Blue 1 - Blueberin

Phytochemistry of Blueberry Leaves

Phytochemical composition of Blueberry leaves extract: Caffeoylquinic + Hydroxycinnamic acid (20% dry weight), Salidroside and its aglycon Tyrosol (0,2-0.4%), Arbutin (0.2%), Coumaric acid (0.4%), Quercetin and Rutin (0,7%) and Malvidine-3-glycoside (0.15%)(Jimenez del Rio) The main key compounds responsible for pharmacological and medicinal properties of blueberry leaves extract are caffeoylquinic acid (CA) and hydroxycinnamic acids (HA) (Durmishidze, 1981).

Blueberry leaves extract and blood sugar

Glucose is a double-edged sword. On the one hand, we cannot live without it - it is the bodies preferred fuel, and the energy we receive from burning it allows us to wake up in the morning and keep moving all day long. The overabundance of glucose in the blood of individuals suffering from diabetes causes some of the most deleterious effects of any disease known. The enzyme glucose-6-phosphatase plays a major role in the homeostatic regulation of blood glucose, which is responsible for the formation of glucose originating from gluconeogenesis and glycogenolysis.

CA was identified as a specific inhibitor of this enzyme in microsomes of liver (Arion et al., 1997; Hemmerle et al., 1997). Inhibition of glucose-6-phosphatase activity in the liver is expected to result in a reduction of hepatic glucose production and may be useful for the reduction of high rates of hepatic glucose output often found in non-insulin dependent diabetes.

Pharmaceutical companies have synthesized caffeoylquinic acid derivatives with glucose-6-phosphatase inhibiting properties in human liver microsomes (Arion et al., 1997) and these synthetic compounds also reduce blood glucose. CA derivative produced concentration-dependent inhibition of glucose synthesis in isolated rat liver (Herling et al., 1998).

The recent discovery that blueberry leaves extract reduce blood sugar level by 26% is not surprising and somewhat expected results. The blueberry leaves extract also reduce plasma triglyceride by 39%, indicating its usefulness in treatment of high blood sugar and plasma triglyceride level (Cirnarella et al., 1996).

Another evidences for blueberry leaves effectiveness in diabetes arrives from human study of Prof of Medicine Abidoff (2006).

Seventy-five healthy volunteers age between 37-66 years were randomly assigned to receive 150mg/three times/day of standardized blueberry leaves standardized extract (Blueberin™) or placebo, to be taken with in 200 ml of water before meals. After meal concentration of blood glucose increased from average 102mg/dl±8md/dl (baseline) to 142mg/dl ±7mg/dl in placebo group, while plasma glucose level in Blueberin™ group increase from 109mg/dl ± 9 mg /dl to 121mg/dl±6mg/dl, indicating that the blueberry leaves extract possess physiologically significant glucose-reducing property.

In another clinical trial twenty-nine patients average age 50 years with type II diabetes were selected to participate in double-blind placebo-controlled 60 days trial (Abidoff, 1999). Subjects were randomly assigned to receive 200mg/three times/day of standardized blueberry leaves extract or placebo with 200 ml of water before meals. Those individuals taking the blueberry leaves extract showed the reduction of plasma glucose levels from approximately 169 mg/dl to 136 mg/dl (p < 0.01), while no such reduction was observed in placebo group.

Furthermore, by the end of the clinical study, those taking Blueberin™ showed a reduction triglyceride and LDL values from 179 ± 95 mg/L to 130 ± 53 mg/L (p < 0.005) and 141 ± 47 mg/cU to 115 ± 34 mg/dl (p < 0.01) respectively. All patients tolerated well blueberry leaves extract at even 400mg/ three times a day (1200mg/day). It is interesting to note that the HA is also involved in the regulation of blood glucose level. Cheng & Liu (2000) demonstrated that the administration of hydroxycinnamic acid reduced blood glucose level. In animal model Hsu et al., (2000) provided evidences that HA lower plasma glucose in diabetic rats

