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Positive molecular identification of medicinal plants is necessary to their misuse. Dendrobii Caulis is a famous Chinese herbal medicine traditionally used in ophthalmology. However, the identification of the species based on morphology is difficult. In this study, three Dendrobium species were collected and differentiated by the sequences of partial rDNA (ribosomal DNA) sections which contain the ITS 1, 5.8S, and ITS 2 regions. The Dendrobium species used in this reseach included Dendrobium tosaense, Dendrobium officinale, and Dendrobium moniliforme. The length of PCR products are 632 nt for D. tosaense, 627 nt for D. officinale, and 628 nt for D. moniliform, respectively. The similarities of the rDNA region between the species pairs shown range from 91% to 95%. The results we present here may be helpful in identifying other species of Dendrobium.

Anoectochilus formosanus is an expensive medicinal folk herb in Taiwan. The plant has been used to treat hypertension, diabetes, and hepatitis by native people for a long time. In this study, 4 typical lines of A. formosanus, red-stem, green-stem, narrow-leaf, and round-leaf, were collected and screened to identify the line-specific SSR (single sequence repeat) markers. In addition, we found the narrow-leaf line exhibited the highest polysaccharide content (3.39 ± 0.74%, polysaccharide wt/plant dried wt) which is more than twofold of the lowest content of the red-stem line (1.27 ± 0.41%). In the culture program construction, the extent of the somaclonal variation among 20 regenerated shoots from the same parental individual of a narrow-leaf line was revealed using the amplified fragment length polymorphism (AFLP) technique. The aim of this study is to preliminarily investigate the genetic variation of A. formosanus through polymorphic marker screening, which will benefit to the cultivation of A. formosanus with high polysaccharide content in future work. The results showed that 6 out of 81 primers screened, generating a total of 26 markers, were able to differentiate the 4 lines in the inter single sequence repeat (ISSR) analysis. On the other hand, 17 sets of AFLP primers were chosen to detect the somatic variation among the excised shoots derived from the same individual. The genetic variation among the regenerated shoots ranged from 0.00% to 5.43%. The primer pair B6 exerts the most powerful selection ability in the AFLP analysis among 20 somaclonal samples used, whereas the E4 primer pair the lowest. The AFLP primer pairs become useful tools for selecting the lowest somaclonal variation from the regenerated shoots of A. formosanus under the tissue culture program.

Hispolon (HIS) is an active polyphenol compound derived from Phellinus linteus (Berkeley & Curtis), and our previous study showed that HIS effectively inhibited inflammatory responses in macrophages [Yang, L.Y., S.C. Shen, K.T. Cheng, G.V. Subbaraju, C.C. Chien and Y.C. Chen. Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages. J. Ethnopharmacol. 156: 61–72, 2014]; however, its effect on neuronal inflammation is still undefined. In this study, HIS concentration- and time-dependently inhibited lipopolysaccharide (LPS)- and lipoteichoic acid (LTA)-induced inducible nitric oxide (NO) synthase (iNOS)/NO production with increased heme oxygenase (HO)-1 proteins in BV-2 microglial cells. Accordingly, HIS protected BV-2 cells from LPS- or LTA-induced apoptosis, characterized by decreased DNA ladder formation, and caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage in BV-2 cells. Similarly, the NOS inhibitor, N-nitro-L-arginine methyl ester (NAME), inhibited LPS- or LTA-induced apoptosis of BV-2 cells, but neither NAME nor HIS showed any inhibition of NO production or cell death induced by the NO donor, sodium nitroprusside (SNP), indicating the involvement of NO in the inflammatory apoptosis of microglial cells. Activation of c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-[Formula: see text]B contributed to LPS- or LTA-induced iNOS/NO production and apoptosis of BV-2 cells, and that was suppressed by HIS. Additionally, HIS possesses activity to induce HO-1 protein expression via activation of extracellular signal-regulated kinase (ERK) in BV-2 cells, and application of the HO inhibitor, tin protoporphyrin (SnPP), or knockdown of HO-1 protein by HO-1 small interfering (si)RNA significantly reversed HIS inhibition of NO production and cell death in BV-2 cells stimulated by LPS. Results of an analysis of the effects of HIS and two structurally related chemicals, i.e. dehydroxy-HIS (D-HIS) and HIS-methyl ester (HIS-ME), showed that HIS expressed the most potent inhibitory effects on iNOS/NO production, JNK activation, and apoptosis in BV-2 microglial cells activated by LPS with increased HO-1 protein expression. Overall these results suggested that HIS possesses inhibitory activity against LPS- or LTA-induced inflammatory responses including iNOS/NO production and apoptosis in BV-2 microglial cells and that the mechanisms involve upregulation of the HO-1 protein and downregulation of JNK/NF-[Formula: see text]B activation. A critical role of hydroxyl at position C3 in the anti-inflammatory actions of HIS against activated BV-2 microglial cells was suggested.

Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/&beta, catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on &beta, catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/&beta, catenin signaling in CRC cells. Notably, ectopic expression of a dominant&ndash, active &beta, catenin mutant (∆N90-&beta, catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/&beta, catenin signaling axis.

