Blueberry Leaf Extract Research Articles

Blueberry Leaf Extract Prevents Lacrimal Hyposecretion in Sjögren's Syndrome-like Model of Non-obese Diabetic Mice.

This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1β and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

Inhibition of adult T-cell leukemia cell proliferation by polymerized proanthocyanidin from blueberry leaves through JAK proteolysis.

We have previously reported that the proanthocyanidin (PAC) fraction of blueberry leaf extract (BB‐PAC) inhibits the proliferation of HTLV‐1‐infected adult T‐cell leukemia (ATL) by inducing apoptosis. In the present study, we further analyzed the structure of BB‐PAC and elucidated the molecular mechanism underlying the inhibitory function of HTLV‐1‐infected and ATL cells. After hot water extraction with fractionation with methanol‐acetone, BB‐PAC was found to be concentrated in fractions 4 to 7 (Fr7). The strongest inhibition of ATL cell growth was observed with Fr7, which contained the highest BB‐PAC polymerization degree of 14. The basic structure of BB‐PAC is mainly B‐type bonds, with A‐type bonds (7.1%) and cinchonain I units as the terminal unit (6.1%). The molecular mechanism of cytotoxicity observed around Fr7 against ATL cells was the degradation of JAK1 to 3 and the dephosphorylation of STAT3/5, which occurs by proteasome‐dependent proteolysis, confirming that PAC directly binds to heat shock protein 90 (HSP90). JAK degradation was caused by proteasome‐dependent proteolysis, and we identified the direct binding of PAC to HSP90. In addition, the binding of cochaperone ATPase homolog 1 (AHA1) to HSP90, which is required for activation of the cofactor HSP90, was inhibited by BB‐PAC treatment. Therefore, BB‐PAC inhibited the formation of the HSP90/AHA1 complex and promoted the degradation of JAK protein due to HSP90 dysfunction. These results suggest that the highly polymerized PAC component from blueberry leaves has great potential as a preventive and therapeutic agent against HTLV‐1‐infected and ATL cells.

Биологические эффекты и оценка острой токсичности антикоагулянтов растительного происхождения

The aim of the study – was to study the biological effects and assess acute toxicity in an in vivo experimental study when injecting plant-derived effectors with an anticoagulant effect to laboratory animals. Materials and methods. As the studied effectors, plant anticoagulants obtained from the leaves of blueberry (Vaccinium myrtillus) and peloid, which were obtained in the previous studies, were used. The biological effect of effectors was evaluated by indicators characterizing coagulation and platelet hemostasis when administered to laboratory animals. Acute toxicity was assessed by the survival rate of animals after administration of effectors. Results. Blueberry leaf extract demonstrated an inhibitory effect on the development of reactions of the internal pathway of plasma coagulation and ADP – induced platelet aggregation. The effector obtained from sapropel significantly inhibits both plasma coagulation and platelet reactions. None of the effectors showed acute toxicity. Conclusion. As a result of this study, we consider the most promising peptide for further study, derived from peloid, as a promising direct-acting anticoagulant that affects the final pathway of plasma coagulation. The anticoagulant from blueberry leaves did not demonstrate the expected activity in the in vivo experiment, presumably due to the presence of impurities that neutralize its anticoagulant effect, and needs additional purification and evaluation.

Effects of phenolic compounds from blueberry leaves on the thermal decomposition of trimethylamine oxide in squid extract

Abstract The effects of chlorogenic acid and quercetin-3-D-galactoside on the decomposition of trimethylamine oxide (TMAO) in squid extract and the main mechanism of inhibition of thermal decomposition were studied. The results indicated that chlorogenic acid and quercetin-3-D-galactoside could inhibit decomposition of TMAO in squid extract. The amount of TMAO was increased by 11.79 and 15.76% in squid extract treated with chlorogenic acid and quercetin-3-D-galactoside from 0 and 2.5 g/L, respectively. The contents of trimethylamine (TMA), dimethylamine (DMA), and formaldehyde (FA) were significantly decreased with increasing contents of chlorogenic acid and quercetin-3-D-galactoside. There were many free radicals in squid extract at high temperatures; however, the free radical signals were weakened after the addition of chlorogenic acid and quercetin-3-D-galactoside therein. This implied that chlorogenic acid and quercetin-3-D-galactoside could inhibit the thermal decomposition of TMAO in squid extract, which was associated with the scavenging of their free radicals. This result provides a theoretical basis for the development and utilization of blueberry leaf extract as an efficient FA inhibitor for aquatic products.

