Exploring immunoregulatory properties of a phenolic-enriched maple syrup extract through integrated proteomics and in vitro assays.

Our laboratory has established a comprehensive program to investigate the phytochemical composition and nutritional/medicinal properties of phenolic-enriched maple syrup extract (MSX). Previous studies support MSX's therapeutic potential in diverse disease models, primarily through its anti-inflammatory effects. We recently demonstrated MSX's ability to regulate inflammatory signaling pathways and modulate inflammatory markers and proteins in a lipopolysaccharide (LPS)-induced peritonitis mouse model. However, MSX's immunoregulatory properties remain unknown. Herein, we investigated MSX's immunoregulatory properties for the first time using an integrated approach, combining data-dependent acquisition (DDA) and data-independent acquisition (DIA) strategies in a proteomic analysis of spleen tissue collected from the aforementioned peritonitis mouse model. Additionally, we conducted immune cell activation assays using macrophages and T lymphocytes. The DIA analysis unveiled a distinctive expression pattern involving three proteins-Krt83, Thoc2, and Vps16-which were present in both the control and MSX-treated groups but absent in the LPS-induced model group. Furthermore, proteins Ppih and Dpp9 exhibited significant reductions in the MSX-treated group. Ingenuity pathway analysis indicated that MSX may modulate several critical signaling pathways, exerting a suppressive effect on immune responses in various cell types involved in both innate and adaptive immunity. Our in vitro cell assays supported findings from the proteomics, revealing that MSX significantly reduced the levels of interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in LPS-stimulated human macrophage cells, as well as the levels of IL-2 in anti-CD3/anti-CD28-induced Jurkat T cells. Taken together, our investigations provide evidence that MSX exerts immune regulatory effects that impact both innate and adaptive immunity, which adds to the data supporting MSX's development as a functional food.

Uncovering the anti-inflammatory mechanisms of phenolic-enriched maple syrup extract in lipopolysaccharide-induced peritonitis in mice: insights from data-independent acquisition proteomics analysis.

Our group has previously reported on the phytochemical composition and biological activities of a phenolic-enriched maple syrup extract (MSX), which showed promising anti-inflammatory effects in several disease models including diabetes and Alzheimer's disease. However, the efficacious doses of MSX and its molecular targets involved in the anti-inflammatory effects are not fully elucidated. Herein, the efficacy of MSX in a peritonitis mouse model was evaluated in a dose-finding study and the underlying mechanisms were explored using data-independent acquisition (DIA) proteomics assay. MSX (at 15, 30 and 60 mg kg-1) alleviated lipopolysaccharide-induced peritonitis by reducing the levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α) in the serum and major organs of the mice. Furthermore, DIA proteomics analyses identified a panel of proteins that were significantly altered (both up- and down-regulated) in the peritonitis group, which were counteracted by the MSX treatments. MSX treatment also modulated several inflammatory upstream regulators including interferon gamma and TNF. Ingenuity pathway analysis suggested that MSX may modulate several signaling pathways in the processes of initiation of cytokine storm, activation of liver regeneration, and suppression of hepatocyte apoptosis. Together, these proteomic and in vivo findings indicate that MSX could regulate inflammation signaling pathways and modulate inflammatory markers and proteins, providing critical insight to its therapeutic potential.

A Network Pharmacology Approach to Identify Potential Molecular Targets for Cannabidiol's Anti-Inflammatory Activity.