Tumor-infiltrating lymphocytes are related to positive clinical prognoses in numerous cancer types. Programmed death ligand 1 (PD-L1), a mediator of the PD-1 receptor, plays an inhibitory role in cancer immune responses. PD-L1 upregulation can impede infiltrating T-cell functions in lung adenocarcinoma (LUAD), a lung cancer subtype. However, associations between the expression of PD-L1 and infiltration of B cells (a major immunoregulatory cell) remain unknown. Therefore, we investigated the role of infiltrating B cells in LUAD progression and its correlation with PD-L1 expression. The Cancer Genome Atlas (TCGA) LUAD data set was used to explore associations among B-cell infiltration, PD-L1 expression, clinical outcome, and gene landscape. Gene set enrichment analysis was used to explore putative signaling pathways and candidate genes. The drug enrichment analysis was used to identify candidate genes and the related drugs. We found that high B-cell infiltration was correlated with better prognoses; however, PD-L1 may interfere with the survival advantage in patients with high B-cell infiltration. The gene landscape was characterized comprehensively, with distinct PD-L1 levels in cell populations with high B-cell infiltration. We obtained five upregulated signaling pathways from the gene landscape: apoptosis, tumor necrosis factor (TNF)-α signaling via nuclear factor (NF)-κB, apical surface, interferon-α response, and KRAS signaling. Moreover, four candidate genes and their related target drugs were also identified, namely interleukin-2β receptor (IL2RB), IL-2γ receptor (IL2RG), Toll-like receptor 8 (TLR8), and TNF. These findings suggest that tumor-infiltrating B cells could act as a clinical factor in anti-PD-L1 immunotherapy for LUAD.

Background Currently, more than one-third of the global population is overweight or obese, which is a risk factor for major causes of death including cardiovascular disease, numerous cancers, and diabetes. Kinsenoside, a major active component of Anoectochilus formosanus exhibits antihyperglycemic, antihyperliposis, and hepatoprotective effects and can be used to prevent and manage obesity. Purpose This study examined the catabolic effects of kinsenoside on lipolysis in adipocytes transformed from C3H10T1/2 cells. Study design/methods The lipolytic effect of kinsenoside in C3H10T1/2 adipocytes was evaluated by oil-red O staining and glycerol production. The underlying mechanisms were assessed by Western blots, chromatin immunoprecipitation (IP), Co-IP, EMSA and siRNAs verification. Results We demonstrated that kinsenoside increased both adipose triglyceride lipase (ATGL)-mediated lipolysis, which was upregulated by AMP-activated protein kinase (AMPK) activation, and the hydrolysis of triglycerides to glycerol and fatty acids that require transportation into mitochondria for further β-oxidation. We also demonstrated that kinsenoside increased the phosphorylation of peroxisome proliferator-activated receptor alpha (PPARα) and CRE-binding protein (CREB), and the protein levels of silent information regulator T1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and carnitine palmitoyltransferase I (CPT1) through an AMPK-dependent mechanism. SIRT1 deacetylated PGC-1α, facilitating AMPK-mediated PGC-1α phosphorylation and increasing the interaction of PPARα with its coactivator, PGC-1α. This interaction elevated the expression of CPT1, a shuttle for the mitochondrial transport of fatty acids, in kinsenoside-treated cells. In addition, AMPK-phosphorylation-mediated CREB activation caused kinsenoside-mediated PGC-1α upregulation. Conclusion AMPK activation not only elevated ATGL expression for lipolysis but also induced CPT1 expression for further mitochondrial translocation of fatty acids. The results suggested that the mechanism underlying the catabolic effects of kinsenoside on lipolysis and increased CPT1 induction was mediated through an AMPK-dependent pathway.

MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM) development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose) polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA), higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2), another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.

Ethnopharmacological relevance Phellinus linteus (Berkeley & Curtis), a well-known medical fungus, has long been used as a traditional medicine in Oriental countries to treat various diseases, and hispolon (HIS) is one of its bioactive components. HIS is known to possess potent antineoplastic and antiviral properties; however, its effect on inflammatory apoptosis is still undefined. Materials and methods RAW264.7 macrophages were incubated with HIS for 30 min followed by LPS, LTA, or PGN stimulation for 12 h. The expression of indicated proteins AP-1 and NF-κB transcriptional activities was examined by Western blotting using specific antibodies. Levels of NO and ROS were examined by Griess reaction, and DCHF-DA staining via flow cytometric analysis, respectively. AP-1 and NF-κB transcriptional activities were detected by luciferase reporter assay. Knockdown of HO-1 protein expression was performed by transfection of macrophages with HO-1 siRNA. Pharmacological inhibitors including ROS scavenger NAC, JNK inhibitor SP600125, NF-κB inhibitor BAY117082 were applied for mechanism study. Results HIS showed concentration-dependent inhibition of LPS, LTA, and PGN-induced iNOS protein expressions and NO production by RAW264.7 macrophages. Accordingly, HIS protected RAW264.7 cells from LPS-, LTA-, and PGN-induced apoptosis. Increased HO-1 by HIS was detected at both protein and mRNA levels along with an increase in intracellular peroxide, and this was inhibited by the translational inhibitor, cycloheximide (CHX), the transcriptional inhibitor, actinomycin D (Act D), and the reactive oxygen species scavenger, N-acetylcysteine (NAC). A mechanistic study indicated that inhibition of c-Jun N-terminal kinase (JNK) protein phosphorylation, and activator protein (AP)-1 and nuclear factor (NF)-κB activation were involved in the anti-inflammatory actions of HIS in macrophages. A structure-activity relationship analysis showed that HIS expressed the most potent effect of inhibiting iNOS and apoptosis elicited by LPS, LTA, and PGN with a significant increase in HO-1 protein in macrophages. Conclusions Evidence supporting HIS prevention of inflammatory apoptosis via blocking NO production and inducing HO-1 protein expression in macrophages is provided, and the hydroxyl at position C3 is a critical substitution for the anti-inflammatory actions of HIS.