Blueberries and Type 2 Diabetes

The incidence of type 2 diabetes has reached epidemic levels in developed countries. It is estimated that by 2025, 380 million people will have diabetes (International Diabetes Federation, 2009). Among the disorders included in metabolic syndrome, diabetes is what concerns doctors the most. Some clinicians considered it a cardiovascular disease. Research has shown that patients with type 2 diabetes, but had no prior history of myocardial infarction, have similar risk of having a cardiac event as patients without type 2 diabetes but who are known to have an underlying coronary disease (Haffner, 1998). Insulin resistance plays an important role in the development of diabetes. Thus, therapeutic regimen includes insulin sensitizers such as thiazolidinediones and metformin, and insulin secretors such as sulfonylureas. Metformin, a widely prescribed drug for diabetes, is derived from the natural compound guanidine isolated from Galega officinalis (Bailey and Day, 2004). Metformin exemplifies the value of nature as a source of drugs for the treatment of diseases, and in this case, diabetes.

Blueberries have been used as traditional anti-diabetic medicine for many years (Jellin et al., 2005). In Canada, blueberry extracts are commercially available and used as a complementary treatment for diabetes (Martineau et al., 2006). Early indication of the use of blueberry leaf extract for the treatment of diabetes could be derived from a clinical study of Watson (1928), although it was concluded from this study that the extract exerted beneficial effects only in certain cases of diabetes, and had limited application in the treatment of diabetes. More recently, Martineau et al., (2006) performed an extensive study and explored the anti-diabetic potential of low bush blueberry. In this work, the ethanolic extracts of the root, stem, leaf, and fruit were tested for anti diabetic activity using various cell-based assays. This group found: 1) blueberry extracts had insulin like activity. Overnight incubation of C2C12 muscle cells with 12.5 mL of root, stem and leaf extracts increased glucose transport by 15-25%. In 3T3-L1 adipocyte cells, an increase of 75% was observed; 2) proliferation of b-cells was observed with the fruit extract only, which gave a 2.8 fold increase in 3H-thymidine incorporation by b TC-tet cells; 3) leafs extract showed a glitazone-like activity. Incubation of leaf extracts resulted in an increase in lipid accumulation in 3T3-L1 cells by 6.5 fold, similar to that of rosiglitazone (6.8 fold increase); 4) stem, leaf and fruit extracts conferred protection against glucose toxicity. The extract decreased apoptosis by 20-33% in PC12-AC cells.

In a 4-week clinical study, Abidov et al. (2006) investigated the effect of Blueberin, a phytomedicine containing 250 mg of leaf extracts of a blueberry related species (Vaccinium arctostaphylos L.; synonym, Caucasian whortleberry), on fasting plasma glucose, levels of the enzymes alanine aminotransferases (ALT), aspartate aminotransferases (AST), glutamyltransferase (GGT), as well as serum levels of inflammatory C-Reactive proteins (CRP), in forty-two volunteer subjects diagnosed with Type 2 diabetes. Blueberin-treated group showed a reduction of fasting glucose, which correlated with reduction of serum CRP. The Blueberin group also had a significantly reduced plasma levels of ALT, AST and GGT, indicating that in addition to anti-diabetic effects, Blueberin also has anti-inflammatory properties. In another clinical trial (Nemes-Nagy et al., 2008) thirty type 1 diabetic children were treated with a dietary supplement containing blueberry and sea buckthorn concentrate for two months, and the effects on glycated haemoglobin, C peptide and two antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were evaluated. Results obtained indicated that this dietary supplement had a beneficial effect in the treatment of type 1 diabetic children.

Serratia vaccinii, a new strain of bacteria isolated from blueberry fruits, when used in the fermentation of blueberry juice, resulted in an increase in the phenolic content and antioxidant activity of the juice (Martin and Matar, 2005).

In an in vivo study, biotransformed blueberry juice incorporated in the drinking water of KKAy mice (phenotypically obese with development of hyperleptinemia, insulin resistance, hyperinsulinemia, diabetes, dyslipidaemia, and hypertension) protected the young mice from developing glucose intolerance and diabetes (Vuong et al., 2009). The biotransformed blueberry juice also caused a significant reduction in mice body weight gains. These results suggested the juice had anti-diabetic and strong anti-obesity potential. In one other study, blueberry powder incorporated in the high-fat diet of male C57BL/6J mice protected against inflammation of adipose tissue (DeFuria et al., 2009). Adipose tissue inflammation promotes insulin resistance and other obesity complications. Inflammatory genes (tumor necrosis factor-a, interleukin-6, monocyte chemo attractant protein 1, inducible nitric oxide synthase) were upregulated in the adipose tissues of mice fed with high-fat diet, while in mice fed diet fortified with blueberry powder gene up regulation was attenuated or non-existent. Together with other results obtained on adipocyte physiology, the data from this study suggested dietary blueberry could provide metabolic benefits to combat obesity-associated pathology (Cassia et al., 2015).

