Scopoletin fluorescence

Tobacco (Nicotiana tabacum) osmotic stress, baffled agitation scopoletin fluorescence [180]... 

Competing hydrogen donors to horseradish peroxidase must be absent from the sample or perfusion medium, otherwise the decrease in scopoletin fluorescence would not represent the actual rate of hydrogen peroxide formation. A drawback of these assays is that continuous measurement of hydrogen peroxide in neutrophil lysates is not feasible because NADPH interferes with the catalytic action of the peroxidase. 

Oxidant production is measured with the fluorogenic substrate para-hydroxyphenylacetic acid (PHPA) in the presence of superoxide dismutase and peroxidase (9). Under these conditions, superoxide is converted to H2O2 by the superoxide dismutase, and two molecules of PHPA are converted to a fluorescent diadduct by H2O2 and peroxidase. Similar assays have been devised using homovanillic acid (16) or scopoletin (17) instead of PHPA. 

Fig. 2 Increase in fluorescence of the genuine fluorescence (A) by treatment with Benedict s reagent (B) and immersion in a paraffin solution (C) and reduction of emission intensities with time for the two cumarins umbelliferone and scopoletin (curves).

H202 Determination. The analytical method for determination of H202 measures the loss in fluorescence of scopoletin by the peroxidase-mediated decomposition of H202 (11, 61-64). Separate standard curves were determined for each water or culture studied because the slope of the curve is affected by dissolved organic carbon (65, 66). 

For coumarins in orange fruits (115), the HPLC used a Zorbax Rx C8 (250-mm X 4.6-mm ID, 5 fim) column maintained at 25°C, and analysis was performed by binary-gradient elution using 0.1% HOAc in acetonitrile (eluent A) and 0.1% HOAc in HzO (eluent B). In the author s lab, standard coumarins could be separated by isocratic elution on Zorbax Rx C8 column with acetonitrile-0.1% HOAc in water (35 65) at 1.0 ml/min, as presented in previous work (1). The eluate from the column was passed to a UV detector (UV 330 nm) and then into a fluorescence detector (excitation at 340 nm, emission at 425 nm). As for the specificity, some of the coumarins do not have native fluorescence. Nine coumarins are separated under UV 330 nm, and three coumarins could not be detected with fluorescence detection. Detailed conditions for coumarin analysis in foods and absorption spectra of coumarins obtained by online diode array detector with HPLC were presented by Lee and Widmer (1). Since coumarins exhibit strong absorption in the ultraviolet region, absorption at approximately 313 nm has been used to estimate the dilution of cold-pressed lemon oil with distilled oil (12). Analysis of umbelliferone (7-hydroxy-coumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) in citrus fruits was performed using... 

Methods for detecting hydrogen peroxide are based on the peroxidase assay systems. The most common employs horseradish peroxidase (HRP) which uses hydrogen peroxide to oxidize scopoletin into a non-fluorescent product (Loschen et al., 1971), originally described by An-dreae (1955). 

Certain fluorescent compounds, such as the coumarin derivative, scopoletin, can act as hydrogen donors in the oxidative reaction catalysed by horseradish peroxidase (Udenfriend, 1969), an enzyme that exhibits substrate specificity for hydrogen peroxide... 

Maehly and Chance, 1954). The rate of decrease in the fluorescence intensity, through oxidation of scopoletin demonstrates directly the rate of formation of hydrogen peroxide. During its oxidation by horseradish peroxidase, fluorescence is lost with a stoichiometry directly proportional to peroxide concentration. 

Calibration of the system The scopoletin concentration is adjusted in such a way that its fluorescence exceeds the substrate-induced fluorescence at least tenfold. Any marked decrease in fluorescence intensity therefore demonstrates an oxidation of scopoletin by H202 via horseradish peroxidase. Hence, after suitable calibration of the system, the rate of loss of fluoresence can be used to measure the rate of hydrogen peroxide production. 

The system is standardized in the absence of cells with known amounts of peroxide either generated as H202 or from glucose in the medium and glucose oxidase or added directly as ethyl peroxide. The relationship between fluorescence intensity of 2 pM scopoletin and peroxide concentration is shown in Fig. 3.13. 

The change in fluorescence intensity produced on reaction of scopoletin with hydrogen peroxide must largely exceed the variance in fluorescence arising from, for example, intracellular metabolism. 

Sensitive samples are often destroyed when subjected to measurement by fluorescence. This sensitivity should be borne in mind when performing a vahdation and, if appropriate, it should be stated in the testing procedures that the chromatogram should be measured only once. Our own investigations into the determination of dried extract of nettle root showed that a second determination of scopoletine on the same chromatogram lane gave a value that had decreased by ca. 5 %. 