Indomethacin (IN) is a widely used nonsteroidal anti-inflammatory drug. In this study, four photoproducts of IN (IN1–IN4) were produced and isolated from photoirradiated IN. This study investigated the abilities of IN and its photoproducts to scavenge hydroxyl radicals and inhibit xanthine oxidase (XO). The hydroxyl radical-scavenging activity was measured in vitro by electron spin resonance spectrometry using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trapping agent. Enzyme activity was measured by continuous monitoring of uric acid formation, using xanthine as a substrate. The results showed that, among all the related products, IN has the strongest hydroxyl radical-scavenging (IC 50 = 65 μM) and XO inhibitory (IC 50 = 86 μM) effects. To further understand the stereochemistry of the reactions between these IN derivatives and XO, we performed computer-aided molecular modeling. IN was the most potent inhibitor with the most favorable interaction in the reactive site. Various photoproducts exhibited affinity toward XO as a result of the absence of hydrogen bonding with molybdopterin domain.

Baicalin is a flavonoid compound purified from the medicinal plantScutellaria baicalensisGeorgi and has been reported to stimulate surfactant protein (SP)-A gene expression in human lung epithelial cell lines (H441). The aims of this study were to determine whether maternal baicalin treatment could increase lung surfactant production and induce lung maturation in fetal rats. This study was performed with timed pregnant Sprague-Dawley rats. One-day baicalin group mothers were injected intraperitoneally with baicalin (5 mg/kg/day) on Day 18 of gestation. Two-day baicalin group mothers were injected intraperitoneally with baicalin (5 mg/kg/day) on Days 17 and 18 of gestation. Control group mothers were injected with vehicle alone on Day 18 of gestation. On Day 19 of gestation, fetuses were delivered by cesarean section. Maternal treatment with 2-day baicalin significantly increased saturated phospholipid when compared with control group and total phospholipid in fetal lung tissue when compared with control and 1-day baicalin groups. Antenatal treatment with 2-day baicalin significantly increased maternal growth hormone when compared with control group. Fetal lung SP-A mRNA expression and maternal serum corticosterone levels were comparable among the three experimental groups. Maternal baicalin treatment increases pulmonary surfactant phospholipids of fetal rat lungs and the improvement was associated with increased maternal serum growth hormone. These results suggest that antenatal baicalin treatment might accelerate fetal rat lung maturation.

A method was developed for rapid identification of Dendrobium species by a dot blot hybridization assay. The dot blot system is based on species-specific amplified fragments derived from the internal transcribed spacer (ITS) region of different Dendrobium species as target DNA, which were blotted as dots on the nylon membrane. A small aliquot of the ITS amplified product from a selected species or mixed ITS DNA from several species was labeled by peroxidase and used as a probe for hybridization to the dot blot membrane. Simply visualizing the positive hybridization reaction between the probe DNA and the target DNA could identify the selected species. The D. officinal-specific and D. nobile-specific ITS-based probes could separately identify D. officinal and D. nobile. Eight Dendrobium species, including D. salaccense, D. brymerianum, D. ellipsophyllum, D. chrysanthum, D. officinale, D. thyrsiflorum, D. nobile and D. chrysotoxum could be simultaneously detected using their complex ITS amplified products as a probe, and the ITS DNA probe amount for detection by the dot blot membrane is very small, just 2.5 ng. The assay can also be used to identify the resource species of Fengdou Shihu and Huangcao Shihu from the commercial market. The identification based on the ITS sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the polymerase chain reaction (PCR)-dot blot hybridization assay can be considered a reliable method for general identification of commercial Shihu.