Introduction: Published preclinical and clinical studies support the anti-inflammatory activity of CBD, but the molecular targets (e.g., genes and proteins) that are involved in its mechanisms of action remain unclear. Herein, a network-based pharmacology analysis was performed to aid in the identification of potential molecular targets for CBD's anti-inflammatory activity. Materials and Methods: Target genes and proteins were obtained from several online databases, including Swiss target prediction, Online Mendelian Inheritance in Man, and the DrugBank database. A compound-target-disease network was constructed with Cytoscape tool, and a network of protein-protein interactions was established with the Search Tool for the Retrieval of Interacting Genes/Proteins database. Lead proteins identified from the compound-target-disease network were further studied for their interactions with CBD by computational docking. In addition, biological pathways involved in CBD's anti-inflammatory activity were identified with the Gene Ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes analysis. Results: A panel of proteins, including cellular tumor antigen p53, NF-kappa-B essential modulator, tumor necrosis factor (TNF) receptor, transcription factor p65, NF-kappa-B p105, NF-kappa-B inhibitor alpha, inhibitor of nuclear factor kappa-B kinase subunit alpha, and epidermal growth factor receptor, were identified as lead targets involved in CBD's anti-inflammatory activity. This finding was further supported by molecular docking, which showed interactions between the lead proteins and CBD. In addition, several signaling pathways, including TNF, toll-like receptor, mitogen-activated protein kinases, nuclear factor kappa-light-chain-enhancer of activated B cells, and nucleotide-binding oligomerization domain-like receptors, were identified as key regulators in the mediation of CBD's anti-inflammatory activity. Conclusion: A network-based pharmacology analysis identified potential molecular targets and signaling pathways for CBD's anti-inflammatory activity. Findings from this study add to the growing body of data supporting the utilization of CBD as a promising anti-inflammatory natural product.

Anti-neuroinflammatory effects of a food-grade phenolic-enriched maple syrup extract in a mouse model of Alzheimer's disease.

Objectives: Alzheimer's disease (AD) is a growing global health crisis exacerbated by increasing life span and an aging demographic. Convergent lines of evidence, including genome-wide association studies, strongly implicate neuroinflammation in the pathogenesis of AD. Several dietary agents, including phenolic-rich foods, show promise for the prevention and/or management of AD, which in large part, has been attributed to their anti-inflammatory effects. We previously reported that a food-grade phenolic-enriched maple syrup extract (MSX) inhibited neuroinflammation in vitro but whether these effects are translatable in vivo remain unknown. Herein, we assessed MSX's ability to attenuate early neuroinflammation in a transgenic mouse model of AD.Methods: The effects of MSX on AD-related neuroinflammation was evaluated by orally administering MSX (100 and 200 mg/kg/day for 30 days) to the 3xTg-AD mouse model of AD. The expression of inflammatory markers in mouse brains were analyzed with LC-MS/MS with SWATH acquisition.Results: 3xTg-AD mice dosed orally with MSX have decreased expression of several inflammatory proteins, including, most notably, the AD risk-associated protein 'triggering receptor expressed on myeloid cells-2' (TREM2), and stimulator of interferon genes TMEM173, and suppressor of cytokine signaling-6 (SOCS6). However, this decrease in inflammation did not coincide with a decrease in pathogenic amyloid generation or lipid peroxidation.Discussion: These data demonstrate that oral administration of this maple syrup derived natural product reduces key neuroinflammatory indices of AD in the 3xTg-AD model of AD. Therefore, further studies to investigate MSX's potential as a dietary intervention strategy for AD prevention and/or management are warranted.

Hepatoprotective and anti-inflammatory effects of a standardized pomegranate (Punica granatum) fruit extract in high fat diet-induced obese C57BL/6 mice.

Diets rich in fats are linked to elevated systemic inflammation, which augments the progression of inflammatory-related disorders including non-alcoholic fatty liver disease (NAFLD) and neurodegenerative diseases. A phenolic-enriched pomegranate fruit extract (PE) was investigated for its hepatoprotective and anti-inflammatory effects in male C57BL/6 mice fed either a high-fat diet or a standard rodent diet with or without 1% of PE for 12 weeks. Mouse livers and hippocampi were evaluated for the expression of genes associated with NAFLD and inflammation by multiplexed gene analysis. PE alleviated diet-induced fatty liver and suppressed hepatic lipid regulating genes including Cd36, Fas, Acot2 and Slc27a1. In addition, PE suppressed gene expression of pro-inflammatory cytokines including Il-1α, Il-7, Il-11, Ifnα, Tnfα and Lepr in the hippocampi. Our findings support the protective effects of PE against high-fat diet-induced hepatic and neurological disease.

Thymocid®, a Standardized Black Cumin (Nigella sativa) Seed Extract, Modulates Collagen Cross-Linking, Collagenase and Elastase Activities, and Melanogenesis in Murine B16F10 Melanoma Cells.

