Acetylation Enhances the Anticancer Activity and Oral Bioavailability of 5-Demethyltangeretin.

A kind of hydroxylated polymethoxyflavone (PMFs) existing in the citrus genus, 5-Demethyltangeretin (5-DTAN), has been reported to possess several bioactivities in vitro and in vivo. The aim of this study was to investigate whether acetylation could enhance the anticancer activity and oral bioavailability of 5-DTAN. PC-3 human prostate cancer cells were treated with tangeretin (TAN), 5-DTAN, and 5-acetylated TAN (5-ATAN), and the results showed that the cytotoxic effect 5-ATAN (IC50 value of 5.1 µM) on the cell viability of PC-3 cells was stronger than that of TAN (IC50 value of 17.2 µM) and 5-DTAN (IC50 value of 11.8 µM). Compared to 5-DTAN, 5-ATAN treatment caused a more pronounced DNA ladder, increased the sub-G1 phase population, and induced G2/M phase arrest in the cell cycle of PC-3 cells. We also found that 5-ATAN triggered the activation of caspase-3 and the progression of the intrinsic mitochondrial pathway in PC-3 cells, suggesting the induction of apoptosis. In a cell wound healing test, 5-ATAN dose-dependently reduced the cell migration, and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was decreased after 48 h of 5-ATAN treatment. Moreover, oral administration of 5-ATAN showed a significantly stronger inhibitory effect on tumor size and tumor weight in tumor-bearing nude mice than those of vehicle or the 5-DTAN group (p < 0.05). Furthermore, pharmacokinetic results showed that single-dose oral administration of 5-ATAN exhibited a higher maximum concentration (Cmax) and area under the curve (AUC) of 5-DTAN in plasma than that of 5-DTAN. More extensive distribution of 5-DTAN to most tissues of mice was also observed in mice treated with 5-ATAN for 7 days. In conclusion, acetylation strongly enhances the anticancer activity and oral bioavailability of 5-DTAN and could be a promising strategy to promote the potential bioactivities of natural products.

The Effect of Far-Infrared Therapy on the Peritoneal Expression of Glucose Degradation Products in Diabetic Patients on Peritoneal Dialysis.

Peritoneal dialysis (PD) is a treatment modality for end-stage renal disease (ESRD) patients. Dextrose is a common osmotic agent used in PD solutions and its absorption may exacerbate diabetes mellitus, a common complication of ESRD. PD solutions also contain glucose degradation products (GDPs) that may lead to encapsulating peritoneal sclerosis (EPS), a severe complication of PD. A previous study showed that far-infrared (FIR) therapy improved a patient's gastrointestinal symptoms due to EPS. Due to limited literature on the matter, this study aims to investigate dialysate GDPs and peritoneal function in diabetic patients on PD. Thirty-one PD patients were enrolled and underwent 40 min of FIR therapy twice daily for six months. We demonstrated the effect of FIR therapy on the following: (1) decrease of methylglyoxal (p = 0.02), furfural (p = 0.005), and 5-hydroxymethylfurfural (p = 0.03), (2) increase of D/D0 glucose ratio (p = 0.03), and (3) decrease of potassium levels (p = 0.008) in both DM and non-DM patients, as well as (4) maintenance and increase of peritoneal Kt/V in DM and non-DM patients, respectively (p = 0.03). FIR therapy is a non-invasive intervention that can decrease dialysate GDPs in PD patients by improving peritoneal transport rate and solute removal clearance, while also maintaining dialysis adequacy.

Citrus depressa peel extract acts as a prebiotic to reduce lipid accumulation and modulate gut microbiota in obese mice.