Effect of Blueberin on fasting glucose, C-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double blind, placebo controlled clinical study.

Abidov M, Ramazanov A, Jimenez Del Rio M, Chkhikvishvili I. Georgian Med News. 2006 Dec;(141): 66-72.

In a 4-week randomised placebo-controlled clinical trial we investigated the effect of 300 mg Blueberin, a phytomedicine containing 250 mg Blueberry leaves (Vaccinium arctostaphylos L, Ericaceae) extract providing minimum 50 mg 3,4-caffeoylquinic (chlorogenic) acid, and 50 mg myricetin, on fasting plasma glucose, alanine aminotransferases (ALT), aspartate aminotransferases (AST), glutamyltransferase (GGT) enzymes levels, and serum inflammatory C-Reactive proteins (CRP) in forty-two volunteer subjects (46+/-15 year of age, BMI 25+/-3 kgs/(m2)) diagnosed with Type 2 diabetes. During the 4-week trial, the Blueberin supplement was administered three times per day, 15-30 minutes prior to a meal along with 100 ml of water. Results of this trial revealed that the supplementation of Blueberin reduced fasting plasma glucose from 143+/-5,2mg/L to 104+/-5,7 mg/L (p<0,001), whereas there were no statistically significant changes in the Placebo group from 138+/-4,8 mg/L to 126+/-5,1mg/L (p>0,05). The reduction of fasting glucose was correlated with the reduction of serum CRP and in the Blueberin group from 5,18+/-1,4 mg/l to 2,14+/-1,8 mg/L (p<0,05), whereas in the Placebo group CRP levels were not significantly reduced from 5,11+/-1,7 mg/l to 4,94+/-1,1mg/L (p>0,05). Furthermore, the Blueberin also significantly reduced the levels of plasma enzymes ALT, AST and GGT, indicating that, in addition to anti-diabetes effects, the Blueberin also possess pharmacologically relevant anti-inflammatory properties.

Blueberries and Metabolic Syndrome

Metabolic syndrome is a cluster of metabolic disorders that is associated with an increased risk of cardiovascular events (Daskalopoulou et al., 2006). Type 2 diabetes, hypertension and dyslipidaemia are among the metabolic disorders included in the syndrome. In 2007, 76 million Americans were diagnosed with metabolic syndrome (American Heart Association, 2009). Because metabolic syndrome is tightly correlated with obesity, this number is more likely to increase due to the growing number of obese persons that is escalating to epidemic levels, which the Western society faces today.

The U.S. National Cholesterol Education Program Adult Treatment Panel III has set diagnostic criteria for metabolic syndrome, defined as an individual having at least three of the five clinical parameters listed in CHART1.

Chart 1: Parameters for the detection of the metabolic syndrome

- Waist circumference ≥ 40 inches for men or 35 inches for women

- Triglycerides ≥ 150mg/dL

- HDL cholesterol ≥ 40mg/dL for men or ≥ 50mg/dL for women

- Blood pressure ≥ 130/85 mm Hg

- Fasting glucose ≥ 110 mg/dL

In spite of efforts from academia and private companies, there is no magic pill that could collectively treat all the disorders encompassed under metabolic syndrome. Currently, the disorders are treated individually and the approaches are limited to drugs used for the treatment of obesity and type 2 diabetes (Flordellis et al., 2005). This approach has several disadvantages including non-specificity and cost. In one study, the effect of sibutramine, a weight loss drug, on some of the symptoms associated with the metabolic syndrome was evaluated (James et al., 2000). Sibutramine decreased the triglyceride and VLDL-cholesterol levels, and increased the HDL cholesterol levels. These effects contributed positively in the treatment of lipid imbalance in patients with metabolic syndrome. However, sibutramine does not treat other cardiovascular risks such as hypertension. Hypertension, a condition defined as having blood pressure of 140/90 mm Hg or higher, is a major risk factor for stroke and myocardial infarction. Coronary heart disease and stroke make up the largest percentage of cardiovascular diseases and are leading causes of death in the US (Lloyd-Jones et al., 2009).

