Amelioration of Cyclosporine A-Induced Acute Nephrotoxicity by Cordyceps cicadae Mycelia via Mg+2 Reabsorption and the Inhibition of GRP78-IRE1-CHOP Pathway: In Vivo and In Vitro.

Fruiting bodies of Cordyceps cicadae (CC) have been reported to have a therapeutic effect in chronic kidney disease. Due to the rare and expensive resources from natural habitats, artificially cultivated mycelia using submerged liquid cultivation of CC (CCM) have been recently developed as an alternative to scarce sources of CC. However, little is known regarding potential protective effects of CCM against cyclosporine A (CsA)-induced acute nephrotoxicity in vivo and in vitro. In this study, male Sprague-Dawley rats were divided into six groups: control, CCM (40 mg and 400 mg/kg, orally), CsA (10 mg/kg, oral gavage), and CsA + CCM (40 mg and 400 mg/kg, orally). At the end of the study on day 8, all rats were sacrificed, and the blood and kidneys retrieved. CsA-induced acute nephrotoxicity was evident by increased levels of blood urea nitrogen (BUN). Levels of the endoplasmic reticulum (ER) resident chaperone glucose regulated protein 78 (GRP 78) were increased significantly in rats with acute nephrotoxicity. BUN and GRP 78 were significantly ameliorated in synchronous oral groups of CCM (40 or 400 mg/kg) plus CsA. Examination of hematoxylin and eosin stained kidney tissues revealed that the combined treatment of CCM slightly improved vacuolization in renal tubules upon CsA-induced damage. CsA-induced down-regulation of protein expression of magnesium ion channel proteins and transient receptor potential melastatin 6 and 7 were abolished by the combined treatment of CCM. CCM has the potential to protect the kidney against CsA-induced nephrotoxicity by reducing magnesium ion wasting, tubular cell damage, and ER stress demonstrated further by human renal proximal tubular epithelial cell line HK-2. Our results contribute to the in-depth understanding of the role of polysaccharides and nucleobases as the main secondary metabolites of CCM in the defense system of renal functions in CsA-induced acute nephrotoxicity.

Inhibitory Effect of Styrylpyrone Extract of Phellinus linteus on Hepatic Steatosis in HepG2 Cells.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 µg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1ß) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Innovative Purification Method of Ovatodiolide from Anisomeles indica to Induce Apoptosis in Human Gastric Cancer Cells.

Ovatodiolide (Ova), found in the plant Anisomeles indica (AI), has been reported to have an anti-proliferation effect in various cancer cells. However, little information is available regarding the anti-cancer effect of Ova in human gastric cancer cells. In this study, we investigated the inhibitory effects and the mechanisms of action responsible for these effects on human AGS cell lines from a newly developed purification technique for Ova from AI extract. Extract obtained at the optimum condition of 95% ethanol extraction of AI was sequentially partitioned by using different polarity solvents. Enriched content of Ova (35.9% purity) from the n-hexane fraction was then applied to the purification by using centrifugal partition chromatography (CPC) in a two-phase solvent system consisting of n-hexane:ethyl acetate:methanol:water (1.0:1.0:1.0:1.0, v/v/v/v) to reach purity over >95.0%. In evaluation of the anti-proliferation effect on AGS cells, Ova induced cell apoptosis with IC50 values of 13.02 and 6.18 µM at 24 and 48 h, respectively, and arrested the cells at the G2/M phase. Quantification of Bax/Bcl2 mRNA expressions using qPCR showed a 2.5-fold increase in the Ova (5 µM)-treated cells at 48 h than in the control group. Specific protein expression data warrant further research to further confirm the proposed Ova-induced apoptotic pathway in AGS cells.

