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1.  Improving the Concentrations of the Active Components in the Herbal Tea Ingredient, Uraria crinita: The Effect of Post-harvest Oven-drying Processing 
Scientific Reports  2017;7:38763.
Uraria crinita is widely used as a popular folk drink; however, little is known about how the post-harvest operations affect the chemical composition and bioactivity of UC. We assessed three drying methods (Oven-drying, Air-drying, Sun-drying), as well as the Oven-drying temperature using metabolomics approaches and bioactivity assays. The samples processed at 40 degree show a greater effect on the levels of estrogen receptor-alpha activity and nuclear factor erythroid 2–related factor 2 activity, anti-oxidative activity, and cyclooxygenase-2 inhibition compared with the other samples. A multivariate analysis showed a clear separation between the 40 degree Oven-dried samples and the other samples, which is consistent with the results of bioactivity assay. These results are ascribed to at least two-fold increase in the concentrations of flavonoids, spatholosineside A and triterpenoids in the oven-dried samples compared with the other groups. The proposed Oven-drying method at 40 degree results in an improved quality of UC.
PMCID: PMC5227699  PMID: 28079108
2.  Pentoxifylline ameliorates non-alcoholic fatty liver disease in hyperglycaemic and dyslipidaemic mice by upregulating fatty acid β-oxidation 
Scientific Reports  2016;6:33102.
Nonalcoholic fatty liver disease (NAFLD), which includes simple steatosis, steatohepatitis, fibrosis, and cirrhosis, is characterised by abnormal fat accumulation in the liver in the absence of excessive alcohol intake. In patients with type 2 diabetes (T2D), concurrent NAFLD might increase the risk of chronic kidney disease and the mortality rate. Although several studies have examined the effectiveness of pentoxifylline (PTX) in NAFLD treatment, no results are available to verify the effectiveness of PTX in treating T2D associated with NAFLD. In this study, we developed a combined high-fat diet-induced obesity and low-dose streptozocin-induced hyperglycaemia mouse model to mimic the concurrent NAFLD and T2D pathological condition. By combining physiological assessments, pathological examinations, metabolomics studies on blood, urine, and liver, and measurements of gene and protein expression, we elucidated the effectiveness and the underlying mechanism of action of PTX in the hyperglycaemic and dyslipidaemic mice. Our results revealed that PTX ameliorated NAFLD in the hyperglycaemic and dyslipidaemic mice by upregulating fatty acid β-oxidation. Furthermore, the glycolysis pathway and branched-chain amino acid-related pathways in these mice were restored by PTX.
PMCID: PMC5017161  PMID: 27612024
3.  Rapid and sensitive detection of rare cancer cells by the coupling of immunomagnetic nanoparticle separation with ELISA analysis 
This study presents a rapid and sensitive method for detecting cancer cells occurring at low concentration. The method involves the simultaneous detection of two biomarkers of T helper cancer cells. One biomarker conjugates with immunofunctionalized magnetic nanoparticles (MNPs), enabling the separation of the T helper cells from a mixed population of cells. The other biomarker is used for detection during enzyme-linked immunosorbent assay (ELISA) analysis. The specific T helper cells can be quantified according to their ELISA absorbance values following magnetic separation. The experimental results demonstrate that immunofunctionalized MNPs can function as magnetic sensors and separate specific T helper cells from a mixed population with high efficiency and high specificity. Coupled with the ELISA technique, the immunofunctionalized MNPs can simultaneously detect rare cells. Results indicated increasing absorbance with increasing T cell number (from 10 to 106). The total detection time was less than 15 minutes, even at a low T cell count. The advantages of the proposed method for detecting specific cells at low concentration include ease of preparation, low cost, rapid detection, and high sensitivity. The proposed system can be adopted to detect circulating tumor cells in early tumor stages for diagnostic or prognostic purposes.