High costs of medications combined with complex diseases inadequately treated leave a place for the use of alternative therapies such as nutraceuticals and functional foods. Numerous epidemiological studies (e.g., Agudo et al., 2007; Benetou et al,. 2008; Ellingsen et al., 2008) have provided results that emphasize the importance of fruits and vegetables as being part of the daily diet for better health, and for the prevention of degenerative diseases including cancer and neurological disorders (Ames et al., 1993). Most of the phytonutrients found in fruits and vegetables are antioxidants. Therefore, the protective effects of fruit- and vegetable-rich diets could be attributed to the antioxidant compounds. A large clinical study conducted for 7.5 years that included 5220 adults provided results in support of recommendations to consume antioxidant-rich foods to reduce the risk of metabolic syndrome (Czernichow et al., 2009). This study also found that antioxidant supplements were effective in reducing the risk of metabolic syndrome. Apart from their positive effects on metabolic disorders, dietary antioxidants also provide other health benefits such as prevention of cancer (Ohigashi and Murakami, 2004; Khan et al., 2008). Antioxidants inhibit the initiation or propagation of oxidizing chain reactions that cause oxidative damage to lipids, proteins and nucleic acids (Yu, 1994). In addition to the well-known antioxidants vitamins C and E, the flavonoids, which are ubiquitous in fruits and vegetables, have also been demonstrated to be effective antioxidants and to modulate various biological pathways (Benavente- Garcia et al., 2008; Soory, 2009). These polyphenols reduce oxidative stress by directly scavenging free radical species, chelating transition metals, quenching singlet oxygen, or inhibiting oxidative enzymes (Cos et al., 2000).

Among twenty-four fruits investigated, blueberries were found to have the highest total antioxidant capacity (TAC) in an oxygen radical absorbance capacity assay (13427 TAC/serving; Wu et al., 2004). In a cell-based assay, wild blueberries also showed the highest cellular antioxidant activity (Wolfe et al., 2008). Wild blueberries (Vaccinium angustifolium) are originally from North America and Canada. Maine is the largest producer in the world with blueberries being cultivated on over 60,000 acres (Yarborough, 2009). There are over 400 species of blueberries. Highbush blueberry, Vaccinium corymbosum, is the most commercialized species growing on over 100,000 acres in the US and Canada (Nesom, 2001). Highbush blueberry is cultivated predominantly in the northern states while in the southern states rabbit eye blueberry (Vaccinium ashei) is the species mostly produced. In Europe, bilberry (Vaccinium myrtillus) is the most common species.

In view of their strong antioxidant activity and in relation to recent reports that antioxidant-rich foods reduce the risk of metabolic syndrome, various studies on blueberries that could have applications for the treatment of the disorders encompassing metabolic syndrome are reviewed here.

Cassia S. Mizuno and Agnes M. Rimando*

United States Department of Agriculture, Agricultural Research Service,
Natural Products Utilization Research Unit
University, MS 38677-8048

Blueberry Constituents and Metabolic Syndrome Disorders

As earlier alluded to, the polyphenol content may be related to the lipid lowering activity of blueberries. Previous works have demonstrated in vitro and in vivo that polyphenols inhibited FAS activity (Li et al., 2002; Ikeda et al., 2005) and increased fatty acid beta-oxidation (Murase et al., 2002; 2005). Blueberries contain higher levels of polyphenols than most fruits and vegetables (Prior et al., 1998), which also provide strong antioxidant property of these fruits (Wang, 2000; Neto, 2007). The “phenolics” that are mostly referred to in much of the studies on blueberries are the anthocyanins. However, phenolic stilbene compounds, such as resveratrol and pterostilbene, have also been identified in blueberries (Rimando et al., 2004; Rimando and Barney, 2005). Resveratrol has long been associated with the “French Paradox,” and has been shown in various tests to protect the cardiovascular system by multidimensional way (reviewed by Das and Das, 2007). Pterostilbene has likewise been shown to lower VLDL- and LDL cholesterol, while increasing HDL-cholesterol, in hamsters fed high-fat diet fortified with pterostilbene (at a dose of 2.5 mg/kg body weight). Additionally, pterostilbene also lowered serum glucose levels. These activities were correlated with activation of the nuclear transcription factor PPARa (Rimando et al., 2005).

