Non-targeted metabolite profiling highlights the potential of strawberry leaves as a resource for specific bioactive compounds.

BACKGROUND: The non-edible parts of horticultural crops, such as leaves, contain substantial amounts of valuable bioactive compounds which are currently only little exploited. For example, strawberry (Fragaria × ananassa) leaves may be a promising bioresource for diverse health-related applications. However, product standardization sets a real challenge, especially when the leaf material comes from varying cultivars. The first step towards better quality control of berry fruit leaf-based ingredients and supplements is to understand metabolites present and their stability in different plant cultivars, so this study surveyed the distribution of potentially bioactive strawberry leaf metabolites in six different strawberry cultivars. Non-targeted metabolite profiling analysis using LC/qTOF-ESI-MS with data processing via principal component analysis and k-means clustering analysis was utilized to examine differences and commonalities between the leaf metabolite profiles. RESULTS: Quercetin and kaempferol derivatives were the dominant flavonol groups in strawberry leaves. Previously described and novel caffeic and chlorogenic acid derivatives were among the major phenolic acids. In addition, ellagitannins were one of the distinguishing compound classes in strawberry leaves. In general, strawberry leaves also contained high levels of octadecatrienoic acid derivatives, precursors of valuable odour compounds. CONCLUSION: The specific bioactive compounds found in the leaves of different strawberry cultivars offer the potential for the selection of optimized leaf materials for added-value food and non-food applications. © 2016 Society of Chemical Industry.

Pinosylvin-mediated protection against oxidative stress in human retinal pigment epithelial cells.

PURPOSE: In this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to modulate oxidative stress in human RPE cells. PS, a stilbenoid polyphenol, occurs in high concentrations in bark byproducts and therefore represents an attractive bioactive compound for health-promoting applications. METHODS: First, we evaluated the toxicity range of PS by exposing ARPE-19 cells to 0.1-200 µM concentrations of PS for 24 h followed by the cell viability test. In the next stage, the ARPE-19 cells were preincubated in PS for 24 h followed by hydroquinone (HQ) exposure without PS for another 24 h. The cell viability test was conducted after HQ exposure. To elucidate the potential mechanisms behind PS-mediated protection against oxidative stress, the ARPE-19 cells were treated with 5 µM PS for 6 h, and mRNA was extracted at four time points (2 h, 6 h, 12 h, 24 h) to determine changes in the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), sequestosome 1 (p62/SQSTM1), heme oxygenase-1 (HO-1), and glutathione S-transferase pi 1 (GSTP1) genes. To clarify the molecular mechanism behind PS-mediated protection further, the ARPE-19 cells were transfected with p62 and Nrf2 siRNAs for 24 h, and the roles of p62, Nrf2, and its target gene HO-1 in conferring protection against oxidative stress were studied with quantitative real-time PCR (qRT-PCR) and the cell viability test. RESULTS: PS treatment at concentrations of 5 and 10 µM significantly enhanced cell survival from oxidative stress. The expression levels of an enzyme with antioxidative, anti-inflammatory, and immunomodulatory properties, HO-1, were increased by PS treatment and correlated strongly with cell survival. PS treatment did not elevate the expression levels of Nrf2 or its target genes, p62 or GSTP1, even though it had a clear effect on the expression of HO-1, another gene controlled by Nrf2. RNA interference analysis further confirmed the important role of Nrf2 and HO-1 in PS-mediated protection against oxidative stress whereas the role of p62 seemed to be insignificant at the gene expression and cell viability levels. CONCLUSIONS: Our results suggest that PS treatment conferred protection against oxidative stress through the induction of HO-1 in human RPE cells. Consequently, PS-stilbene compounds, which can be isolated in significant amounts from bark waste, may possess health-promoting properties against aging-related diseases associated with oxidative stress such as age-related macular degeneration (AMD) and Alzheimer's disease. These natural compounds may offer opportunities for high-value use of bark waste in diverse health-related applications.

