Xianli Wu

Anthocyanins: Structural characteristics that result in unique metabolic patterns and biological activities

Interest in anthocyanins has increased immensely during the past decade. From these studies, it is clear that anthocyanins have unique properties: Anthocyanins are absorbed intact and absorption can be saturated; acylation of anthocyanins lowers their apparent absorption; anthocyanidin diglycosides in the form of sambubioside or rutinoside impart increased stability to the anthocyanin molecule; and the quantities excreted in urine are less than 0.1% of intake. However, 60–90% of the anthocyanins may disappear from the gastrointestinal tract within 4 h after a meal. What happens to the bulk of the anthocyanins that disappear is not clear. Degradation accounts for a part of this disappearance, but differs for the various aglycones and may be modified further by the nature of the aglycone glycosylation, which further complicates our understanding of this process. Anthocyanins may play an important role in health promotion in terms of obesity prevention, cardiovascular health, anti-inflammatory and anti-cancer effects.

Whole Berries versus Berry Anthocyanins: Interactions with Dietary Fat Levels in the C57BL/6J Mouse Model of Obesity

Male C57BL/6J mice received diets with either 10% of calories from fat (LF) or a high-fat diet [45% (HF45) or 60% (HF60) calories from fat] for 92 days (expt 1) or 70 days (expt 2). These were given with or without freeze-dried powders from whole blueberries (BB) or strawberries (SB) (expt 1) or purified anthocyanin extracts from BB or SB (expt 2). Body composition was determined utilizing Echo MRI. Berries added to the LF diet did not alter weight gain, final body weights, body fat, or protein (percent body weight) or diet (grams) or energy (kilocalories) intake. However, in both HF45- and HF60-fed mice, weight gain, final weights, body fat (percent), and epididymal fat weights increased and body protein decreased ( p < 0.01) compared to LF mice. In mice fed HF45 diet plus BB, body weight gains, body fat (percent of BW), and epididymal fat weights were significantly greater than those in the HF45-fed controls, whereas weights of mice fed SB HF were similar to those of HF controls. SB or BB feeding did not alter glucose tolerance, although glucose tolerance decreased with age and in HF45 versus LF mice. Baseline plasma glucose was lower in SB- versus HF45-fed mice. After 8 weeks, mice fed the HF60 diet plus purified anthocyanins from BB in the drinking water had lower body weight gains and body fat than the HF60-fed controls. Anthocyanins fed as the whole blueberry did not prevent and may have actually increased obesity. However, feeding purified anthocyanins from blueberries or strawberries reduced obesity.

Plasma Antioxidant Capacity Changes Following a Meal as a Measure of the Ability of a Food to Alter In Vivo Antioxidant Status

Objective: Determine 1) if consumption of a meal of different fruits or berries increases plasma hydrophilic (H-) or lipophilic (L-) antioxidant capacity (AOC) measured as Oxygen Radical Absorbance Capacity (ORACFL); 2) if including macronutrients in the meal alters postprandial changes in AOC; and 3) if preliminary recommendations can be developed for antioxidant intake. Methods: Changes in plasma AOC following consumption of a single meal of berries/fruits (blueberry, dried plum, dried plum juice, grape, cherry, kiwifruit and strawberry) were studied in 5 clinical trials with 6–10 subjects per experiment. In two studies with blueberry or grape, additional macronutrients (carbohydrate, fat, protein) were included in the control and treatment meals. Blood samples collected before and after the meal were analyzed for AOC. Results: Consumption of dried plums or dried plum juice did not alter either the H- or L-AOC area under the curve (AUC). Consumption of blueberry in 2 studies and of mixed grape powder [12.5 (Study #1), 39.9 (Study #4) and 8.6 (Study #5) mmole Trolox Equivalents (TE) AOC, respectively] increased hydrophilic AOC AUC. L-AOC increased following a meal of blueberry containing 12.5 mmole TE AOC (Study #1). Consumption of 280 g of cherries (4.5 mmol TE AOC) increased plasma L-AOC but not H-AOC. The AOC in the control groups in which additional macronutrients (Studies #4 and #5) were added decreased from the postprandial baseline AOC measurement. Conclusion: We have demonstrated that consumption of certain berries and fruits such as blueberries, mixed grape and kiwifruit, was associated with increased plasma AOC in the postprandial state and consumption of an energy source of macronutrients containing no antioxidants was associated with a decline in plasma AOC. However, without further long term clinical studies, one cannot necessarily translate increased plasma AOC into a potential decreased risk of chronic degenerative disease. Preliminary estimates of antioxidant needs based upon energy intake were developed. Consumption of high antioxidant foods with each meal is recommended in order to prevent periods of postprandial oxidative stress.

