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Investigation of phytochemicals in Prunus davidiana fruits led to the isolation of 13 phenolic compounds: five flavonoids ( 1 - 5 ), four phenylpropanoids ( 6-9 ), two lignans ( 10 and 11 ), one benzoic glycoside ( 12 ), and one cyanobenzyl glycoside ( 13 ). All phenolic compounds were reported for the first time from P. davidiana fruits in this study. The chemical structures of 1 - 13 were decided using various spectroscopic analyses such as MS, IR, and NMR. As well, the occurrence of the isolated ones in Prunus species was discussed. All compounds were evaluated for radical scavenging activity using ABTS and DPPH radicals. Compounds 2 , 5 , 6 , 8 , 9 , and 11 had much higher scavenging activity than ascorbic acid, a well-known antioxidant.

Cnidium officinalis rhizomes were immersed in 80% MeOH. The extract was fractionated to water, n-butanol, and ethyl acetate fractions (Fr). Open column chromatography was repeatedly carried out on n-butanol and ethyl acetate Fr using silica gel, octadecyl silica gel, and Sephadex LH-20 as the stationary phase affording five phenyl alkanoids 1–5 including two new ones. The molecular structures including stereochemistry were decided based on spectroscopic interpretation of nuclear magnetic resonance, mass spectrometry, and infrared spectroscopy as well as chemical reaction. Three known compounds, coniferyl alcohol methyl ether (1), vanillin (2), and coniferyl aldehyde (3), were reported in the beginning for this plant by authors. Two new phenyl alkanoids were named, 7-methoxyeugenol and cnidiumoside.

Headaches are common in patients with systemic lupus erythematosus (SLE). It is important to identify the exact cause of headaches in SLE to avoid unnecessary steroid or immunosuppressive therapy like in neuropsychiatric SLE. A 35-year-old woman with SLE suddenly developed severe headache. Magnetic resonance angiography showed multifocal segmental narrowing of cerebral arteries, suggestive of central nervous system vasculitis. However, lack of abnormal enhancement in vessel wall imaging indicated reversible cerebral vasoconstriction syndrome (RCVS) rather than central nervous system vasculitis. The patient was treated with oral nimodipine and she recovered over a period of two months. Following magnetic resonance angiography on day 90 was normal. Herein we report a case of reversible cerebral vasoconstriction syndrome in an SLE patient with literature review.

Background: The major determining factor of prognosis of oral squamous cell carcinoma is cervical lymph node metastasis. 6,8-Diprenylgenistein (6,8-DG), an isoflavonoid isolated from Cudrania tricuspidata has been reported to have anti-microbial and anti-obesity activities. However, its effects on lymphangiogenesis and lymph node metastasis in oral cancer have not yet been reported. Methods: To investigate the in vitro inhibitory effects of 6,8-DG on VEGF-A-induced lymphangiogenesis, we performed the proliferation, tube formation, and migration assay using human lymphatic microvascular endothelial cells (HLMECs). RT-PCR, Western blot, immunoprecipitation, ELISA and co-immunoprecipitation assays were used to investigate the expression levels of proteins, and mechanism of 6,8-DG. The in vivo inhibitory effects of 6,8-DG were investigated using an oral cancer sentinel lymph node (OCSLN) animal model. Results: 6,8-DG inhibited the proliferation, migration and tube formation of rhVEGF-A treated HLMECs. In addition, the in vivo lymphatic vessel formation stimulated by rhVEGF-A was significantly reduced by 6,8-DG. 6,8-DG inhibited the expression of VEGF-A rather than other lymphangiogenic factors in CoCl2-treated SCCVII cells. 6,8-DG inhibited the expression and activation of VEGFR-2 stimulated by rhVEGF-A in HLMECs. Also, 6,8-DG inhibited the activation of the lymphangiogenesis-related downstream signaling factors such as FAK, PI3K, AKT, p38, and ERK in rhVEGF-A-treated HLMECs. Additionally, 6,8-DG inhibited the expression of the hypoxia-inducible factor (HIF-1&alpha, ), which is involved in the expression of VEGF-A in CoCl2-treated SCCVII cells, and 6,8-DG inhibited VEGF-A signaling via interruption of the binding of VEGF-A and VEGFR-2 in HLMECs. In the VEGF-A-induced OCSLN animal model, we confirmed that 6,8-DG suppressed tumor-induced lymphangiogenesis and SLN metastasis. Conclusion: These data suggest that 6,8-DG inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in vitro and in vivo. Furthermore, the inhibitory effects of 6,8-DG are probably mediated by inhibition of VEGF-A expression in cancer cells and suppression of the VEGF-A/VEGFR-2 signaling pathway in HLMEC. Thus, 6,8-DG could be novel and valuable therapeutic agents for metastasis prevention and treatment of oral cancer.