Black cumin (Nigella sativa) seed extract has been shown to improve dermatological conditions, yet its beneficial effects for skin are not fully elucidated. Herein, Thymocid®, a chemically standardized black cumin seed extract, was investigated for its cosmeceutical potential including anti-aging properties associated with modulation of glycation, collagen cross-linking, and collagenase and elastase activities, as well as antimelanogenic effect in murine melanoma B16F10 cells. Thymocid® (50, 100, and 300 µg/mL) inhibited the formation of advanced glycation end-products (by 16.7-70.7%), collagen cross-linking (by 45.1-93.3%), collagenase activity (by 10.4-92.4%), and elastases activities (type I and III by 25.3-75.4% and 36.0-91.1%, respectively). In addition, Thymocid® (2.5-20 µg/mL) decreased melanin content in B16F10 cells by 42.5-61.6% and reduced cellular tyrosinase activity by 20.9% (at 20 µg/mL). Furthermore, Thymocid® (20 µg/mL for 72 h) markedly suppressed the mRNA expression levels of melanogenesis-related genes including microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP1), and TYRP2 to 78.9%, 0.3%, and 0.2%, respectively. Thymocid® (10 µg/mL) also suppressed the protein expression levels of MITF (by 15.2%) and TYRP1 (by 97.7%). Findings from this study support the anti-aging and antimelanogenic potential of Thymocid® as a bioactive cosmeceutical ingredient for skin care products.

Cytoprotective effects of a proprietary red maple leaf extract and its major polyphenol, ginnalin A, against hydrogen peroxide and methylglyoxal induced oxidative stress in human keratinocytes.

Phytochemicals from functional foods are common ingredients in dietary supplements and cosmetic products for anti-skin aging effects due to their antioxidant activities. A proprietary red maple (Acer rubrum) leaf extract (Maplifa™) and its major phenolic compound, ginnalin A (GA), have been reported to show antioxidant, anti-melanogenesis, and anti-glycation effects but their protective effects against oxidative stress in human skin cells remain unknown. Herein, we investigated the cytoprotective effects of Maplifa™ and GA against hydrogen peroxide (H2O2) and methylglyoxal (MGO)-induced oxidative stress in human keratinocytes (HaCaT cells). H2O2 and MGO (both at 400 µM) induced toxicity in HaCaT cells and reduced their viability to 59.2 and 61.6%, respectively. Treatment of Maplifa™ (50 µg mL-1) and GA (50 µM) increased the viability of H2O2- and MGO-treated cells by 22.0 and 15.5%, respectively. Maplifa™ and GA also showed cytoprotective effects by reducing H2O2-induced apoptosis in HaCaT cells by 8.0 and 7.2%, respectively. The anti-apoptotic effect of Maplifa™ was further supported by the decreased levels of apoptosis associated enzymes including caspases-3/7 and -8 in HaCaT cells by 49.5 and 19.0%, respectively. In addition, Maplifa™ (50 µg mL-1) and GA (50 µM) reduced H2O2- and MGO-induced reactive oxygen species (ROS) by 84.1 and 56.8%, respectively. Furthermore, flow cytometry analysis showed that Maplifa™ and GA reduced MGO-induced total cellular ROS production while increasing mitochondria-derived ROS production in HaCaT cells. The cytoprotective effects of Maplifa™ and GA in human keratinocytes support their potential utilization for cosmetic and/or dermatological applications.

Phenolic-enriched maple syrup extract protects human keratinocytes against hydrogen peroxide and methylglyoxal induced cytotoxicity.

Reactive carbonyl species including methylglyoxal (MGO) are oxidation metabolites of glucose and precursors of advanced glycation end products (AGEs). They are important mediators of cellular oxidative stress and exacerbate skin complications. Published data supports that certain phenolic compounds can exert cellular protective effects by their antioxidant activity. A phenolic-enriched maple syrup extract (MSX) was previously reported to show protective effects against AGEs- and MGO-induced cytotoxicity in human colon cells but its skin protective effects remain unknown. The protective effects of MSX were evaluated against hydrogen peroxide (H2 O2 )- and MGO-induced cytotoxicity in human keratinocytes (HaCaT cells). Cellular viability and antioxidant activity were evaluated by the luminescent cell viability CellTiter-Glo assay and the reactive oxygen species (ROS) assay, respectively. A single-cell gel electrophoresis (Comet assay) was used to measure the strand breaks in the DNA of HaCaT cells. MSX (at 50 µg/mL) ameliorated H2 O2 - and MGO-induced cytotoxicity by increasing cell viability by 21.5% and 25.9%, respectively. MSX reduced H2 O2 - and MGO-induced ROS production by 69.4% and 56.6%, respectively. MSX also reduced MGO-induced DNA damage by 47.5%. MSX showed protective effects against H2 O2 - and MGO-induced cytotoxicity in HaCaT cells supporting its potential for dermatological and/or cosmeceutical applications.

