Evolution of congenital haemophilia care in Taiwan.

Haemophilia care in Taiwan has come a long way over the past 35 years, from the absence of specialised haemophilia treatment centres before 1984 to the establishment of treatment centers in the majority of medical centers, the listing of haemophilia as a catastrophic illness with full treatment reimbursement by the Taiwan National Health Insurance (NHI), and the implementation of full NHI coverage for prophylaxis therapy. This has led to outcome improvements such as reduced bleed-related morbidity and mortality, fewer viral infections, and enhanced overall multi-modality care. Most people with haemophilia (PWH) are now able to live normal, active lives. Early diagnosis has improved through increased awareness, physician education, and prenatal diagnosis; while comprehensive care, including state of the art rehabilitation and orthopaedic management for haemophilic arthropathy, eradication therapy for chronic hepatitis C, and better treatments for human immunodeficiency virus, allows PWH to enjoy a better quality of life and improved survival. Efforts are now being made to raise prophylaxis rates through full NHI reimbursement and the use of extended half-life recombinant factor products. Overall, Taiwan has made great strides in haemophilia care and we would like to share these experiences for the benefit of all healthcare providers involved in haemophilia care.

Composition Profiling and Authenticity Assessment of Camellia Oil Using High Field and Low Field 1H NMR.

Camellia oil (CA), mainly produced in southern China, has always been called Oriental olive oil (OL) due to its similar physicochemical properties to OL. The high nutritional value and high selling price of CA make mixing it with other low-quality oils prevalent, in order to make huge profits. In this paper, the transverse relaxation time (T2) distribution of different brands of CA and OL, and the variation in transverse relaxation parameters when adulterated with corn oil (CO), were assessed via low field nuclear magnetic resonance (LF-NMR) imagery. The nutritional compositions of CA and OL and their quality indices were obtained via high field NMR (HF-NMR) spectroscopy. The results show that the fatty acid evaluation indices values, including for squalene, oleic acid, linolenic acid and iodine, were higher in CA than in OL, indicating the nutritional value of CA. The adulterated CA with a content of CO more than 20% can be correctly identified by principal component analysis or partial least squares discriminant analysis, and the blended oils could be successfully classified by orthogonal partial least squares discriminant analysis, with an accuracy of 100% when the adulteration ratio was above 30%. These results indicate the practicability of LF-NMR in the rapid screening of food authenticity.

Preparation and evaluation of PEGylated asiatic acid nanostructured lipid carriers on anti-fibrosis effects.

Liver fibrosis is a major pathological feature of chronic liver diseases, and effective therapies are limited at present. Asiatic acid (AA) is a triterpenoid isolated from Centella asiatica, which exhibits efficient anti-inflammatory and anti-oxidative activities. However, AA shows very low plasma levels after oral administration. In this study, AA loading PEGylated nanostructured lipid carriers (P-AA-NLCs) were prepared. P-AA-NLCs were characterized for particle size distribution, polydispersity index, entrapment efficiency, X-ray powder diffraction (XRD) pattern, differential scanning colorimeter (DSC), and transmission electron microscopy (TEM). The intestinal absorption, in vivo distribution, pharmacokinetics, and anti-fibrosis effects of P-AA-NLC were studied compared with that of AA-NLC. In situ single-pass intestinal perfusion model shows that there are significant differences in absorption between the free and NLCs formulation. The Peff values of P-AA-NLC were significantly enhanced in all four intestinal segments compared to AA-NLC and free AA (p < .05). fa% and Ka showed similar trends, suggesting the PEGylated NLC can improve the gastrointestinal absorption of the drug. The pharmacokinetic studies presented that P-AA-NLC prolonged blood circulation times with a 1.5-fold higher relative bioavailability compared with AA-NLC. In vivo distribution experiments demonstrated that the fluorescence concentration in the liver was higher than that in other organs and the fluorescence intensity in the liver of DIR-P-NLC was about 1.3 times that of DIR-NLC. In addition, oral administration of P-AA-NLC can significantly attenuate CCl4-induced liver fibrosis and functional impairment in a dosage-dependent manner, including an increase in the albumin (ALB) and decrease in aspartate aminotransferase (AST) and alanine transaminase (ALT). Moreover, the MDA and HYP in liver tissue were downregulated, while the SOD activity was upregulated. In conclusion, P-AA-NLC can increase gastrointestinal absorption of AA and enhance anti-liver fibrosis effects in SD rats.

Metabolic Effect of Dietary Taurine Supplementation on Grouper (Epinephelus coioides): A 1H-NMR-Based Metabolomics Study.

