Diphenylboric Acid 2-aminoethyl Ester Research Articles

Influence of Molecular Structures on Fluorescence of Flavonoids and Their Detection in Mammalian Cells.

Flavonoids are being increasingly applied for the treatment of various diseases due to their anti-cancer, anti-oxidant, anti-inflammatory, and anti-viral properties. However, it is often challenging to detect their presence in cells and tissues through bioimaging, as most of them are not fluorescent or are too weak to visualize. Here, fluorescence possibilities of nine naturally occurring analogous flavonoids have been investigated through UV/visible spectroscopy, molecular structure examination, fluorescent images in mammalian cells and their statistical analysis employing aluminum chloride and diphenylboric acid 2-aminoethyl ester as fluorescence enhancers. It is found that, in order to form a stable fluorescent complex with an enhancer, flavonoids should have a keto group at C4 position and at least one -OH group at C3 or C5 position. Additionally, the presence of a double bond at C2–C3 can stabilize extended quinonoid structure at the cinnamoyl moiety, which thereby enhances the complex stability. A possible restriction to the free rotation of ring B around C1′–C2 single bond can contribute to the further enhancement of fluorescence. Thus, these findings can act as a guide for distinguishing flavonoids capable of exhibiting fluorescence from thousands of their analogues. Finally, using this technique, flavonoids are detected in neuroblastoma cells and their time course assay is conducted via fluorescence imaging. Their cellular uptake efficiency is found to be high and differential in nature and their distribution throughout the cytoplasm is clearly detected.

An analyses of flavonoids present in the inflorescence of sunflower

Flavonoids are low molecular weight secondary metabolites present ubiquitously in plants. They are known to have diverse metabolic functions, including regulation of growth and development, protection against biotic and abiotic stress, male fertility and pollen–stigma interaction. Present investigations deal with the importance of flavonoids in reproductive biology of sunflower (Helianthus annuus L. 1753), a member of family Asteraceae. A relationship among flavonoids, phenols and reactive oxygen species has been established in the reproductive component of sunflower. Flavonoids have been quantified, localized and compared using diphenylboric acid 2-aminoethyl ester fluorescent probe, high-performance liquid chromatography, thin-layer chromatography and spectrophotometry. Present investigations reveal that flavonoids and phenols are present in all the components of inflorescence—stigma, pollen, ray and disc florets. Quercetin is the main flavonoid identified in all the components of the inflorescence. Quercetin and kaempferol have been identified in pollen grains. These investigations further reveal that flavonoids act as antioxidants, have a developmental role in stigma, metabolic role in pollen and also play a signalling role in pollen–stigma interactions.

A Method for Visualizing Fluorescence of Flavonoid Therapeutics in Vivo in the Model Eukaryote Dictyostelium Discoideum

Naturstoff reagent A (diphenylboric acid 2-aminoethyl ester [DPBA]) has been used historically in plant science to observe polyphenolic pigments, such as flavonoids, whose fluorescence requires enhancement to be visible by microscopy. Flavonoids are common dietary constituents and are the focus of considerable attention because of their potential as novel therapies for numerous diseases. The molecular basis of therapeutic activity is only gradually being established, and one strand of such research is making use of the social amoeba Dictyostelium discoideum. We extended the application of DPBA to flavonoid imaging in these preclinical studies, and report the first method for use of DPBA in this eukaryotic model microbe and its applicability alongside subcellular markers. This in vivo fluorescence imaging provided a useful adjunct to parallel chemical and genetic studies.

Flavonols Induced by 5-Aminolevulinic Acid Are Involved in Regulation of Stomatal Opening in Apple Leaves

Flavonols, the main flavonoids in plant leaves, have newly been proposed as a H2O2 scavenger in guard cells. However, whether 5-aminolevulinic acid (5-ALA) is involved in regulating stomatal movement of apple (Malus × domestica Borkh. ‘Fuji’) leaves through flavonol accumulation remains unclear. In this study, using diphenylboric acid 2-amino ethyl ester (DPBA, a flavonol fluorescent dye) and a laser scanning confocal microscope, we observed that 5-ALA pretreatment significantly improved flavonol accumulation in guard cells of apple leaves, especially around the nucleus. Then we demonstrated that 5-ALA pretreatment inhibited ABA-induced stomatal closure via decreasing reaction oxygen species (ROS) accumulation in guard cells. Pretreatment with quercetin or kaempferol, two main kinds of flavonols in plants, also inhibited ABA-induced stomatal closure by decreasing ROS content in the guard cells. Furthermore, exogenous flavonols could suppress H2O2-induced stomatal closure in apple leaves. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is involved in the inhibitory effect of ALA on ABA-induced ROS accumulation and stomatal closure in apple leaves.