Abeliophyllum distichum (Oleaceae), which is the only species in the monotypic genus and is grown only on the Korean peninsula, has a high scarcity value. Its five variants (white, pink, round, blue, and ivory) have different morphological characteristics in terms of the color of petals and sepals or shape of the fruits. Despite its high value, there has been no study on variant classification except in terms of their morphological characteristics. Thus, we performed a volatile component analysis of A. distichum flowers and multivariate data analyses to reveal the relationship between fragments emitted from five variants of A. distichum flowers with their morphological characteristics. As a result, 66 volatile components of this plant were identified by headspace solid-phase microextraction gas chromatography&ndash, mass spectrometry (HS-SPME-GC-MS), showing unique patterns for each set of morphological characteristics, especially the color of the petals. These results suggest that morphological characteristics of each variant are related to the volatile composition.

Because cinnamon is orally ingested, its biological activity is likely to influence the small intestinal epithelium and the microbiota along the intestinal tract. We investigated small intestinal epithelial gene expression associated with immunity and lipid metabolism and measured IgA level in the small intestines and sera of mice fed cinnamon. We also analyzed microbial changes in the small and large intestines and feces using pyrosequencing of the 16 s rRNA gene. Cinnamon increased the gene expression of Muc2, RegIIIγ, and Pigr, which are necessary for defense against gut bacteria. It also increased the gene expression of GATA4 and Slc27a2, which are implicated in lipid absorption. Cinnamon increased luminal IgA level but not serum IgA and decreased the class Gammaproteobacteria (classified within the phylum Proteobacteria), particularly the genus Pseudomonas, in the large intestine. Cinnamon thus affected immunity and lipid absorption in the small intestinal epithelium and microbial composition in the intestines.

The present study assessed the indoor level of pesticide residue contamination at a total of 45 dwelling facilities in 5 cities of South Korea from June to November 2014. Pesticide residue contamination was assessed by measuring the frequency and concentration of chlorpyrifos, dichlorvos, and cypermethrin residues in airborne particles, indoor dust, and surface wipes. A preparatory test showed a decreasing tendency in the concentrations of pesticide residues in indoor air over time: from 0.458 to 0.073 mg/m3 in dichlorvos for 4 weeks and from below 0.050 mg/m3 to non-detection in the other substances for 2 weeks. Then, pesticide residues were detected indoors 4 weeks after outdoor chemical control, implying the infiltration of pesticide particles from outdoors. Airborne particles of dichlorvos were found at a higher level (74.4% of samples at a concentration of 0.053 mg/m3), whereas those of the other substances were detected at lower levels (6.1% at 0.002 mg/m3 in chlorpyrifos and 9.4% at 0.022 mg/m3 in cypermethrin). There was no consistent tendency in the indoor levels of pesticide residue contamination according to dwelling types or indoor height. The indoor levels of dichlorvos residue contamination were lower in industrial districts than in urban or rural districts: 63.9% and 0.013 mg/m3 for airborne particles, 13.3% and 0.002 μg/g for indoor dust, and 6.7% and 0.001 mg/cm2 for surface wipes, respectively. There were no significant differences in the indoor levels of pesticide residue contamination between urban and rural districts. The current study found that most dwelling facilities managed their indoor levels of pesticide residue contamination below permissible exposure limit (PEL, 1.0 mg/m3) or threshold limit value (TVL, 0.1 mg/m3), whereas some facilities did not. So, we suggest that certain guidelines should be drawn up regarding the indoor environment management.