The anti-diabetic effect of anthocyanins in blueberries was evaluated in vivo using diabetic C57bl/6J mice (Grace et al., 2009). Phenolic rich and anthocyanin enriched fractions were administered, using a micro-emulsifying drug delivery system (Labrasol), at 500mg/Kg body weight. The phenolic fraction decreased blood glucose levels by 33% while the anthocyanin fraction exhibited better hypoglycemic activity (51% decrease in blood glucose levels), which suggested that the hypoglycemic activity was due to the anthocyanins. It must be noted that hypoglycemic activity was not significant when administered without Labrasol. Pure delphinidin-3-Oglucoside and pure malvidin-3-O glucoside, at 300 mg/Kg body weight, both formulated with Labrasol, were then investigated. Malvidin-3-O-glucoside, but not delphinidin-3-O-glucoside, was found to be significantly hypoglycemic (Grace et al., 2009). Purified anthocyanins from blueberries incorporated in the drinking water were also shown to reduce obesity in C57BL/6J mice in an 8-week study (Prior et al. 2008). Interestingly, it was also found that the mice fed high-fat (45% calorie) diet (HF45) mixed with extracts from blueberries, body weight gains, body fat (per cent of BW), and epididymal fat weights were significantly greater than those in the HF45-fed controls.

Besides phenolic compounds, blueberries also contain fibres (Avila da Silveira. 2007; Jeong et al., 2008). Research has shown that binding of dietary fibres to bile acids increased their faecal elimination, which was considered a possible mechanism by which fibres decreased cholesterol levels (Lund, 1989; Anderson and Siesel, 1990). Based on equal dry matter, the bile acid binding of blueberries was higher than all the fruits tested. Considering equal total polysaccharides blueberry and plums presented the best binding (Kahlon and Smith, 2006).

Vascular-cardio diseases

This research article demonstrates how blueberries and blueberry leaf extract (Blueberin) prevent plaque build-up in arteries, thereby reducing vascular-cardio diseases. Different effect of anthocyanin’s and phenolic acids from wild blueberry (Vaccinium angustifolium) on monocytes adhesion to endothelial cells in a TNF-α stimulated pro-inflammatory environment

Monocyte adhesion to the vascular endothelium is a crucial step in the early stages of atherogenesis. Atherosclerosis (also known as arteriosclerotic vascular disease or ASVD) is a specific form of arteriosclerosis in which an artery-wall thickens as a result of invasion and accumulation of white blood cells (WBCs)(form cell) and proliferation of intimal-smooth-muscle cell creating a fibro fatty plaque.

A study by Del Bo et al, investigated the capacity of an anthocyanin (ACN) and phenolic acid (PA)-rich fraction (RF) of a wild blueberry, single ACNs (cyanidin, malvidin, delphinidin) and related metabolites (protocatechuic, syringic and gallic aid) to counteract monocytes (THP-1) adhesion to endothelial cells (HUVECs) in a tumor necrosis α (TNF-α) mediated pro-inflammatory environment.

HUVECs were incubated with different concentrations (from 0.01 to 10μg mL-1) of the compounds for 24h. Labeled monocytic THP-1 cells were added to HUVECs and their adhesion was induced by TNF-α (100 ng mL-1). ACN-RF reduced THP-1 adhesion to HUVECs with a maximum effect at 10μg mL-1 (-33%). PA-RF counteracted THP-1 adhesion at 0.01, 0.1 and 1μg mL-1 (-45%, -48.7% and -27.6%, respectively), but not at maximum concentration. Supplementation with gallic acid reduced THP-1 adhesion to HUVECs with a maximum effect at 1μg mL-1 (-29.9%), while malvidin-3-glucoside and syringic acid increased the adhesion. No effect was observed for the other compounds.