Metabolite profiling of leek (Allium porrum L) cultivars by (1) H NMR and HPLC-MS.

INTRODUCTION: Leek (Allium ampeloprasum var. porrum) is consumed as a vegetable throughout the world. However, little is known about the metabolites of leek cultivars, especially those with potentially important beneficial properties for human health. OBJECTIVE: We provide new information for the overall metabolite composition of several leek cultivars grown in Europe by using HPLC-MS and (1) H NMR. METHODS: The use of a novel CTLS/NMR (constrained total-line-shape nuclear magnetic resonance) approach was found to be capable of reliable quantification, even with overlapping metabolite signals in the (1) H NMR of plant metabolites. Additionally, a new application for leek flavonoids was optimised for HPLC-MS. RESULTS: The total concentration of carbohydrates (glucose, fructose, kestose/nystose and sucrose) and nine amino acids varied by fourfold in leek juice from different cultivars, while the total concentrations of four organic acids were similar in all cultivars. All the quantified flavonols were kaempferol derivatives or quercetin derivatives and threefold differences in flavonol concentrations were detected between cultivars. CONCLUSION: In this study, various phytochemical profiles were determined for several leek cultivars by (1) H NMR spectroscopy with CTLS combined with HPLC-MS. The wide variation in bioactive compounds among commercial leek cultivars offers promising opportunities for breeders to raise the levels of important biochemical compounds in leek breeding lines, and also provides some objective measure for quality assurance for the leek industry.

Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells.

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.

Red clover (Trifolium pratense L.) isoflavones: root phenolic compounds affected by biotic and abiotic stress factors.

BACKGROUND: Phenolic compounds have recently received considerable attention for their ability to protect plant and human cells from oxidative stress-induced damage. Red clover (Trifolium pratense L.) is a rich source of isoflavonoids with multiple potential protective functions. The aim of this study was to identify and characterise phenolic compounds in red clover roots by high-performance liquid chromatography and mass spectrometry and to study the effects of stress factors and growth stage on root phenolics. RESULTS: A total of 28 phenolic compounds were tentatively identified in red clover roots. The most abundant phenolics in pot-grown roots were formononetin glycoside malonate (G-M) (1.51-4.26 mg g(-1)), formononetin (2.21-3.57 mg g(-1)) and biochanin A (1.73-2.17 mg g(-1)), whereas field-grown roots were rich in formononetin-G-M (3.90-4.27 mg g(-1)), maackiain-G-M (2.35-3.02 mg g(-1)) and pseudobaptigenin-G-M (1.80-2.58 mg g(-1)). Concentrations were affected by the growth stage. Ozone exposure slightly affected the total phenolic content in roots and also had minor effects on individual compounds. CONCLUSION: Elevated ozone, cultivation regime and growth stage affected the levels of phenolics in red clover roots, suggesting sensitivity of root phenolics to biotic and abiotic stress conditions. The high levels of phenolics found in roots even in late autumn may be utilised in many applications.

Benzothiadiazole induces the accumulation of phenolics and improves resistance to powdery mildew in strawberries.

Benzothiadiazole (BTH) enhanced the accumulation of soluble and cell-wall-bound phenolics in strawberry leaves and also improved the resistance to powdery mildew infection under greenhouse conditions. The most pronounced change was seen in the levels of ellagitannins, which increased up to 2- to 6-fold 4 days after the BTH application, but persisted only in the inoculated plants. The induction of phenolic metabolism by BTH was also reflected in the fruits, several compounds being increased in inoculated, BTH-treated plants. Basal salicylic acid (SA) content was high in strawberry leaves, but increased in a similar fashion to other phenolics after the treatments. Several phenolic compounds were identified in strawberries for the first time. For example, ellagic acid deoxyhexose, three agrimoniin-like ellagitannins, sanguiin H-10- and lambertianin C-like ellagitannins in the leaves, ellagic acid, p-coumaric acid, gallic acid, and kaempferol hexose in the cell-wall-bound fraction of the leaves, and kaempferol malonylglucoside in the fruits. The findings show that BTH can enhance the accumulation of phenolics in strawberry plants which may then be involved in the BTH-induced resistance to powdery mildew.