Flow Cytometric Method for the Detection of Flavonoids in Cell Lines

Here, we describe an easy-to-use flow cytometric method using diphenylboric acid 2-amino ethyl ester (DPBA) stain for the detection of flavonoids in cells from human/animal origin. Flavonoid bioavailability and bioactivity depend on structure, conjugation and the cell type to which they are presented. We have studied cellular uptake of five flavonoids with different structures and conjugation forms. First, parameters including fixation method, technical and batch variability, and concentration were optimized. Second, uptake of two aglycones—quercetin and hesperetin—and their corresponding glycosides—rutin and hesperidin—in Caco-2 cells was compared. Third, the aglycone quercetin, glycoside rutin, and glucuronide baicalin were added to the Caco-2, HepG2, and CHO-K1 cell lines at 1, 10, and 20 µM concentrations and cellular uptake was measured after 1, 4, and 7 h. We conclude that quercetin was taken up by cells in a dose-dependent way, and that HepG2 cells had the highest uptake factors, followed by CHO-K1 and Caco-2 cells. Confocal microscopy showed cell type–dependent localization of quercetin in the cell membrane and cytoplasm. No uptake of flavonoid glycosides was detected. This flow cytometric method can be used for future research unravelling mechanisms behind flavonoid bioactivity in health and disease at the cellular level.

The Biosynthetic Activities of Primary and Secondary Metabolites in Suspension Cultures of Aquilaria microcarpa

Two types of suspension-cultured Aquilaria microcarpa cells, friable and aggregated, were selectively generated. The biosynthetic activities of primary and secondary metabolites in target cells were detected using laser scanning microscopy (LSM) imaging with diphenylboric acid 2-amino ethyl ester (DPBA) and 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) staining. Scanned friable cells produced weakly fluorescent images revealing low productivity of metabolites. On the other hand, scanning of aggregated cells produced clear fluorescent images depicting the accumulations of flavonoids and lipids. Furthermore, abundant deposition of an unknown resinous compound in extracellular portion of aggregated cells could be visualized. The resinous compound was white to whitish-gray in color and highly sedimented in the medium. Based on these observations, we focused our investigation of metabolite productivity on aggregated suspension cells. Some prominent extracellular compounds were detected in the used liquid medium, as well as in the resinous residue within the medium. The characteristics of these metabolites were investigated in detail via gas chromatography-mass spectrometry (GC-MS) analysis.

Rapid quantification of cellular flavonoid levels using quercetin and a fluorescent diphenylboric acid 2-amino ethyl ester probe

Flavonoids are abundant in fruits and vegetables and have various biological activities. A rapid, sensitive method for assessing cellular quercetin levels is necessary because cellular levels are often low. A new method for measuring cellular flavonoid levels is proposed using diphenylboric acid 2-aminoethyl ester (DPBA), a fluorescent probe, and quercetin, a representative flavonoid. This spectrofluorometry-based assay is simple and allows rapid quantification of the cellular quercetin content with high sensitivity. CHO-K1 cells were incubated with quercetin for 5 min, and then the cellular quercetin concentration was assayed using DPBA spectrofluorometry. Results, compared with high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), and conventional spectrophotometry measurements, were highly correlated with HPLC data (R>0.99). The method is more sensitive than TLC and conventional spectrophotometry. Spectrofluorometric measurement using DPBA is a simple, rapid, and sensitive method for assessing cellular quercetin levels.

