Yann-Lii Leu

Antiviral Effect of Epigallocatechin Gallate on Enterovirus 71

Oxidative stress is known to be a determinant of a hosts susceptibility to pathogens. Natural compounds with antioxidant activity may provide a preventive measure against infection. Tea polyphenols were evaluated for their ability to inhibit enterovirus 71 (EV71) replication in Vero cell culture. Among the polyphenolic compounds tested, epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG) potently inhibited replication of EV71. EGCG and GCG reduced the titer of infectious progeny virus by 95%. Quantitative RT-PCR analysis also revealed that EGCG suppressed replication of genomic RNA. It was accompanied by an increased cytoprotective effect. EGCG and GCG caused 5-fold increase in the viability of EV71-infected cells. The viral inhibitory effect correlated well with the antioxidant capacity of polyphenol. Mechanistically, EV71 infection led to increased oxidative stress, as shown by increased dichlorofluorescein and MitoSOX Red fluorescence. Upon EGCG treatment, reactive oxygen species (ROS) generation was significantly reduced. Consistent with this, EV71 replication was enhanced in glucose-6-phosphate dehydrogenase deficient cells, and such enhancement was largely reversed by EGCG. These findings suggest that EGCG may suppress viral replication via modulation of cellular redox milieu.

Effect of enhancers and retarders on percutaneous absorption of flurbiprofen from hydrogels

The effect of enhancers/retarders on the transdermal absorption of flurbiprofen from cellulose hydrogels was studied in vitro. The release rate of flurbiprofen and the viscosity of hydrogel matrices were also examined. The flux of flurbiprofen from cellulose hydrogels approximated that from aqueous buffers, whereas the skin reservoir of flurbiprofen was lower with hydrogels. Incorporation of the cosolvents, propylene glycol (PG) and ethanol, did not significantly increase skin absorption of flurbiprofen. Ethanol even reduced the skin reservoir of the drug. Oleic acid, an unsaturated fatty acid, produced the largest skin reservoir of the drug when incorporated into the hydrogels. D-Limonene, a cyclic monoterpene, showed the greatest ability to enhance the flux of flurbiprofen. However, phospholipids as retarders markedly reduced the skin absorption of flurbiprofen. The mechanisms by which enhancers/retarders govern flurbiprofen permeation were elucidated by in vitro permeation studies using various skin types (enhancers/retarders-pretreated skin, stratum corneum (SC)-stripped skin, and delipidized skin) and histological examination. The results suggest different mechanisms and skin structural modifications caused by different enhancers/retarders.

Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component.

BACKGROUND Indigo naturalis has shown efficacy in treating psoriasis in our previous clinical studies. OBJECTIVES To investigate the potential effect of indigo naturalis on regulating keratinocyte proliferation and differentiation. METHODS Skin samples from six patients were analyzed for proliferating cell nuclear antigen (PCNA) and involucrin expression by immunohistochemical staining. In addition, indigo naturalis extracts from 10 to 500 microg/ml were added to cultured keratinocytes and cell viability determined. Real-time RT-PCR, Western blotting analysis and indirect immunofluorescent labeling were used to investigate the messenger (m)RNA and protein expressions of PCNA and involucrin. Finally, high-performance liquid chromatography (HPLC) was used to identify major components of indigo naturalis and their in vitro effects compared. RESULTS Immunohistochemical results demonstrated decreased PCNA and increased involucrin in psoriatic lesions after indigo naturalis treatment. Cultured keratinocytes decreased after indigo naturalis treatment, while G(0)/G(1) arrest was observed to dose-dependently increase. Staining revealed decreased PCNA-stained nuclei and increased cytosolic involucrin in treated keratinocytes. Decreased PCNA and increased involucrin at both the mRNA and protein levels were confirmed. Both major components, indirubin and indigo, could cause G(0)/G(1) phase arrest; however, only indirubin modulated the expressions of PCNA and involucrin similar to indigo naturalis. CONCLUSIONS Together, these findings indicate that the anti-psoriatic effects of indigo naturalis are mediated, at least in part, by modulating the proliferation and differentiation of keratinocytes, with indirubin as the major active component.