Citrus peels contain abundant polyphenols, particularly flavonoids, and have been shown to exert lipid accumulation decreasing ability. In this study, Citrus depressa peel applied to oven drying and extracted with ethanol extract as CDEE to analyze its flavonoids compositions and investigated its effects on a high-fat diet (HFD)-induced obese mice model. CDEE contained several flavonoids such as hesperidin, sinesentin, nobiletin, tangeretin, 5-demethylnobiletin, and 5-demethyltangeretin. The mice fed an HFD, and administration of 2% CDEE to could decrease weight gain, abdominal fat weight, inguinal fat weight, and the adipocyte size, and CDEE also reduced serum total cholesterol (TCHO), triacylglycerol (TG) compared with mice fed only on HFD. CDEE hindered lipid accumulation through a decreased fatty acid synthase (FAS) protein expression via upregulation of the protein expression of AMP-activated protein kinase α (AMPKα). Moreover, CDEE modulated gut microbiota that altered by HFD through an increased abundance of Lactobacillus reuteri compared with the HFD group. The results demonstrated that CDEE helps decrease lipid accumulation through the AMPK pathway, which also indicates a prebiotic-like effect on gut microbiota.

Tangeretin and 4'-demethyltangeretin prevent damage to mouse hepatocytes from oxidative stress by activating the Nrf2-related antioxidant pathway via an epigenetic mechanism.

Polymethoxyflavones (PMFs) in citrus fruits have a variety of biological activities, including antioxidant, anti-inflammatory, anticancer, and anti-atherosclerotic effects. The liver is the major detoxifying organ of the human body; however, factors such as acetaminophen (APAP) overdose may increase oxidative stress in liver cells and lead to severe liver failure. In this study we examined the effects of tangeretin (TAN), a common citrus PMF, and its metabolite 4'-demethyltangeretin (4'-OH-TAN) on activation of the Nrf2 antioxidant system in mouse AML-12 hepatocytes through regulation by epigenetic mechanisms. The ability of TAN and 4'-OH-TAN to inhibit APAP-induced hepatotoxicity was also evaluated. The results showed that TAN and 4'-OH-TAN significantly increased the mRNA and protein levels of Nrf2 and Nrf2-mediated antioxidant and detoxifying enzymes (UGT1A, HO-1, and NQO1) in AML-12 cells. TAN and 4'-OH-TAN also suppressed protein expression of histone deacetylases (HDACs) and DNA methyltransferases (DMNTs) and reduced DNA methylation of the nrf2 promoter. Furthermore, TAN and 4'-OH-TAN prevented APAP-induced injury and inhibited APAP-induced ROS generation in AML-12 cells. Based on these results, we conclude that TAN and 4'-OH-TAN may increase the antioxidant capacity of liver cells by regulating epigenetic alteration to activate the Nrf2-related antioxidant system, thereby preventing liver cells from being damaged by APAP-induced oxidative stress.

Proteomics for species authentication of cod and corresponding fishery products.

Seafood substitutions is a global problem and come under the spotlight in recent years. In Taiwan, Greenland halibut is usually substituted for the cod because of its lower price. Nowadays, DNA technology is widely used for fish species identifications; however, it still has concern about the DNA of processed fishery products might be destroyed. This study was designed to develop a proteomic-based method for fish and fishery product authentication by using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) with Sequential window acquisition of all theoretical fragment ion spectra (SWATH). The protein biomarkers from the meat of Alaska pollock, Atlantic cod, and Greenland halibut were identified and validated for species authentication of cod and corresponding fishery products, which might prevent consumer substitutions and fish product mislabeling. Besides, the E. coli proteins can be measured from existing SWATH-MS data though retrospective analysis successfully, it might present the quality of fish meat.

Probabilistic risk and benefit assessment of nitrates and nitrites by integrating total diet study-based exogenous dietary exposure with endogenous nitrite formation using toxicokinetic modeling.