A preliminary study revealed that a 10 µg/mL n-BuOH fraction of Malva verticillata aerial parts significantly enhanced splenocyte proliferation and induced significant enhancement of natural-killer (NK) cell activity against tumor cells (YAC-1). This study was initiated to identify the principal components that exhibited these activities, and four glycerides were isolated through repeated SiO2 and ODS column chromatography. Structures of compounds 1–4 were determined to be (2S)-1-O-palmitoyl glyceride, (2S)-1-O-stearoyl glyceride, (2S)-1-O-linolenoyl glyceride, and (2S)-1,2-di-O-linoleoyl glyceride, respectively. Compounds 1–3 showed potential immune-enhancing activity in murine splenocyte and natural-killer (NK) cells at 10 µM. In contrast, compound 4 showed weak activity, indicating the monoacyl glycerides (1–3) are more effective than diacyl glyceride (4). Also, the longer the carbon number of the fatty acid in monoacyl glyceride, the better the activity, and the monoacyl glyceride including an unsaturated fatty acid (3) is more effective than the glycerides including the saturated fatty acids (1–2).

Objective: The aim of this is to compare the immunotherapeutic effects of human colorectal cancer antigen GA733-2 fused to the Fc fragment of antibody (GA733-2-Fc) and to Fc and endoplasmic reticulum (ER) retention motif KDEL (GA733-2-Fc-KDEL). Materials and Methods: Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were produced from infiltrated Nicotiana benthamiana leaves and purified by affinity chromatography. Glycan structures were determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The allergic and immunogenic responses of recombinant GA733-2-Fc and GA733-2-Fc-KDEL were estimated in an intraperitoneally immunized mouse. The tumor regression effect of recombinant GA733-2-Fc and GA733-2-Fc-KDEL was examined using a colorectal carcinoma CT-26 animal model. Results: Recombinant GA733-2-Fc contained plant-specific glycan structures including β(1,2)-xylose and α(1,3)-fucose whereas recombinant GA733-2-Fc-KDEL contained oligomannose type glycan structures. Mice immunized intraperitoneally with recombinant GA733-2-Fc and GA733-2-Fc-KDEL elicited strong GA733-2-Fc-specific immunoglobulin G (IgG) and IgA serum antibody responses. Recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE. Recombinant GA733-2-Fc-KDEL increased the production of interferon-γ. Intraperitoneal preimmunization with recombinant GA733-2-Fc and GA733-2-Fc-KDEL regressed tumor growth in a colorectal carcinoma CT-26 animal model. The tumor regression effect induced by recombinant GA733-2-Fc-KDEL was greater than that induced by recombinant GA733-2-Fc. The human and mouse colorectal carcinoma cell binding activities of recombinant GA733-2-Fc-KDEL-immunized sera were higher than those of recombinant GA733-2-Fc. Conclusions: Our results suggest that GA733-2-Fc conjugated to ER-retention motif KDEL is a more efficient antigen to prevent tumor growth induced by colorectal carcinoma and minimize an allergic response.

In Platycodi Radix (root of Platycodon grandiflorum), there are a number of platycosides that consist of a pentacyclic triterpenoid aglycone and two sugar moieties. Due to the pharmacological activities of platycosides, it is critical to assess their contents in PR, and develop an effective method to profile various platycosides is required. In this study, an analytical method based on ultra performance liquid chromatography coupled with quadrupole time-of-flight/mass spectrometry (UPLC-QTOF/MS) with an in-house library was developed and applied to profile various platycosides from four different Platycodi Radix cultivars. As a result, platycosides, including six isomeric pairs, were successfully analyzed in the PRs. In the principal component analysis, several platycosides were represented as main variables to differentiate the four Platycodi Radix cultivars. Their different levels of platycosides were also represented by relative quantification. Finally, this study indicated the proposed method based on the UPLC-QTOF/MS can be an effective tool for identifying the detail characterization of various platycosides in the Platycodi Radix.

The fruits of Prunus davidiana were extracted with 80 % aqueous methanol at room temperature. The concentrated extract was partitioned as ethyl acetate (EtOAc), n-butyl alcohol, and water fractions. From the EtOAc fraction, three triterpenoids were isolated through the repeated silica gel (SiO2) and octadecyl SiO2 column chromatographies. Based on physico-chemical and spectroscopic data including nuclear magnetic resonance, mass spectrometry, and infrared, the compounds were identified to be ursolic acid (1), corosolic acid (2), and α-amyrin (3).

