Accumulation Of Rutin Research Articles

The redox thermodynamics and kinetics of flavonoid rutin adsorbed at glassy carbon electrodes by stripping square wave voltammetry

The adsorptive accumulation of rutin (RU) at glassy carbon (GC) electrodes in 10% ethanol+90% 1moldm−3 HClO4 aqueous solution is studied by using cyclic (CV) and square wave (SWV) voltammetries. The Frumkin adsorption isotherm best described the specific interaction of rutin with carbon electrodes. By fitting the experimental data, values of −31.9kJmol−1 and 0.54±0.02 were obtained for the Gibbs free energy of adsorption and the interaction parameter, respectively. SWV fully characterized the thermodynamics and kinetics of the surface redox process, using a combination of the “quasi-reversible maximum” and the “splitting of SW peaks” methods. Average values of 0.644±0.003V and 0.44±0.02 were obtained for the formal potential and the anodic transfer coefficient, respectively. Moreover, a formal rate constant of 6.1×102s−1 was obtained. SWV was also employed to generate calibration curves. The lowest concentration of RU experimentally measured for a signal-to-noise ratio of 3:1 was 2×10−8moldm−3 (12ppb).

Expression of flavonoid biosynthesis genes vis-à-vis rutin content variation in different growth stages of Fagopyrum species

Buckwheat is one of the field crops with the highest concentration of rutin, an important flavonoid of medicinal value. Two species of buckwheat, Fagopyrum esculentum and Fagopyrum tataricum, are the major sources of rutin. Seeds of latter contain 40–50× higher rutin compared to the former. The physiological and molecular bases of rutin content variation between Fagopyrum species are not known. The current study investigated the differences in rutin content in seeds and in other tissues and growth stages of two Fagopyrum species, and also correlated those differences with the expression of flavonoid pathway genes. The analysis of rutin content dynamics at different growth stages, S1–S9 (from seed germination to mature seed formation) of Fagopyrum species revealed that rutin content was higher during seedling stages of F. tataricum (3.5 to 4.6-fold) compared to F. esculentum and then increased exponentially from stages S3 to S6 (different leaf maturing stages and inflorescence) of F. esculentum, whereas it fluctuated in F. tataricum. The rutin content was highest in the inflorescence stage (S6) of both species, with a relatively higher biosynthesis and accumulation during post-flowering stages of F. tataricum compared to F. esculentum. The expression of flavonoid pathway genes, through qRT-PCR, in different growth stages vis-à-vis rutin content variation showed differential expression for four genes, PAL, CHS, CHI and FLS with the amounts of transcripts relatively higher in F. tataricum compared to F. esculentum, thereby, correlating these genes with the biosynthesis and accumulation of rutin. The expression of PAL was highest, 7.69 and 8.96-fold in Stages 2 (seedling stage) and 9 (fully developed seeds) of F. tataricum compared to F. esculentum, respectively. The expression of the CHS gene correlated with the rutin content because it was highest in the flowers (S6) and fully developed seeds (S9) of both Fagopyrum species, with relatively higher transcript amounts (2.13 and 3.19-fold, respectively) in F. tataricum (IC-329457) compared to F. esculentum (IC-540858). This study provides useful information on molecular and physiological dynamics of rutin biosynthesis and accumulation in Fagopyrum species and the correlation of expression of flavonoid biosynthesis genes with the rutin content can be useful in planning for genetic improvement.

Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMYB12, leads to insect resistance.

Flavonoids synthesized by the phenylpropanoid pathway participate in myriad physiological and biochemical processes in plants. Due to the diversity of secondary transformations and the complexity of the regulation of branched pathways, single gene strategies have not been very successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis (Arabidopsis thaliana) transcription factor, AtMYB12, in tobacco (Nicotiana tabacum), which resulted in enhanced expression of genes involved in the phenylpropanoid pathway, leading to severalfold higher accumulation of flavonols. Global gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMYB12 regulated a number of pathways, leading to flux availability for the phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial microRNA reversed insect resistance of the AtMYB12-expressing tobacco plants. Our study suggests that AtMYB12 can be strategically used for developing safer insect pest-resistant transgenic plants.