Effects of Juice Matrix and Pasteurization on Stability of Black Currant Anthocyanins during Storage.

The effects of juice matrix and pasteurization on the stability of total phenols and especially total and individual anthocyanins were examined in black currant (BC) juice and mixtures with apple, persimmon, and peach juices at 4 °C and 20 °C. Total phenol content decreased in all juices at both temperatures but there was a trend to lower levels in unpasteurized over pasteurized juices. Differences in the decline of total anthocyanins between pasteurized and unpasteurized juices varied according to the juice type and the storage temperature. At 4 °C storage, anthocyanins declined in all juices according to pseudo 1st-order kinetics and there were only small differences in the rates between pasteurized and unpasteurized juices. However, at 20 °C, although pasteurized and unpasteurized BC juices and pasteurized mixed juices followed pseudo 1st-order kinetics, there was a different pattern in unpasteurized mixed juices; a rapid initial decline was followed by a slowing down. The effect of the added juice on anthocyanin decline was also different at either temperature. At 4 °C, the anthocyanins decreased faster in mixed juices than BC juice alone, but at 20 °C, at least in pasteurized mixed juices, the decline was similar or even slower than in BC juice; there were only small differences among the 3 mixed juices. At 20 °C, in pasteurized and unpasteurized BC juices, the rate of decrease was essentially the same for all 4 individual anthocyanins but in the mixed juices the 2 glucosides decreased significantly faster than the 2 rutinosides.

High-performance liquid chromatography analysis of black currant (Ribes nigrum L.) fruit phenolics grown either conventionally or organically.

Black currants (Ribes nigrum L.) contain a diverse range of phenolics and possess a high antioxidant activity, which makes them an interesting target for the functional food industry. In this study, phenolic profiles of organically and conventionally grown black currant fruits, collected from commercial farms within a climatically similar area, were compared. Compounds were identified using UV/vis and mass spectroscopy techniques and quantified with high-performance liquid chromatography equipped with UV/vis detection. Several different conjugates of hydroxycinnamic acids, flavonols, and anthocyanins were quantified. Statistically significant differences between farms were found for almost all compounds. Differences between the highest and the lowest measured values of major phenolic compounds of different phenolic classes ranged from 24 to 77%. Principal component analysis quite effectively separated farms from each other but did not cluster them according to cultivation technique. Thus, it was concluded that the biochemical quality of organically grown black currant fruits does not differ from those grown conventionally.

Influence of fertilization, mulch color, early forcing, fruit order, planting date, shading, growing Environment, and genotype on the contents of selected phenolics in strawberry (Fragaria x ananassa Duch.) fruits.

The influence of agricultural practices (fertilization, mulch color, early forcing, and planting date), environment (light and growing area), cultivar, and fruit order on the selected phenolic content and antioxidant activity in strawberry (Fragaria x ananassa Duch.) fruits was studied. Three different levels of fertilization were given to plants in the fertilization experiment. The lowest fertilization level increased the contents of flavonols and ellagic acid from 19 to 57%. Between cultivars, up to 4-fold differences were found in the flavonol content, and it also varied according to growing environment. Planting date in glasshouse production was important for the phenolic content, and a statistically significant interaction was found between planting date and fruit order. Fruit order caused at highest 1.5-2.0-fold differences in the contents of phenolics. Interestingly, compared with other phenolics, anthocyanins were affected differently by many factors. Thus, the findings show that minor cultivation changes can increase the content of phenolics, especially in under-glass production where conditions can be easily manipulated.

Metabolic profiling discriminates between strawberry (Fragaria×ananassa Duch.) cultivars grown in Finland or Estonia.