Taurine is an indispensable amino acid for many fish species and taurine supplementation is needed when plant-based diets are used as the primary protein source for these species. However, there is limited information available to understand the physiological or metabolic effects of taurine on fish. In this study, 1H nuclear magnetic resonance (NMR)-based metabolomic analysis was conducted to identify the metabolic profile change in the fish intestine with the aim to assess the effect of dietary taurine supplementation on the physiological and metabolomic variation of fish, and reveal the possible mechanism of taurine's metabolic effect. Grouper (Epinephelus coioides) were divided into four groups and fed diets containing 0.0%, 0.5%, 1.0%, and 1.5% taurine supplementation for 84 days. After extraction using aqueous and organic solvents, 25 significant taurine-induced metabolic changes were identified. These metabolic changes in grouper intestine were characterized by differences in carbohydrate, amino acid, lipid and nucleotide. The results reflected both the physiological state and growth of the fish, and indicated that taurine supplementation significantly affects the metabolome of fish, improves energy utilization and amino acid uptake, promotes protein, lipid and purine synthesis, and accelerates fish growth.

Ginseng stem-leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine.

Pseudorabies, a herpesvirus infection, is mainly controlled by using attenuated live vaccines. In this study, the effect of ginseng stem and leaf saponins (GSLS) in combination with selenium (Se; in the form of sodium selenite) on vaccination against attenuated pseudorabies virus (aPrV) was evaluated. It was found that GSLS and Se have an adjuvant effect and that a combination of GSLS and Se stimulates significantly enhanced immune responses than does GSLS or Se alone. Following oral administration of GSLS, mice immunized with an attenuated PrV vaccine diluted in Se-containing physiological saline solution (PSS) provoked a significantly stronger gB-specific serum antibodies response (IgG, IgG1 and IgG2a), enhanced lymphocyte proliferation and cytolytic activity of NK cells, along with higher production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) by splenocytes. Notably, the combination of GSLS and Se conferred a much higher resistance to fPrV challenge after immunization of the mice with aPrV vaccine. This study offers convincing experimental evidence that an injection of Se with oral GSLS is a promising adjuvant combination that improves the efficacy of vaccination against PrV and deserves further study regarding improvement of responses to other animal vaccines.

Stereoselectivity evaluation of chiral chitosan microspheres delivery system containing rac-KET in vitro and in vivo.

The influence of chiral excipient D-chitosan (CS) on the stereoselective release of racemic ketoprofen (rac-KET) microspheres has been investigated in comparison to those microspheres containing individual enantiomers in vitro and in vivo. Stereoselectivity was observed in vitro release test, with R-KET release slightly higher than that of S-KET, especially in 3% rac-KET loading microspheres. Stereoselectivity is dependent on the content of chiral excipient and pH of release medium. A molecular docking study between CS and KET enantiomers further revealed that S-KET has a stronger interaction with CS compared to R-KET. Moreover, the plasma concentration of KET enantiomers in rats shows substantial differences, as the plasma levels of S-KET were higher than those of R-KET. Plasma levels of enantiomers from the R-KET microspheres had similar stereoselectivity as rac-KET microspheres. The S/R ratio of rac-KET microspheres was significantly lower than that of rac-KET suspension (regular-release formulation) (p<.05), and the differences is 3-5 fold. Besides, rates of R-KET converted to S-KET exhibited differences between rac-KET microspheres and suspension. Similar results were also found between R-KET microspheres and suspension. All investigations suggest that the chitosan interacting preferentially with S-KET to R-KET significantly affect the stereoselective pharmacokinetics of rac-KET from chitosan microspheres in rats.

Phase-contrast imaging for body composition measurement.

PURPOSE: In this paper, we propose a novel method for human body composition measurement, especially for the bone mineral density (BMD) measurement. The proposed method, using the absorption and differential phase information retrieved from X-ray grating-based interferometer (XGBI) to measure the BMD, has potential to replace dual-energy X-ray absorptiometry (DEXA), which is currently widely used for body composition measurement. METHODS: The DEXA method employs two absorption images acquired at two different X-ray spectra (high energy and low energy) to calculate the human body composition. In this paper, a new method to calculate BMD using a single X-ray measurement is proposed. XGBI is a relatively new X-ray technique that provides absorption, phase and scattering information simultaneously using a single X-ray spectrum. With the absorption and differential phase information retrieved from XGBI, BMD can be measured using only one single X-ray spectrum. Numerical simulations are performed with a body phantom of bone (Cortical, ICRU-44) surrounded by soft tissue (Soft, ICRU-44). BMD is calculated with both the DEXA method and the proposed method. RESULTS: Results show that BMD can be measured accurately with the proposed method; moreover, better signal-to-noise ratio (SNR) is obtained compared to DEXA. CONCLUSION: With the proposed method, BMD can be measured with XGBI setup. Further, the proposed method can be realized using current X-ray phase-contrast imaging (XPCI) apparatus without any hardware modification, suggesting that this technique can be a promising supplementary function to current XPCI equipment.