Jasmonate-Dependent and -Independent Pathways Mediate Specific Effects of Solar Ultraviolet B Radiation on Leaf Phenolics and Antiherbivore Defense

Ultraviolet B (UV-B) radiation, a very small fraction of the daylight spectrum, elicits changes in plant secondary metabolism that have large effects on plant-insect interactions. The signal transduction pathways that mediate these specific effects of solar UV-B are not known. We examined the role of jasmonate signaling by measuring responses to UV-B in wild-type and transgenic jasmonate-deficient Nicotiana attenuata plants in which a lipoxygenase gene (NaLOX3) was silenced (as-lox). In wild-type plants, UV-B failed to elicit the accumulation of jasmonic acid (JA) or the bioactive JA-isoleucine conjugate but amplified the response of jasmonate-inducible genes, such as trypsin proteinase inhibitor (TPI), to wounding and methyl jasmonate, and increased the accumulation of several phenylpropanoid derivatives. Some of these phenolic responses (accumulation of caffeoyl-polyamine conjugates) were completely lacking in as-lox plants, whereas others (accumulation of rutin and chlorogenic acid) were similar in both genotypes. In open field conditions, as-lox plants received more insect damage than wild-type plants, as expected, but the dramatic increase in resistance to herbivory elicited by UV-B exposure, which was highly significant in wild-type plants, did not occur in as-lox plants. We conclude that solar UV-B (1) uses jasmonate-dependent and -independent pathways in the elicitation of phenolic compounds, and (2) increases sensitivity to jasmonates, leading to enhanced expression of wound-response genes (TPI). The lack of UV-B-induced antiherbivore protection in as-lox plants suggests that jasmonate signaling plays a central role in the mechanisms by which solar UV-B increases resistance to insect herbivores in the field.

Nanomolar detection of rutin based on adsorptive stripping analysis at single-sided heated graphite cylindrical electrodes with direct current heating

Abstract A single-sided heated graphite cylindrical electrode (ss-HGCE) was designed. Compared to previous alternative current (AC) heating, much simpler and cheaper direct current (DC) heating supplier was adopted for the first time to perform adsorptive accumulation of rutin at ss-HGCE at elevated electrode temperature. This offers great promise for low cost, miniaturization and high compatibility with portability. The square wave voltammetry (SWV) stripping peak current was enhanced with increasing the electrode temperature only during preconcentration step. This enhancement was contributed to the forced thermal convection induced by heating the electrode rather than the bulk solution, which is able to improve mass transfer and facilitate adsorption hence enhance stripping response. A detection limit of 1.0 × 10−9 M (S/N = 3) could be obtained at an electrode temperature of 48 °C during 5 min accumulation, one magnitude lower than that at 28 °C (room temperature). This is the lowest value at carbon-based electrodes for rutin determination as we know. Such novel method was also successfully used to determine rutin in pharmaceutical tablets.

Variation of bioactive substances in Hypericum montbretii during plant growth

The present study was conducted to determine phenologic and morphogenetic variation of chlorogenic acid and flavonoids, as rutin, hyperoside, apigenin-7-O-glucoside, quercitrin, quercetin and viteksin content of Hypericum montbretii growing in Turkey. Wild growing plants were harvested at vegetative, floral budding, full flowering, fresh fruiting and mature fruiting stages and dissected into stem, leaf and reproductive tissues and assayed for bioactive compounds by HPLC method. Accumulation of rutin and quercetin was not detected in plant parts of H. montbretii during plant growth. Chlorogenic acid and hyperoside content in whole plant was decreased linearly with advancing of development stages and reached their highest level at vegetative stage. On the contrary, apigenin-7-O-glucoside, quercitrin and viteksin content in whole plant increased during the course of seasonal development and the highest level of those compounds was observed at the stage of full flowering. Leaves did not produce apigenin-7-O-glucoside, while viteksin was not detectable in stem and reproductive tissues. Depending on development stages, reproductive parts had the highest level of apigenin-7-O-glucoside and leaves produced major amount of chlorogenic acid, hyperoside and viteksin whereas accumulation of quercitrin was prevailed in stem tissue. Such kind of data could be useful for elucidation of the chemotaxonomical significance of these compounds and medicinal evaluation of this species.