Medicinal plants have long been used as a source of therapeutic agents. They are thought to be important anti-aging ingredients in prophylactic medicines. The aim of this study was to screen extracts from Taiwanese plant materials for phenolic contents and measure the corresponding matrix metalloproteinase-9 (MMP-9) activity. We extracted biological ingredients from eight plants native to Taiwan (Alnus formosana, Diospyros discolor, Eriobotrya deflex, Machilus japonica, Pyrrosia polydactylis, Pyrus taiwanensis, Vitis adstricta, Vitis thunbergii). Total phenolic content was measured using the Folin-Ciocalteu method. MMP-9 activities were measured by gelatin zymography. The extracted yields of plants ranged from 3.7 % to 16.9 %. The total phenolic contents ranged from 25.4 to 36.8 mg GAE/g dry material. All of these extracts (except Vitis adstricta Hance) were shown to inhibit MMP-9 activity of WS-1 cell after ultraviolet B irradiation. These findings suggest that total phenolic content may influence MMP-9 activity and that some of the plants with higher phenolic content exhibited various biological activities that could serve as potent inhibitors of the ageing process in the skin. This property might be useful in the production of cosmetics.

DNA topoisomerase I (TOP1) levels of several human neoplasms are higher than those of normal tissues. TOP1 inhibitors are widely used in treating conventional therapy-resistant ovarian cancers. However, patients may develop resistance to TOP1 inhibitors, hampering chemotherapy success. In this study, we examined the mechanisms associated with the development of camptothecin (CPT) resistance in ovarian cancers and identified evodiamine (EVO), a natural product with TOP1 inhibiting activity that overcomes the resistance. The correlations among TOP1 levels, cancer staging, and overall survival (OS) were analyzed. The effect of EVO on CPT-resistant ovarian cancer was evaluated in vitro and in vivo. TOP1 was associated with poor prognosis in ovarian cancers (p = 0.024). EVO induced apoptosis that was detected using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The tumor size decreased significantly in the EVO treatment group compared with the control group (p < 0.01) in a xenograft mouse model. Effects of drugs targeting TOP1 for prognosis and therapy in CPT-resistant ovarian cancer are anticipated. EVO with TOP1 can be developed as an antiproliferative agent for overcoming CPT resistance in ovarian cancers.

The water extract of Anoectochilus formosanus Hayata showed a potent tumor inhibitory activity in BALB/c mice after subcutaneous transplantation of CT-26 murine colon cancer cells. The tumor-inhibition ratios of mice pre-administered with A. formosanus for 2 days before tumor transplantation, and treated further for 12 consecutive days, were 55.4% and 58.9% at the oral dose of 50 and 10 mg/mouse per day, respectively. Even for the tumor-bearing mice, after oral administration of the water extract of A. formosanus for 12 consecutive days, the tumor inhibition ratios were still 23.8% and 40.5% at doses of 50 and 10 mg/mouse, respectively. Because the low-concentration water extract of A. formosanus does not show direct cytotoxicity in CT-26 tumor cells, we observed further that oral administration of the water extract of A. formosanus may activate murine immune responses, such as stimulating the proliferation of lymphoid tissues and activating the phagocytosis of peritoneal macrophages against Staphylococcus aureus. This study suggests that the antitumor activity of A. formosanus may be associated with its potent immunostimulating effect. It is worth further analyzing the immunomodulating component purified from A. formosanus, and evaluating its potential value for the treatment of human cancers.

Plant phenolic compounds isolated from a 70% aqueous acetone extract of the leaves of Malus doumeri A. CHEV. var. formosana (KAWAK. & KOIDZ.) S. S. YING, a type of Taiwanese indigenous plant, were evaluated for potential application in the field of skin care. A phytochemical investigation of the active fractions resulted in the isolation of seven compounds of which the structures were identified by spectroscopic characterization. In the present study, the isolated phenolic compounds were evaluated for their free radical-scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the superoxide radicals, anti-elastase, and for their anti-matrix metalloproteinase-1 (MMP-1) activity in human skin fibroblast cells. Of these compounds, 3-hydroxyphloridzin (2), 3-hydroxyphloretin (6), and quercetin (7) exhibited the strongest DPPH and superoxide radical-scavenging activities. The IC50 values of these compounds were 9.2, 7.7, and 15.4 microM, respectively, for the DPPH radical, and 25.0, 19.6, and 42.6 microM, respectively, for the superoxide radical. 3-Hydroxyphloridzin (2) and 3-hydroxyphloretin (6) also showed xanthine oxidase inhibitory activity, with IC(50) values of 52.1 and 22.4 muM, respectively. In the test for elastase inhibitory activity, phloretin (5) and 3-hydroxyphloretin (6) were the most potent compounds. Phloretin (5), 3-hydroxyphloretin (6), and quercetin (7) showed better inhibition of MMP-1 production in fibroblast cells. To the best of our knowledge, this is the first time that the active phenolic compounds from M. doumeri var. formosana have been isolated, reported, and described. The above results suggest that the extract of M. doumeri var. formosana containing phenolic compounds could be suitable naturally occurring active constituents for use in anti-aging or cosmetic products.