Hepatoprotective Effect of Antrodia cinnamomea Mycelium in Patients with Nonalcoholic Steatohepatitis: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: Nonalcoholic steatohepatitis (NASH) has become a prominent liver disease in contemporary society because of the changing dieting styles. Complicated syndromes often accompanied by obesity and diabetes makes no standard treatment for NASH. Therefore, we investigated the potential role of Antrodia cinnamomea mycelium (ACM) as nutraceutical supplementation in the treatment of NASH in this 6-month randomized, double-blind, placebo-controlled study. METHOD: 28 Participants were treated with three capsules per day containing either 420 mg of ACM or 420 mg of starch as a placebo. The participants were required to follow a predetermined regular visit to hospital every three months during the intervention period (6 months). During each study visit, subjects underwent anthropometric measurements and blood testing for biochemical analysis, immune function assay, inflammatory cytokines assay, and FibroMax test. RESULTS: The ACM supplemented group had a significant improvement in steatosis and decreased in the inflammatory marker of TNF-α after three and six months. NASH patients who received ACM showed a significant decrease in the SteatoTest mean value from 0.66 at baseline to 0.49 at 6 months (p < 0.029) and the ActiTest mean value decreased from 0.46 at baseline to 0.30 at 6 months (p < 0.029). CONCLUSION: This is the first clinical investigation that explores the hepatoprotective effect of A. cinnamomea mycelium in patients with NASH. No participants experienced any adverse events during the study, which suggested that ACM is a safe alternative treatment for NASH.

Potential Protection Effect of ER Homeostasis of N6-(2-Hydroxyethyl)adenosine Isolated from Cordyceps cicadae in Nonsteroidal Anti-Inflammatory Drug-Stimulated Human Proximal Tubular Cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage. Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N6-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK-2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex® LH-20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK-2 cells. The HK-2 cells were pretreated with HEA (10-20 µM) and then insulted with the NSAIDs diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF-6, PERK, IRE1α, CDCFHOP, IL1ß, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.

Djulis (Chenopodium formosanum) and Its Bioactive Compounds Protect Human Lung Epithelial A549 Cells from Oxidative Injury Induced by Particulate Matter via Nrf2 Signaling Pathway.

The protective effects of water extracts of djulis (Chenopodium formosanum) (WECF) and their bioactive compounds on particulate matter (PM)-induced oxidative injury in A549 cells via the nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling were investigated. WECF at 50-300 µg/mL protected A549 cells from PM-induced cytotoxicity. The cytoprotection of WECF was associated with decreases in reactive oxygen species (ROS) generation, thiobarbituric acid reactive substances (TBARS) formation, and increases in superoxide dismutase (SOD) activity and glutathione (GSH) contents. WECF increased Nrf2 and heme oxygenase-1 (HO-1) expression in A549 cells exposed to PM. SP600125 (a JNK inhibitor) and U0126 (an ERK inhibitor) attenuated the WECF-induced Nrf2 and HO-1 expression. According to the HPLC-MS/MS analysis, rutin (2219.7 µg/g) and quercetin derivatives (2648.2 µg/g) were the most abundant bioactive compounds present in WECF. Rutin and quercetin ameliorated PM-induced oxidative stress in the cells. Collectively, the bioactive compounds present in WECF can protect A549 cells from PM-induced oxidative injury by upregulating Nrf2 and HO-1 via activation of the ERK and JUN signaling pathways.

Antrodan Alleviates High-Fat and High-Fructose Diet-Induced Fatty Liver Disease in C57BL/6 Mice Model via AMPK/Sirt1/SREBP-1c/PPARγ Pathway.

Non-alcoholic fatty liver disease (NAFLD) and -steatohepatitis (NASH) imply a state of excessive fat built-up in livers with/or without inflammation and have led to serious medical concerns in recent years. Antrodan (Ant), a purified ß-glucan from A. cinnamomea has been shown to exhibit tremendous bioactivity, including hepatoprotective, antihyperlipidemic, antiliver cancer, and anti-inflammatory effects. Considering the already well-known alleviating bioactivity of A. cinnamomea for the alcoholic steatohepatitis (ASH), we propose that Ant can be beneficial to NAFLD, and that the AMPK/Sirt1/PPARγ/SREBP-1c pathways may be involved in such alleviations. To uncover this, we carried out this study with 60 male C57BL/6 mice fed high-fat high-fructose diet (HFD) for 60 days, in order to induce NAFLD/NASH. Mice were then grouped and treated (by oral administration) as: G1: control; G2: HFD (HFD control); G3: Ant, 40 mgkg (Ant control); G4: HFD+Orlistat (10 mg/kg) (as Orlistat control); G5: HFD+Ant L (20 mg/kg); and G6: HFD+Ant H (40 mg/kg) for 45 days. The results indicated Ant at 40 mg/kg effectively suppressed the plasma levels of malondialdehyde, total cholesterol, triglycerides, GOT, GPT, uric acid, glucose, and insulin; upregulated leptin, adiponectin, pAMPK, Sirt1, and down-regulated PPARγ and SREBP-1c. Conclusively, Ant effectively alleviates NAFLD via AMPK/Sirt1/CREBP-1c/PPARγ pathway.