The leaves of eggplant (Solanum melongena L.) were extracted with 80 % aqueous MeOH, and the concentrated extract was partitioned with n-hexane, EtOAc, n-BuOH, and water fractions. From the n-BuOH fraction, five compounds were isolated through the repeated silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatographies. On the basis of physic-chemical and spectroscopic data including mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance, they were identified to be caffeic acid (1), chlorogenic acid (2), cryptochlorogenic acid (3), panasenoside (4), and (6R,7E,9R)-4,7-megastigmadien-3-one-9-β-D-glucopyranoside (5). Compounds 3 and 4 were isolated for the first time from the leaves of S. melongena L. in this study.

A red pigment produced by Zooshikella ganghwensis, was purified by solvent extraction and fractionation. The pigment was identified to be prodigiosin on the basis of spectroscopic data such as 1H-NMR, 13C-NMR, DEPT, 2D-NMR, IR, and FAB-MS. Prodigiosin inhibited matrix metalloproteinase-1 gene expression and the growth of human cancer cell lines, SK-BR-3, HCT-116, SK-OV-3, HeLa, HepG2, and SK-MEL-5.

Background:Gout shows a seasonal variation that widely differs among geographic areas, and we previously reported a seasonal effect on gout in Korea. However, we had no information regarding changes in diet and had only limited laboratory data because this was a retrospective study in patients receiving a urate-lowering therapy. Therefore, we designed this prospective study to elucidate the seasonality and associated factors of gout attacks in Korea.Objectives:To evaluate the seasonality and associated factors of the incidence of gout attacks in Korea.Methods:We prospectively enrolled patients with gout attacks who were treated at nine rheumatology clinics between January 2015 and July 2018 and followed them for 1-year. Demographic data, clinical and laboratory features, and meteorological data including seasonality were collected.Results:Two hundred-five patients (males, 94.1%) were enrolled. The proportion of patients with initial gout attacks was 46.8% (n = 96). The mean age, body mass index, attack duration, and serum uric acid level at enrollment were 50.5 years, 26.1, 10.2 days, and 7.3 mg/dL, respectively. Gout attacks were most common during spring (43.4%,P< 0.001) and in March (23.4 %,P< 0.001). A similar pattern of seasonality was observed in the group with initial gout attacks. Alcohol was the most common provoking factor (39.0%), particularly during summer (50.0%). The mean diurnal temperature change on the day of the attack was highest in the spring (10.3°C), followed by winter (9.1°C), summer (8.1°C), and fall (8.0°C) (P= 0.027). The mean change in humidity between the 2 consecutive days (the day before and the day of the attack) was significantly different among the seasons (3.4%, spring; 0.2%, summer; 0.4%, fall; -3.9%, winter;P= 0.015). One hundred twenty-five (61%) patients completed 1-year follow-up (51% in the initial attack group). During the follow-up period, 51 gout flares developed (18 in the initial attack group). No significant seasonal variation in the follow-up flares was found.Conclusion:In this prospective study, the most common season and month of gout attacks in Korea were spring and March, respectively. Alcohol was the most common provoking factor, particularly during summer. Diurnal temperature changes on the day of the attack and humidity changes from the day before the attack to the day of the attack were associated with gout attack in our cohort.References:[1]Choi HJ, Lee CH, Lee JH et al. Seasonality of gout in Korea: a multicenter study. J Korean Med Sci 2015;30:240-4.Disclosure of Interests: :None declared

In the food industry and herbal markets, it is critical to control the quality of processed Panax ginseng products. In this study, ultra-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UPLC-QTOF/MS)-based metabolomics was applied for the quality evaluation of white ginseng (WG), tae-geuk ginseng (TG), red ginseng (RG), and black ginseng (BG). Diverse metabolites including ginsenosides were profiled by UPLC-QTOF/MS, and the datasets of WG, TG, RG, and BG were then subjected to multivariate analyses. In principal component analysis (PCA), four processed ginseng products were well-differentiated, and several ginsenosides were identified as major components of each product. S-plot also characterized the metabolic changes between two processed ginseng products, and the major ginsenosides of each product were found as follows: WG (M-Rb1, M-Rb2, M-Rc, Re, Rg1), TG (Rb2, Rc, Rd, Re, Rg1), RG (Rb1, Rb2, Rc, Rd, Re, Rg1), and BG (Rd, Rk1, Rg5, Rg3). Furthermore, the quantitative contents of ginsenosides were evaluated from the four processed ginseng products. Finally, it was indicated that the proposed metabolomics approach was useful for the quality evaluation and control of processed ginseng products.