[Oral absorption of asiatic acid nanoparticles modified with PEG].

A solvent diffusion method was used to prepare pegylated asiatic acid (AA) loaded nanostructured lipid carriers (p-AA-NLC), and the ligated intestinal circulation model was established to observe the absorption and distribution in small intestine. The concentration of AA in bile after oral administration of p-AA-NLC was detected by HPLC in healthy SD rats to indirectly evaluate the oral absorption promoting effect of PEG-modified namoparticles. The results showed that the penetration of p-AA-NLC was enhanced significantly and the transport capacity was increased greatly in small intestinal after PEG modification. As compared with the normal nanoparticles (AA-NLC), the Cmax of the drug excretion was increased by 76%, the time to reach the peak (tmax ) was decreased and the elimination half-life t1/2 was doubled in the rats after oral administration of p-AA-NLC, and the AUC0→t was 1.5 times of the AA-NLC group, indicating that the oral bioavailability of AA-NLC was significantly improved by hydrophilic modification of PEG.

Liver, blood microdialysate and plasma pharmacokinetics of matrine following transdermal or intravenous administration.

Matrine is contained in several herbs used in traditional Chinese medicine, named Sophora alopecuroides, Sophora flavescens or Sophora subprostrata. In vitro and in vivo studies have focused on the treatment of chronic hepatitis or liver fibrosis using matrine. However, little is known about its liver pharmacokinetic profile. In this study pharmacokinetics of matrine in rat organs and tissues, such as liver, blood and skin were studied after intravenous (40 mg/kg) or transdermal administration (6 mg/cm2, 5 cm2). Samples were collected at timed intervals for measurement of matrine by a HPLC-UV method. The pharmacokinetic parameters were calculated by non-compartmental analysis using DAS 2.0. The AUC(0-t) values in the liver, blood microdialysates and plasma after intravenous administration were 395.91±74.48, 848.86±146.35 and 1304.07±305.92 min·mg/l, respectively. Following transdermal administration, the AUC(0-t) value in the liver, blood, plasma and skin microdialysates were 695.30±233.79, 1096.07±390.71, 2767.57±518.48 and 42735.77±27938.33 min·mg/l, respectively. Here, we show a promising delivery system for matrine that could replace traditional administration, and a better understanding of the transdermal pharmacokinetics of matrine, which may be helpful for further clinical and laboratory studies.

[Response surface method for optimization of asiatic acid nanoparticles modified with PEG and its enhancing effects on intestinal absorption].

A solvent diffusion method was used to prepare pegylated asiatic acid (AA) loaded nanostructured lipid carriers (p-AA-NLC). Then central composite design-response surface method was used to obtain optimum condition for preparation technology of p-AA-NLC, where PEG/lipid ratio was 8.0% and AA/lipid ratio was 22.0%. Under the optimum condition, the system had particle size of (111.2±2.9) nm, Zeta potential of (-37.1±0.9) mV, drug loading of (15.4±0.2)% and entrapment efficiency greater than 90%. The deviations between observed values and predicated values were all below 5%, indicating that the established model had a good predictability. Meanwhile, a low-speed single pass perfusion model of rat in situ was set up to estimate the absorption kinetics of p-AA-NLC in small intestine, where the effective permeability (Peff), absorption rate constant (Ka) and other parameters were used to evaluate the drug absorption. It turned out that Peff and Ka in p-AA-NLC group were significantly higher than those in unmodified group (P<0.05), indicating that asiatic acid loaded nanostructured lipid carriers (AA-NLC) could enhance the effects on intestinal absorption after being modified with hydrophilic PEG.

Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in U87-MG Astrocytes.

Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti-Alzheimer's disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for ischemic stroke treatment.

Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC.

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV-vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C(18) column using gradient elution in a water-methanol system. Detection was set at UV wavelength of 248nm. A calibration curve ranging from 0.01 to 1.5microg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01microg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8microg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at -20 degrees C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T(1/2), 4.29h; T(max), 2.70h; C(max), 0.74microg/mL; AUC(0-t) and AUC(0-infinity), 3.74 and 3.82microgh/mL, respectively.