Anti-Metastatic Effects of Antrodan with and without Cisplatin on Lewis Lung Carcinomas in a Mouse Xenograft Model.

Antrodan, a unique protein-bound polysaccharide derived from the fungal mycelia of Antrodia cinnamomea, has been reported to exhibit antitumor and anti-metastatic effects on Lewis lung carcinoma (LLC) cells through direct action and immunomodulation in vitro. In this study, we investigated the combined treatment of antrodan with an anti-cancer drug-cisplatin-and its underlying molecular mechanisms of action in a mouse xenograft tumor model. C57BL/6 mice were implanted (s.c.) with LLCs for nine days, before administration with only antrodan (20 mg/kg and 40 mg/kg; p.o.) daily, only cisplatin (1 mg/kg; i.p.) twice per week, or a combination of both for an additional 28 days. As expected, antrodan on its own significantly inhibited metastasis of lung and liver tissues, while treatment with cisplatin only merely inhibited metastasis of the liver. Antrodan exhibited efficient adjuvant therapy in combination with cisplatin, by inhibiting the activities of the plasma urokinase plasminogen activator (uPA) and the liver matrix metalloproteinase 9 (MMP-9), as well as by inhibiting the phosphorylation of p38 and extracellular signal-regulated kinase 2 (ERK2) in lung and liver tissues. In addition, antrodan effectively ameliorated cisplatin-induced kidney dysfunction when treated combinatorially, as evidenced by a decrease in cisplatin-induced blood urea nitrogen (BUN) levels in plasma and in the level of p38 phosphorylation in the kidney. Mechanistically, the actions of antrodan on its own involved (i) reducing the activities of uPA and MMP-2 and -9 in plasma; (ii) reducing protein expression of MMP-2/9, and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 in lung and liver tissues; and (iii) enhancing immune system functions resulting in the promotion of an anti-metastatic response through immunomodulation, by increasing interferon-γ (IFN-γ) levels and decreasing interleukin-6 (IL-6) levels in plasma. These results demonstrated that antrodan provides a novel, complementary therapeutic strategy against cancer metastasis, by attenuating the activities of MMP-2 and -9 through the modulation of STAT3/MAPK/ERK/JNK signaling pathways, and of the host's immune system.

Erinacine A-Enriched Hericium erinaceus Mycelium Produces Antidepressant-Like Effects through Modulating BDNF/PI3K/Akt/GSK-3ß Signaling in Mice.

Antidepressant-like effects of ethanolic extract of Hericium erinaceus (HE) mycelium enriched in erinacine A on depressive mice challenged by repeated restraint stress (RS) were examined. HE at 100, 200 or 400 mg/kg body weight/day was orally given to mice for four weeks. After two weeks of HE administration, all mice except the control group went through with 14 days of RS protocol. Stressed mice exhibited various behavioral alterations, such as extending immobility time in the tail suspension test (TST) and forced swimming test (FST), and increasing the number of entries in open arm (POAE) and the time spent in the open arm (PTOA). Moreover, the levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were decreased in the stressed mice, while the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were increased. These changes were significantly inverted by the administration of HE, especially at the dose of 200 or 400 mg/kg body weight/day. Additionally, HE was shown to activate the BDNF/TrkB/PI3K/Akt/GSK-3ß pathways and block the NF-κB signals in mice. Taken together, erinacine A-enriched HE mycelium could reverse the depressive-like behavior caused by RS and was accompanied by the modulation of monoamine neurotransmitters as well as pro-inflammatory cytokines, and regulation of BDNF pathways. Therefore, erinacine A-enriched HE mycelium could be an attractive agent for the treatment of depressive disorders.

The Inhibitory Effects of Djulis (Chenopodium formosanum) and Its Bioactive Compounds on Adipogenesis in 3T3-L1 Adipocytes.