Anti-amebic effects of Chinese rhubarb (Rheum palmatum) leaves' extract, the anthraquinone rhein and related compounds.

Entamoeba histolytica infects 50 million people worldwide and causes 55 thousand fatalities every year. Current anti-amebic drugs (e.g. paromomycin) work either at the level of the intestinal lumen (where trophozoites proliferate via cell divisions) or on the invasive trophozoites that have penetrated the gut or colonized internal organs (e.g. metronidazole). Some of these drugs are highly toxic to patients, have generated trophozoite resistance, or caused mutations and cancer in laboratory animals. Thus, alternative anti-amebic compounds need to be identified to minimize the side effects (on patients) or resistance (by amebas) to current treatments. The literature suggests that anthraquinones (chemicals found in medicinal plants) have antibacterial, antiparasitic, anti-inflammatory and antioxidant properties. Here we provide experimental evidence that Chinese rhubarb (Rheum palmatum) leaves' extract (rich in the anthraquinone rhein) inhibits E. histolytica trophozoite growth in vitro. In addition, from a set of ten isolated/synthetic anthraquinones (which we suspected to have anti-amebic properties), four analogs (rhein; AHHDAC = 1-amino-4-hydroxy-9, 10-dioxo-9, 10-dihydro-anthracene-2-carboxylic acid; unisol blue AS; and sennoside B) efficiently inhibited amebic growth at EIC50 concentrations comparable to metronidazole. The mechanism of action of these compounds still needs to be determined, although anthraquinones might enhance the production of toxic oxygen metabolites as it has been suggested for various protists (e.g. Leishmania, Plasmodium, Trypanosoma). Our research is the first to explore anti-amebic effects of Chinese rhubarb leaves' extract and isolated/synthetic anthraquinones on pathogenic Entamoeba.

Barringtogenol C-type Triterpenoid Saponins from the Stem Bark of Norway Maple (Acer Platanoides).

Four new barringtogenol C-type triterpenoid saponins, namely acerplatanosides A - D (1: -4: ), along with 22 known compounds (5: -26: ), were isolated from the stem bark of Norway maple (Acer platanoides). Their structures were elucidated on the basis of comprehensive spectroscopic analyses and chemical hydrolysis. This is the first report of triterpenoid saponins isolated from Norway maple. Compounds 1, 3: , and 4: showed cytotoxicity against 4 human cancer cell lines with IC50 values ranging from 9.4 to 39.5 µM.

Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073.

Urinary tract infections (UTIs) caused by Escherichia coli create a large burden on healthcare and frequently lead to recurrent infections. Part of the success of E. coli as an uropathogenic bacterium can be attributed to its ability to form quiescent intracellular reservoirs in bladder cells and its persistence after antibiotic treatment. Cranberry juice and related products have been used for the prevention of UTIs with varying degrees of success. In this study, a group of cranberry pectic oligosaccharides (cPOS) were found to both inhibit quiescence and reduce the population of persister cells formed by the uropathogenic strain, CFT073. This is the first report detailing constituents of cranberry with the ability to modulate these important physiological aspects of uropathogenic E. coli. Further studies investigating cranberry should be keen to include oligosaccharides as part of the 'active' cocktail of chemical compounds.

Jamun (Eugenia jambolana Lam.) Fruit Extract Prevents Obesity by Modulating the Gut Microbiome in High-Fat-Diet-Fed Mice.