Noni (Morinda citrifolia) fruit polysaccharide films containing blueberry (Vaccinium corymbosum) leaf extract as an antioxidant packaging material

Novel biodegradable noni (Morinda citrifolia) fruit polysaccharide (NPS) films containing blueberry (Vaccinium corymbosum) leaf extract (BLE) at various concentrations (0.4, 0.7, and 1.0%) were prepared and characterized. The prepared NPS films had good tensile strength and Young's modulus, whereas the incorporation of BLE into the films as an antioxidant decreased both the values. The addition of BLE effectively enhanced the antioxidant activities of the NPS films, which was the result of phenolic compounds present in BLE. Moreover, the NPS films with BLE had greater water vapor permeability than that of the neat NPS films, which was closely related to microstructural changes in the films. Increased surface roughness of the films following the addition of BLE was also observed in micrographs of the film surface and average roughness. These results suggest that NPS films containing BLE have the potential to serve as antioxidant packaging material.

The Anti-Proliferative and Anti-Invasive Effect of Leaf Extracts of Blueberry Plants Treated with Methyl Jasmonate on Human Gastric Cancer In Vitro Is Related to Their Antioxidant Properties.

Gastric cancer is the third main cause of cancerous tumors in humans in Chile. It is well-accepted that a diet rich in antioxidant plants could help in fighting cancer. Blueberry is a fruit crop with a high content of antioxidants. Methyl jasmonate (MeJA) is a phytohormone involved in plant defenses under stress conditions. The exogenous application of MeJA can improve the antioxidant properties in plants. We studied in vitro and in vivo anticancer action on human gastric cancer (cell line AGS) and the antioxidant properties of extracts from blueberry plants untreated and treated with MeJA. The results demonstrated that leaf extracts displayed a higher inhibition of cancer cell viability as well as greater antioxidant properties compared to fruit extracts. Besides, MeJA applications to plants improved the antioxidant properties of leaf extracts (mainly anthocyanins), increasing their inhibition levels on cell viability and migration. It is noteworthy that leaf extract from MeJA-treated plants significantly decreased cancer cell migration and expression of gastric cancer-related proteins, mainly related to the mitogen-activating protein kinase (MAPK) pathway. Interestingly, in all cases the anticancer and antioxidant properties of leaf extracts were strongly related. Despite highlighted outcomes, in vivo results did not indicate significant differences in Helicobacter pylori colonization nor inflammation levels in Mongolian gerbils unfed and fed with blueberry leaf extract. Our findings demonstrated that MeJA increased antioxidant compounds, mainly anthocyanins, and decreased the viability and migration capacity of AGS cells. In addition, leaf extracts from MeJA-treated plants were also able to decrease the expression of gastric cancer-related proteins. Our outcomes also revealed that the anthocyanin-rich fraction of blueberry leaf extracts showed higher in vitro antiproliferative and anti-invasive effects than the crude leaf extracts. However, it is still uncertain whether the leaf extracts rich in anthocyanins of blueberry plants are capable of exerting a chemopreventive or chemoprotective effect against gastric cancer on an in vivo model.