The aim of this study was to provide new insights into the role of the ethanolic extracts of Djulis (Chenopodium formosanum, EECF) and its bioactive compounds in preventing adipogenesis in 3T3-L1 adipocytes. The results demonstrated EECF significantly inhibited oil red O-stained material (OROSM), triglyceride levels and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 adipocytes. The expression of the critical molecules involved in lipid synthesis such as PPARγ, C/EBPα and SREBP-1c was attenuated in EECF-treated cells. According to HPLC-DAD and HPLC-MS/MS analysis, rutin, kaempferol, betanin and another nine compounds were present in EECF. The suppression of lipid accumulation by rutin, kaempferol and betanin occurred by decreasing the gene expression of PPARγ, C/EBPα and SREBP-1c. Taken together, these findings suggest the presence of bioactive compounds in EECF may partly account for the anti-adipogenesis of EECF and EECF is therefore a potentially lipid lowering functional food.

The induction of apoptosis and autophagy by Wasabia japonica extract in colon cancer.

PURPOSE: Wasabia japonica (wasabi) has been shown to exhibit properties of detoxification, anti-inflammation and the induction of apoptosis in cancer cells. This study aimed to investigate the molecular mechanism of the cytotoxicity of wasabi extract (WE) in colon cancer cells to evaluate the potential of wasabi as a functional food for chemoprevention. METHODS: Colo 205 cells were treated with different doses of WE, and the cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Apoptosis and autophagy were detected by 4',6-diamidino-2-phenylindole, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-yanine iodide and staining for acidic vascular organelles (AVOs), along with Western blotting. RESULTS: The results demonstrated that WE induced the extrinsic pathway and mitochondrial death machinery through the activation of TNF-α, Fas-L, caspases, truncated Bid and cytochrome C. WE also induced autophagy by decreasing the phosphorylation of Akt and mTOR and promoting the expression of microtubule-associated protein 1 light chain 3-II and AVO formation. An in vivo xenograft model verified that tumor growth was delayed by WE treatment. CONCLUSION: Our studies revealed that WE exhibits anti-colon cancer properties through the induction of apoptosis and autophagy. These results provide support for the application of WE as a chemopreventive functional food and as a prospective treatment of colon cancer.

Improved Hepatoprotective Effect of Liposome-Encapsulated Astaxanthin in Lipopolysaccharide-Induced Acute Hepatotoxicity.

Lipopolysaccharide (LPS)-induced acute hepatotoxicity is significantly associated with oxidative stress. Astaxanthin (AST), a xanthophyll carotenoid, is well known for its potent antioxidant capacity. However, its drawbacks of poor aqueous solubility and low bioavailability have limited its utility. Liposome encapsulation is considered as an effective alternative use for the improvement of bioavailability of the hydrophobic compound. We hypothesized that AST encapsulated within liposomes (LA) apparently shows improved stability and transportability compared to that of free AST. To investigate whether LA administration can efficiently prevent the LPS-induced acute hepatotoxicity, male Sprague-Dawley rats (n = six per group) were orally administered liposome-encapsulated AST at 2, 5 or 10 mg/kg-day (LA-2, LA-5, and LA-10) for seven days and then were LPS-challenged (i.p., 5 mg/kg). The LA-10 administered group, but not the other groups, exhibited a significant amelioration of serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), blood urea nitrogen (BUN), creatinine (CRE), hepatic malondialdehyde (MDA) and glutathione peroxidase (GSH-Px), IL-6, and hepatic nuclear NF-κB and inducible nitric oxide synthase (iNOS), suggesting that LA at a 10 mg/kg-day dosage renders hepatoprotective effects. Moreover, the protective effects were even superior to that of positive control N-acetylcysteine (NAC, 200 mg/kg-day). Histopathologically, NAC, free AST, LA-2 and LA-5 partially, but LA-10 completely, alleviated the acute inflammatory status. These results indicate that hydrophobic AST after being properly encapsulated by liposomes improves bioavailability and can also function as potential drug delivery system in treating hepatotoxicity.

Sixteen years post radiotherapy of nasopharyngeal carcinoma elicited multi-dysfunction along PTX and chronic kidney disease with microcytic anemia.