Commercial pectins with different degrees of esterification (DE) were reacted with equal volumes of 2 M alkaline hydroxylamine (pH 12.0) at room temperature for 4 h to prepare pectin hydroxamic acids (PHAs; DE94T4, DE65T4, and DE25T4) according to a previously reported method (Hou et al., J. Agric. Food Chem. 2003, 51, 6362-6366) and were used to test the antioxidant and antiradical activities in comparison with those of DE94, DE65, and DE25 pectins. The half-inhibition concentrations, IC(50), of scavenging activity against DPPH were 1.51, 5.43, and 5.63 mg/mL for DE94T4, DE65T4, and DE25T4, respectively, and were much lower than those of corresponding DE pectins under the same concentrations. The scavenging activities of PHAs for DPPH radicals were positively correlated with original DE values of pectin. The optimal pH of DE94T4 for scavenging DPPH radicals was 7.9 or 8.0. Using electron spin resonance (ESR) for scavenging hydroxyl radicals, under the same concentrations of 125 microg/mL, DE94T4, DE65T4, and DE25T4, respectively, exhibited 73.53, 69.01, and 55.17% antiradical activities. PHAs also exhibited protection against hydroxyl radical-mediated DNA damage and anti-human low-density lipoprotein peroxidation tests.

Pulmonary surfactant, a complex lipoprotein, is secreted by alveolar type II cells. It lies at the alveolar air-fluid interface and prevents alveolar collapse by reducing surface tension. The high incidence of respiratory distress syndrome (RDS) in premature infants results principally from a deficiency of pulmonary surfactant. Surfactant protein A (SP-A) is the most abundant surfactant protein and reduces surface tension at the alveolar air-liquid interface in lung cells. In this study, RT-PCR and Western blot analyses of SP-A were performed to evaluate the biological activity of baicalin, a Chinese medicine prescribed extensively for preventing miscarriage. In in vitro experiments, lung adenocarcinoma cell line H441 was cultured with baicalin in varying concentrations and for varying lengths of time. The results show that the expression of SP-A gene was positively affected by baicalin in dose-dependent and time-course manners. The maximal expression of the SP-A gene, 1.7-fold greater than control, is induced at 150 nM of baicalin treated for 48 h.

The 70% aqueous acetone extracts of ten Taiwanese native plants were evaluated by various antioxidant assays, including 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (.OH) radicals, and reducing power assay. In the present study, extracts of Acer buerferianum var. formosanum, Cleyera japonica var. morii, Cyclobalanopsis stenophylla var. stenophylloides, and Machilus zuihoensis exhibited stronger activity against DPPH radicals, and their IC50 values ranged from 5.4 to 8.3 microg/ml. The ten selected extracts effectively inhibited the formation of .OH generated in the Fenton reaction system. Among the extracts whose reducing power activities were determined, A. buerferianum var. formosanum, C. japonica var. morii, C. stenophylla var. stenophylloides, Eriobotrya deflex, and M. zuihoensis showed high activity. The results indicate the 70% aqueous acetone extracts of A. buerferianum var. formosanum, C. japonica var. morii, C. stenophylla var. stenophylloides, and M. zuihoensis with great potency in these assay systems and may be candidates for the development of natural antioxidants.

Currently, more than one-third of the global population is overweight or obese, which is a risk factor for major causes of death including cardiovascular disease, numerous cancers, and diabetes. Kinsenoside, a major active component of Anoectochilus formosanus exhibits antihyperglycemic, antihyperliposis, and hepatoprotective effects and can be used to prevent and manage obesity.This study examined the catabolic effects of kinsenoside on lipolysis in adipocytes transformed from C3H10T1/2 cells.The lipolytic effect of kinsenoside in C3H10T1/2 adipocytes was evaluated by oil-red O staining and glycerol production. The underlying mechanisms were assessed by Western blots, chromatin immunoprecipitation (IP), Co-IP, EMSA and siRNAs verification.We demonstrated that kinsenoside increased both adipose triglyceride lipase (ATGL)-mediated lipolysis, which was upregulated by AMP-activated protein kinase (AMPK) activation, and the hydrolysis of triglycerides to glycerol and fatty acids that require transportation into mitochondria for further β-oxidation. We also demonstrated that kinsenoside increased the phosphorylation of peroxisome proliferator-activated receptor alpha (PPARα) and CRE-binding protein (CREB), and the protein levels of silent information regulator T1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and carnitine palmitoyltransferase I (CPT1) through an AMPK-dependent mechanism. SIRT1 deacetylated PGC-1α, facilitating AMPK-mediated PGC-1α phosphorylation and increasing the interaction of PPARα with its coactivator, PGC-1α. This interaction elevated the expression of CPT1, a shuttle for the mitochondrial transport of fatty acids, in kinsenoside-treated cells. In addition, AMPK-phosphorylation-mediated CREB activation caused kinsenoside-mediated PGC-1α upregulation.AMPK activation not only elevated ATGL expression for lipolysis but also induced CPT1 expression for further mitochondrial translocation of fatty acids. The results suggested that the mechanism underlying the catabolic effects of kinsenoside on lipolysis and increased CPT1 induction was mediated through an AMPK-dependent pathway.

The genetic variability of Astragalus medicine materials sold by twenty randomly selected stores in Taiwan was investigated using RAPD analysis in order to obtain available primers which could clearly differentiate among them. Total DNA isolated from the rhizomes of the samples were used as templates, and sixty 10 mer arbitrary primers were used in the analysis. The aim of the present study is to construct an identification model of molecular biotechniques applicable to Chinese herbal medicines in RAPD analysis. Three of the primers, OPT-03, OPT-13, and OPT-17, revealed polymorphic RAPD fingerprints among the samples of Astragalus membranaceus, and between Astragalus membranaceus and Hedysarum polybotrys samples. SSCP analysis was also conducted on PCR products from the ITS-1 region of ribosomal DNA in order to differentiate the two species.