SCOPE: Published data support that gut microbiota play an important role in the pathological process of obesity and related metabolic disorders. In the current study, it is investigated whether a standardized extract from Jamun (Eugenia jambolana), a widely consumed tropical fruit, could alleviate obesity and alter gut microbial community in high-fat diet (HFD)-fed mice. METHODS AND RESULTS: C57BL/6 mice are fed either a standard diet (SD) or HFD with or without Jamun fruit extract (JFE; 100 mg kg-1 day-1 ) by oral gavage for 8 weeks. JFE supplementation significantly alleviated diet-induced obesity, insulin resistance, and liver steatosis. JFE supplementation also improved HFD-induced gut dysbiosis by restoring the ratio of Firmicutes to Bacteroidetes as revealed by 16S rDNA analyses. The relative abundance of certain genera, as well as levels and proportion of intestinal-derived short-chain fatty acids are improved in JFE-treated mice in comparison to the HFD-fed control group. CONCLUSION: These promising data show the potential association between gut microbiota modulation and metabolism improvement of the JFE administration, and support the utilization and further investigation of Jamun fruit as a dietary intervention strategy for the prevention of obesity and related metabolic disorders.

Pomegranate ellagitannin-gut microbial-derived metabolites, urolithins, inhibit neuroinflammation in vitro.

OBJECTIVES: Urolithins, ellagitannin-gut microbial-derived metabolites, have been reported to mediate pomegranate's neuroprotective effects against Alzheimer's disease (AD), but there are limited data on their effects against neuroinflammation. Herein, we: (1) evaluated whether urolithins (urolithins A and B and their methylated derivatives) attenuate neuroinflammation in murine BV-2 microglia and human SH-SY5Y neurons, and (2) evaluated hippocampus of transgenic AD (R1.40) mice administered a pomegranate extract (PE; 100 or 200 mg/kg/day for 3 weeks) for inflammatory biomarkers. METHODS: Effects of urolithins (10 µM) on inflammatory biomarkers were evaluated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. In a non-contact co-culture cell model, SH-SY5Y cell viability was assessed after exposure to media collected from LPS-BV-2 cells treated with or without urolithins. Effects of urolithins on apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells were assessed. Hippocampal tissues of vehicle and PE-treated transgenic R1.40 mice were evaluated for gene expression of inflammatory biomarkers by qRT-PCR. RESULTS: Urolithins decreased media levels of nitric oxide, interleukin 6 (IL-6), prostaglandin E2, and tumor necrosis factor alpha from LPS-BV-2 microglia. In the co-culture cell model, media from LPS-BV-2 cells treated with urolithins preserved SH-SY5Y cell viability greater than media from cells treated without urolithins. Urolithins mitigated apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells. While not statistically significant, inflammatory biomarkers (TNF-α, COX-2, IL-1, and IL-6) appeared to follow a decreasing trend in the hippocampus of high-dose PE-treated animals compared to controls. DISCUSSION: The attenuation of neuroinflammation by urolithins may contribute, in part, toward pomegranate's neuroprotective effects against AD.

Levodopa-Reduced Mucuna pruriens Seed Extract Shows Neuroprotective Effects against Parkinson's Disease in Murine Microglia and Human Neuroblastoma Cells, Caenorhabditis elegans, and Drosophila melanogaster.

Mucuna pruriens (Mucuna) has been prescribed in Ayurveda for various brain ailments including 'kampavata' (tremors) or Parkinson's disease (PD). While Mucuna is a well-known natural source of levodopa (L-dopa), published studies suggest that other bioactive compounds may also be responsible for its anti-PD effects. To investigate this hypothesis, an L-dopa reduced (<0.1%) M. pruriens seeds extract (MPE) was prepared and evaluated for its anti-PD effects in cellular (murine BV-2 microglia and human SH-SY5Y neuroblastoma cells), Caenorhabditis elegans, and Drosophila melanogaster models. In BV-2 cells, MPE (12.5⁻50 µg/mL) reduced hydrogen peroxide-induced cytotoxicity (15.7-18.6%), decreased reactive oxygen species production (29.1-61.6%), and lowered lipopolysaccharide (LPS)-induced nitric oxide species release by 8.9⁻60%. MPE (12.5-50 µg/mL) mitigated SH-SY5Y cell apoptosis by 6.9-40.0% in a non-contact co-culture assay with cell-free supernatants from LPS-treated BV-2 cells. MPE (12.5-50 µg/mL) reduced 6-hydroxydopamine (6-OHDA)-induced cell death of SH-SY5Y cells by 11.85⁻38.5%. Furthermore, MPE (12.5-50 µg/mL) increased median (25%) and maximum survival (47.8%) of C. elegans exposed to the dopaminergic neurotoxin, methyl-4-phenylpyridinium. MPE (40 µg/mL) ameliorated dopaminergic neurotoxin (6-OHDA and rotenone) induced precipitation of innate negative geotaxis behavior of D. melanogaster by 35.3 and 32.8%, respectively. Therefore, MPE contains bioactive compounds, beyond L-dopa, which may impart neuroprotective effects against PD.

