These findings suggest that C. pyrenoidosa may be useful in inhibiting the absorption of dioxins via food and the reabsorption of dioxins stored already in the body in the intestinal tract, thus preventing accumulation of dioxins within the body.

Effects of chlorella on activities of protein tyrosine phosphatases, matrix metalloproteinases, caspases, cytokine release, B and T cell proliferations, and phorbol ester receptor binding.

A Chlorella powder was screened using 52 in vitro assay systems for enzyme activity, receptor binding, cellular cytokine release, and B and T cell proliferation. The screening revealed a very potent inhibition of human protein tyrosine phosphatase (PTP) activity of CD45 and PTP1C with 50% inhibitory concentration (IC(50)) values of 0.678 and 1.56 microg/mL, respectively. It also showed a moderate inhibition of other PTPs, including PTP1B (IC(50) = 65.3 microg/mL) and T-cell-PTP (114 microg/mL). Other inhibitory activities and their IC(50) values included inhibition of the human matrix metalloproteinases (MMPs) MMP-1 (127 microg/mL), MMP-3 (185 microg/mL), MMP-7 (18.1 microg/mL), and MMP-9 (237 microg/mL) and the human peptidase caspases caspase 1 (300 microg/mL), caspase 3 (203 microg/mL), caspase 6 (301 microg/mL), caspase 7 (291 microg/mL), and caspase 8 (261 microg/mL), as well as release of the cytokines interleukin (IL)-1 (44.9 microg/mL), IL-2 (14.8 microg/mL), IL-4 (49.2 microg/mL), IL-6 (34.7 microg/mL), interferon-gamma (31.6 microg/mL), and tumor necrosis factor-alpha (11 microg/mL) from human peripheral blood mononuclear cells. Chlorella also inhibited B cell proliferation (16.6 microg/mL) in mouse splenocytes and T cell proliferation (54.2 microg/mL) in mouse thymocytes. The binding of a phorbol ester, phorbol 12,13-dibutyrate, to its receptors was also inhibited by Chlorella with an IC(50) of 152 microg/mL. These results reveal potential pharmacological activities that, if confirmed by in vivo studies, might be exploited for the prevention or treatment of several serious pathologies, including inflammatory disease and cancer.