Anthracycline anticancer agents are widely used in the cancer chemotherapy for hepatocelluar carcinoma. However, accurate kinetic analyses of the hepatocellular uptake and efflux of the drugs have not been reported. We, therefore, investigated the hepatobiliary transport of epirubicin, an anthracycline derived antibiotic, after intravenous (i.v.) infusion in rats. The hepatic uptake mechanisms of epirubicin were also investigated in isolated rat hepatocytes. To analyze epirubicin levels in the biological samples, we used an HPLC-based method which has been validated for a kinetic study by suitable criteria. The uptake process of epirubicin by the hepatocytes revealed one saturable component, with a Km of 99.1 ?g/mL and Vmax of 3.70 ?g/min/10(6) cells. The initial uptake velocity of epirubicin was significantly inhibited in a temperature-dependent manner. The velocity was also reduced in the presence of metabolic inhibitors such as rotenone or carbonylcyanide-p-(trifluoromethoxy)-phenylhydrazone. Substrates for organic anion transporters such as bromosulfophthalein and taurocholate significantly inhibited the initial uptake velocity of epirubicin. We also attempted to determine the hepatobiliary transport of epirubicin after i.v. infusion in vivo. At steady-state after i.v. infusion of epirubicin (10-160 ?g/min/kg), the drug was extensively accumulated in the liver, followed by excretion into bile. Furthermore, the CLbile,plasma and CLbile,liver decreased with a corresponding increase in the Css,plasma and Css,liver. In conclusion, present studies using isolated rat hepatocytes and in vivo i.v. infusion demonstrate that epirubicin is likely to be taken up into liver cells via organic anion transporting polypeptides, and that its biliary excretion might be mediated via specific transporters.
Rhizoctonia solani (R. solani), a soil-borne necrotrophic pathogen, causes various plant diseases. Rhizoctonia solani is a mitosporic fungus, the sclerotium of which is the primary inoculum and ensures survival of the fungus during the offseason of the host crop. Since the fungus does not produce any asexual or sexual spores, understanding the biology of sclerotia is important to examine pathogen ecology and develop more efficient methods for crop protection. Here, one- and two-dimensional gel electrophoresis (1-DE and 2-DE, respectively) were used to examine protein regulation during the maturation of fungal sclerotia. A total of 75 proteins (20 proteins from 1-DE using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) and 55 proteins from 2-DE using MALDI-TOF MS or MALDI-TOF/TOF MS) were differentially expressed during sclerotial maturation. The identified proteins were classified into ten categories based on their biological functions, including genetic information processing, carbohydrate metabolism, cell defense, amino acid metabolism, nucleotide metabolism, cellular processes, pathogenicity and mycotoxin production, and hypothetical or unknown functions. Interestingly, two vacuole function-related proteins were highly up-regulated throughout sclerotial maturation, which was confirmed at the transcript level by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. These findings contribute to our understanding of the biology of R. solani sclerotia.
The Korean prostrate spurge Euphorbia supina is a weed that has been used in folk medicine in Korea against a variety of diseases. Nine polyphenols were characterized for this plant by using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and the results were compared with the literature data. The individual components were validated using the calibration curves of structurally related external standards and quantified for the first time by using the validated method. Correlation coefficients (r (2)) were >0.9907. The limit of detection and limit of quantification of the method were >0.028?mg/L and 0.094?mg/L, respectively. Recoveries measured at 50?mg/L and 100?mg/L were 76.1-102.8% and 85.2-98.6%, respectively. The total amount of the identified polyphenols was 3352.9?±?2.8?mg/kg fresh plant. Quercetin and kaempferol derivatives formed 84.8% of the total polyphenols. The antioxidant activities of the flavonoids were evaluated in terms of 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging activity, and the reducing power showed a dose-dependent increase. Cell viability was effectively suppressed at polyphenol mixture concentrations >250?mg/L.
The type and content of plant polyphenols can be influenced by maturity. Korean chokeberry (Aronia melanocarpa) leaves of three different maturities (young, mature, and aged) were extracted with 70% aqueous methanol. The polyphenols in the leaves were analysed for the first time using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and comparison with reported data. Among the 12 characterised components, five flavonoids, 3, 4, and 10-12, and a dicaffeoylquinic acid derivative, 6, were characterised for the first time in chokeberry leaves. Each polyphenol component was validated and quantified using a representative polyphenol standard of the same group. The antioxidant activity of the three different mature leaf extracts was determined. The antioxidant activity was highest for young leaves, followed by mature and aged leaves. The results suggest that younger chokeberry leaves may be more favourable for processing a higher quality functional tea due to their higher polyphenol content.