Phenolic-enriched blueberry-leaf extract attenuates glucose homeostasis, pancreatic β-cell function, and insulin sensitivity in high-fat diet–induced diabetic mice

Blueberry fruit exhibits strong antioxidant activity owing to the presence of anthocyanin. As blueberry-leaf extract (BLE) contains chlorogenic acid and flavonol glycosides, we hypothesized that phenolic-enriched BLE would improve glucose homeostasis and insulin sensitivity. In this study, we examined whether BLE administration decreases the glucose levels and enhances the pancreatic function in mice with high-fat diet (HFD)-induced obesity and diabetes. C57BL/6J mice were divided into the following four groups: control diet (CD), HFD (60 kcal% fat diet), BLE (HFD with 1% BLE wt/wt diet), and yerba mate extract (YME; HFD with 0.5% YME wt/wt diet). Dietary BLE and YME reduced glucose tolerance, body weight, and plasma glucose, glycated hemoglobin, insulin, homeostasis model assessment of insulin resistance, triglyceride (TG), and non-esterified fatty acid levels. Compared with those of the HFD group, BLE was found to significantly reduce the pancreatic islet size and insulin content. Moreover, it increased the mRNA levels of pancreatic β-cell proliferation-related genes, Ngn3, MafA, Pax4, Ins1, and Ins2, and pancreatic insulin signaling-related genes, IRS-1, IRS-2, PIK3ca, PDK1, PKCε, and GLUT-2, and decreased the transcriptional expression of the β-cell apoptosis-related gene, FoxO1. Both BLE and YME improved insulin sensitivity by inhibiting TG synthesis and enhancing lipid utilization in the liver and white adipose tissue (WAT). In pancreatic MIN6 β-cells, BLE and its main component (chlorogenic acid) increased β-cell proliferation and promoted insulin signaling. Overall, BLE enriched with phenolic compounds has the capacity to prevent HFD-induced glucose tolerance and hyperglycemia by improving the pancreatic β-cell function.

Chronic intake of high-dose of blueberry leaf extract does not augment the harmful effects of ethanol in rats.

Excessive alcohol consumption is a risk factor for liver diseases. Enhancement of alcohol metabolism could be an effective strategy to prevent these adverse effects since it promotes the clearance of ethanol and acetaldehyde from the serum. Polyphenol-rich products have shown to protect against alcohol-related liver damage. Blueberry leaves have attracted attention as they are rich polyphenols such as proantocyanidins and chlorogenic acid. In this study, we investigated the effects of a high dose of blueberry leaf extract (BLEx) on alcohol metabolism during chronic intake of ethanol. Seven-week old Sprague-Dawley (SD) rats were divided into four groups: normal liquid diet group (NLD), normal liquid diet + BLEx group (NLD + BLEx), alcohol liquid diet group (ALD), and alcohol liquid diet + BLEx (ALD + BLEx). Then, rats were fed experimental diet for 5 weeks and at the end of feeding period, body weight, food intake, liver weight, indices of liver injury, expression and activity of alcohol metabolism-related and anti-oxidative enzymes, and levels of carbonyl protein, triglyceride (TG), and total cholesterol (T-Chol) were measured. Body weight and food intake decreased, whereas liver aldehyde dehydrogenase (ALDH) activity, liver microsomal cytochrome P450 2E1 (CYP2E1) protein and mRNA expression, and heme oxygenase 1 (HO-1) mRNA expression were upregulated by ethanol intake. Dietary BLEx, however, did not affect any of these ethanol-related changes. Indices of liver injury, expression and activity of other alcohol metabolism-related enzymes, liver carbonyl protein, TG, and T-Chol levels were not altered by ethanol and BLEx. Thus, chronic BLEx intake does not ameliorate the harmful effects of ethanol.

Blueberry Leaf Polyphenols Prevent Body Fat Accumulation in Mice Fed High-fat, High-sucrose Diet.