Metabolic profiling analysis with LC-ESI-QTOF-MS was utilized to separate and identify 186 putative major metabolites demonstrating significantly different levels in 15 strawberry cultivars grown in Finland or Estonia. Principal component analysis showed close clustering of genetically related samples grown in Estonia, and hierarchical cluster analyses highlighted differences and similarities in their metabolic profiles driving separation between cultivars with specific metabolic phenotypes. Phenolic acids, flavonoids, flavan-3-ol derivatives, terpenes, and many types of glycosidically bound aroma and flavor precursors showed clear variation between strawberry cultivars. These cultivar-specific differences in the levels of major potentially bioactive phytochemicals in strawberries suggests that cultivar selection is essential for breeding strawberry cultivars with optimal phytochemical compositions contributing to possible functional properties and good cultivation and sensory qualities.

Nontargeted metabolite profiles and sensory properties of strawberry cultivars grown both organically and conventionally.

Strawberry (Fragaria × ananassa Duch.) contains many secondary metabolites potentially beneficial for human health, and several of these compounds contribute to strawberry sensory properties, as well. In this study, three strawberry cultivars grown both conventionally and organically were subjected to nontargeted metabolite profiling analysis with LC-qTOF-ESI-MS and to descriptive sensory evaluation by a trained panel. Combined metabolome and sensory data (PLS model) revealed that 79% variation in the metabolome explained 88% variation in the sensory profiles. Flavonoids and condensed and hydrolyzable tannins determined the orosensory properties, and fatty acids contributed to the odor attributes of strawberry. Overall, the results indicated that the chemical composition and sensory quality of strawberries grown in different cultivation systems vary mostly according to cultivar. Organic farming practices may enhance the accumulation of some plant metabolites in specific strawberry genotypes. Careful cultivar selection is a key factor for the improvement of nutritional quality and marketing value of organic strawberries.

Polyphenol Stilbenes: Molecular Mechanisms of Defence against Oxidative Stress and Aging-Related Diseases.

Numerous studies have highlighted the key roles of oxidative stress and inflammation in aging-related diseases such as obesity, type 2 diabetes, age-related macular degeneration (AMD), and Alzheimer's disease (AD). In aging cells, the natural antioxidant capacity decreases and the overall efficiency of reparative systems against cell damage becomes impaired. There is convincing data that stilbene compounds, a diverse group of natural defence phenolics, abundant in grapes, berries, and conifer bark waste, may confer a protective effect against aging-related diseases. This review highlights recent data helping to clarify the molecular mechanisms involved in the stilbene-mediated protection against oxidative stress. The impact of stilbenes on the nuclear factor-erythroid-2-related factor-2 (Nrf2) mediated cellular defence against oxidative stress as well as the potential roles of SQSTM1/p62 protein in Nrf2/Keap1 signaling and autophagy will be summarized. The therapeutic potential of stilbene compounds against the most common aging-related diseases is discussed.

Polyphenols in strawberry (Fragaria × ananassa) leaves induced by plant activators.

Strawberry leaves contain high amounts of diverse phenolic compounds potentially possessing defensive activities against microbial pathogens and beneficial properties for human health. In this work, young strawberry plants were treated with two plant activators, S-methylbenzo-1,2,3-thaidiazole-7-carbothiate (BTH) and birch wood distillate. Phenolic compounds from activator-treated and control leaves were subjected to quantitative analyses by HPLC-DAD, HPLC-ESI-MS, and microQTOF ESI-MS. Thirty-two different phenolic compounds were detected and characterized, and 21 different ellagitannins constituted the largest group of compounds in the strawberry leaves (37.88-45.82 mg/g dry weight, 47.0-54.3% of total phenolics). Treatment with BTH resulted in higher levels of individual ellagitannins, whereas treatment with birch wood distillate strongly increased the levels of chlorogenic acid in strawberry leaves compared with the control. The results suggest that different plant activators may be useful tools for the activation of different branches in the phenylpropanoid biosynthesis in strawberry.