Anatomical and histochemical characterization of in vitro haustorium from roots of Castilleja tenuiflora

In vitro induction of haustoria from Castilleja tenuiflora Benth. was achieved by applying 25 μM catechin, 25 μM vanillin, or 25 μM H2O2. Of the treatments tested, 25 μM vanillin was the strongest inducer of haustoria in C. tenuiflora roots in vitro (up to 3 haustoria per root). Haustorium development occurred laterally and was observable 14 d after inducer application. It was characterized by elongation of the epidermal cells and division of the inner cortical cells which also possessed abundant nuclei. Histochemical analysis using 3,3-diaminobenzidine (DAB) and diphenylboric acid 2-aminoethyl ester (DBPA) indicated that the formation of haustoria was associated with the accumulation of H2O2 and flavonoids.

GRAVITY PERSISTENT SIGNAL 1 (GPS1) Reveals Novel Cytochrome P450s Involved in Gravitropism

Gravity is an important environmental factor that affects growth and development of plants. In response to changes in gravity, directional growth occurs along the major axes and lateral branches of both shoots and roots. The gravity persistent signal (gps) mutants of Arabidopsis thaliana were previously identified as having an altered response to gravity when reoriented relative to the gravity vector in the cold, with the gps1 mutant exhibiting a complete loss of tropic response under these conditions. Thermal asymmetric interlaced (TAIL) PCR was used to identify the gene defective in gps1. Gene expression data, molecular modeling and computational substrate dockings, quantitative RT-PCR analyses, reporter gene fusions, and physiological analyses of knockout mutants were used to characterize the genes identified. Cloning of the gene defective in gps1 and genetic complementation revealed that GPS1 encodes CYP705A22, a cytochrome P450 monooxygenase (P450). CYP705A5, a closely related family member, was identified as expressed specifically in roots in response to gravistimulation, and a mutation affecting its expression resulted in a delayed gravity response, increased flavonol levels, and decreased basipetal auxin transport. Molecular modeling coupled with in silico substrate docking and diphenylboric acid 2-aminoethyl ester (DBPA) staining indicated that these P450s are involved in biosynthesis of flavonoids potentially involved in auxin transport. The characterization of two novel P450s (CYP705A22 and CYP705A5) and their role in the gravity response has offered new insights into the regulation of the genetic and physiological controls of plant gravitropism.

Flavone deglycosylation increases their anti‐inflammatory activity and absorption

Flavones have reported anti-inflammatory activities, but the ability of flavone-rich foods to reduce inflammation is unclear. Here, we report the effect of flavone glycosylation in the regulation of inflammatory mediators in vitro and the absorption of dietary flavones in vivo. The anti-inflammatory activities of celery extracts, some rich in flavone aglycones and others rich in flavone glycosides, were tested on the inflammatory mediators tumor necrosis factor α (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in lipopolysaccharide-stimulated macrophages. Pure flavone aglycones and aglycone-rich extracts effectively reduced TNF-α production and inhibited the transcriptional activity of NF-κB, while glycoside-rich extracts showed no significant effects. Deglycosylation of flavones increased cellular uptake and cytoplasmic localization as shown by high-performance liquid chromatography (HPLC) and microscopy using the flavonoid fluorescent dye diphenylboric acid 2-aminoethyl ester (DPBA). Celery diets with different glycoside or aglycone contents were formulated and absorption was evaluated in mice fed with 5 or 10% celery diets. Relative absorption in vivo was significantly higher in mice fed with aglycone-rich diets as determined by HPLC-MS/MS (where MS/MS is tandem mass spectrometry). These results demonstrate that deglycosylation increases absorption of dietary flavones in vivo and modulates inflammation by reducing TNF-α and NF-κB, suggesting the potential use of functional foods rich in flavones for the treatment and prevention of inflammatory diseases.

The soybean F3′H protein is localized to the tonoplast in the seed coat hilum

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3'-hydroxylase (F3'H) gene (sf3'h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3'h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3'h1 was not detected in To7G. A truncated sf3'h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3'H was detected using microsomal fractions from yeast transformed with sf3'h1 from To7B, but not from To7G. These results indicate that sf3'h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3'h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3'h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3'H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

C(a2+)-dependent glutamate release involves two classes of endoplasmic reticulum Ca(2+) stores in astrocytes.