BACKGROUND: The hypothalamic-pituitary (h-p) unit is a particularly radiosensitive region in the central nervous system. As a consequence, radiation-induced irreversible, progressively chronic onset hypopituitarism (RIH) commonly develops after radiation treatments and can result in variably impaired pituitary function, which is frequently associated with increased morbidity and mortality. CASE PRESENTATION: A 38-year-old male subject, previously having received radiotherapy for treatment of nasopharygeal carcinoma (NPCA) 16 years ago, appeared at OPD complaining about his failure in penile erection, loss of pubic hair, atrophy of external genitalia: testicles reduced to 2×1.5 cm; penile size shrunk to only 4 cm long. Characteristically, he showed extremely lowered human growth hormone, (HGH, 0.115 ng/mL), testosterone (<0.1 ng/mL), total thyroxine (tT4: 4.740 g/mL), free T4 (fT4, 0.410 ng/mL), cortisol (2.34 g/dL); lowered LH (1.37 mIU/mL) and estradiol (22 pg/mL); highly elevated TSH (7.12 IU/mL). As contrast, he had low end normal ACTH, FSH, total T3, free T3, and estriol; high end normal prolactin (11.71 ng/mL), distinctly implicating hypopituitarism-induced hypothyroidism and hypogonadism. serologically, he showed severely lowered Hb (10.6 g/dL), HCT (32.7%), MCV (77.6 fL), MCH (25.3 pg), MCHC (32.6 g/dL), and platelet count (139×103/L) with extraordinarily elevated RDW (18.2%), together with severely lowered ferritin (23.6 ng/mL) and serum iron levels; highly elevated total iron binding capacity (TIBC, 509 g/dL) and transferrin (363.4 mg/dL), suggesting microcytic anemia. Severely reduced estimated glomerular filtration rate (e-GFR) (89 mL/mim/1.73 m2) pointed to CKD2. Hypocortisolemia with hyponatremia indicated secondary adrenal insufficiency. Replacement therapy using androgen, cortisol, and Ringer's solution has shown beneficial in improving life quality. CONCLUSIONS: To our believe, we are the first group who report such complicate PTX dysfunction with adrenal cortisol insufficiency concomitantly occurring in a single patient.

Comparison of the apoptotic effects of supercritical fluid extracts of Antrodia cinnamomea mycelia on hepatocellular carcinoma cells.

Antrodia cinnamomea (AC) has been widely used as a folk medicine in the prevention and treatment of liver diseases, such as hepatitis, hepatic fibrosis, and hepatocellular carcinoma. Previous studies have indicated that triterpenoids and benzenoids show selective cytotoxicity against human hepatoma cell lines. The aim of the study was to compare the triterpenoid content of extract and the extract-induced cytotoxicity in HepG2 cells from mycelia extracts of solid state cultured AC obtained by supercritical fluid extraction (SFE) and the conventional solvent extraction method. SFE with CO2 mixed with a constant amount of ethanol co-solvent (10% of CO2 volume) applied at different temperatures and pressures (40, 60 and 80 °C and, 20.7, 27.6 and 34.5 Mpa) was also compared in the study. Although the extraction yield of triterpenoids (59.7 mg/g) under the optimal extraction conditions of 34.5 MPa (5000 psi)/60 °C (designated as sample S-5000-60) was equivalent to the extraction yield using conventional liquid solvent extraction with ethanol (ETOH-E) at room temperature (60.33 mg/g), the cytotoxicity of the former against the proliferation of HepG2 cell line measured as the inhibition of 50% of cell growth activity (IC50) at dosages of 116.15, 57.82 and 43.96 µg/mL was superior to that of EtOH-E at 131.09, 80.04 and 48.30 µg/mL at 24, 48 and 72 h, respectively. Additionally, we further proved that the apoptotic effect of S-5000-60 presented a higher apoptosis ratio (21.5%) than ETOH-E (10.5%) according to annexin V-FITC and propidium iodide double staining assay results. The high affinity and selectivity of SFE on bioactive components resulted in a higher extraction efficiency than conventional solvent extraction. The chemical profile of the obtained extracts from solid state cultivated mycelium of AC was also determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS), whereby three benzenoids and four triterpenoids were found for the first time in SFE extracts with 4,7-dimethoxy-5-methyl-l,3-benzodioxole (5.78 mg/g) being the most abundant component, followed by 2,4-dimethoxy-6-methylbenzene-1,3-diol (3.03 mg/g) and dehydroeburicoic acid (0.40 mg/g).

Extraction of antioxidant components from Bidens pilosa flowers and their uptake by human intestinal Caco-2 cells.