These results suggest that ACNs/PA-RF may prevent atherogenesis while the effects of the single ACNs and metabolites are controversial and merit further exploration

Blueberry and Hyperlipidaemia

The correlation between cholesterol levels and risk of coronary heart diseases has been previously established. Hyperlipidaemia is the major cause of atherosclerosis, which is one of the leading causes of cardiac deaths worldwide. Atherosclerosis is the accumulation of fat in the blood vessels resulting in narrowing of the lumen of artery. Free hydroxy radicals are the major cause of oxidative damage to low density lipoproteins (LDL), which are responsible for the development of atherosclerosis in hyperlipidemic patients (Parthasarathy, 1992). Cignarella et al., (1996) reported the lipid lowering activity of blueberries for the first time. Blueberry leaf extracts administered orally to streptozotocin-diabetic rats decreased plasma triglycerides levels by 39%. The results were confirmed using different models of hyperlipidaemia. Extracts of blueberry leaves also showed lipid-lowering activity in Yoshida rats, a genetic model of hypertriglyceridemia. Supplementation of pig basal diets (containing 70% of soya, oats and barley) with blueberries reduced blood lipid levels. At 2% of blueberries, total cholesterol was lowered by 11.7%, LDL by 15.1% and HDL by 8.3% (Kalt et al., 2008).

The lipid lowering effect of blueberries was attenuated when plant based components was decreased from 70 to 20%, indicating that blueberry and dietary components might be interacting synergistically to lipid lowering effect. The hypolipidemic activity of blueberries was also demonstrated in another study using bile acids binding assay (Kahlon and Smith, 2006). The effect of blueberries on the lipid metabolism of OLETF (Otsuka Long-Evans Tokushima Fatty) rats, an animal model of type 2 diabetes with obesity, has also been investigated. Serum cholesterol and phospholipids levels were decreased, in a dose dependent manner, in OLEFT rats fed freeze-dried blueberry leaves. The hepatic levels of cholesterol and phospholipids were also lowered, but not significantly (Nagao et al., 2008). Biological assays analysing the effects on the activity of enzymes involved in lipid metabolism suggested that the hypolipidemic activity of the leaves might be due to the inhibition of fatty acid synthase, a key enzyme in fatty acid synthesis, and to the activation of carnitine palmitoyl-transferase, an important enzyme involved in fatty acid betaoxidation (Nagao et al. 2008). In a study using male Wistar rats the effects of daily intake of blueberries with those of spontaneous exercise were compared (Hamazu et al. 2005). Serum HDL-cholesterol level in rats fed with diet supplemented with blueberry paste every day was higher than those of the control rats, in one part of the study. Another part of the study the rats were maintained in high-fat diet. The calcium/elastin level (an index of arterial calcification) was found to be lowest in rats that were supplied with blueberries daily. Blueberries and spontaneous exercise decreased the risk of arteriosclerosis differently.

Dietary supplementation with blueberries has also been shown to protect from ischemic brain damage (Wang et al., 2005). Adult male Sprague-Dawley rats were fed with blueberry- (together with a group fed spinach-, and another group fed spirulina) enriched diets for four weeks. Animals on blueberry (or spinach or spirulina) diets had a significant reduction in the volume of infarction in the cerebral cortex, and an increase in post-stroke locomotor activity compared to the control animals. Another study has also shown that blueberries protected the brain against damage from ischemia (Sweeney et al., 2002). Rats were fed a diet fortified with 14.3% lowbush blueberries, and stroke was simulated by ligation of the left common carotid artery (ischemia), followed by hypoxia. In rats on blueberry-supplemented diet, hypoxia-ischemia resulted in only 17 ± 2% loss (while control rats lost 40 ± 2%) of neurons in the hippocampus of the left cerebral hemisphere, as compared to the right hemisphere. Neuroprotection was observed in the CA1 and CA2 regions, but not CA3 region, of the hippocampus. Results from this study suggested that ischemic stroke outcomes could be improved by inclusion of blueberries in the diet.

