Tissue bioavailability of anthocyanins from whole tart cherry in healthy rats.

Our aim was to confirm and identify the presence of tart cherry anthocyanins in several target tissues of healthy rats. Liquid chromatography-mass spectrometry analysis was employed for detection and characterisation of anthocyanin metabolites. It was shown that four native anthocyanins, namely cyanidin 3-glucosylrutinoside, cyanidin 3-rutinoside, cyanidin 3-rutinoside 5-ß-D-glucoside, and peonidin 3-rutinoside were differentially distributed among targeted tissues of rats. Bladder and kidney contained more total anthocyanins than all other tissues analysed. It was also revealed that the bioavailability pattern of these native anthocyanins among tissues is varied. The highest concentration of individual anthocyanin cyanidin 3-glucosylrutinoside (2339 picograms/gram of tissue) was detected in bladder, followed by cyanidin 3-rutinoside 5-ß-d-glucoside (916 picograms/gram) in the liver of rats. Although the diverse distribution of tart cherry anthocyanins in different rat tissues still requires further explanation, it may provide an evidentiary link between tissue bioavailability and health-enhancing properties of anthocyanins at target sites.

Blueberry intake alters skeletal muscle and adipose tissue peroxisome proliferator-activated receptor activity and reduces insulin resistance in obese rats.

Metabolic syndrome can precede the development of type 2 diabetes and cardiovascular disease and includes phenotypes such as obesity, systemic inflammation, insulin resistance, and hyperlipidemia. A recent epidemiological study indicated that blueberry intake reduced cardiovascular mortality in humans, but the possible genetic mechanisms of this effect are unknown. Blueberries are a rich source of anthocyanins, and anthocyanins can alter the activity of peroxisome proliferator-activated receptors (PPARs), which affect energy substrate metabolism. The effect of blueberry intake was assessed in obesity-prone rats. Zucker Fatty and Zucker Lean rats were fed a higher-fat diet (45% of kcal) or a lower-fat diet (10% of kcal) containing 2% (wt/wt) freeze-dried whole highbush blueberry powder or added sugars to match macronutrient and calorie content. In Zucker Fatty rats fed a high-fat diet, the addition of blueberry reduced triglycerides, fasting insulin, homeostasis model index of insulin resistance, and glucose area under the curve. Blueberry intake also reduced abdominal fat mass, increased adipose and skeletal muscle PPAR activity, and affected PPAR transcripts involved in fat oxidation and glucose uptake/oxidation. In Zucker Fatty rats fed a low-fat diet, the addition of blueberry also significantly reduced liver weight, body weight, and total fat mass. Finally, Zucker Lean rats fed blueberry had higher body weight and reduced triglycerides, but all other measures were unaffected. In conclusion, whole blueberry intake reduced phenotypes of metabolic syndrome in obesity-prone rats and affected PPAR gene transcripts in adipose and muscle tissue involved in fat and glucose metabolism.

Regular tart cherry intake alters abdominal adiposity, adipose gene transcription, and inflammation in obesity-prone rats fed a high fat diet.

Obesity, systemic inflammation, and hyperlipidemia are among the components of metabolic syndrome, a spectrum of phenotypes that can precede the development of type 2 diabetes and cardiovascular disease. Animal studies show that intake of anthocyanin-rich extracts can affect these phenotypes. Anthocyanins can alter the activity of tissue peroxisome proliferator-activated receptors (PPARs), which affect energy substrate metabolism and inflammation. However, it is unknown if physiologically relevant, anthocyanin-containing whole foods confer similar effects to concentrated, anthocyanin extracts. The effect of anthocyanin-rich tart cherries was tested in the Zucker fatty rat model of obesity and metabolic syndrome. For 90 days, rats were pair-fed a higher fat diet supplemented with either 1% (wt/wt) freeze-dried, whole tart cherry powder or with a calorie- and macronutrient-matched control diet. Tart cherry intake was associated with reduced hyperlipidemia, percentage fat mass, abdominal fat (retroperitoneal) weight, retroperitoneal interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) expression, and plasma IL-6 and TNF-alpha. Tart cherry diet also increased retroperitoneal fat PPAR-alpha and PPAR-gamma mRNA (P = .12), decreased IL-6 and TNF-alpha mRNA, and decreased nuclear factor kappaB activity. In conclusion, in at-risk obese rats fed a high fat diet, physiologically relevant tart cherry consumption reduced several phenotypes of metabolic syndrome and reduced both systemic and local inflammation. Tart cherries may reduce the degree or trajectory of metabolic syndrome, thereby reducing risk for the development of type 2 diabetes and heart disease.

Chronic intake of a phytochemical-enriched diet reduces cardiac fibrosis and diastolic dysfunction caused by prolonged salt-sensitive hypertension.