Phenolics from Eugenia jambolana seeds with advanced glycation endproduct formation and alpha-glucosidase inhibitory activities.

Published data suggest that dietary-derived phenolics exert beneficial effects against hyperglycemia-mediated diseases, such as diabetes, through inhibiting the formation of advanced glycation endproducts (AGEs) and carbohydrate hydrolyzing enzyme activities. In the course of our investigation on the edible berry, Eugenia jambolana (known as Jamun), 21 phenolics (1-21) were isolated and identified from its seeds. Among these, one compound (1) is new and eleven compounds (3, 6, 9-13, 17, and 19-21) are being reported from E. jambolana for the first time. The anti-AGE activities of thirteen pure isolates (2-7, 9-12, 14, 15, and 20) were either comparable or superior to the synthetic anti-glycation agent, aminoguanidine, at three test concentrations (20, 50, and 100 µM) in the BSA-fructose assay. Most of these phenolics with anti-AGE activity exhibited potent free radical scavenging activity in the DPPH assay, and attenuated intracellular levels of LPS-induced reactive oxygen species in RAW264.7 macrophage. In addition, compounds 2-6, and 14 showed superior α-glucosidase inhibitory activity (IC50 = 5.0-21.2 µM) compared to the clinical α-glucosidase inhibitor, acarbose (IC50 = 289.9 µM). This is the first report of the anti-AGE effects of compounds 2-6 and 9-12, and α-glucosidase inhibitory activities of compounds 3-6, 9, 11 and 14. The current study supports the role of phenolics in the antidiabetic properties attributed to this edible berry, and warrants further animal studies to evaluate their potential as dietary agents for the prevention and/or therapy of hyperglycemia-mediated diseases.

Hypoglycemic and hypolipidemic effects of triterpenoid-enriched Jamun (Eugenia jambolana Lam.) fruit extract in streptozotocin-induced type 1 diabetic mice.

The edible berries of Eugenia jambolana Lam. (known as Jamun) are consumed in various parts of the world. Our previous studies revealed that a triterpenoid-enriched Jamun fruit extract (TJFE) showed beneficial effects on glucose homeostasis in non-diabetic mice. Herein, the anti-diabetic effects of TJFE (100 mg kg-1 by oral gavage for ten days) were evaluated in streptozotocin (STZ)-induced type 1 diabetic mice. TJFE significantly attenuated STZ-induced hyperglycemia and glucose intolerance, suppressed the abnormal elevation of hepatic gluconeogenesis, and improved dyslipidemia in the mice. Histopathology and mechanism-based studies revealed that TJFE preserved the architecture and function of pancreatic islets, attenuated insulin secretion deficiency, enhanced insulin/Akt signaling transduction, reduced lipogenic gene expression, and prevented the abnormal activation of Erk MAPK in the liver tissues of the STZ-induced diabetic mice. The current study adds to previously published data supporting the potential beneficial effects of this edible fruit on diabetes management.

Liquid chromatography coupled with time-of-flight tandem mass spectrometry for comprehensive phenolic characterization of pomegranate fruit and flower extracts used as ingredients in botanical dietary supplements.