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway

BackgroundFloral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis.ResultsThe main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives.ConclusionThis systems biology program combined chemical analysis, genomics and bioinformatics to elucidate the scent biosynthesis pathway and identify the relevant genes. It integrates the forward and reverse genetic approaches to knowledge discovery by which researchers can study non-model plants.

Lipid Nano/Submicron Emulsions as Vehicles for Topical Flurbiprofen Delivery

The application of lipid nano/submicron emulsions as topical drug carrier systems for the percutaneous absorption of flurbiprofen was investigated. The lipid emulsions were made up of isopropyl myristate (IPM), soybean oil, or coconut oil as the oil phase, egg lecithin as the predominant emulsifier, and double-distilled water as the external phase. Stearylamine (SA) and deoxycholic acid (DA) also were used to produce positively and negatively charged emulsions. To evaluate the physicochemical properties of the lipid emulsions, particle size by laser light scattering, the image of atomic force microscopy, and relaxation time values by Nuclear Magnetic Resonance (NMR) were determined. The in vitro permeation data showed that incorporation of SA significantly reduced the topical delivery of flurbiprofen. On the other hand, incorporation of DA exhibited no or a negligible effect on drug permeation. Enhancement of drug absorption was observed when adding oleic acid as part of the oil phase. The in vivo topical application of flurbiprofen from selected lipid emulsions showed a similar trend to the in vitro status. Furthermore, the intersubject variability was considerably reduced by lipid emulsions than by aqueous suspensions in both the in vitro and in vivo experiments. The irritant profiles of lipid emulsions showed that IPM elicited higher irritation than soybean oil. The incorporation of oleic acid also produced skin disruption. The results in the present study suggest the feasibility of lipid emulsions for the topical delivery of flurbiprofen.

Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils

ETHNOPHARMACOLOGICAL RELEVANCE Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. AIM OF THE STUDY Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. MATERIALS AND METHODS Superoxide anion (O2(.-)) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. RESULTS QD significantly inhibited O2(.-) generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. CONCLUSIONS Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O2(.-) generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization.

Transdermal delivery of selegiline from alginate–Pluronic composite thermogels

The present work was carried out to design a practical, controlled-release transdermal system for selegiline using thermosensitive hydrogels. The copolymers of alginate and Pluronic F127 (PF127) were used to design thermogels by either physical blending (A+P) or chemical grafting (AP). The thermogels were characterized in terms of the sol-gel temperature, scanning electron microscopy (SEM), degradation ratio, and skin permeation behavior. The chemical grafting of alginate to PF127 could delay the sol-gel temperature from 24.1 to 30.4°C, which is near the temperature of the skin surface. The gelling temperature of the physical mixture of alginate and PF127 (A+P) did not significantly differ. The porosity of the A+P structure was greater compared to that of the AP structure. AP thermogels were regularly degraded, with 60% of the gel matrix remaining after a 48-h incubation. PF127 and A+P hydrogels showed almost no degradation. The results of skin permeation across porcine skin and nude mouse skin suggested that the thermogels could produce sustained selegiline release, with AP showing the most-sustained permeation. AP hydrogels exhibited linear permeation properties for the transdermal delivery of selegiline. Inter-subject variations in skin permeation were reduced by incorporation of the thermogel. Such a thermosensitive hydrogel can be advantageous as a topical therapeutic formulation for selegiline.

In vitro topical application and in vivo pharmacodynamic evaluation of nonivamide hydrogels using Wistar rat as an animal model

Nonivamide, a so-called synthetic capsaicin, is a substitute for capsaicin which has a similar chemical structure and pharmacological activities as those of capsaicin. The purposes of this study were to explore the in vivo pharmacodynamic responses of nonivamide in hydrogels using Wistar rat as an animal model and to correlate the in vivo results with in vitro topical application. The incorporation of Pluronic F-127 polymer into hydrogels resulted in retarded release of nonivamide. Chitosan and carboxymethylcellulose hydrogels produced higher levels of in vitro nonivamide permeation and skin distribution. The in vivo effects of nonivamide on skin perturbation and vasodilation were found to differ depending on dose and duration after topical application. Quantification of transepidermal water loss was demonstrated to correlate with the measured in vitro skin distribution of nonivamide. The various doses of nonivamide in the hydrogels did not markedly influence erythematous reactions of skin as determined by colorimetric measurements. Hydrogel formulations of nonivamide delivered more drug to the skin and produced greater pharmacodynamic activities than did cream bases of capsaicin.