Background Propionibacterium acnes ( P. acnes ), a gram-positive anaerobic bacterium, plays a critical role in the development of inflammatory lesion as a result of cytokines production by keratinocytes and macrophages activation. However, effect of P. acnes on iNOS/NO and COX-2/PGE 2 production in macrophages is still uninvestigated. Objective This study aimed at determining the reactive oxygen species (ROS), inducible nitric oxide (NO) synthase (iNOS)/nitric oxide (NO), and cyclooxygenase (COX)-2/prostaglandin (PG)E 2 produced by macrophages upon P. acnes infection, and dissecting the mechanism of P. acnes -stimulated multiplicity of infection (MOI)-dependent increases in iNOS and COX-2 protein expressions in accordance with the elevation of NO and PGE 2 production by RAW264.7 macrophages. Methods Using an in vitro cell culture system, the effects of P. acnes on iNOS/NO, COX-2/PGE 2 , ROS production, ERK/JNK, and AP-1/NF-κB activation were examined via Western blotting, a flow cytometric analysis, and luciferase assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, diphenylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (U0126 and SP600125) were applied to investigate the mechanism. Results We found that P. acnes exposures increased iNOS/NO and COX-2/PGE 2 expression in RAW264.7, J774A.1, and peritoneal macrophages via a MOI-dependent manner. Increased ROS production, ERK/JNK protein phosphorylation, and elevated AP-1/NF-κB luciferase activity are identified in P. acnes -induced iNOS/NO and COX-2/PGE 2 production. Additionally, hispolon but not its analogs, hispolon methylether or dehydroxyhispolon, showed significant inhibition of P. acnes -induced iNOS/NO and COX-2/PGE 2 production, indicating an important role of OH at C5 for hispolon's inhibition of P. acnes -induced inflammatory events in macrophages. Conclusion ROS-dependent stimulation of ERK, JNK, NF-κB, and AP-1 activation contributes to P. acnes -induced iNOS/NO and COX-2/PGE 2 in macrophages, and chemicals such as hispolon possessing ability to block iNOS/NO and COX-2/PGE 2 production reserve potential to be further developed for treatment of the early phase of inflammation elicited by P. acnes .

Dermal papilla cells (DPCs) control the development of hair follicles via cell–cell interactions and extracellular molecules. Colchicine affected active anagen DPCs to result in hair loss in the clinical setting. The purpose of this study was to identify the retro-modulator released by DPCs exposed to sub-toxic dose of colchicine and elucidate its effect on dermal papilla culture. The molecular-weight cutoff ultrafiltration and HPLC were used to purify the components of colchicine-treated DPC secretomes and examined their ability to down-regulate the growth and alkaline phosphatase (ALP) activity of DPCs. The active product was identified by in-gel trypsin digestion, nano-LC-ESI-MS/MS and validated by Western blot to be histone H4 ( P62804 ), which inhibited the proliferation and diminished the ALP activity of cultured DPCs. Treating DPCs with recombinant histone H4 reproduced the growth inhibition effect whereas adding antibody to immunoneutralize histone H4 abolished this growth inhibitory consequence. DPCs with high ALP activity can induce the neogenesis of hair follicles and support the hair fiber growth in vivo . Our results indicated that sub-lethal colchicine can inactivate DPCs through releasing histone H4. Through the investigation of the retro-modulation of histone H4 on dermal papillae may give implications for understanding the mechanism of colchicine-induced hair disorder.

Successful fertilization is tightly regulated by capacitation and decapacitation processes. Without appropriate decapacitation regulation, sperm would undergo a spontaneous acrosome reaction which leads to loss of fertilization ability. Seminal plasma is known to negatively regulate sperm capacitation. However, the suppressive mechanisms still remain unclear. In this study, we demonstrate the decapacitation mechanism of mouse seminal vesicle autoantigen (SVA) might target membrane sphingomyelin (SPM) and regulate plasma membrane Ca 2þ ATPase (PMCA) activity. The SVA was shown to suppress sperm capacitation induced by a broad panel of capacitation factors (bovine serum albumin (BSA), PAF, and cyclodextrin (CD)). Furthermore, SVA significantly decreased [Ca 2þ ]i and NaHCO3-induced [cAMP]i. Cyclic AMP agonists bypassed the SVA’s suppressive ability. Importantly, the SVA may regulate PMCA activity which was evidenced by the fact that the SVA decreased the [Ca 2þ ]i and intracellular pH (pHi) of sperm; meanwhile, a PMCA inhibitor (carboxyeosin) could reverse SVA’s suppression of [Ca 2þ ]i. The potential target of the SVA on membrane SPM/lipid rafts was highlighted by the high binding affinity of SPM‐SVA (with a Kd of � 3 mM) which was close to the IC50 of SVA’s suppressive activity. Additionally, treatment of mink lung epithelial cells with the SVA enhanced plasminogen activator inhibitor (PAI)-1 expression stimulated by tumor growth factor (TGF)-b and CD. These observations supported the membrane lipid-raft targeting of SVA. In summary, in this paper, we demonstrate that the decapacitation mechanism of the SVA might target membrane sphingolipid SPM and regulate PMCA activity to lower [Ca 2þ ]i, thereby decreasing the [cAMP]i level and preventing sperm pre-capacitation. J. Cell. Biochem. 111: 1188‐1198, 2010. 2010 Wiley-Liss, Inc.