In Vitro and in Vivo Antioxidant and Anti-inflammatory Capacities of an Antioxidant-Rich Fruit and Berry Juice Blend. Results of a Pilot and Randomized, Double-Blinded, Placebo-Controlled, Crossover Study

This study investigated the in vitro and in vivo antioxidant and anti-inflammatory properties of a juice blend (JB), MonaVie Active, containing a mixture of fruits and berries with known antioxidant activity, including acai, a palm fruit, as the predominant ingredient. The phytochemical antioxidants in the JB are primarily in the form of anthocyanins, predominantly cyanidin 3-rutoside, cyanidin 3-diglycoside, and cyanidin 3-glucoside. The cell-based antioxidant protection of erythrocytes (CAP-e) assay demonstrated that antioxidants in the JB penetrated and protected cells from oxidative damage ( p < 0.001), whereas polymorphonuclear cells showed reduced formation of reactive oxygen species ( p < 0.003) and reduced migration toward three different pro-inflammatory chemoattractants: fmlp ( p < 0.001), leukotriene B4 ( p < 0.05), and IL-8 ( p < 0.03). A randomized, double-blinded, placebo-controlled, crossover trial with 12 healthy subjects examined the JBs antioxidant activity in vivo. Blood samples at baseline, 1 h, and 2 h following consumption of the JB or placebo were tested for antioxidant capacity using several antioxidant assays and the TBARS assay, a measure of lipid peroxidation. A within subject comparison showed an increase in serum antioxidants at 1 h ( p < 0.03) and 2 h ( p < 0.015), as well as inhibition of lipid peroxidation at 2 h ( p < 0.01) postconsumption.

Purified Blueberry Anthocyanins and Blueberry Juice Alter Development of Obesity in Mice Fed an Obesogenic High-Fat Diet†

Male C57BL/6J mice (25 days of age) were fed either a low-fat diet (10% kcal from fat) (LF) or a high-fat diet (45% kcal from fat) (HF45) for a period of 72 days. Blueberry juice or purified blueberry anthocyanins (0.2 or 1.0 mg/mL) in the drinking water were included in LF or HF45 treatments. Sucrose was added to the drinking water of one treatment to test if the sugars in blueberry juice would affect development of obesity. Total body weights (g) and body fat (%) were higher and body lean tissue (%) was lower in the HF45 fed mice compared to the LF fed mice after 72 days, but in mice fed HF45 diet plus blueberry juice or blueberry anthocyanins (0.2 mg/mL), body fat (%) was not different from those mice fed the LF diet. Anthocyanins (ACNs) decreased retroperitoneal and epididymal adipose tissue weights. Fasting serum glucose concentrations were higher in mice fed the HF45 diet. However, it was reduced to LF levels in mice fed the HF45 diet plus 0.2 mg of ACNs/mL in the drinking water, but not with blueberry juice. beta cell function (HOMA-BCF) score was lowered with HF45 feeding but returned to normal levels in mice fed the HF45 diet plus purified ACNs (0.2 mg/mL). Serum leptin was elevated in mice fed HF45 diet, and feeding either blueberry juice or purified ACNs (0.2 mg/mL) decreased serum leptin levels relative to HF45 control. Sucrose in drinking water, when consumption was restricted to the volume of juice consumed, produced lower serum leptin and insulin levels, leptin/fat, and retroperitoneal and total fat (% BW). Blueberry juice was not as effective as the low dose of anthocyanins in the drinking water in preventing obesity. Additional studies are needed to determine factors responsible for the differing responses of blueberry juice and whole blueberry in preventing the development of obesity.

Purified berry anthocyanins but not whole berries normalize lipid parameters in mice fed an obesogenic high fat diet.

Male C57BL/6 mice received diets with either 10% of kcal from fat, or a high fat diet [45% (HF45) or 60% (HF60) kcal from fat]. Diets were prepared with or without freeze-dried powders (10%) from whole blueberries (BB), strawberries (SB), Concord grape or black raspberry. In the 2nd study, purified anthocyanins (ACNs) from SB or BB were added to the drinking water of the treatments fed the HF60 diet. In Study 1, serum triglycerides were increased by feeding the HF45 diet but were elevated further when black raspberry or BB was included in the HF45 diet. Liver total lipids and triglycerides were increased in mice fed HF45 diet and inclusion of any of the berry powders in the HF45 diet did not alter concentrations compared to HF45 controls. In the 2nd study, mice fed the HF60 diet plus purified ACNs from BB in the water had lower body weight gains and body fat than the HF60 fed. Serum cholesterol and triglyceride levels were elevated with the HF60 diet and decreased to control levels when ACNs from either SB or BB were included in the drinking water. Serum leptin levels were consistently decreased to control low fat levels in those ACN treatments in which measures of body fat were decreased. Administering purified ACNs from BB and strawberry via drinking water prevented the development of dyslipidemia and obesity in mice, but feeding diets containing whole berries or purple corn (PC) ACNs did not alter the development of obesity.

The purple cauliflower arises from activation of a MYB transcription factor.

Anthocyanins are responsible for the color of many flowers, fruits, and vegetables. An interesting and unique Purple (Pr) gene mutation in cauliflower (Brassica oleracea var botrytis) confers an abnormal pattern of anthocyanin accumulation, giving the striking mutant phenotype of intense purple color in curds and a few other tissues. To unravel the nature of the Pr mutation in cauliflower, we isolated the Pr gene via a combination of candidate gene analysis and fine mapping. Pr encoded a R2R3 MYB transcription factor that exhibited tissue-specific expression, consistent with an abnormal anthocyanin accumulation pattern in the mutant. Transgenic Arabidopsis (Arabidopsis thaliana) and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype, confirming the isolation of the Pr gene. Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding flavonoid 3’-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower. Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant. The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables, and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal.