Thermosensitive Hydrogels Composed of Hyaluronic Acid and Gelatin as Carriers for the Intravesical Administration of Cisplatin

The aim of this work was to evaluate the use of thermosensitive hydrogels for intravesical cisplatin delivery into the bladder. Poly(N-isopropylacrylamide) (PNIPAM) was grafted onto hyaluronic acid (HA) to synthesize an HPN copolymer, which was further grafted with gelatin to form an HPNG copolymer. A 3% concentration of HPN and HPNG was sufficient to exert a thermosensitive response, whereas a concentration of 8% was needed for PNIPAM to form the hydrogel. The physicochemical and drug delivery properties were examined by scanning electron microscopy (SEM), the lower critical solution temperature (LCST), hydration ratio, and in vitro cisplatin release. The incorporation of HA and gelatin produced a different microstructure compared to the parent PNIPAM hydrogel. Gelatin conjugation increased the fibrous structure in the matrix. The LCSTs of PNIPAM, HPN, and HPNG were 32.3, 32.0, and 30.7°C, respectively. The copolymers showed an eightfold increase in the hydration capacity compared to PNIPAM, with no significant difference in values between HPN and HPNG. The release of cisplatin from an aqueous solution (control) was nearly complete after 8 h, compared to 85, 80, and 52% release from PNIPAM, HPN, and HPNG, respectively. In vivo evaluation of cisplatin levels in bladder tissues was performed following intravesical instillation in rats. When the dwell time was extended to 6 h, PNIPAM showed a sevenfold enhancement in the drug concentration in the bladder wall. HPNG also showed a twofold increase in the drug concentration. The administration of cisplatin by the HPN carrier did not change the drug accumulation compared to the control. Confocal laser scanning microscopic results confirmed the trend of drug absorption from various systems. A histological examination showed no adverse change in the urothelium with HPN or HPNG application. PNIPAM caused partial desquamation of umbrella cells. The thermosensitive hydrogels prepared in this study may be promising carriers for targeted drug delivery to the bladder.

Inhibitory effects of Scutellaria baicalensis extract on hepatic stellate cells through inducing G2/M cell cycle arrest and activating ERK-dependent apoptosis via Bax and caspase pathway.

ETHNOPHARMACOLOGICAL RELEVANCE The bioactive components extracted from Scutellaria baicalensis Georgi (SB) have been widely used for anti-cancer, anti-oxidation, anti-inflammation and modulating the immune response. AIM OF THE STUDY The purpose of this study is to verify the inhibitory effect and the underlying mechanisms of Scutellaria baicalensis ethanol extract (SBEE) on activated hepatic stellate cells which play a central role in liver fibrogenesis. MATERIALS AND METHODS Dimethylnitrosamine (DMN)-administrated rat model was applied to evaluate the anti-fibrotic effect of SBEE in vivo. Flow cytometric analysis and immunoblotting were then used to further investigate the molecular mechanisms by which Scutellaria baicalensis extract induces HSC-T6 cell death. RESULTS Hepatic collagen contents and alpha-smooth muscle actin levels were remarkably reduced by treating with SBEE. 100 μg/mL SBEE-induced apoptosis of HSC-T6 cell was characterized with elevated levels of activated caspase-3, poly-(ADP-ribose) polymerase (PARP) cleavage, and release of cytochrome c into the cytosol in a time-dependent manner. A 24h treatment of SBEE induced G(2)/M cell cycle arrest with increased expression of p21 and downregulation of cdc2 and cyclin B1 protein levels. Again, SBEE induced bax expression with concomitant decrease of bcl-2 and upregulated the p53 and MAPK signaling in HSC-T6 cells. CONCLUSIONS These findings demonstrated that SBEE could prevent hepatic fibrosis by promoting ERK-p53 pathways which may in turn cause G(2)/M cell cycle arrest and activate caspase system resulting in final apoptosis of HSC-T6 cells.