Genomic-environmental associations in wild cranberry (Vaccinium macrocarpon Ait.).

Understanding the genetic basis of local adaptation in natural plant populations, particularly crop wild relatives, may be highly useful for plant breeding. By characterizing genetic variation for adaptation to potentially stressful environmental conditions, breeders can make targeted use of crop wild relatives to develop cultivars for novel or changing environments. This is especially appealing for improving long-lived woody perennial crops such as the American cranberry (Vaccinium macrocarpon Ait.), the cultivation of which is challenged by biotic and abiotic stresses. In this study, we used environmental association analyses in a collection of 111 wild cranberry accessions to identify potentially adaptive genomic regions for a range of bioclimatic and soil conditions. We detected 126 significant associations between SNP marker loci and environmental variables describing temperature, precipitation, and soil attributes. Many of these markers tagged genes with functional annotations strongly suggesting a role in adaptation to biotic or abiotic conditions. Despite relatively low genetic variation in cranberry, our results suggest that local adaptation to divergent environments is indeed present, and the identification of potentially adaptive genetic variation may enable a selective use of this germplasm for breeding more stress-tolerant cultivars.

Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding.

Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection 'Ben Lear,' which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for 'Ben Lear' inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop.

Nontargeted Metabolomic Study on Variation of Phenolics in Different Cranberry Cultivars Using UPLC-IM - HRMS.

The metabolomic profiles of American cranberry ( Vaccinium macrocarpon) fruits and their variation among 10 diverse cultivars were investigated by ultraperformance liquid chromatography ion-mobility high-resolution mass spectrometry (UPLC-IM - HRMS). Over 80 metabolites, belonging to various phenolic compound groups, were putatively characterized. An HRMS data matrix consisting of 4778 unique ions across the 10 cultivars was built and analyzed by orthogonal projections to latent structures discriminant analysis (OPLS-DA). The 10 cultivars segregated into 4 clusters on the basis of their metabolomic similarities, which largely reflected their genetic backgrounds. Anthocyanins exhibited the most extensive variations among all the cultivars, reflecting the effects of cranberry breeding selection on fruit color. Flavonols, phenolic acid derivatives, and proanthocyanidins also varied among the different cultivars. The nontargeted metabolomic comparison using multivariate analysis proved to be efficient and robust for determining specific metabolite differences among the cultivars.

Influence of Degree-of-Polymerization and Linkage on the Quantification of Proanthocyanidins using 4-Dimethylaminocinnamaldehyde (DMAC) Assay.

Proanthocyanidins (PACs) are naturally occurring flavonoids possessing health beneficial bioactivities. Their quantification often utilizes the 4-dimethylaminocinnamaldehyde (DMAC) spectrophotometric assay with the assumption that molar absorption coefficients (MACs) are similar across the various PAC species. To assess the validity of this assumption, individual PAC monomers and oligomers were examined for their absorbance response with DMAC. Our results have shown that PAC dimers and trimers with interflavan linkage variations exhibited differential absorbance response. Absence of A-type linkage between the terminal and second units in PAC molecule not only impacts absorbance intensity at 640 nm but also elicits a prominent secondary 440 nm absorbance peak. Cranberry (A-type) and cocoa (B-type) oligomeric PACs exhibited differential absorbance (MACs) relationship with degree-of-polymerization. Thus, PAC structural variations have considerable impact on the resulting MAC. The use of DMAC assay in PAC quantification, especially in comparing across specific oligomers and compositions, should not assume MACs are similar.

Variation in proanthocyanidin content and composition among commonly grown North American cranberry cultivars (Vaccinium macrocarpon).

BACKGROUND: Cranberry fruit (Vaccinium macrocarpon) is rich in polyphenols, particularly oligomeric proanthocyanidins (PACs) possessing antimicrobial and antioxidant properties. PACs may play a role in resistance to fruit rot. Although many cranberry cultivars are grown for use in foods, beverages and nutraceuticals, data on PAC content among cultivars is limited. Eight cultivars were sampled from four growing regions during the 2010 season and analyzed for PAC content and composition. RESULTS: MALDI-TOF MS showed that isolated PACs had similar oligomer profiles among cultivars. The major constituents were A-type (epi)catechin oligomers of two to eight degrees of polymerization. Total PAC content ranged between 18 and 92 g PAC kg⁻¹ dried fruit, quantified as procyanidin A2 by the dimethylaminocinnamaldehyde method. Among the cultivars sampled, Howes had the highest total PACs (76-92 g kg⁻¹), followed by Mullica Queen and Early Black (48-82 g kg⁻¹). Ben Lear, a disease-susceptible variety, was significantly lower in PACs than the other cultivars (P < 0.001). CONCLUSIONS: Several traditional and newer cultivars of cranberry from various growing regions in North America are excellent sources of PACs, particularly the Howes, Mullica Queen and Early Black cultivars. PAC content may play a role in keeping quality.