Blueberry leaf is currently a popular dietary supplement. Effects of dietary blueberry leaf and its active components on body fat accumulation were examined. C57BL/6J mice were fed high-fat, high-sucrose diet with or without 3% blueberry leaf extract (BLEx) or 3% concentrated-polyphenolic BLEx (CP BLEx) for 8 weeks. Compared to mice fed a high-fat, high-sucrose diet without blueberry leaf, BLEx and CP BLEx significantly reduced body weight and adipose tissue weight gain. Adipocytes were also smaller and and liver lipid accumulatioin was significantly inhibited in mice fed either BLEx or CP BLEx. These effects tended to be more pronounced in mice fed CP BLEx compared to in mice fed BLEx. Together, results suggest that blueberry leaf inhibits body fat accumulation typically observed in mice fed a high-fat, high-sucrose diet, and that inhibition is attributable to polyphenolic components in leaf extracts.

Mechanistic insights to the cardioprotective effect of blueberry nutraceutical extract in isoprenaline-induced cardiac hypertrophy

BackgroundLowbush blueberry extract (Vaccinium angustifolium) is abundant with polyphenols (such as chlorogenic acid) with high antioxidant profile. It has received great interest due to its protective role in many disorders such as heart diseases and neurological disorders. HypothesisWe hypothesized that blueberry leaf extract might have a protective effect against cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis. MethodBlueberry leaf nutraceutical extract was administered orally to male albino rats at three different doses (25, 50 and 100 mg/kg/day of the extract, equivalent to 3.4, 6.8 and 13.6 mg of chlorogenic acid, respectively) once daily for 28 consecutive days against a dose of isoprenaline (ISO) (5 mg/kg) for 14 days. ResultsThe results indicated that isoprenaline induced significant myocardial damage, characterized by conduction abnormalities, increased heart-to-body weight ratio, increased serum CKMB, AST, c-TnI and LDH. Pretreatment with blueberry extract at a dose of 50 mg/kg/day (equivalent to 6.8 mg chlorogenic acid) protected against ISO-induced ECG changes, leakage of cardiac enzymes and histopathological changes. Also, ISO caused significant glutathione depletion, lipid peroxidation and reduction in activities of antioxidant catalase enzyme. These effects were prevented by pretreatment with blueberry extract. Additionally, ISO elicited inflammatory effects by increasing the expression of NF-κB, COX-2, TNF-α and IL-6 while pretreatment with blueberry extract significantly inhibited these inflammatory responses. Furthermore, ISO induced fibrosis by increasing the level of TGF-β while pretreatment with blueberry extract significantly reduced it. ConclusionThese findings indicate that blueberry leaf extract possessed a potent protective effect against ISO-induced cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis.

Effects of blueberry leaf and stem extracts on hepatic lipid levels in rats consuming a high-sucrose diet

Background: Blueberry stems, a by-product of blueberry leaf tea production, are typically discarded. We evaluated the effects of hot-water extracts of rabbiteye blueberry (Vaccinium virgatum Aiton; RB species) leaves and stems on hepatic lipid levels in rats consuming a high-sucrose diet.Methods: Male Sprague-Dawley rats were divided into groups that received a control high-sucrose diet alone or supplementation with 2.0% blueberry leaf extract or 0.5% or 2.0% blueberry stem extract. Blood and hepatic lipid levels, hepatic lipogenic enzyme activity, and hepatic quercetin metabolites were evaluated after 28 days of ad libitum consumption.Results: Supplementation with the extracts did not affect body weight gain, food intake, liver and white adipose tissue weights, or serum lipid levels. Hepatic triglyceride and total cholesterol levels were reduced in the groups that received 2.0% supplementation of either extract. Hepatic malic enzyme activity was also reduced in those groups. Quercetin and its glycosides, the major polyphenols identified in the extracts, accumulated in the liver as quercetin aglycone and quercetin metabolites. Conclusion: We demonstrated how daily consumption of blueberry leaf and stem extracts can decrease hepatic lipid levels, potentially downregulating malic enzyme activity. These effects were intensive in leaf extracts. The active compounds existed in both extracts may be quercetin and its glycosides. Therefore, blueberry stems and leaves may be an attractive candidate novel functional food.Keywords: Blueberry leaf; blueberry stem; quercetin; hepatic lipid; rat; functional food

Vaccinium ashei leaves extract alleviates insulin resistance via AMPK independent pathway in C2C12 myotube model