The impacts of dietary nitrates and nitrites on human health have been a controversial topic for many years. However, the risk and benefit assessment of nitrates and nitrites is complicated by the large variation in nitrate and nitrite intake among people and the endogenous nitrite formation in the body. This study conducted a probabilistic risk-benefit assessment of dietary nitrates and nitrites based on internal dose by integrating exogenous and endogenous exposures with human trial data on cardiovascular benefits. A total diet study was carried out to quantify the age-specific dietary intakes of nitrates and nitrites. A previously well-validated human toxicokinetic model was used to predict internal doses for different age groups. In addition, the integrated approach was applied to different populations from different countries/regions based on reported exposure estimates to conduct a comprehensive risk-benefit assessment of dietary nitrates and nitrites. The results demonstrated that vegetable consumption was the main contributor to the internal nitrate and nitrite levels in all age groups. Exposure to nitrates and nitrites exceeding acceptable daily intakes in a variety of foods showed cardiovascular benefits. The probabilistic risk assessment showed that the exposure to nitrates and nitrites did not pose an appreciable health and safety risk. Therefore, the present results suggest that dietary nitrates and nitrites have clear cardiovascular benefits that may outweigh potential risks. Our analysis contributes significantly to addressing the controversy regarding risks and benefits from dietary nitrates and nitrites, and our approach could be applied to other dietary constituents with the potential for both risks and benefits.

A Tangeretin Derivative Inhibits the Growth of Human Prostate Cancer LNCaP Cells by Epigenetically Restoring p21 Gene Expression and Inhibiting Cancer Stem-like Cell Proliferation.

Prostate cancer ranks the second in incidence and the fifth in mortality cancer in male globally. Citrus polymethoxyflavonoids (PMFs), such as tangeretin (PMF1), have been found to exhibit various biological activities. Here, we evaluated the inhibitory effects and mechanism of synthetic 5,4'-didemethyltangeretin (PMF2) on human prostate cancer LNCaP cells. We found that PMF2 inhibited the growth of LNCaP cells (GI50 14.6 µM) more strongly than PMF1, and it was less cytotoxic against the normal human prostate RWPE-1 cells. PMF2 upregulated Bad and Bax, downregulated Bcl-2, and activated caspase-3 and PARP in the LNCaP cells, thereby inducing apoptosis. PMF2 also suppressed the anchorage-independent growth of the LNCaP cells. It triggered p21 gene expression by demethylation of the p21 promoter region, and inhibited the protein expressions of DNMT 3B and HDACs 1, 2, and 4/5/9 by epigenetic regulations. We further found that PMF2 showed interactions with DNMTs 1, 2, and 3A ex vivo, which might inhibit DNMT activity. Additionally, PMF2 decreased the anchorage-independent growth of isolated LNCaP cancer stem-like cells (CSLCs) with high CD166 mRNA expression. These results indicated that PMF2 might inhibit the growth of human prostate cancer cells through different mechanisms, suggesting that PMF2 could be an innovative agent for prostate cancer therapy and prevention.

Pharmacokinetics, bioavailability, tissue distribution and excretion of tangeretin in rat.

Tangeretin, 4',5,6,7,8-pentamethoxyflavone, is one of the major polymethoxyflavones (PMFs) existing in citrus fruits, particularly in the peels of sweet oranges and mandarins. Tangeretin has been reported to possess several beneficial bioactivities including anti-inflammatory, anti-proliferative and neuroprotective effects. To achieve a thorough understanding of the biological actions of tangeretin in vivo, our current study is designed to investigate the pharmacokinetics, bioavailability, distribution and excretion of tangeretin in rats. After oral administration of 50 mg/kg bw tangeretin to rats, the Cmax, Tmax and t1/2 were 0.87 ± 0.33 µg/mL, 340.00 ± 48.99 min and 342.43 ± 71.27 min, respectively. Based on the area under the curves (AUC) of oral and intravenous administration of tangeretin, calculated absolute oral bioavailability was 27.11%. During tissue distribution, maximum concentrations of tangeretin in the vital organs occurred at 4 or 8 h after oral administration. The highest accumulation of tangeretin was found in the kidney, lung and liver, followed by spleen and heart. In the gastrointestinal tract, maximum concentrations of tangeretin in the stomach and small intestine were found at 4 h, while in the cecum, colon and rectum, tangeretin reached the maximum concentrations at 12 h. Tangeretin excreted in the urine and feces was recovered within 48 h after oral administration, concentrations were only 0.0026% and 7.54%, respectively. These results suggest that tangeretin was mainly eliminated as metabolites. In conclusion, our study provides useful information regarding absorption, distribution, as well as excretion of tangeretin, which will provide a good base for studying the mechanism of its biological effects.