Anthracnose caused by Colletotrichum gloeosporioides is one of the most serious postharvest diseases of citrus fruit. Fruit peels infected with C. gloeosporioides and the peels of healthy fruit were analysed for flavonoids, using high performance liquid chromatography-tandem mass spectroscopy, to evaluate variations in flavonoid levels in Citrus benikoji Hort. ex. Tan. Seventeen flavonoids were characterised from the fruit peels. The flavonoids were validated using structurally related standards and quantified. Among the 17 flavonoids, the concentration of component 3 was the highest and that of component 10 was the lowest. During 8days after inoculation, the concentration of the seven flavonoids 1-3, 5, 6, 13, and 14 increased gradually up to day 8. Flavonoid 4 was detected from day 7. The seven minor flavonoid components, 8-12, 15, and 16 increased to day 5 and then decreased. However, flavonoids level variations were not significantly different from that of the non-infected fruits during the experimental period. The monitoring suggested that the constitutively formed seven polymethoxyflavones (8-12, 15, and 16) may act as phytoanticipins in the defence mechanism against the fungus, and that hespertin 7-O-glucoside (4), produced de novo on day 6 after infection, may function as a phytoalexin.
Soybean sprouts (Kongnamool) are one of the most popular and nutritive traditional vegetables in East Asia. Anthracnose caused by Colletotrichum gloeosporioides is one of the most serious diseases of soybean sprouts. In order to obtain basic information for breeding and/or selecting soybean genotypes with increased natural defense against anthracnose, phenolic compounds were profiled for healthy and infected soybean (Glycine max Merr.) sprouts by using high-performance liquid chromatography coupled with tandem mass spectrometry.
As watermelon is farmed primarily by protected and successive cultivation techniques, a number of pesticides are required for the control of pests and diseases. To evaluate the harmful effects of pesticides in watermelon and to guarantee consumers safety, a rapid screening process for pesticides is required. A LC-MS/MS method was applied for the direct quantitation of 44 pesticide residues in watermelon. A Zorbax XDB-C(18) column was selected for analysis, with a mobile phase consisting of a gradient system of water and 5 mM methanolic ammonium formate. MS/MS experiments were performed in ESI positive ion and multiple reaction monitoring modes. The LOQs were in the range of 1-26 microg/kg, thereby indicating good sensitivity. Most of the recoveries ranged between 70-131% with RSDs < or = 20%. We suggested that the amount of pesticide residues such as pyroquilon (pyn), boscalid (bd), and dimethomorph (di) in amides (AM) and cinosulfuron (ci) in ureas (UR) may have been overestimated for the pesticides owing to increased alpha-error risk, whereas the amounts of pesticide residues, such as imibenconazole (ie) in the triazoles (TR) and fenpyroximate (fee) in the imidazoles (IM), may have been underestimated as the result of increased beta-error risk. The current method allowed for the rapid quantitation and identification of low pesticide levels in the watermelon samples. No pesticide residues were detected in any of the surveyed watermelons obtained from eight local markets in the Republic of Korea. Statistical analysis of the recoveries classified the 44 pesticides into nine groups and three overall categories.
Light-emitting diodes (LEDs) are an efficient alternative to traditional lamps for plant growth. To investigate the influence of LEDs on flowering and polyphenol biosynthesis in the leaves of chrysanthemum, the plants were grown under supplemental blue, green, red, and white LEDs. Flower budding was formed even after a longer photoperiod than a critical day length of 13.5 h per day under blue light illumination. The weights of leaves and stems were highest under the white light illumination growth condition, whereas the weight of roots appeared to be independent of light quality. Among nine polyphenols characterized by high-performance liquid chromatography-tandem mass spectroscopy, three polyphenols were identified for the first time in chrysanthemum. A quantitation and principal component analysis biplot demonstrated that luteolin-7-O-glucoside (2), luteolin-7-O-glucuronide (3), and quercetagetin-trimethyl ether (8) were the highest polyphenols yielded under green light, and dicaffeoylquinic acid isomer (4), dicaffeoylquinic acid isomer (5), naringenin (7), and apigenin-7-O-glucuronide (6) were greatest under red light. Chlorogenic acid (1) and 1,2,6-trihydroxy-7,8-dimethoxy-3-methylanthraquinone (9) were produced in similar concentrations under both light types. The white and blue light appeared inefficient for polyphenol production. Taken together, our results suggest that the chrysanthemum flowering and polyphenol production are influenced by light quality composition.
Phospholipids (PLs) in 57 varieties of soybeans were profiled by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry and principal component analysis (PCA) to discriminate PL-rich soybeans. The PL calibration curves showed linearity with correlation coefficients >0.9964. The recoveries at 5 mg/L spiked level ranged from 72.8 to 86.7% and those at 12.5 mg/L from 78.2 to 85.1%. The repeatability at a 5 mg/L spiked level ranged from 2.5 to 7.0% and those at 12.5 mg/L from 1.2 to 3.9%. The average total PL content in the 57 soybean varieties was about 35.3 mg/kg. The total PL content was the highest in Aodaiz (35, 48.7 ± 1.4 mg/kg) and the lowest in Poongsannamul (56, 16.0?±?0.7 mg/kg). The PCA showed that RS-78sun (42), Gyeongsang #1 (3) and Aodaiz (35) are the most improved varieties of the investigated 57 varieties from the viewpoint of PL content.
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