Kudzu (Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep) is a perennial leguminous vine, and its root and flower have been used for herbal medicine in Asia for a long time. Most dietary flavonoids are reported to be concentrated in its root, not in its aerial parts including leaves. In this study, we investigated whether kudzu leaf and its major constituent, robinin (kaempferol-3-O-robinoside-7-O-rhanmoside) possessed anti-inflammatory activity. To test this hypothesis, we used peritoneal macrophages isolated from BALB/c mice and stimulated the cells with lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. Compared with kudzu root extract, its leaf extract was more potent in inhibiting the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-α, and interleukin-6. Kudzu leaf extract decreased LPS-induced activation of c-Jun N-terminal kinase (JNK) and TANK-binding kinase 1(TBK1) with no effects on nuclear factor-κB and activator protein 1 transcriptional activity. Also, kudzu leaf extract inhibited LPS/IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) activation partly via an altered level of STAT1 expression. Robinin, being present in 0.46% of dry weight of leaf extract, but almost undetected in the root, decreased iNOS protein involving modulation of JNK and STAT1 activation. However, robinin showed no impact on other inflammatory markers. Our data provide evidence that kudzu leaf is an excellent food source of as yet unknown anti-inflammatory constituents.

The sprouts of red kohlrabi (Brassica oleracea var. gongylodes) were extracted with methanol, and the concentrated extracts were successively partitioned with ethyl acetate, n-butyl alcohol (n-BuOH), and H2O. From the n-BuOH fraction, four phenylpropanoids were isolated through repeated silica gel and ODS column chromatographies. On the basis of physico-chemical and spectroscopic data including NMR, MS, and IR, the isolated compounds were identified as 3-(3,4,5-trimethoxyphenyl)-2E-propenoic acid methyl ester (1), (E)-sinapic acid methyl ester (2), (E)-sinapoyl glucoside (3), and lawsoniaside B (4). Moreover, the compounds dose-dependently inhibited NO production in RAW 264.7 cells with IC50 values of 41.22 (1), 38.32 (3), and 22.65 (4) μM, respectively. With further studies, these compounds may be potentially useful in developing new anti-inflammatory agents.

Korean ginseng (Panax ginseng C.A. Meyer) contains several types of ginsenosides, which are considered the major active medicinal components of ginseng. The types and quantities of ginsenosides found in ginseng may differ, depending on the location of cultivation, making it necessary to establish a reliable method for distinguishing cultivation locations of ginseng roots. P. ginseng roots produced in different regions of Korea, China, and Japan have been unintentionally confused in herbal markets owing to their complicated plant sources. PCA and PLS-DA using RRLC-QTOF/MS data was able to differentiate between ginsengs cultivated in Korea, China, and Japan. The chemical markers accountable for such variations were identified through a PCA loadings plot, tentatively identified by RRLC-QTOF/MS and partially verified by available reference standards. The classification result can be used to identify P. ginseng origin.

Principal components of aquatic plants responsible for inhibiting the growth of Microcystis aeruginosa, a blue-green alga (cyanobacterium) were determined. Methanol extracts of Brasenia schreberi showed strong inhibitory activity against the growth of the cyanobacterium. Two purified flavonoids were afforded through repeated column chromatographies. The compounds were determined as quercetin (1) and quercetin 3-O-(6″-galloyl)-β-d-glucopyranoside, tellimoside (2) by the interpretation of spectroscopic data including NMR, MS, and IR. Compounds 1 and 2 were first isolated from Brasenia schreberi. In addition, compound 2 showed very strong inhibitory activity against the growth of M. Aeruginosa.

From the heartwood of Lindera glauca Blume three new diarylpropanoids have been isolated and identified, which were named lindeglaucol, lindeglaucone, and lindeglaucoside A. Chipped heartwood was extracted repeatedly with 80% aqueous methanol (MeOH) at room temperature, and the concentrated methanolic extract was successively partitioned with ethyl acetate (EtOAc), n-butanol, and H2O. From the EtOAc fraction, three new diarylpropanoids and a known chalconoid were isolated through repeated column chromatography with silica gel, octadecyl silica gel, and Sephadex LH-20. The chemical structures of the compounds were elucidated by interpretation of extensive spectroscopic data such as nuclear magnetic resonance, fast atom bombardment mass spectroscopy, and infrared. In addition, a known chalconoid, cilicicone B, was isolated for the first time from the heartwood of L. glauca.