Blueberry (Vaccinium ashei) leaf has recently attracted attention as an anti-obesity food component. In this study, we examined the effects of blueberry leaf extract (BLEx) on insulin signaling in C2C12 differentiated myoblasts. The results showed that BLEx promotes the intracellular uptake of 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]−2-deoxy-D-glucose (2-NBDG) and phosphorylation of Akt under insulin stimulation. In addition, pretreatment with BLEx ameliorated TNF-α-nduced insulin resistance with regard to 2-NBDG uptake and Akt phosphorylation. Moreover, BLEx prevented the TNF-α−induced activation of JNK and NF-kB pathways and phosphorylation of IRS-1 at serine residue. BLEx failed to induce phosphorylation of AMPK as well as did not prevent the restoration of 2-NBDG uptake under TNF-α–induced insulin resistance. Overall, skeletal muscle is a putative target for the anti-diabetic effect of BLEx by amelioration of insulin resistance.

Vaccinium angustifolium (lowbush blueberry) leaf extract increases extravillous trophoblast cell migration and invasion in vitro.

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V.angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V.angustifolium leaf extract (0 to 2×104 ng/ml) on trophoblast cell migration (n=4) and invasion (n=4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n=3) and trypan blue exclusion assays (n=3), respectively. At 20ng/ml, V.angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p<.01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n=3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted.

Making good use of the byproducts of cultivation: green synthesis and antibacterial effects of silver nanoparticles using the leaf extract of blueberry.

In this study, we made an effort to use blueberry leaves extract as a reducing and a capping agent to biosynthesize silver nanoparticles (AgNPs). The successful formation of AgNPs was confirmed with UV-visible spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. Furthermore, their antimicrobial activities against four kinds of multi drug resistant clinical pathogens and aquatic pathogens were investigated. The results showed that as-prepared AgNPs exhibited broad-spectrum antibacterial activities. The antibacterial effects of the AgNPs based on the structural damage and DNA degradation of the tested bacteria were also studied. The green synthesis of AgNPs using blueberry leaf extract, which provided a new idea on the use of the byproducts of blueberry harvesting.

Quercetin glycosides and chlorogenic acid in highbush blueberry leaf decoction prevent cataractogenesis in vivo and in vitro: Investigation of the effect on calpains, antioxidant and metal chelating properties

The present study investigates whether highbush blueberry leaf polyphenols prevent cataractogenesis and the underlying mechanisms. Chlorogenic acid, quercetin, rutin, isoquercetin and hyperoside were quantified in Vaccinium corymbosum leaf decoction (BBL) using HPLC-DAD. Wistar rats were injected subcutaneously with 20 μmol selenite (Na2SeO3)/kg body weight on postnatal (PN) day 10 (Se, n = 8–10/group) only or also intraperitoneally with 100 mg dry BBL/kg body weight on PN days 11 and 12 (SeBBL group, n = 10). Control group received only normal saline (C). Cataract evaluation revealed that BBL significantly prevented lens opacification. It, also, protected lens from selenite oxidative attack and prevented calpain activation, as well as protein loss and aggregation. In vitro studies showed that quercetin attenuated porcine lens turbidity caused by SeO32− or Ca2+ and interacted efficiently with those ions according to UV–Vis titration experiments. Finally, rutin, isoquercetin and hyperoside moderately inhibited pure human μ-calpain. Conclusively, blueberry leaf extract, a rich source of bioactive polyphenols, prevents cataractogenesis by their strong antioxidant, chelating properties and through direct/indirect inhibition of lens calpains.

Antioxidative Activity of Blueberry Leaf Extract Prevents High-fat Diet-induced Obesity in C57BL/6 Mice.