A new biflavonoid, 2,3-dihydro-4',4'''-di-O-methylamentoflavone (5), and five known compounds, (-)-catechin (1), quercetin (2), 2,3-dihydrosciadopitysin (3), sciadopitysin (4), and isoginkgetin (6), were isolated from Podocarpus macrophyllus var. macrophyllus (Podocarpaceae). These compounds were evaluated their ability to inhibit cellular tyrosinase activity and for their melanin inhibitory activity in human epidermal melanocytes (HEMn). In the melanin synthesis assay, 2,3-dihydro-4',4'''-di-O-methylamentoflavone (5) showed a potent anti-tyrosinase effect with IC(50)=0.098 mM in HEMn. It also significantly decreased both protein and mRNA levels of the tyrosinase-related protein-2 (TRP-2) by Western blot and quantitative real-time PCR (qRT-PCR) analysis. These findings suggest that the new compound, 2,3-dihydro-4',4'''-di-O-methylamentoflavone (5), is the most active component of P. macrophyllus var. macrophyllus in inhibiting pigmentation and that this inhibition is exerted through inhibition of transcription of the genes encoding TRP2.

For identification of genuine and counterfeit samples of Cordyceps sinensis, an RAPD (random amplified polymorphic DNA) analysis was performed. In the study, twelve samples were collected, of which six were genuine and six were counterfeit samples. Genuine samples of Cordyceps sinensis contain two parasitic larvae produced from each of the three provinces, Sichuan (SC), Chinghai (CH), and Tibet (TB). The counterfeits contain two products of each of the three species, Cordyceps hawkesii, Stachys geobombycis, and Stachys sieboldii. In the RAPD analysis, twenty arbitrary decamer primers were screened to obtain primers appropriate for differentiating the genuine and counterfeit samples. The result of twelve RAPD markers generated from four primers, OPT-08, OPT-12, OPT-13, and OPT-17, were selected. The primers can easily distinguish between genuine and counterfeit samples of Cordyceps sinensis.

RAPD (random amplified polymorphic DNA) markers were developed to distinguish Anoectochilus formosanus from Anoectochilus koshunensis and their putative hybrids. Morphological differentiation of these two species beyond the flowering period is difficult. RAPD markers provide a rapid and easy tool for identification of the two Anoectochilus species. In the study, forty arbitrary decamer primers were screened, and nineteen species-specific RAPD markers generated from polymerase chain reactions (PCR) with eight random primers were obtained. Nine were specific to A. formosanus and ten to A. koshunensis. Two primers, OPC-08 and OPL-07, produced two markers, one specific to A. formosanus and the other specific to A. koshunensis, which simultaneously appeared in the hybrids pattern. The RAPD markers can be applied both to identification of A. formosanus and A. koshunensis species and to assessment of the extent fo hybridization in hybrids between them. This information facilitates the breeding program process.

In this study, the RAPD (random amplified polymorphic DNA) technique was employed for the first time to determine the components in a Chinese herbal prescription. Forty decamer oligonucleotide primers were screened in the RAPD analysis to identify three Chinese medicines, the dried root of Astragalus membranaceus (Fisch.) Bge., the dried root of Ledebouriella seseloides Wolff, and the dried rhizome of Atractylodes macrocephala Koidz, in a Chinese prescription. Only primer OPP-10 simultaneously generated three distinct markers were each specific to one component. The marker with 200 bp is specific to Astragalus membranaceus; the 440 bp marker is specific to Atractylodes macrocephala; and the remaining marker with 500 bp was present in Ledebouriella seseloides. The presence of the three herbal medicines in the mixed sample, the Chinese prescription, was determined when the primer OPP-10 RAPD reaction was performed. The technique was proved to contribute to the identification of components in the Chinese medicinal preparations.

The genetic relationships among six strains of rhizobia, including three strains of Rhizobium fredii and three strains of Bradyrhizobium japonicum, was determined using random amplified polymorphic DNA (RAPD) technique. In this study, 46 arbitrary 10mer primers were employed for RAPD, generating a total of 251 informative fragments. A dendrogram of phylogenetic relationships among the six strains was constructed. The results indicated that geographical distribution may affect phylogeny, as there were closer relationships among the four Taiwanese strains, SB138, SB562, SB368 and SB651, than between these strains and USDA192, which originated from mainland China. The strain USDA110, obtained from the United States, was used in the parsimony analysis. The greatest similarity (55.6%), existed between two strains of B. japonicum, SB562 and SB138, which both, and the lowest R. fredii (44.4%) between two strains of R. fredii, SB368 and USDA192. We also found a RAPD marker specific to the four Taiwanese SB strains used in the study. The RAPD technique is a potential tool for the identification of the genetics and systematics of different populations.