Anthocyanin-enriched bilberry and blackcurrant extracts modulate amyloid precursor protein processing and alleviate behavioral abnormalities in the APP/PS1 mouse model of Alzheimer's disease.

A growing body of epidemiological evidence suggests that fruit and vegetable juices containing various phenolic compounds can reduce the risk of Alzheimer's disease (AD). As the altered amyloid precursor protein (APP) processing leading to increased ß-amyloid (Aß) production is a key pathogenic feature of AD, we elucidated the effects of different polyphenols on neuroprotection and APP processing under different in vitro stress conditions. The effects of these compounds were also investigated in transgenic AD mice (APdE9). Free radical toxicity and apoptosis were induced in human SH-SY5Y neuroblastoma cells overexpressing APP751. Menadione-induced production of reactive oxygen species was significantly decreased upon treatment with myricetin, quercetin or anthocyanin-rich extracts in a dose-dependent manner. However, these extracts did not affect caspase-3 activation, APP processing or Aß levels upon staurosporine-induced apoptosis. APdE9 mice fed with anthocyanin-rich bilberry or blackcurrant extracts showed decreased APP C-terminal fragment levels in the cerebral cortex as compared to APdE9 mice on the control diet. Soluble Aß40 and Aß42 levels were significantly decreased in bilberry-fed mice as compared to blackcurrant-fed mice. Conversely, the ratio of insoluble Aß42/40 was significantly decreased in blackcurrant-fed mice relative to bilberry-fed mice. Both berry diets alleviated the spatial working memory deficit of aged APdE9 mice as compared to mice on the control diet. There were no changes in the expression or phosphorylation status of tau in APdE9 mice with respect to diet. These data suggest that anthocyanin-rich bilberry and blackcurrant diets favorably modulate APP processing and alleviate behavioral abnormalities in a mouse model of AD.

Leaf phenolic compounds in red clover (Trifolium pratense L.) induced by exposure to moderately elevated ozone.

Red clover (Trifolium pratense L.), an important feed crop in many parts of the world, is exposed to elevated ozone over large areas. Plants can limit ozone-induced damages by various defence mechanisms. In this work, changes in the concentrations of antioxidant phenolic compounds induced by slightly elevated levels of ozone were determined in red clover leaves by high-performance liquid chromatography and mass spectrometry. 31 different phenolics were identified and the most abundant isoflavones and flavonoids were biochanin A glycoside malonate (G-M), formononetin-G-M and quercetin-G-M. Elevated ozone (mean 32.4 ppb) increased the total phenolic content of leaves and also had minor effects on the concentrations of individual compounds. Elevated ozone increased the net photosynthesis rate of red clover leaves before visible injuries by 21-23%. This study thus suggests that the concentrations of phenolics in red clover leaves change in response to slightly elevated ozone levels.

Variation in the anthocyanin concentration of wild populations of crowberries (Empetrum nigrum L subsp. hermaphroditum).

Crowberry (Empetrum nigrum L.) is a relatively under-utilized wild berry that occurs widely throughout the northern hemisphere such as in Canada, Eurasia, and northern Europe. In this work, the anthocyanins of crowberries were analyzed from four geographically distinct crowberry populations in Finland using HPLC-DAD and HPLC-ESI/MS/MS. A total number of 15 anthocyanins were detected; 15 (11 structure elucidated) in all samples in order to profile-specific anthocyanin compositions throughout Finland. The major anthocyanin found in the samples collected from central and eastern Finland was delphinidin 3-galactoside accounting for more than 24% of the total anthocyanin content, while the cyanidin 3-galactoside was the major anthocyanin in the northernmost and in the western samples. Significant variation in the concentrations of different anthocyanins between and within crowberry populations were found suggesting that the synthesis of anthocyanins is modified by site-specific environmental conditions. The suitability of the crowberries as a potential source of health-promoting ingredients for incorporation into pharmaceutical and food industrial products is highlighted in this work due to the diverse anthocyanin profile.