Purified cranberry proanthocyanidines (PAC-1A) cause pro-apoptotic signaling, ROS generation, cyclophosphamide retention and cytotoxicity in high-risk neuroblastoma cells.

Optimized purification of oligomeric proanthocyanidines (PAC) from cranberry generated PAC-1A which selectively affected the viability of various neuroblastoma (NB) cell lines representing a spectrum of high-risk NB features. PAC-1A caused a loss of mitochondrial transmembrane depolarization potential (∆Ψm) and increased generation of reactive oxygen species (ROS) which was directly correlated to the modulation of apoptotic marker proteins in SMS-KCNR cells. PAC-1A reduced the expression of pro-survival (Bcl-2, MCL-1, Bcl-xL) and increased levels of pro-apoptotic (Bax, Bad, Bid) Bcl family proteins, upregulated the activity of SAPK/JNK MAPK and downregulated expression or activity of PI3K/AKT/mTOR pathway components. PAC-1A increased the cellular uptake/retention of cyclophosphamide (CP). PAC-1A and CP synergistically increased cytotoxicity and expression of pro-apoptotic markers, reduced cellular glutathione (GSH) and superoxide dismutase (SOD) levels. Additional features of PAC-1A as an anticancer drug as shown in SMS-KCNR NB cells include delay of cell cycle progression and induction of cell death via TNF-family death receptor activity, thus, targeting both the extrinsic and intrinsic pathway of apoptosis. PAC-1A partially blocked the cell cycle in G2/M phase which correlated with a decrease of the G0/G1 subpopulation, upregulation of cyclin D1 and downregulation of CDK6 and p27 expression. In summary, PAC-1A has demonstrated chemotherapeutic potential to treat a broad spectrum of NBs including highly malignant tumors that show resistance to standard chemotherapeutics and apoptotic stimuli.

Anti-angiogenic activity of cranberry proanthocyanidins and cytotoxic properties in ovarian cancer cells.

Cranberry extracts may provide beneficial health effects in the treatment of various diseases, including cancer. However, the underlying molecular mechanisms of antineoplastic properties are not understood. We report the effect of a proanthocyanidin (PAC)-rich isolate from cranberry (PAC-1) as a therapeutic agent with dual activity to target both ovarian cancer viability and angiogenesis in vitro. PAC-1 treatment of chemotherapy-resistant SKOV-3 cells blocked cell cycle progression through the G2/M phase, increased the generation of reactive oxygen species (ROS), and induced apoptosis through activation of intrinsic and extrinsic pathway components. Cytotoxicity of PAC-1 was partially based on ROS generation and could be blocked by co-treatment with antioxidant glutathione. PAC-1 reduced the cell viability of both SKOV-3 ovarian cancer cells and HUVEC endothelial cells in a dose-dependent manner and blocked the activation of the pro-survival factor AKT. Furthermore, PAC-1 blocked vascular endothelial growth factor (VEGF)-stimulated receptor phosphorylation in endothelial cells, which correlated with the inhibition of endothelial tube formation in vitro. Our findings suggest that PAC-1 exerts potent anticancer and anti-angiogenic properties and that highly purified PAC from cranberry can be further developed to treat ovarian cancer in combinational or single-agent therapy.

Cranberry proanthocyanidins are cytotoxic to human cancer cells and sensitize platinum-resistant ovarian cancer cells to paraplatin.

Polyphenolic extracts of the principal flavonoid classes present in cranberry were screened in vitro for cytotoxicity against solid tumor cells lines, identifying two fractions composed principally of proanthocyanidins (PACs) with potential anticancer activity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) analysis of the proanthocyanidins (PACs) fractions indicated the presence of A-type PACs with 1-4 linkages containing between 2-8 epicatechin units with a maximum of 1 epigallocatechin unit. PACs exhibited in vitro cytotoxicity against platinum-resistant human ovarian, neuroblastoma and prostate cancer cell lines (IC50 = 79-479 microg/mL) but were non-cytotoxic to lung fibroblast cells (IC50 > 1000 microg/ml). SKOV-3 ovarian cancer cells treated with PACs exhibited classic apoptotic changes. PACs acted synergistically with paraplatin in SKOV-3 cells. Pretreatment of SKOV-3 cells with PACs (106 microg/ml) resulted in a significant reduction of the paraplatin IC50 value. Similarly, in a BrdU incorporation assay, co-treatment of SKOV-3 cells with PACs and paraplatin revealed reduced cell proliferation at lower concentrations than with either individually. In SKOV-3 cell cultures co-treated with PAC-1 and paraplatin, an HPLC analysis indicated differential quantitative presence of various PAC oligomers such as DP-8, -9, -11 and -14 indicating either selective binding or uptake. Cranberry proanthocyanidins exhibit cell-line specific cytotoxicity, induce apoptotic markers and augment cytotoxicity of paraplatin in platinum-resistant SKOV-3 ovarian cancer cells.