The stem woods of Lindera obtusiloba Blume were extracted in 80% aqueous methanol and the concentrated extract was partitioned with ethyl acetate (EtOAc), butanol (n-BuOH), and H2O, successively. From the EtOAc and n-BuOH fractions, five compounds were isolated through the repeated silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatographies. On the basis of spectroscopic data including mass spectrometry, IR, 1H-NMR, 13C-NMR, distortionless enhancement by polarization transfer, and two-dimensional-NMR gradient correlated spectroscopy (gCOSY), gradient heteronuclear single quantum correlation (gHSQC), gradient heteronuclear multiple bonding connectivity (gHMBC), the chemical structures of the compounds were determined as asarinin (1), (+)-catechin (2), (-)-epicatechin (3), hyperin (4), and nudiposide (5). Compounds 1 and 5 were isolated for the first time from the stem wood of L. obtusiloba Blume.

Arabidopsis thaliana sterol glycosyltransferase (SGT), UGT80A2, was expressed from stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Recombinant SGT was detected in both intracellular and extracellular fractions with a molecular mass of approximately 76 kDa. Secreted recombinant SGT accounted for approximately 60% of the total recombinant SGT production. Recombinant SGT in the extracellular fractions was purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. Radiometrical assay using uridine diphospho-d-[U-14C]glucose (UDP-14C-glucose) as a sugar donor and sterols, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDP-glycosyltransferase activity and could attach 14C-glucose to β-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with β-sitosterol, which was similar to the recombinant SGT produced by a bacterial expression system. The transfer of 14C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated 14C-glucosetransferred β-sitosterol and stigmasterol reactants.

In order to distinguish the cultivation area of Panax ginseng, principal component analysis (PCA) using quantitative and qualitative data acquired from HPLC was carried out. A new HPLC method coupled with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous quantification of ten major ginsenosides, namely Rh₁, Rg₂, Rg₃, Rg₁, Rf, Re, Rd, Rb₂, Rc, and Rb₁ in the root of P. ginseng C. A. Meyer. Simultaneous separations of these ten ginsenosides were achieved on a carbohydrate analytical column. The mobile phase consisted of acetonitrile-water-isopropanol, and acetonitrile-waterisopropanol using a gradient elution. Distinct differences in qualitative and quantitative characteristics for ginsenosides were found between the ginseng roots produced in two different Korean cultivation areas, Ganghwa and Punggi. The ginsenoside profiles obtained via HPLC analysis were subjected to PCA. PCA score plots using two principal components (PCs) showed good separation for the ginseng roots cultivated in Ganghwa and Punggi. PC1 infl uenced the separation, capturing 43.6% of the variance, while PC2 affected differentiation, explaining 18.0% of the variance. The highest contribution components were ginsenoside Rg₃ for PC1 and ginsenoside Rf for PC2. Particularly, the PCA score plot for the small ginseng roots of six-year old, each of which was light than 147 g fresh weight, showed more distinct discrimination. PC1 infl uenced the separation between different sample sets, capturing 51.8% of the variance, while PC2 affected differentiation, also explaining 28.0% of the variance. The highest contribution component was ginsenoside Rf for PC1 and ginsenoside Rg2 for PC2. In conclusion, the HPLC-ELSD method using a carbohydrate column allowed for the simultaneous quantifi cation of ten major ginsenosides, and PCA analysis of the ginsenoside peaks shown on the HPLC chromatogram would be a very acceptable strategy for discrimination of the cultivation area of ginseng roots.

The sprouts of Brassica oleracea var. gongylodes were extracted with 100% MeOH and the concentrated extract was partitioned with EtOAc, n-BuOH and , successively. From the EtOAc fraction, three sterols were isolated through the repeated silica gel and ODS column chromatographies. On the basis of physico-chemical and spectroscopic data including NMR, MS, and IR, the chemical structures of the sterols were determined as -sitosterol (1), brassicasterol (2), and 7-ketobrassicasterol (3). Compound 1 is usually observed in plant. Compounds 2 is observed in Brassica sp., and compounds 3 have very rarely occurred in natural source including plant.