B In UV-36.5 nm, the un.sub,stituted coumarin, in contrast to coumarins with -OH, -OCH,, sLib,stituents or fiirano- or pyrano-coumarins, shows a typical green-blue fluorescence only after treatment with KOH reagent. Scopoletin (Tl) and umbelliferone (T3) are present in low concentrations only, seen as blue fluorescent zones at Rj 0.25 and 0.4, rc.spcctively (1-3). 

The metlianolic extracts of Abrotani herba (1) and Fabiani herba (2) are characterized by the prominent blue fluorescent coumarin aglycoiie zone at R, 0.95 (scopoletin, isofraxidin and umbelliferone). They are differentiated by the violet-blue fluoresceiit isofraxidin-7-0 and scopoletin-7-O-glucosides in the range of0.4-0.45 and an additional zone at R 0.7 in sample 1 and the scopoletin-7-O-priinveroside at Rf 0.15 in extract 2. 

The caffeic acid derivatives with chlorogenic add at R, 0.45 (Tl) and isochlorogenic acids at RfO. 7-0.8 are more concentrated in Abrotani herba (1) than in Fabiani herba (2). The later shows rutin at Rf — 0.4 as a prominent orange zone and tlie violet-blue Zone of scopoletin-7-O-primveroside at Rf 0.15. Blue coumarin and orange fluorescent flavonoid aglycones move with the solvent front,... 

Fraxini cortex (2) has two prominent blue fluorescent coumarin aglycone.s such as fraxidin, fraxinol and isofraxidin in the R, range 0.1 -0.25, directly below the scopoletin test T4. 

The coumarin glycosides of Fraxini cortex (2) are detected with NP/PEG reagent as four to five intense, bright-blue fluorescent zones (UV-365 nm) in the range 0.35-0.75, such as fraxin (R, 0.25/T2), and the coumarin aglycones at R, 0.8-0.95 with fraxetin (yellow/ R[ — 0.8, T3), isofraxidin and scopoletin (blue/Rj 0.8-0,95, T4). 

In the polar system B scopoletin moves almost with the solvent front (R, 0.95). The scopoletin gluco.side is found at R( 0.4 directly below chlorogenic acid (T2/R, 0.45). Scopoliae radix (1) shows five to six blue fluorescent zones from the start up to R, 0.5. Similar, hut less concentrated zones are found in Belladonnae (2) and Mandragorae radix (3) extract,s. 

Treatment with KOH reagent intensifies the fluorescence of the coumarins such as scopoletin at the solvent front and scopoletin-7-O-glucoside at R, 0.45. 

C Ammi majoris fructus (2) is characterized by furanocoumarins, seen as ten to 12 blue fluorescent zones between the start and R, 0.65, with prominent zones in the lower R, range. Bergapten, xanthotoxin (e g. scopoletin) and i riiperatorin move into the R, range 0.45-0.55, and visnadin to R, 0.6. Ammi visnagae fructus (1) shows from the start up to K, 0.6 white-blue zones (e.g. visnagin, T2). 

B KOH reagent intensifies the fluorescence of herniarin and scopoletin to a bright blue. Both compounds are detectable in samples (l)-(3). 

The presence of the blue fluorescent scopoletin (R, 0.7/Tl) and the dark-green fluore.scent biflavonoid ainentoflavone (Rf 0.4/T2) is characteristic for Viburni prunifolii cortex (1). 

The Hedera helix samples (1,3,5) are characterized by a series of prominent blue fluorescent zones of phenol carboxylic adds and coumarins in the R, range 0.45-0.95 e.g. chlorogenic acid (T2) and scopoletin-7-O-glucoside at R, 0.45-0.5, the... 

Watanabe et ah (82) also observed the accumulation of scopoletin glucoside in boron-deficient tobacco leaves. The leaves from boron-deficient plants contained a 20-fold increase in the glucoside compared with normal tobacco plants. Other blue fluorescent compounds accumulating in boron-deficient tissue have also been identified (83). Boron-deficient culture medium used for sunflower stem callus and root tissue culture resulted in less lignification than in normal tissue 41). Recently,... 

Ascorbic acid inhibits catalase in concentrations as low as 2 X 10 moll. Very low concentration of Cu " " ions accelerates the rate of inhibition. The inhibition of ascorbic acid to catalase occurs in the presence of ions. The activity of catalase can be monitored by the decrease of H2O2 substrate or increase of O2. For fluorimetric detection of H2O2, the decrease in fluorescence of scopoletin or the increase in fluorescence of diaceyldichlorofluorescein upon reaction with H2O2 are preferably monitored. 

All Herba and Folium Artemisiae extracts 1-7 are characterized by a very homogenous TLC-profile with blue, green and orange fluorescent zones with scopoletin (T5) and chlorogenic acid (T6) as dominant zones in all samples. Hyperoside (T3) is detectable in samples 1-3 and 7. The blue-violet zone at I =0.45 in samples 1-3 may be assigned to scopolin. 

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