Salt-sensitive hypertension is common in the aged population. Increased fruit and vegetable intake reduces hypertension, but its effect on eventual diastolic dysfunction is unknown. This relationship is tested in the Dahl Salt-Sensitive (Dahl-SS) rat model of salt-sensitive hypertension and diastolic dysfunction. Table grape powder contains phytochemicals that are relevant to human diets. For 18 weeks, male Dahl-SS rats were fed one of five diets: low salt (LS), a low salt + grape powder (LSG), high salt (HS), a high salt + grape powder (HSG), or high salt + vasodilator hydralazine (HSH). Compared to the HS diet, the HSG diet lowered blood pressure and improved cardiac function; reduced systemic inflammation; reduced cardiac hypertrophy, fibrosis, and oxidative damage; and increased cardiac glutathione. The HSH diet similarly reduced blood pressure but did not reduce cardiac pathogenesis. The LSG diet reduced cardiac oxidative damage and increased cardiac glutathione. In conclusion, physiologically relevant phytochemical intake reduced salt-sensitive hypertension and diastolic dysfunction.

Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry.

Elevated plasma lipids, glucose, insulin, and fatty liver are among components of metabolic syndrome, a phenotypic pattern that typically precedes the development of Type 2 diabetes. Animal studies show that intake of anthocyanins reduces hyperlipidemia, obesity, and atherosclerosis and that anthocyanin-rich extracts may exert these effects in association with altered activity of tissue peroxisome proliferator-activated receptors (PPARs). However, studies are lacking to test this correlation using physiologically relevant, whole food sources of anthocyanins. Tart cherries are a rich source of anthocyanins, and whole cherry fruit intake may also affect hyperlipidemia and/or affect tissue PPARs. This hypothesis was tested in the Dahl Salt-Sensitive rat having insulin resistance and hyperlipidemia. For 90 days, Dahl rats were pair-fed AIN-76a-based diets supplemented with either 1% (wt:wt) freeze-dried whole tart cherry or with 0.85% additional carbohydrate to match macronutrient and calorie provision. After 90 days, the cherry-enriched diet was associated with reduced fasting blood glucose, hyperlipidemia, hyperinsulinemia, and reduced fatty liver. The cherry diet was also associated with significantly enhanced hepatic PPAR-alpha mRNA, enhanced hepatic PPAR-alpha target acyl-coenzyme A oxidase mRNA and activity, and increased plasma antioxidant capacity. In conclusion, physiologically relevant tart cherry consumption reduced several phenotypic risk factors that are associated with risk for metabolic syndrome and Type 2 diabetes. Tart cherries may represent a whole food research model of the health effects of anthocyanin-rich foods and may possess nutraceutical value against risk factors for metabolic syndrome and its clinical sequelae.

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA.

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.

The production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure.

In vitro shoot cultures of Hypericum perforatum derived from wild populations grown in Armenia have a wide variation of hypericin and pseudohypericin metabolite content. We found that a germ line denoted as HP3 produces six times more hypericin and fourteen times more pseudohypericin than a second line labeled HP1. We undertook a structural comparison of the two lines (HP1 and HP3) in order to see if there are any anatomical or morphological differences that could explain the differences in production of these economically important metabolites. Analysis by LM (light microscopy), SEM (scanning electron microscopy), and TEM (transmission electron microscopy) reveals that the hypericin/pseudohypericin-containing black glands located along the margins of the leaves consist of a peripheral sheath of flattened cells surrounding a core of interior cells that are typically dead at maturity. The peripheral cells of the HP3 glands appear less flattened than those of the HP1 glands. This may indicate that the peripheral cells are involved in hypericin/pseudohypericin production. Furthermore, we find that these peripheral cells undergo a developmental transition into the gland's interior cells. The fact that the size of the peripheral cells may correlate with metabolite production adds a new hypothesis for the actual site of hypericin synthesis.

Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots.

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate.

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g(-1) dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.

Identification and characterization of an auxin-inducible protein kinase, VrCRK1, from mungbean.

An auxin-inducible protein kinase, VrCRK1, was isolated by a differential reverse transcriptase-polymerase chain reaction, using mRNAs extracted from auxin-treated mungbean hypocotyls. VrCRK1 exhibits high homology with plant CDPKs over catalytic domains, however, it does not have any calcium-binding EF-hand which is typically shown in plant CDPKs. Auxin treatment increased the expression level of VrCRK1. However, the increased level was reduced to basal level by treatment with PCIB, an auxin inhibitor. When extracts of mungbean hypocotyls were immunoprecipitated and the resultant immunoprecipitates were used as the enzyme source, kinase activity of VrCRK1 was found, and such activity was also increased by auxin treatment. In transgenic tobacco plants that express VrCRK1, the transcript levels of some auxin-dependent genes were elevated as much as those in wild type plants treated with auxin. These results indicate that gene expression of VrCRK1 is specifically induced by auxin, and that VrCRK1 may play a role in auxin signaling via protein phosphorylation.