The fresh ginseng roots were extracted with aqueous methanol, and the obtained extracts were partitioned using ethyl acetate, n-butanol, and water, successively. The repeated silica gel and octadecyl silica gel column chromatogaraphy for n-butanol fraction afforded four diol ginseng saponins, ginsenosides Rb₁, Rb₂, Rc, and Rd. The physicochemical, spectroscopic, and chromatographic characteristics of these ginsenosides were measured and compared with those reported in the literature. Some of the peak assignments in previously published ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectra were inaccurate. This study employed two-dimensional NMR experiments, including ¹H?¹H correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond connectivity, to determine exact peak assignments.

The fruits of Capsicum annuum were extracted using 80% aqueous MeOH, and fractionated using EtOAc and water. Repeated column chromatography using silica gel, octadecyl silica gel, and Sephadex LH-20 for the EtOAc fraction led to the isolation of a new lignan glycoside and a known one, icariside E(5). From the results of spectroscopic data, including EIMS, FABMS, UV, IR, (1)H and (13)C-NMR, DEPT, and 2D-NMR (COSY, HSQC, HMBC), the chemical structure of the new lignan glycoside was determined as (8R)-isodehydrodiconiferyl alcohol-4'-O-(6''-vanilloyl)-beta-D-glucopyranoside named vanilloylicariside E(5). All isolated compounds were tested for antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Icariside E(5) (2) and (8R)-Isodehydrodiconiferyl alcohol (3) exhibited a strong scavenging effect on DPPH (2: IC(50)=42.1 microM, 3: IC(50)=4.5 microM).

The leaves of Capsicum annuum L. were extracted with 80% aqueous MeOH, and the concentrated extract was partitioned successively with EtOAc, n-BuOH, and H2O. Three acyclic diterpenoids were isolated from the EtOAc fraction through repeated silica gel column chromatography. The physico-chemical and spectroscopic analyses including NMR, EI-MS, and IR indicated that the three compounds correspond to α-tocopherol (1), phytol (2), and vitamin E quinone (3). This is the first time these compounds have been isolated from the leaves of C. annuum.

β-Secretase (β-SEC) is one of the prime targets for therapeutic intervention of Alzheimer’s disease. We previously described the expression of human β-SEC in Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase (ST). To extend this work we investigated the kinetics of recombinant β-secretase and the feasibility of recombinant β-secretase for its inhibitor screening application. Recombinant β-SECs were purified from cultures of Tn5B1-4βSEC and Tn5B1-4βSEC/GalT-ST cells. Recombinant β-SEC from Tn5B1-4βSEC/GalT-ST cells exhibited a higher kcat/KM value compared to the control Tn5B1-4βSEC cells. We also attempted to screen the compounds inhibiting the activity of recombinant β-secretase among the metabolites isolated from Chrysanthemum coronarium L. Based on the inhibition analysis using purified β-SEC from Tn5B1-4βSEC/GalT-ST cells, 3-O-[β-D-galactopyranosyl (1→6)-α-D-galactopyranosyl]-glyceride was the best inhibitor among the compounds tested. The concentration at half-maximum inhibition values (IC50) of this compound was estimated to be 2.8 μM.

Expression of the human cyclooxygenase 1 (COX-1) In Trichoplusia ni BTI Tn 5B1-4 cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase (ST6Gal) was examined. Southern blot analysis indicated that the glycosyl-transferase genes were integrated into the Tn 5B1-4 cell genome. A lectin blot analysis also indicated that the recombinant COX-1s from the Tn 5B1-4/COX-1/GalT-ST cells contained the glycan residues of β1,4-linked galactose and α2,6-linked sialic acid. The specific peroxidase activity of the recombinant sialylated COX-1 from Tn 5B1-4/COX-1/GalT-ST cells was 23,930 units/mg, indicating an increase of approximately 17% compared with the non-sialylated control (20,500 units/mg) from the Tn 5B1-4/COX-1 cells.

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.