Colon Cancer

Phenolic Compounds from Blueberries Can Inhibit Colon Cancer Cell Proliferation and Induce Apoptosis
Research has shown that diets rich in phenolic compounds may be associated with lower risks of several chronic diseases including cancer. This study systematically evaluated the bioactivities of phenolic compounds in rabbit eye blueberries and assessed their potential antiproliferation and apoptosis induction effects using two colon cancer cell lines, HT-29 and Caco-2. Polyphenols in three blueberry cultivars, Briteblue, Tifblue, and Powderblue, were extracted and freeze-dried. The extracts were further separated into phenolic acids, tannins, flavonols, and anthocyanin’s using an HLB cartridge and LH20 column. Some individual phenolic acids and flavonoids were identified by HPLC with >90% purity in anthocyanin fractions. The dried extracts and fractions were added to the cell culture medium to test for antiproliferation activities and induction of apoptosis. Flavonol and tannin fractions resulted in 50% inhibition of cell proliferation at concentrations of 70−100 and 50−100 μg/mL in HT-29 and Caco-2 cells, respectively. The phenolic acid fraction showed relatively lower bioactivities with 50% inhibition at 1000 μg/mL. The greatest antiproliferation effect among all four fractions was from the anthocyanin fractions. Both HT-29 and Caco-2 cell growth was significantly inhibited by >50% by the anthocyanin fractions at concentrations of 15−50 μg/mL. Anthocyanin fractions also resulted in 2−7 times increases in DNA fragmentation, indicating the induction of apoptosis. The effective dosage levels are close to the reported range of anthocyanin concentrations in rat plasma. These findings suggest that blueberry intake may reduce colon cancer risk

(Yi et al., 2005).

Prostate Cancer - Androgen Sensitive

Differential effects of blueberry proanthocyanidins on androgen sensitive and insensitive human prostate cancer cell lines
Blueberries are rich in health-promoting polyphenolic compounds including proanthocyanidins. The purpose of this study was to determine if proanthocyanidin-rich fractions from both wild and cultivated blueberry fruit have the same inhibitory effects on the proliferation of LNCaP, an androgen-sensitive prostate cancer cell line, and DU145, a more aggressive androgen insensitive prostate cancer cell line. When 20μg/ml of a wild blueberry proanthocyanidin fraction (fraction 5) was added to LNCaP media, growth was inhibited to 11% of control with an IC50 of 13.3μg/ml. Two similar proanthocyanidin-rich fractions from cultivated blueberries (fractions 4 and 5) at the same concentration inhibited LNCaP growth to 57 and 26% of control with an IC50 of 22.7 and 5.8μg/ml, respectively. In DU145 cells, the only fraction that significantly reduced growth compared to control was fraction 4 from cultivated blueberries with an IC50 value of 74.4μg/ml, indicating only minor inhibitory activity. Differences in cell growth inhibition of LNCaP and DU145 cell lines by blueberry fractions rich in proanthocyanidins indicate that blueberry proanthocyanidins have an effect primarily on androgen-dependent growth of prostate cancer cells. Possible molecular mechanisms for growth inhibition are reviewed (Schmidt et al., 2006).

Prostate cancer

Inhibition of matrix metalloproteinase activity in DU145 human prostate cancer cells by flavonoids from lowbush blueberry (Vaccinium angustifolium): possible roles for protein kinase C and mitogen-activated protein-kinase-mediated events
Regulation of the matrix metalloproteinases (MMPs) is crucial to regulate extracellular matrix (ECM) proteolysis which is important in metastasis. This study investigated the mechanism(s) by which three flavonoid-enriched fractions from lowbush blueberry (Vaccinium angustifolium) down-regulate MMP activity in DU145 human prostate cancer cells. Metalloproteinase activity was evaluated from cells exposed to “crude,” anthocyanin-enriched (AN) and proanthocyanidin-enriched (PAC) fractions. Differential down-regulation of MMPs was observed. The activity of the endogenous tissue inhibitors of metalloproteinases (TIMPs) from these cells was also evaluated. Increases in TIMP-1 and TIMP-2 activity were observed in response to these fractions. The possible involvement of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways in the flavonoid-mediated decreases in MMP activity was observed. These findings indicate that blueberry flavonoids may use multiple mechanisms in down-regulating MMP activity in these cells (Matchett et al., 2005).