Mechanisms of suppression of nitric oxide production by 3-O-methylquercetin in RAW 264.7 cells.

Rhamnus nakaharai Hayata (Rhamnaceae) is used as a folk medicine in Taiwan for treating constipation, inflammation, tumors, and asthma. 3-O-Methylquercetin (3-MQ), a main constituent of the plant, has been reported to have potential for use in the treatment of asthma. The mechanisms of anti-inflammation of 3-MQ are still unclear. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) through iNOS expression in RAW 264.7 cells, a mouse macrophage cell line, may reflect the degree of inflammation and may provide a measure for assessing the effect of drugs on the inflammatory process. Therefore, we were interested in investigating the mechanisms of suppression of NO production by 3-MQ in RAW 264.7 cells. 3-MQ (1-10 microM) concentration-dependently inhibited LPS (100 ng/mL)-induced NO production in RAW 264.7 cells. The IC(50) value was calculated to be 4.23 microM. 3-MQ (1-10 microM) significantly and concentration-dependently inhibited LPS (100 ng/mL)-induced iNOS protein and mRNA expressions in cells. The IC(50) values were calculated to be 4.36 and 6.53 microM, respectively. There was no significant difference among these three IC(50) values of 3-MQ. In conclusion, 3-MQ may exert its anti-inflammatory effect through the inhibition of iNOS DNA transcription.

3-O-methylquercetin more selectively inhibits phosphodiesterase subtype 3.

Rhamnus nakaharai Hayata (Rhamnaceae), has been used as a folk medicine in Taiwan for treating constipation, inflammation, tumors and asthma. 3-O-methylquercetin (3-MQ), a main constituent of the plant, has been reported to inhibit total cAMP- and cGMP-phosphodiesterase (PDE) of guinea pig trachealis. Therefore we were interested in investigating the inhibitory effect of 3-MQ on various PDE isozymes from guinea pig lungs and hearts. Isolated guinea pig lungs and hearts were homogenized and centrifuged. The supernatant was chromatographed over a column of Q-sepharose, and eluted with various concentrations of NaCl. In the following order, PDE subtypes 1, 5, 2, 4 from lungs, and 3 from hearts were separated. The IC 50 values of 3-MQ on these isozymes were 31.9, 86.9, 18.6, 28.5 and 1.6 microM, respectively. 3-MQ (10-100 microM) non-competitively inhibited PDE2, but competitively inhibited PDE4. 3-MQ (1-10 microM) also competitively inhibited PDE3. However, 3-MQ (10-100 microM) did not competitively inhibit PDE1 and 5, although it had a tendency to competitively inhibit PDE1 at concentrations of 10 - 30 microM. The present results showed that K i value of 3-MQ was similar to that of milrinone in PDE3, and was not significantly different from that of Ro 20 - 1724 in PDE4, respectively. In conclusion, 3-MQ was revealed to be a selective and competitive PDE3/PDE4 inhibitor, although its inhibitory effect on PDE4 was not potent. Therefore, 3-MQ may have a potential in the treatment of asthma beside its antiviral activity.

S-Isopetasin, a sesquiterpene of Petasites formosanus, allosterically antagonized carbachol in isolated guinea pig atria.

We investigated the antimuscarinic effect of S-isopetasin in isolated guinea pig atria to clarify whether it preferentially acts on muscarinic M 2 or M 3 receptors. The tension changes of isolated atria were isometrically recorded on a polygraph. S-Isopetasin at 50 and 100 microM significantly inhibited baselines of contractile tension and heart rate, but atropine at 1 microM enhanced both. S-Isopetasin (10 - 100 microM) did not significantly alter the concentration-negative inotropic response curves of carbachol (CCh) in left atria. S-Isopetasin (10 - 100 microM) allosterically antagonized negative inotropic and chronotropic responses induced by CCh in spontaneously beating right atria, based on the slopes of Schild plots significantly differing from unity. On the contrary, atropine (0.01 - 1 microM) competitively antagonized all the above responses to CCh. The pA 2 values of S-isopetasin were significantly less than that of S-isopetasin in guinea pig trachealis, suggesting that S-isopetasin may preferentially act on tracheal muscarinic M 3, but not cardiac muscarinic M 2 receptors. However, atropine preferentially acts neither. This finding reveals that S-isopetasin may have benefit in the treatment of asthma.