Background:Health beneficial effects of blueberry have been well documented. Obesity is health hazard that is associated with metabolic abnormalities. We investigated the effect of blueberry leaf extract (BBLE) on high-fat diet (HFD)-induced obesity in C57BL/6J mice.Methods:C57BL/6 mice were fed HFD with or without BBLE for 10 weeks. Body weight, serum parameter, and adipose tissues morphology were assessed. The expression of mRNA associated with adipogenesis was measured using real-time polymerase chain reaction (RT-PCR) analysis.Results:Administration of BBLE to mice challenged with HFD significantly decreased the body weight gain, the levels of plasma triglyceride (TG) and liver lipid peroxidation, and reduced the adipocyte size and improved hepatic status compared with the group treated with HFD only. BBLE treatment significantly improved glucose control compared with the HFD group. Moreover, BBLE showed an inhibitory effect on adipocyte differentiation in obese mice together with significant decrease in the lipid accumulation by downregulating gene expression of adipocyte-specific transcription factors, such as peroxisome proliferation-activity receptor and acetyl coenzyme A carboxylase and upregulating the mRNA expression of adiponectin, which are critical for adipogenesis.Conclusion:BBLE suppressed the body weight gain in the HFD-fed C57BL/6 mice. Intake of BBLE reduced body weight in HFD-fed mice by 20%. Furthermore, BBLE supplementation significantly decreased the TG level in the liver and inhibited leptin secretion. BBLE supplementation also improved insulin resistance. Therefore, BBLE is a possible agent to prevent obesity.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression.

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.

Distinguishing Vaccinium species by chemical fingerprinting based on NMR spectra, validated with spectra collected in different laboratories.

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R(2) = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.

Blueberry (Vaccinium virgatum) Leaf Extracts Protect Against Aβ-Induced Cytotoxicity and Cognitive Impairment

The ethylacetate (EtOAc) fraction of blueberry leaf extract was investigated to examine the in vivo antiamnesic effects against amyloid β protein (Aβ)-induced learning and memory deficit. The fraction showed the highest antioxidant activities, and the generation of intracellular reactive oxygen species was significantly decreased. Cell viability assays revealed the in vitro cytoprotective effects of the fraction, and the cytoplasmic lactate dehydrogenase release into the medium was dose-dependently inhibited. In addition, a chlorogenic acid was identified as a predominant phenolic compound by high-performance liquid chromatography analysis. Antiamnesic effects were evaluated by using in vivo the Y-maze and passive avoidance tests, and preadministration of the fraction attenuated Aβ-induced memory impairment in both in vivo experiments. Acetylcholinesterase prepared from mice brain was inhibited by the fraction, and malondialdehyde generation in the brain homogenate was also decreased. These findings suggest that the EtOAc fraction of blueberry leaf extract could possess a wide range of physiological effects against neurodegenerative diseases.

Qualitative and quantitative differentiation of species from Vaccinium by NMR spectroscopy in automation

The genus Vaccinium contains approximately 450 species including bilberry, blueberry, cranberry, lingonberry, and huckleberry. The fruit of Vaccinium are widely eaten and touted for their health benefits, attributed to antioxidants and micronutrients. Blueberry leaf extract is a traditional medicine of the Cree people, believed to have anti-diabetic properties. Extracts from Vaccinium plants are commonly added to foods, dietary supplements, and cosmetics. Given their widespread use in humans, it is important to have methods to verify the identity and purity of Vaccinium extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for characterizing mixtures such as plant extracts in full automation. Discrimination of different species, absolute identification & quantification of key compounds and verification if the determined parameters are in the allowed range is the basis for the quality control of a sample. The automation of this process minimizes failures in the analysis. A comprehensive report makes the results easy to interpret. An NMR spectrum, such as a 1D1H spectrum, shows the superposition of the characteristic signals of all of the compounds in the mixture. When enough samples of a specific material are available, chemometric models can be build. Then new samples can be classified against the model to determine whether they represent the same material. The result is represented in different ways, e.g. as quantile plot. Identifying and quantitating of key compounds from a mixture spectrum can be based on spectral analysis or using PLS-1. Both results can be used to verify if the sample is in the allowed range. We will demonstrate the power of these techniques using examples from our ongoing study of Vaccinium.