Bidens pilosa L. var. radiata (BPR, Asteraceae) is a commonly used folk medicine for treating various disorders such as diabetes, inflammation and hypertension. Recent studies to determine its chemical composition have revealed three di-O-caffeoylquinic acids (DiCQAs) and three polyacetylene glucosides (PGAs) to be among the major bioactive markers. To obtain the major compounds of these two chemical classes, the ethyl acetate fraction (EM) obtained using liquid-liquid partition from the methanol extract resulted in a fraction with the highest total phenolic and total flavonoid contents and antioxidant activities in radical scavenging and ferric reducing power assays. To assess the bioavailability of EM, we examined the in vitro uptake using the Caco-2 human colonic cell line. The apparent permeability coefficient (Papp) for each of the compounds within PGAs measured in both apical (AP) to basolateral (BL) and BL to AP was found to preferentially appear BL to AP direction, indicated that a basolateral to apical efflux system was detected in the study. DiCQAs had a lower efflux ratio than those from PGAs (2.32-3.67 vs. 6.03-78.36). Thus, it strongly implies that most of the DiCQAs are better absorbed than the PGAs.

Physicochemical characteristics and anti-inflammatory activities of antrodan, a novel glycoprotein isolated from Antrodia cinnamomea mycelia.

Antrodia cinnamomea (AC) is a unique fungus found inhabiting the rotten wood of Cinnamomum kanehirai. A submerged liquid culture of AC has been developed and its bioproducts have been used to meet the market demand for natural fruiting bodies. AC exhibits anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. Previously, we isolated polysaccharide AC-2 from AC mycelia by means of alkali extraction with subsequent acid precipitation and found it had a pronounced anti-inflammatory effect. In this study, a novel polysaccharide named "antrodan" was obtained by further purification of AC-2 using Sepharose CL-6B column chromatography. Antrodan exhibited a molecular weight of 442 kD and contained a particularly high content of uronic acid (152.6±0.8 mg/g). The protein content was 71.0%, apparently, higher than the carbohydrate content (14.1%), and thus antrodan was characterized as a glycoprotein. Its total glucan content was 15.65%, in which ß-glucan (14.20%) was prominently higher than α-glucan (1.45%). Its FTIR confirmed the presence of ß-linkages between sugars, and intramolecular amide bonds between sugars and amino acids. Its 1H-NMR spectrum showed that antrodan was a complex union of α- and ß-glucans, which had (1→4)-linked α-Glcp and (1→3)-linked ß-Glcp linkages to the carbohydrate chains via asparagine linked to protein site. Biologically, antrodan was confirmed to be totally non-detrimental to RAW 264.7 cell line even at dose as high as 400 µg/mL. It showed potent suppressing effect on the lipopolysaccharide-induced inflammatory responses in RAW 264.7 cell line. Moreover, antrodan significantly reduced the nitrogen oxide production at doses as low as 18.75 µg/mL.

Unique bioactive polyphenolic profile of guava (Psidium guajava) budding leaf tea is related to plant biochemistry of budding leaves in early dawn.

BACKGROUND: Guava leaf tea (GLT), exhibiting a diversity of medicinal bioactivities, has become a popularly consumed daily beverage. To improve the product quality, a new process was recommended to the Ser-Tou Farmers' Association (SFA), who began field production in 2005. The new process comprised simplified steps: one bud-two leaves were plucked at 3:00-6:00 am, in the early dawn period, followed by withering at ambient temperature (25-28 °C), rolling at 50 °C for 50-70 min, with or without fermentation, then drying at 45-50 °C for 70-90 min, and finally sorted. RESULTS: The product manufactured by this new process (named herein GLTSF) exhibited higher contents (in mg g(-1), based on dry ethyl acetate fraction/methanolic extract) of polyphenolics (417.9 ± 12.3) and flavonoids (452.5 ± 32.3) containing a compositional profile much simpler than previously found: total quercetins (190.3 ± 9.1), total myricetin (3.3 ± 0.9), total catechins (36.4 ± 5.3), gallic acid (8.8 ± 0.6), ellagic acid (39.1 ± 6.4) and tannins (2.5 ± 9.1). CONCLUSION: We have successfully developed a new process for manufacturing GLTSF with a unique polyphenolic profile. Such characteristic compositional distribution can be ascribed to the right harvesting hour in the early dawn and appropriate treatment process at low temperature, avoiding direct sunlight.

Improvement of lipopolysaccharide-induced hepatic injuries and inflammation with mulberry extracts.