Plant phenolic compounds isolated from a 70% aqueous acetone extract of the leaves of Malus doumeri A. CHEV. var. formosana (KAWAK.KOIDZ.) S. S. YING, a type of Taiwanese indigenous plant, were evaluated for potential application in the field of skin care. A phytochemical investigation of the active fractions resulted in the isolation of seven compounds of which the structures were identified by spectroscopic characterization. In the present study, the isolated phenolic compounds were evaluated for their free radical-scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the superoxide radicals, anti-elastase, and for their anti-matrix metalloproteinase-1 (MMP-1) activity in human skin fibroblast cells. Of these compounds, 3-hydroxyphloridzin (2), 3-hydroxyphloretin (6), and quercetin (7) exhibited the strongest DPPH and superoxide radical-scavenging activities. The IC50 values of these compounds were 9.2, 7.7, and 15.4 microM, respectively, for the DPPH radical, and 25.0, 19.6, and 42.6 microM, respectively, for the superoxide radical. 3-Hydroxyphloridzin (2) and 3-hydroxyphloretin (6) also showed xanthine oxidase inhibitory activity, with IC(50) values of 52.1 and 22.4 muM, respectively. In the test for elastase inhibitory activity, phloretin (5) and 3-hydroxyphloretin (6) were the most potent compounds. Phloretin (5), 3-hydroxyphloretin (6), and quercetin (7) showed better inhibition of MMP-1 production in fibroblast cells. To the best of our knowledge, this is the first time that the active phenolic compounds from M. doumeri var. formosana have been isolated, reported, and described. The above results suggest that the extract of M. doumeri var. formosana containing phenolic compounds could be suitable naturally occurring active constituents for use in anti-aging or cosmetic products.

Anoectochilus formosanus (AF) is a popular folk medicine in Taiwan whose pharmacological effects have been characterized. In this work we investigated the antioxidant properties of an aqueous extract prepared from AF. The AF extract was capable of scavenging H2O2 in a dose-dependent man- ner. We induced oxidative stress in HL-60 cells, either by the addition of hydro- gen peroxide (H2O2) or by the xanthine/xanthine oxidase reaction. Apoptosis caused by oxidative damage was displayed by DNA fragmentation on gel elec- trophoresis, and the apoptotic fraction was quantified with flow cytometry. The cell damage induced by oxidative stress was prevented by the plant extract in a concentration-dependent manner. Furthermore, the proteolytic cleavage of poly(ADP-ribose) polymerase during the apoptotic process was also inhibited by AF extract. Our results provide the basis for determining an AF extract to be an antioxidant.

We investigated the effects of maternal administration of Anoectochilus formosanus extract and dexamethasone on lung maturation in preterm rats. A. formosanus group mothers were tube-fed A. formosanus extract (300 mg/kg body wt./day) for 7 days from days 12-18 of gestation. Dexamethasone group mothers were injected intraperitoneally with dexamethasone (0.2 mg/kg body wt.) in saline on day 18 of gestation. Control group mothers were similarly injected with saline alone. On day 19 of gestation, fetuses were delivered by cesarean section. A. formosanus treatment significantly increased the fetal lung/body weight ratio, as compared to dexamethasone treatment. Saturated phosphatidylcholine levels in fetal lung tissue and growth hormone levels in maternal serum were significantly increased in the A. formosanus- and dexamethasone-treated groups as compared to controls. The histological appearance of preterm rat lungs revealed extensive branching of intermediate airways, denser mesenchyme, and more epithelial tubules in the dexamethasone and A. formosanus groups as compared with the control group. These results suggest that antenatal A. formosanus treatment may play a role in accelerating fetal rat lung maturation.

In a previous study, we evaluated the effect of baicalin on the expression of SP-A (surfactant protein A), which was developmentally regulated in an alveolar type II cell, H441. SP-A is encoded by two similar genes, SP-A1 and SP-A2, in humans. The maximal induction of SP-A1 gene of H441 occurred at treating 150 nM of baicalin for 48 h. In the present study, cDNA subtraction analysis is performed to examine the differential expression in H441 cell upon baicalin treatment with a view to investigating the regulatory mechanism. The mRNA of H441 cell incubated with 150 nM baicalin for 48 h was compared to that of blank control. Two PCR products were obtained through subtractive cDNA amplification. A product encoding cytochrome c oxidase was demonstrated to be a differential signal by RT-PCR analysis, and the other was a false positive. The induction of cytochrome c oxidase might increase ATP level in cell, and consequently elevates cAMP, which upregulates surfactant synthesis and secretion.

Structure of spatozoate, n-butyl 2-benzoyloxymethylbenzoate 1, a metabolite of Spatoglossum variabile has been revised into benzyl n-butyl phthalate 2 by synthesis. NMR spectral data of synthetic 2 agrees well with those reported for the natural spatozoate.