The production of hypericins and hyperforin by in vitro cultures of St. John's wort (Hypericum perforatum).

St. John's wort ( Hypericum perforatum L.) is a herbaceous perennial distributed throughout the World that has been widely used in traditional medicine. H. perforatum produces several types of biologically active compound, including the hypericins--a family of light-activated anthraquinones, localized within specialized glands found predominantly on flowers and leaves--and the hyperforins--a family of prenylated acylphloroglucinols localized in the reproductive structures of the plant. Hypericins are known to be toxic to mammals and display antiviral and anticancer activity, but the role of these compounds within the plant is unknown. Hyperforins display potent antimicrobial activity and are thought to be the primary bioactive ingredient for anti-depressive effects of the herb. The introduction of H. perforatum from Europe into the U.S.A. occurred in the 17th Century. Since the plant is considered a noxious weed, few efforts have been carried out to analyse populations in the context of secondary-metabolite concentrations. But in terms of secondary-metabolite studies, H. perforatum is an ideal model system to study the biosyntheses of aromatic polyketides and regulation of those pathways by environmental and genetic influences. This is due, in part, to the ease of conducting these studies in plant tissue culture. This review describes the progress of secondary-metabolite studies currently underway using H. perforatum. Specifically, this Review focuses on the production and regulation of the hypericins and the hyperforin in wild populations, field cultivation, greenhouse studies and plant tissue culture. Additionally, factors optimizing compound production--particularly in in vitro cultures--are presented and reviewed.

Antioxidant capacity of polyphenolic extracts from leaves of Crataegus laevigata and Crataegus monogyna (Hawthorn) subjected to drought and cold stress.

Crataegus laevigata and Crataegus monogyna (hawthorn) were subjected to drought and cold stress treatments, and polyphenolic extracts from control and stress-treated plants were assayed for antioxidant capacities using a modified version of the Total Antioxidant Status Assay (Randox, San Francisco, CA). In addition, these plants were analyzed for levels of flavanol-type substance [(-)-epicatechin] and flavonoid (vitexin 2' '-O-rhamnoside, acetylvitexin 2' '-O-rhamnoside, and hyperoside) constituents that are important metabolites in hawthorn herbal preparations used to treat patients with heart disease. Drought and cold stress treatments caused increases in levels of (-)-epicatechin and hyperoside in both Crataegus species. Such treatments also enhanced the antioxidant capacity of the extracts. The results from this study thus indicate that these kinds of stress treatments can enhance the levels of important secondary metabolites and their total antioxidant capacities in leaves of Crataegus.

Upregulation of isoflavonoids and soluble proteins in edible legumes by light and fungal elicitor treatments.

OBJECTIVE: In this study, our working hypothesis was that continuous light and fungal elicitation treatment of legume seedlings would lead to enhanced levels of isoflavonoids and soluble proteins. RESULTS: Based on short-term light and dark treatments, isoflavonoid (genistein, genistin, daidzein, and daidzin) and soluble protein concentrations were significantly upregulated in the "light" environment compared to the "dark" environment for all edible legume species (kudzu vine, soybean, garbanzo bean, fava bean, mung bean, adzuki bean) that were tested. Kudzu seedlings showed the highest levels of both isoflavonoids and soluble proteins after light-elicited upregulation compared to the other legumes analyzed. All legumes showed less up-regulation of isoflavonoid synthesis when treated with Phytophtora sojae fungal elicitor. Oligosaccharide fungal elicitor caused no such upregulation. CONCLUSIONS: The findings in this study show that edible legume seedlings have enhanced levels of isoflavonoids and soluble proteins when they are grown in the light compared to the conventional practice of growing such seedlings in the dark. This will clearly result in significant improvement in their nutritive and medicinal value.

Changes in phosphorylation of 50 and 53 kDa soluble proteins in graviresponding oat (Avena sativa) shoots.

The present work indicates that phosphorylation of a 50 kDa soluble protein is involved in the gravitropic response in graviresponsive pulvini of oat (Avena sativa) stems. This 50 kDa protein shows a differential pattern of phosphorylation between lower and upper halves of pulvini both in vivo and in vitro. The differential phosphorylation of this protein is detected only when stem segments are gravistimulated for short and long time periods. The differential phosphorylation of the 50 kDa protein occurs as early as 5 min after the initiation of gravistimulation. This corresponds closely to the presentation time of 5.2 min. This differential phosphorylation pattern was changed by treatments with cycloheximide, implying that a newly-synthesized protein is involved in the differential phosphorylation during the gravitropic response. An autophosphorylation experiment shows that the 50 kDa protein has kinase activity. The phosphorylation patterns of a 53 kDa protein were similar to those of the 50 kDa protein, but were only expressed in vitro. These findings indicate that the differential phosphorylation of the 50 (and 53 kDa) soluble proteins in graviresponding oat shoots may be an important component of the gravity signal transduction pathway.