Dihydroflavonol 4-reductase (DFR) is part of an important step in the flavonoid biosynthesis pathway of anthocyanins. We isolated theDFR gene fromBrassica rapa ssp.pekinesis (Chinese cabbage), and found that biosynthesis of anthocyanins in tobacco could be modulated by introducing a heterologousDFR Bra gene. RT-PCR showed that transgenic plants had abnormal flower colors compared with the wild-type controls, a response that was correlated with the level ofDFR Bra transcription. HPLC analysis revealed that the isolated DFRBra cDNA mainly produced cyanidin as well as a small amount of pelargonidin in the anthocyanin biosynthesis pathway of those tobacco plants.

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6-phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P2 levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P2 were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P2 contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.

A new flavonolignan glucoside, tricin-4′-O-(erythro-β-guaiacylglyceryl)ether 9′′-O-β-D-glucopyranoside (9′′-O-glucopyranosyl salcolin B), was isolated from the aerial parts of Oryza sativa L. The structure of the compound was established on the basis of NMR, FAB-MS, and IR spectroscopic data.

Flowers of Erigeron annuus L. were extracted with 80% aqueous MeOH, and the concentrated extract was partitioned with EtOAc, n-BuOH, and H2O. Repeated silica gel and ODS column chromatography of the EtOAc fraction led to the isolation of a sterol, through activity-guided fractionation, using ACAT inhibitory activity measurements. From the physico-chemical data, including NMR, MS, and IR, the chemical structure of the compound was determined to be an ergosterol peroxide (1), which has been isolated for the first time from this plant. This compound exhibited hACAT-1 and Lp-PLA2 inhibitory effects, with inhibitory values of 51.6 +/- 0.9 and 51.7 +/- 1.2%, at a treatment concentration of 0.23 mM.

Cell-wall invertase (CIN) catalyzes the hydrolysis of sucrose into glucose and fructose for the supply of carbohydrates to sink organs via an apoplastic pathway. To study the CIN genes in rice (Oryza sativa L.), we isolated cDNA clones showing amino acid similarity to the plant cell wall invertase proteins from a search of rice sequence databases. Profile analyses revealed that the cloned genes are expressed in unique patterns in various organs. For example, transcripts of OsCIN1, OsCIN2, OsCIN4, and OsCIN7 were detected in immature seeds whereas OsCIN3 gene expression was flower-specific. Further transcript analysis of these genes expressed in developing seeds indicated that OsCIN1, OsCIN2, and OsCIN7 might play an important role involving sucrose partitioning to the embryo and endosperm. Sucrose, a substrate of CINs, induced the accumulation of OsCIN1 transcripts in excised leaves and OsCIN2 in immature seeds, while the level of OsCIN5 was significantly down-regulated in excised leaves treated with sucrose. Infecting the tissues with rice blast (Magnaporthe grisea) as a biotic stressor increased the expression of OsCIN1, OsCIN4, and OsCIN5, suggesting that these genes may participate in a switch in metabolism to resist pathogen invasion. These results demonstrate that OsCIN genes play diverse roles involving the regulation of metabolism, growth, development, and stress responses.

To investigate the effects of sucrose-phosphate synthase (SPS) on carbon partitioning, transgenicArabidopsis plants transformed withSynechocystis SPS were constructed. The integration, copy number and expression level were confirmed by Southern and Northern blot analyses. SPS activity in leaves from the transgenic and wild type plants was not significantly different. The level of sucrose and starch in the leaves of transgenic plant were slightly decreased compared to wild type. The glucose and fructose contents were increased up to two-fold compared to wild type during the light period. It is our speculation that the decreased sucrose level of the transgenic plant might be caused by the high acid invertase.

Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni2+-affinity fractionation to a specific activity of 24 800 U mg−1 protein. The peak level of recombinant Cox 2 production was 1.6 μg (107 cells)−1, seven days after induction with 0.5 mM CuSO4. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively.

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant endostatin was approximately 0.35 μg ml−1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l−1 at 6 days of cultivation.

Recombinant rotavirus VP6 production was 0.49 mg l−1 when 10 mM sodium butyrate was added to the suspension culture of transgenic tomato cells after 6 days' incubation. Recombinant VP6 production increased to 0.73 mg l−1, when the culture was supplemented with 10 mM sodium butyrate on day 6 and 9 after incubation. Total protein extracts of transgenic tomato cells from sodium butyrate-supplemented culture were examined by two-dimensional electrophoresis: 14 proteins were up-regulated and 7 down-regulated.