PMCID: PMC3390995  PMID: 22787392
ELISA; magnetic nanoparticles; immunoassay; cancer cell
4.  Antidepressant-Like Activity of the Ethanolic Extract from Uncaria lanosa Wallich var. appendiculata Ridsd in the Forced Swimming Test and in the Tail Suspension Test in Mice 
This study investigated the antidepressant activity of ethanolic extract of U. lanosa Wallich var. appendiculata Ridsd (ULEtOH) for two-weeks administrations by using FST and TST on mice. In order to understand the probable mechanism of antidepressant-like activity of ULEtOH in FST and TST, the researchers measured the levels of monoamines and monoamine oxidase activities in mice brain, and combined the antidepressant drugs (fluoxetine, imipramine, maprotiline, clorgyline, bupropion and ketanserin). Lastly, the researchers analyzed the content of RHY in the ULEtOH. The results showed that ULEtOH exhibited antidepressant-like activity in FST and TST in mice. ULEtOH increased the levels of 5-HT and 5-HIAA in cortex, striatum, hippocampus, and hypothalamus, the levels of NE and MHPG in cortex and hippocampus, the level of NE in striatum, and the level of DOPAC in striatum. Two-week injection of IMI, CLO, FLU and KET enhanced the antidepressant-like activity of ULEtOH. ULEtOH inhibited the activity of MAO-A. The amount of RHY in ULEtOH was 17.12 mg/g extract. Our findings support the view that ULEtOH exerts antidepressant-like activity. The antidepressant-like mechanism of ULEtOH may be related to the increase in monoamines levels in the hippocampus, cortex, striatum, and hypothalamus of mice.
PMCID: PMC3332070  PMID: 22567032
5.  Early Detection for Cases of Enterovirus- and Influenza-Like Illness through a Newly Established School-Based Syndromic Surveillance System in Taipei, January 2010 ~ August 2011 
PLoS ONE  2015;10(4):e0122865.
School children may transmit pathogens with cluster cases occurring on campuses and in families. In response to the 2009 influenza A (H1N1) pandemic, Taipei City Government officials developed a School-based Infectious Disease Syndromic Surveillance System (SID-SSS). Teachers and nurses from preschools to universities in all 12 districts within Taipei are required to daily report cases of symptomatic children or sick leave requests through the SID-SSS. The pre-diagnosis at schools is submitted firstly as common pediatric disease syndrome-groups and re-submitted after confirmation by physicians. We retrieved these data from January 2010 to August 2011 for spatio-temporal analysis and evaluated the temporal trends with cases obtained from both the Emergency Department-based Syndromic Surveillance System (ED-SSS) and the Longitudinal Health Insurance Database 2005 (LHID2005). Through the SID-SSS, enterovirus-like illness (EVI) and influenza-like illness (ILI) were the two most reported syndrome groups (77.6% and 15.8% among a total of 19,334 cases, respectively). The pre-diagnosis judgments made by school teachers and nurses showed high consistency with physicians’ clinical diagnoses for EVI (97.8%) and ILI (98.9%). Most importantly, the SID-SSS had better timeliness with earlier peaks of EVI and ILI than those in the ED-SSS. Furthermore, both of the syndrome groups in these two surveillance systems had the best correlation reaching 0.98 and 0.95, respectively (p<0.01). Spatio-temporal analysis observed the patterns of EVI and ILI both diffuse from the northern suburban districts to central Taipei, with ILI spreading faster. This novel system can identify early suspected cases of two important pediatric infections occurring at schools, and clusters from schools/families. It was also cost-effective (95.5% of the operation cost reduced and 59.7% processing time saved). The timely surveillance of mild EVI and ILI cases integrated with spatial analysis may help public health decision-makers with where to target for enhancing surveillance and prevention measures to minimize severe cases.
PMCID: PMC4398411  PMID: 25875080
6.  The correlation between the presence of viremia and clinical severity in patients with enterovirus 71 infection: a multi-center cohort study 
BMC Infectious Diseases  2014;14:417.
Enterovirus 71 (EV71) is a great disease burden across the whole world, particularly in Southeast Asia. However, in recent decades, the pathogenesis of severe EV71 infection was not well understood. This study was aimed to investigate the correlation between the presence of viremia and the clinical severity of EV71 infection.
We organized a prospective cohort study and enrolled laboratory-confirmed EV71 cases in six tertiary care hospitals in Taiwan during the EV71 epidemic from 2011 to 2012. Blood samples were collected once in the acute stage, on the first day of admission. We used real-time RT-PCR to detect EV71 viremia. Demographical and clinical data were collected and the clinical severity was categorized into four grades. Data analysis was performed to identify the risk factors of viremia and the correlation between viremia and clinical severity of EV71 infection.
Of the total 224 enrolled patients, 59 (26%) patients were confirmed to have viremia. Two-thirds (68%) of viremic cases were detected within the first three days of infection. Viremia occurred more frequently in children under the age of one year old (odds ratios [OR] 4.82, p < 0.001) but the association between the presence of viremia and complicated EV71 infection was not found (OR 1.02, p = 0.96). In the viremia group, patients had significantly more severe complications if viremia was detected after the third day of disease onset (26% vs. 5%, p = 0.03).