Anticarcinogenic

Anticarcinogenic Activity of Strawberry, Blueberry, and Raspberry Extracts to Breast and Cervical Cancer Cells
Freeze-dried fruits of two strawberry cultivars, Sweet Charlie and Carlsbad, and two blueberry cultivars, Tifblue and Premier were sequentially extracted with hexane, 50% hexane/ethyl acetate, ethyl acetate, ethanol, and 70% acetone/water at ambient temperature. Each extract was tested separately for in vitro anticancer activity on cervical and breast cancer cell lines. Ethanol extracts from all four fruits strongly inhibited CaSki and SiHa cervical cancer cell lines and MCF-7 and T47-D breast cancer cell lines. An unfractionated aqueous extract of raspberry and the ethanol extract of Premier blueberry significantly inhibited mutagenesis by both direct-acting and metabolically activated carcinogens (Wedge et al., 2001).

Inhibits Cancer Cell Proliferation

Inhibition of Cancer Cell Proliferation and Suppression of TNF-induced Activation of NFκB by Edible Berry Juice
Berries contain several phytochemicals, such as phenolic acids, proanthocyanidins, anthocyanin’s and other flavonoids. There has been growing interest in a variety of potential chemopreventive activities of edible berries. The potential chemopreventive activity of a variety of small berries cultivated or collected in the province of Québec, Canada were evaluated here. Materials and Methods: Strawberry, raspberry, black currant, red currant, white currant, gooseberry, high-bush blueberry, low-bush blueberry, velvet leaf blueberry, serviceberry, blackberry, black chokeberry, sea buckthorn and cranberry were evaluated for antioxidant capacity, anti-proliferative activity, anti-inflammatory activity, induction of apoptosis and cell cycle arrest. Results: The growth of various cancer cell lines, including those of stomach, prostate, intestine and breast, was strongly inhibited by raspberry, black currant, white currant, gooseberry, velvet leaf blueberry, low-bush blueberry, sea buckthorn and cranberry juice, but not (or only slightly) by strawberry, high-bush blueberry, serviceberry, red currant, or blackberry juice. No correlation was found between the anti-proliferative activity of berry juices and their antioxidant capacity (p>0.05). The inhibition of cancer cell proliferation by berry juices did not involve caspase-dependent apoptosis, but appeared to involve cell-cycle arrest, as evidenced by down-regulation of the expression of cdk4, cdk6, cyclin D1 and cyclin D3. Of the 13 berries tested, juice of 6 significantly inhibited the TNF-induced activation of COX-2 expression and activation of the nuclear transcription factor NFκB (Boivin et al., 2007).

References

Abidov M, Ramazanov A, Jimenez Del Rio M, Chkhikvishvili I. (2006). Effect of Blueberin on fasting glucose, C-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double-blind, placebo controlled clinical study. Georgian Med News, (141):66-72.

Boivin D, Blanchette M, Barrette S, et al. Anticancer Research. March-April 2007 vol. 27 no. 2 937-48

Cassia S. Mizuno and Agnes M. Rimando. United States Department of Agriculture, Agricultural Research Service,
Natural Products Utilization Research Unit
University, 2015, MS 38677-8048

Del Bo C, Roursgaard M, Porrini M, et al. Molecular Nutrition & Food Research DOI: 10.1002/mnfr.201600178

Georgian Med News. 2006 Dec;(141): 66-72. Institute of Immunopathology, Center of Modern Medicine, Russian Academy of Natural Sciences, Moscow, Russia

Jimenez del Rio, Miguel, Blueberry Leaves Extract: Diabetes & More. President Natural Polyphenols SA Las Palmas, Canary Islands, Spain. http://diabetes.boomja.com/mobile/ITEM-Blueberry-Leaves-Extract-Diabetes-and-More-39876.html

Matchett MD, MacKinnon, L, Sweeney MI, Gottschall-Pass KT, & Hurta, RAR. NRC Institute. DOI: 10.1016/j.jnutbio.2005.05.014.

Matchett MD, MacKinnon, L, Sweeney MI, Gottschall-Pass KT, Hurta, RAR. (2006). Inhibition of matrix metalloproteinase activity in DU145 human prostate cancer cells by flavonoids from lowbush blueberry (Vaccinium angustifolium): possible roles for protein kinase C and mitogen-activated protein-kinase-mediated events. The Journal of Nutritional Biochemistry. doi: 10.1016/j.jnutbio.2005.05.014.

Schmidt BM, Erdman Jr JW, & Lila MA. Cancer Letters. Volume 231, Issue 2, Pages 240-246 (18 January 2006)

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