Astrocytes can modulate synaptic transmission by releasing glutamate in a Ca(2+)-dependent manner. Although the internal Ca(2+) stores have been implicated as the predominant source of Ca(2+) necessary for this glutamate release, the contribution of different classes of these stores is still not well defined. To address this issue, we cultured purified solitary cortical astrocytes and monitored changes in their internal Ca(2+) levels and glutamate release into the extracellular space. Ca(2+) levels were monitored by using the Ca(2+) indicator fluo-3 and quantitative fluorescence microscopy. Glutamate release was monitored by an L-glutamate dehydrogenase-linked detection system. Astrocytes were mechanically stimulated with a glass pipette, which reliably caused an increase in internal Ca(2+) levels and glutamate release into the extracellular space. Although we find that the presence of extracellular Cd(2+), a Ca(2+) channel blocker, significantly reduces mechanically induced glutamate release from astrocytes, we confirm that internal Ca(2+) stores are the predominant source of Ca(2+) necessary for this glutamate release. To test the involvement of different classes of internal Ca(2+) stores, we used a pharmacological approach. We found that diphenylboric acid 2-aminoethyl ester, a cell-permeable inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist, greatly reduced mechanically induced glutamate release. Additionally, the preincubation of astrocytes with caffeine or ryanodine also reduced glutamate release. Taken together, our data are consistent with dual IP(3)- and caffeine/ryanodine-sensitive Ca(2+) stores functioning in the control of glutamate release from astrocytes.

Flavonoid-related regulation of auxin accumulation in Agrobacterium tumefaciens-induced plant tumors.

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.

Effect of fluorescence intensifiers on the fluorodensitometric determination of flavones and flavonols after detection with diphenylboric acid 2-aminoethyl ester

The measurement of the fluorescence emitted by flavones and flavonols in situ on thin-layer chromatography plates can be improved by dipping the developed plates in appropriate solutions. The effect of different substances on the intensity and stability of the fluorescence of 14 flavone and 26 flavonol derivatives detected with diphenylboric acid 2-aminoethyl ester (Naturstoffreagens A, NA) is described. Silicone oil or paraffin is most appropriate for lipophilic flavonoids while the more hydrophilic polyethylene glycol 4000 is most appropriate for less lipophilic aglycones as well as glycosides. Authentic reference standards in appropriate concentrations must be measured on the same plate as the experimental sample since flavonoids demonstrate specific fluorescence behaviour and because in situ fluorodensitometry measurements are difficult to reproduce. The correlation between fluorescence and molecular structure is discussed briefly. The test conditions established are an improvement on the accepted in situ fluorodensitometry determination of flavonoids.

PHENOLIC VARIATION IN LEAF POSITION IN APPLE

The foliar phenolic content of 21 apple cultivars was evaluated. Ten leaves were sampled randomly from three positions on current-season terminal shoots. Shoots were divided as tip, middle, and basal positions. The phenolic content was determined by spectrophotometric method (390 nm) using diphenylboric acid 2-aminoethyl ester as the reagent and caffeic acid as standard. Cultivars varied significantly in phenolic content. `Stark Ultra Red' had the highest amount, and `Liberty' had the lowest amount. Significant variations in the phenolic content due to leaf position were observed. Phenolic content was highest in leaves from the tip position, and it decreased toward the basal portion of the shoot. Factors affecting the phenolic content of apple cultivars will be investigated to determine apple × insect interactions.

Diphenyltin dichloride as a chromogenic reagent for the detection of flavonoids on thin-layer plates

Among several complexation reagents, diphenyltin dichloride (DTC) proved to be a useful chromogenic compound for both the qualitative and quantitative analysis of flavones and flavonols on thin-layer plates by forming fluorescent complexes of different colour. For qualitative analysis, comparative detection with DTC and diphenylboric acid 2-aminoethyl ester can indicate the position of the glycosidated hydroxyl of kaempferol and luteolin glycosides. Fluorimetric densitometry showed high sensitivity to flavonols with a free 3-hydroxyl group and flavones containing two adjacent hydroxyl groups in ring B.