Human glycemic response and phenolic content of unsweetened cranberry juice.

This cross-sectional study determined the phenolic composition of an over-the-counter cranberry juice (CBJ) with high-performance liquid chromatography and examined the effects of low- and normal-calorie CBJ formulations on the postprandial glycemic response in healthy humans. The CBJ used in this study contained seven phenolic acids, with 3- and 5-caffeoylquinic acid being the primary components, and 15 flavonol glycosides, with myricetin-3-galactoside and quercetin-3-galactoside being the most prevalent. CBJ proanthocyanidins consisted of three different tetramers and a heptamer, which were confirmed with matrix-assisted laser desorption ionization-time of flight-mass spectrometry analysis. Participants received one of the following six treatments: nothing (no water/beverage), water (480 mL), unsweetened low-calorie CBJ (38 Cal/480 mL), normal-calorie CBJ (280 Cal/480 mL), isocaloric normal calorie (high fructose corn syrup [HFCS]), or isocaloric low-calorie beverages. No significant differences in postprandial blood glucose or insulin were observed in the groups receiving nothing, water, or low-calorie treatments. In contrast, the ingestion of normal-calorie CBJ and normal-calorie control beverage resulted in significantly higher blood glucose concentrations 30 minutes postprandially, although the differences were no longer significant after 180 minutes. Plasma insulin of normal-calorie CBJ and control (HFCS) recipients was significantly higher 60 minutes postprandially, but not significantly different 120 minutes postprandially. CBJ ingestion did not affect heart rate or blood pressure. This study suggests that the consumption of a low-calorie CBJ rich in previously uncharacterized trimer and heptamer proanthocyanidins is associated with a favorable glycemic response and may be beneficial for persons with impaired glucose tolerance.

Antidepressant and antistress activity of GC-MS characterized lipophilic extracts of Ginkgo biloba leaves.

Lipophilic extracts of Ginkgo biloba L. leaves were tested for their possible role on rodent models of depression and stress. Lipophilic extracts of Ginkgo leaves (LEG) at (50 and 100 mg/kg, p.o.) exhibited dose dependent, significant antidepressant activity in the behavioral despair test and learned helplessness rodent model of depression. The activities were comparable to that of imipramine (15 mg/kg) and EGb 761 (50 mg/kg). In the cold immobilization stress induced gastric ulcer model of stress, only the LEG showed a significant reduction in the ulcer index. GC-MS characterization of this bioactive extract was found to be rich in a group of 6-alkyl salicylates (6-AS), along with a fatty alcohol, fatty acids and cardanols. The n-heptadecenyl salicylate represented 60% of the 6-AS. Notable was the absence of dihydroxy alkylphenols which are linked to allergic reactions similar to the urushiols present in poison ivy. In commercial products of Ginkgo, these dihydroxy phenols as well as the favorable 6-AS are removed during enrichment of flavonol glycosides and terpenic lactones. The current findings suggest that intact carboxylic acid groups containing 6-AS are the bioactive components of the lipophilic extract of Ginkgo leaves with antidepressant and antistress activities.

Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms.

Cranberry fruit is a rich source of polyphenols, and has shown biological activities against Streptococcus mutans. In the present study, we examined the influence of extracts of flavonols (FLAV), anthocyanins (A) and proanthocyanidins (PAC) from cranberry on virulence factors involved in Streptococcus mutans biofilm development and acidogenicity. PAC and FLAV, alone or in combination, inhibited the surface-adsorbed glucosyltransferases and F-ATPases activities, and the acid production by S. mutans cells. Furthermore, biofilm development and acidogenicity were significantly affected by topical applications of PAC and FLAV (P<0.05). Anthocyanins were devoid of any significant biological effects. The flavonols are comprised of mostly quercetin glycosides, and the PAC are largely A-type oligomers of epicatechin. Our data show that proanthocyanidins and flavonols are the active constituents of cranberry against S. mutans.