5-Demethylnobiletin and 5-Acetoxy-6,7,8,3',4'-pentamethoxyflavone Suppress Lipid Accumulation by Activating the LKB1-AMPK Pathway in 3T3-L1 Preadipocytes and High Fat Diet-Fed C57BL/6 Mice.

Polymethoxyflavones (PMFs) and hydroxylated polymethoxyflavones (HPMFs), such as nobiletin (Nob) and 5-demethylnobiletin (5-OH-Nob), are unique flavonoids that are found exclusively in citrus peels. Nobiletin has been shown to suppress adipogenesis in vitro, but the antiadipogenic activity of 5-OH-Nob has not been investigated. Both nobiletin and 5-OH-Nob have poor aqueous solubility and low oral bioavailability. We employed chemical modification to produce the acetyl derivative of 5-OH-Nob, that is, 5-acetyloxy-6,7,8,3',4'-pentamethoxyflavone (5-Ac-Nob), to improve its bioavailability and bioactive efficiency. We found that 5-Ac-Nob reduced triacylglycerol (TG) content to a greater extent than 5-OH-Nob in 3T3-L1 preadipocytes. Orally administered 5-Ac-Nob resulted in a significant reduction in body weight, intra-abdominal fat, plasma and liver TG levels, and plasma cholesterol level in high fat diet-induced obese male C57BL/6J mice. The 5-Ac-Nob treatment decreased lipid accumulation by triggering the adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway to alter transcriptional factors or lipogenesis-related enzymes in vivo and in vitro.

An integrated electrophoretic mobility control device with split design for signal improvement in liquid chromatography-electrospray ionization mass spectrometry analysis of aminoglycosides using a heptafluorobutyric acid containing mobile phase.

Electrophoretic mobility control (EMC) was used to alleviate the adverse effect of the ion-pairing agent heptafluorobutyric acid (HFBA) in the liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis of aminoglycosides. Aminoglycosides separated by LC were directed to a connecting column before their detection via ESI. Applying an electric field across the connecting column caused the positively charged aminoglycosides to migrate toward the mass spectrometer whereas the HFBA anions remained in the junction reservoir, thus alleviating the ion suppression caused by HFBA. To accommodate the flow rate of a narrow-bore column, minimize the effect of electrophoretic mobility on separation, and facilitate the operation, an integrated EMC device with a split design was fabricated. With the proposed EMC device, the signals of aminoglycosides were enhanced by a factor of 5-85 without affecting the separation efficiency or elution order. For the analysis of aminoglycosides in bovine milk, the proposed approach demonstrates a sensitivity that is at least 10 times below the maximum residue limits set by most countries.

Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry.

Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate.

Identification of 5,7,3',4'-tetramethoxyflavone metabolites in rat urine by the isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry.

5,7,3',4'-Tetramethoxyflavone (TMF), one of the major polymethoxyflavones (PMFs) isolated from Kaempferia parviflor , has been reported possessing various bioactivities, including antifungal, antimalarial, antimycobacterial, and anti-inflammatory activities. Although several studies on the TMF have been reported, the information about the metabolism of TMF and the structures of TMF metabolites is still not yet clear. In this study, an isotope-labeling method was developed for the identification of TMF metabolites. Three isotope-labeled TMFs (5,7,3',4'-tetramethoxy[3'-D(3)]flavone, 5,7,3',4'-tetramethoxy[4'-D(3)]flavone, and 5,7,3',4'-tetramethoxy[5,4'-D(6)]flavone) were synthesized and administered to rats. The urine samples were collected, and the main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry. Five TMF metabolites were unambiguously identified as 3'-hydroxy-5,7,4'-trimethoxyflavone, 7-hydroxy-5,3',4'-trimethoxyflavone sulfate, 7-hydroxy-5,3',4'-trimethoxyflavone, 4'-hydroxy-5,7,3'-trimethoxyflavone, and 5-hydroxy-7,3',4'-trimethoxyflavone.