Viremia occurred more frequently in children under the age of one year and viremia detected beyond three days after the onset of disease correlated with more severe disease in EV71 patients.
PMCID: PMC4133623  PMID: 25069383
Enterovirus; Viremia; Severe EV71 infection
7.  Gallic Acid Ameliorated Impaired Glucose and Lipid Homeostasis in High Fat Diet-Induced NAFLD Mice 
PLoS ONE  2014;9(6):e96969.
Gallic acid (GA), a naturally abundant plant phenolic compound in vegetables and fruits, has been shown to have potent anti-oxidative and anti-obesity activity. However, the effects of GA on nonalcoholic fatty liver disease (NAFLD) are poorly understood. In this study, we investigated the beneficial effects of GA administration on nutritional hepatosteatosis model by a more “holistic view” approach, namely 1H NMR-based metabolomics, in order to prove efficacy and to obtain information that might lead to a better understanding of the mode of action of GA. Male C57BL/6 mice were placed for 16 weeks on either a normal chow diet, a high fat diet (HFD, 60%), or a high fat diet supplemented with GA (50 and 100 mg/kg/day, orally). Liver histopathology and serum biochemical examinations indicated that the daily administration of GA protects against hepatic steatosis, obesity, hypercholesterolemia, and insulin resistance among the HFD-induced NAFLD mice. In addition, partial least squares discriminant analysis scores plots demonstrated that the cluster of HFD fed mice is clearly separated from the normal group mice plots, indicating that the metabolic characteristics of these two groups are distinctively different. Specifically, the GA-treated mice are located closer to the normal group of mice, indicating that the HFD-induced disturbances to the metabolic profile were partially reversed by GA treatment. Our results show that the hepatoprotective effect of GA occurs in part through a reversing of the HFD caused disturbances to a range of metabolic pathways, including lipid metabolism, glucose metabolism (glycolysis and gluconeogenesis), amino acids metabolism, choline metabolism and gut-microbiota-associated metabolism. Taken together, this study suggested that a 1H NMR-based metabolomics approach is a useful platform for natural product functional evaluation. The selected metabolites are potentially useful as preventive action biomarkers and could also be used to help our further understanding of the effect of GA in hepatosteatosis mice.
PMCID: PMC4053315  PMID: 24918580
8.  Antioxidant, Analgesic, Anti-Inflammatory, and Hepatoprotective Effects of the Ethanol Extract of Mahonia oiwakensis Stem 
The aim of this study was to evaluate pharmacological properties of ethanol extracted from Mahonia oiwakensis Hayata stems (MOSEtOH). The pharmacological properties included antioxidant, analgesic, anti-inflammatory and hepatoprotective effects. The protoberberine alkaloid content of the MOSEtOH was analyzed by high-performance liquid chromatography (HPLC). The results revealed that three alkaloids, berberine, palmatine and jatrorrhizine, could be identified. Moreover, the MOSEtOH exhibited antioxidative activity using the DPPH assay (IC50, 0.743 mg/mL). The DPPH radical scavenging activity of MOSEtOH was five times higher that that of vitamin C. MOSEtOH was also found to inhibit pain induced by acetic acid, formalin, and carrageenan inflammation. Treatment with MOSEtOH (100 and 500 mg/kg) or silymarin (200 mg/kg) decreased the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels compared with the CCl4-treated group. Histological evaluation showed that MOSEtOH reduced the degree of liver injury, including vacuolization, inflammation and necrosis of hepatocytes. The anti-inflammatory and hepatoprotective effect of MOSEtOH were found to be related to the modulation of antioxidant enzyme activity in the liver and decreases in malondialdehyde (MDA) level and nitric oxide (NO) contents. Our findings suggest that MOSEtOH has analgesic, anti-inflammatory and hepatoprotective effects. These effects support the use of MOSEtOH for relieving pain and inflammation in folk medicine.
PMCID: PMC3588023  PMID: 23364614
Mahonia oiwakensis Hayata; high-performance liquid chromatography; hepatoprotective effect; malondialdehyde
9.  Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications 
BMC Biotechnology  2012;12:27.
Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria.
Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.
On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.
PMCID: PMC3443062  PMID: 22672291

Results 1-9 (9)