Ontogenetic and Seasonal Variation in the Flavonoid Composition of Sophora japonica. Cultivated in Bulgaria

Quantitative changes in the accumulation of the flavonol glycosides rutin and kaempferol 3-rutinoside and the isoflavone glycosides genistin and sophorabioside have been investigated in flowers and fruits of cultivated in Bulgaria Sophora japonica. during ontogenetic and seasonal development. A method for extraction of flavonoids with good recovery was developed (98% for rutin and 97% for kaempferol 3-rutinoside). The subsequent high-performance liquid chromatography separation of flavonoid glycosides was achieved using Hypersil ODS C18 column and UV detection. The mobile phase comprised methanol and phosphate buffer, pH 3.23, and linear gradient elution mode within 25 min was applied. Accumulation of rutin started with initiation of the flower buds, having the maximal content up to 287 mg/g (dry weight) in mid-July during the earliest stages of flower development and tended to have high levels until mid-August. A further decrease in the amount of rutin up to 44 mg/g was found proportionally with the decline of flavonoids throughout and after the flowering period. Rutin was the predominant glycoside and can account for up to 90% of flavonoid glycoside mixture in young buds, whereas sophorabioside (64.7 mg/g, calculated as rutin equivalent) and kaempferol 3-rutinoside (46.8 mg/g) dominated at later development stages of the fruits. No significant differences in flavonoid quantity between the analysis periods of two successive years were observed. This indicated clearly the ontogenetic dependence of flavonoid formation.

Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco

Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene ( SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a p I of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.

Determination of Rutin in Pharmaceutical Compounds and Tea Using Cathodic Adsorptive Stripping Voltammetry

A new sensitive electrochemical procedure is presented for the determination of Rutin based on the accumulation of Rutin onto the surface of hanging mercury drop electrode for 80 s. Then the preconcentrated Rutin-complex was analyzed by cathodic stripping square-wave voltammetry. The effect of various parameters such as pH, concentration of copper, accumulation potential, accumulation time and scan rate on the sensitivity were studied. The optimum conditions for determination of Rutin include pH 6.0, 6.0 ng mL−1 copper(II) concentration, accumulation potential of −0.30 V and scan rate of 0.40 V s−1. Under the optimum conditions and for an accumulation time of 80 s, the measured peak current at about −0.03 V is proportional to the concentration of Rutin over the range of 2.0–85.0 nM. The practical limit of detection is 0.5 nM. The relative standard deviations for six replicate analyses of 10 and 50 nM Rutin are 1.8% and 1.7%, respectively. The method was applied to the determination of Rutin in synthetic, tea, and pharmaceutical samples with satisfactory results. The diffusion coefficient, complex formation constant and number of electron transfer of Rutin–Cu(II) complex was also estimated.

Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols

Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel ( approximately 5-10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials for tomato-based functional food products.

Induced Accumulation and Potential Antioxidative Function of Rutin in Two Cultivars of Nicotiana tabacum L.

The rutin (quercetin-3-rhamnosyl-glucoside) content of two tobacco cultivars (Nicotiana tabacum L.) which differ in their ozone-sensitivity was assayed after exposure to various rutininducing stimuli. In the growth-chamber, UV radiation in combination with white light led to the accumulation of similar amounts of rutin in both cultivars. Treatment with radical producing agents (tert-butylhydroperoxide and paraquat) also led to rutin accumulation. In this case, the rutin content was higher in the tolerant cultivar. The rutin content was also higher in the tolerant cultivar upon exposure of the plants on an out-door stand, even when the UV-part of the sun spectrum was excluded by cut-off filters. The potential role of rutin as antioxidant was tested with an ion leakage assay. Plants with relatively high rutin content were less sensitive towards paraquat-induced ion leakage than plants without rutin. Thus, the higher rutin content of the ozone-tolerant cultivar Bel B may well contribute to its tolerance against oxidative stress.

Diversity of Responses to Ca2+ Ions in the Accumulation of Buckwheat Flavonoids Implying Tissue Specific Biosynthesis

Ca2+ ions reduced stimulatory effect of kinetin on the accumulation of anthocyanins and rutin in excised buckwheat cotyledons and also inhibited formation of these flavonoids in the material supplied with exogenous L-phenylalanine. However, Ca2+ ions markedly enhanced, at low doses, accumulation of C-glycosyl-flavones in the cotyledons when the latter, either treated or not treated with kinetin, were concomitantly fed with that exogenous precursor. Experiments with [1-14C]-L- phenylalanine showed that the observed increase was mainly due to an enhancement of C-glycosyl- flavone biosynthesis from the substrate of endogenous origin while a decrease was observed in its utilization for building anthocyanins and rutin. The results suggest a redistribution of the endogenous resources of L-phenylalanine under blockage of its transport, by Ca2+ ions, to the sites of antho- cyanin and rutin biosynthesis in the outer layers of cells of buckwheat cotyledons. The results imply tissue specific differences in the localization of flavonoid biosynthesis in that plant.