Pomegranate (Punica granatum L.) fruit and flower extracts, which are rich sources of bioactive phenolics, are widely utilized as ingredients in botanical dietary supplements. While the phenolic characterization of extracts of pomegranate fruit has been previously studied by liquid chromatography with tandem mass spectrometry, there is lack of similar data for pomegranate flowers. Herein, liquid chromatography with time-of-flight tandem mass spectrometry was utilized to comprehensively characterize the phenolics present in two pomegranate extracts, previously studied for their in vitro and in vivo biological effects, namely, a patented commercial pomegranate fruit extract (Pomella®), and a pomegranate flower extract. Seventy-one phenolics were characterized in the pomegranate fruit extract with the vast majority identified in the flower extract. However, only the pomegranate fruit extract contained tannin-glucuronides and two punicalagin isomers (a characteristic pomegranate phenolic) were identified in this extract while four were identified in the flower extract. The previously reported compounds, pomellatannin and punicatannins A/B, were identified as unique chemical markers in the pomegranate fruit and flower extracts, respectively. This study will aid in the quality control, authentication, and standardization of these botanical ingredients to evaluate their potential health benefits in future planned pre-clinical and clinical studies. Also, this is the first phenolic characterization of a pomegranate flower extract using liquid chromatography with tandem mass spectrometry.

Ultra-fast liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry for the rapid phenolic profiling of red maple (Acer rubrum) leaves.

The red maple (Acer rubrum) species is economically important to North America because of its sap, which is used to produce maple syrup. In addition, various other red maple plant parts, including leaves, were used as a traditional medicine by the Native Americans. Currently, red maple leaves are being used for nutraceutical and cosmetic applications but there are no published analytical methods for comprehensive phytochemical characterization of this material. Herein, a rapid and sensitive method using liquid chromatography with electrospray ionization time-of-flight tandem mass spectrometry was developed to characterize the phenolics in a methanol extract of red maple leaves and a proprietary phenolic-enriched red maple leaves extract (Maplifa™). Time-of-flight mass spectrometry and tandem mass spectrometry experiments led to the identification of 106 phenolic compounds in red maples leaves with the vast majority of these compounds also detected in Maplifa™. The compounds included 68 gallotannins, 25 flavonoids, gallic acid, quinic acid, catechin, epicatechin, and nine other gallic acid derivatives among which 11 are potentially new and 75 are being reported from red maple for the first time. The developed method to characterize red maple leaves phenolics is rapid and highly sensitive and could aid in future standardization and quality control of this botanical ingredient.

Evaluation of Polyphenol Anthocyanin-Enriched Extracts of Blackberry, Black Raspberry, Blueberry, Cranberry, Red Raspberry, and Strawberry for Free Radical Scavenging, Reactive Carbonyl Species Trapping, Anti-Glycation, Anti-ß-Amyloid Aggregation, and Microglial Neuroprotective Effects.

Glycation is associated with several neurodegenerative disorders, including Alzheimer's disease (AD), where it potentiates the aggregation and toxicity of proteins such as ß-amyloid (Aß). Published studies support the anti-glycation and neuroprotective effects of several polyphenol-rich fruits, including berries, which are rich in anthocyanins. Herein, blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts were evaluated for: (1) total phenolic and anthocyanins contents, (2) free radical (DPPH) scavenging and reactive carbonyl species (methylglyoxal; MGO) trapping, (3) anti-glycation (using BSA-fructose and BSA-MGO models), (4) anti-Aß aggregation (using thermal- and MGO-induced fibrillation models), and, (5) murine microglia (BV-2) neuroprotective properties. Berry crude extracts (CE) were fractionated to yield anthocyanins-free (ACF) and anthocyanins-enriched (ACE) extracts. The berry ACEs (at 100 µg/mL) showed superior free radical scavenging, reactive carbonyl species trapping, and anti-glycation effects compared to their respective ACFs. The berry ACEs (at 100 µg/mL) inhibited both thermal- and MGO-induced Aß fibrillation. In addition, the berry ACEs (at 20 µg/mL) reduced H2O2-induced reactive oxygen species production, and lipopolysaccharide-induced nitric oxide species in BV-2 microglia as well as decreased H2O2-induced cytotoxicity and caspase-3/7 activity in BV-2 microglia. The free radical scavenging, reactive carbonyl trapping, anti-glycation, anti-Aß fibrillation, and microglial neuroprotective effects of these berry extracts warrant further in vivo studies to evaluate their potential neuroprotective effects against AD.

Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

PURPOSES: We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. RESULTS: BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. CONCLUSION: mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.