BACKGROUND: Mulberry water extracts (MWEs), which contain polyphenols including anthocyanins, have been used in traditional Chinese edible food. The hepatoprotective effects and molecular mechanisms of MWEs on acute liver failure induced by lipopolysaccharides (LPS) were investigated in vivo. RESULTS: Rats were administered different doses of MWEs (0.5 and 1 g kg(-1)) 1 h before injection of LPS (5 mg kg(-1)) and then sacrificed 10 h after treatment with LPS. Liver function, inflammatory factors, oxidative stress index and hepatic histopathological alteration were examined in the rats with and without MWE treatment. Pretreatment with MWEs prevented LPS-induced liver damage by preventing associated increases of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALKP), triglycerol (TG), cholesterol and low-density lipoprotein/high-density lipoprotein ratio. MWEs also suppressed oxidative stress to prevent the formation of hepatic malondialdehyde (MDA). Furthermore, the molecular mechanism involved in LPS-induced liver injury was associated with reducing the expression of COX-2, NF-κB and iNOS in liver tissues. CONCLUSION: The results support the investigation of MWEs as a therapeutic candidate for liver injuries and indicate that MWEs exhibit hepatoprotective activities via NF-κB signaling.

The Nephroprotective Effects of Hibiscus sabdariffa Leaf and Ellagic Acid in Vitro and in Vivo Models of Hyperuricemic Nephropathy.

Hyperuricemic nephropathy (HN) is caused by urate crystals that get deposited in the kidney and contribute to renal fibrosis. Uric acid (UA) has been proven to directly cause renal mesangial cell oxidative stress and fibrosis in the pathogenesis of HN. Some antioxidants can be used as chemopreventive agents of HN. Hibiscus sabdariffa leaf extracts (HLE), rich in polyphenol, have been shown to possess hypoglycemic, antioxidant, hypolipidemic, antiatherosclerotic, and anticancer effects. The aim of the study is to examine the inhibitory effect of HLE and its main component ellagic acid (EA) on renal fibrosis. In vitro, mouse renal glomerular mesangial SV40MES13 cells pretreated with UA were demonstrated to trigger obvious morphological changes and viability loss, as well as affect matrix metalloproteinases (MMPs) activities. Noncytotoxic doses of HLE and EA abolished the UA-induced cell injury and MMP-2/9 secretion. In addition, HLE and EA exhibited antioxidant and anti-inflammatory effects on the UA-treated cells with a reduction in transforming growth factor-beta (TGF-ß) production. Next, the UA-activated pro-fibrotic factors, extracellular matrix (ECM) deposition, and epithelial-mesenchymal-transition (EMT) were inhibited by HLE or EA. Mechanistic assays indicated that antifibrotic effects of HLE might be mediated via TGF-ß/Smad signaling, as confirmed by the transfection of Smad7 siRNA. In vivo, HLE and EA supplementations significantly alleviated HN development, which may result from inhibiting adenine-induced TGF-ß production accompanying oxidative stress and inflammation, as well as fibrogenesis. Our data imply that EA-enriched HLE regulates the TGF-ß/Smad signaling, which in turn led to reduced renal mesangial cell injury and fibrosis in HN and provided a new mechanism for its nephroprotective activity.

Protective Effect of Hawthorn Fruit Extract against High Fructose-Induced Oxidative Stress and Endoplasmic Reticulum Stress in Pancreatic ß-Cells.

Hyperglycemia has deleterious effects on pancreatic ß-cells, causing dysfunction and insulin resistance that lead to diabetes mellitus (DM). The possible causes of injury can be caused by glucose- or fructose-induced oxidative and endoplasmic reticulum (ER) stress. Hawthorn (Crataegus pinnatifida) fruit has been widely used as a hypolipidemic agent in traditional herbal medicine. The study aimed to investigate whether high fructose-induced pancreatic ß-cell dysfunction could be reversed through amelioration of ER stress by the treatment of polyphenol-enriched extract (PEHE) from hawthorn fruit. The extract was partitioned using ethyl acetate as a solvent from crude water extract (WE) of hawthorn fruits, followed by column fractionation. The results showed that the contents of total polyphenols, flavonoids and triterpenoids in PEHE could be enhanced by 2.2-, 7.7- and 1.1-fold, respectively, in comparison to the original obtained WE from hawthorn fruit. In ER stress studies, a sharp increase in the inhibitory activity on the gene expression levels of GRP79, ATF6, IRE1α and CHOP involved in ER stress was evident when dosages of PEHE at 50-100 µg/mL were used against high-fructose (150 mM)-treated cells. HPLC-MS/MS analysis showed that polyphenols and flavonoids collectively accounted for 87.03% of the total content of PEHE.