The açaí flavonoid velutin is a potent anti-inflammatory agent: blockade of LPS-mediated TNF-α and IL-6 production through inhibiting NF-κB activation and MAPK pathway

Recent studies have shown that some flavonoids are modulators of proinflammatory cytokine production. In this study, velutin, a unique flavone isolated from the pulp of açaí fruit (Euterpe oleracea Mart.), was examined for its effects in reducing lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW 264.7 peripheral macrophages and mice peritoneal macrophages. Three other structurally similar and well-studied flavones, luteolin, apigenin and chrysoeriol, were included as controls and for comparative purposes. Velutin exhibited the greatest potency among all flavones in reducing TNF-α and IL-6 production. Velutin also showed the strongest inhibitory effect in nuclear factor (NF)-κB activation (as assessed by secreted alkaline phosphatase reporter assay) and exhibited the greatest effects in blocking the degradation of inhibitor of NF-κB as well as in inhibiting mitogen-activated protein kinase p38 and JNK phosphorylation; all of these are important signaling pathways involved in production of TNF-α and IL-6. The present study led to the discovery of a strong anti-inflammatory flavone, velutin. This compound effectively inhibited the expression of proinflammatory cytokines TNF-α and IL-6 in low micromole levels by inhibiting NF-κB activation and p38 and JNK phosphorylation.

Repression of Mammosphere Formation of Human Breast Cancer Cells by Soy Isoflavone Genistein and Blueberry Polyphenolic Acids Suggests Diet-Mediated Targeting of Cancer Stem-Like/Progenitor Cells

Mammary stem cells are undifferentiated epithelial cells, which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we addressed whether dietary factors selectively target mammary epithelial cells that display stem-like/progenitor subpopulations with previously recognized tumor-initiating potential. Using estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cell lines and freshly isolated epithelial cells from MMTV-Wnt-1 transgenic mouse mammary tumors, we demonstrate that sera of adult mice consuming soy isoflavone genistein (GEN) or blueberry (BB) polyphenol-containing diets alter the population of stem-like/progenitor cells, as measured by their functional ability to self-renew and form anchorage-independent spheroid cultures in vitro at low frequency (1-2%). Serum effects on mammosphere formation were dose-dependently replicated by GEN (40 nM >2 μM) and targeted the basal stem-like CD44+/CD24-/ESA+ and the luminal progenitor CD24+ subpopulations in MDA-MB-231 and MCF-7 cells. GEN inhibition of mammosphere formation was mimicked by the Akt inhibitor perifosine and was associated with enhanced tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression. In contrast, a selected mixture of BB phenolic acids was only active in MDA-MD-231 cells and its CD44+/CD24-/ESA+ subpopulation, and this activity was independent of induction of PTEN expression. These findings delineate a novel and selective function of distinct dietary factors in targeting stem/progenitor cell populations in estrogen receptor-dependent and -independent breast cancers.

Obesity Reduces Bone Density Associated with Activation of PPARγ and Suppression of Wnt/β-Catenin in Rapidly Growing Male Rats

Background It is well established that excessive consumption of a high fat diet (HFD) results in obesity; however, the consequences of obesity on postnatal skeletal development have not been well studied. Methodology and Principal Findings Total enteral nutrition (TEN) was used to feed postnatal day 27 male rats intragastrically with a high 45% fat diet (HFD) for four weeks to induce obesity. Fat mass was increased compared to rats fed TEN diets containing 25% fat (medium fat diet, MFD) or a chow diet (low fat diet, LFD) fed ad libitum with matched body weight gains. Serum leptin and total non-esterified fatty acids (NEFA) were elevated in HFD rats, which also had reduced bone mass compared to LFD-fed animals. This was accompanied by decreases in bone formation, but increases in the bone resorption. Bone marrow adiposity and expression of adipogenic genes, PPARγ and aP2 were increased, whereas osteoblastogenic markers osteocalcin and Runx2 were decreased, in bone in HFD rats compared to LFD controls. The diversion of stromal cell differentiation in response to HFD stemmed from down-regulation of the key canonical Wnt signaling molecule β-catenin protein and reciprocal up-regulation of nuclear PPARγ expression in bone. In a set of in vitro studies using pluripotent ST2 bone marrow mesenchymal stromal cells treated with serum from rats on the different diets or using the free fatty acid composition of NEFA quantified in rat serum from HFD-fed animals by GC-MS, we were able to recapitulate our in vivo findings. Conclusions/Significance These observations strongly suggest that increased NEFA in serum from rats made obese by HFD-feeding impaired bone formation due to stimulation of bone marrow adipogenesis. These effects of obesity on bone in early life may result in impaired attainment of peak bone mass and therefore increase the prevalence of osteoporosis later on in life.