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.

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.

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.

Anti-glycation and anti-oxidative effects of a phenolic-enriched maple syrup extract and its protective effects on normal human colon cells.

Oxidative stress and free radical generation accelerate the formation of advanced glycation endproducts (AGEs) which are linked to several chronic diseases. Published data suggest that phenolic-rich plant foods, show promise as natural anti-AGEs agents due to their anti-oxidation capacities. A phenolic-enriched maple syrup extract (MSX) has previously been reported to show anti-inflammatory and neuroprotective effects but its anti-AGE effects remain unknown. Therefore, herein, we investigated the anti-glycation and anti-oxidation effects of MSX using biochemical and biophysical methods. MSX (500 µg mL-1) reduced the formation of AGEs by 40% in the bovine serum albumin (BSA)-fructose assay and by 30% in the BSA-methylglyoxal (MGO) assay. MSX also inhibited the formation of crosslinks typically seen in the late stage of glycation. Circular dichroism and differential scanning calorimeter analyses demonstrated that MSX maintained the structure of BSA during glycation. In the anti-oxidant assays, MSX (61.7 µg mL-1) scavenged 50% of free radicals (DPPH assay) and reduced free radical generation by 20% during the glycation process (electron paramagnetic resonance time scan). In addition, the intracellular levels of hydrogen peroxide induced reactive oxygen species were reduced by 27-58% with MSX (50-200 µg mL-1) in normal/non-tumorigenic human colon CCD-18Co cells. Moreover, in AGEs and MGO challenged CCD-18Co cells, higher cellular viabilities and rapid extracellular signal-regulated kinase (ERK) phosphorylation were observed in MSX treated cells, indicating its protective effects against AGEs-induced cytotoxicity. Overall, this study supports the biological effects of MSX, and warrants further investigation of its potential as a dietary agent against diseases mediated by oxidative stress and inflammation.

Development of a neuroprotective potential algorithm for medicinal plants.

Medicinal plants are promising candidates for Alzheimer's disease (AD) research but there is lack of systematic algorithms and procedures to guide their selection and evaluation. Herein, we developed a Neuroprotective Potential Algorithm (NPA) by evaluating twenty-three standardized and chemically characterized Ayurvedic medicinal plant extracts in a panel of bioassays targeting oxidative stress, carbonyl stress, protein glycation, amyloid beta (Aß) fibrillation, acetylcholinesterase (AChE) inhibition, and neuroinflammation. The twenty-three herbal extracts were initially evaluated for: 1) total polyphenol content (Folin-Ciocalteu assay), 2) free radical scavenging capacity (DPPH assay), 3) ferric reducing antioxidant power (FRAP assay), 4) reactive carbonyl species scavenging capacity (methylglyoxal trapping assay), 5) anti-glycative effects (BSA-fructose, and BSA-methylglyoxal assays) and, 6) anti-Aß fibrillation effects (thioflavin-T assay). Based on assigned index scores from the initial screening, twelve extracts with a cumulative NPA score ≥40 were selected for further evaluation for their: 1) inhibitory effects on AChE activity, 2) in vitro anti-inflammatory effects on murine BV-2 microglial cells (Griess assay measuring levels of lipopolysaccharide-induced nitric oxide species), and 3) in vivo neuroprotective effects on Caenorhabditis elegans post induction of Aß1-42 induced neurotoxicity and paralysis. Among these, four extracts had a cumulative NPA score ≥60 including Phyllanthus emblica (amla; Indian gooseberry), Mucuna pruriens (velvet bean), Punica granatum (pomegranate) and Curcuma longa (turmeric; curcumin). These extracts also showed protective effects on H2O2 induced cytotoxicity in differentiated cholinergic human neuronal SH-SY5Y and murine BV-2 microglial cells and reduced tau protein levels in the SH-SY5Y neuronal cells. While published animal data support the neuroprotective effects of several of these Ayurvedic medicinal plant extracts, some remain unexplored for their anti-AD potential. Therefore, the NPA may be utilized, in part, as a strategy to help guide the selection of promising medicinal plant candidates for future AD-based research using animal models.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

Pomegranate's Neuroprotective Effects against Alzheimer's Disease Are Mediated by Urolithins, Its Ellagitannin-Gut Microbial Derived Metabolites.

Pomegranate shows neuroprotective effects against Alzheimer's disease (AD) in several reported animal studies. However, whether its constituent ellagitannins and/or their physiologically relevant gut microbiota-derived metabolites, namely, urolithins (6H-dibenzo[b,d]pyran-6-one derivatives), are the responsible bioactive constituents is unknown. Therefore, from a pomegranate extract (PE), previously reported by our group to have anti-AD effects in vivo, 21 constituents, which were primarily ellagitannins, were isolated and identified (by HPLC, NMR, and HRESIMS). In silico computational studies, used to predict blood-brain barrier permeability, revealed that none of the PE constituents, but the urolithins, fulfilled criteria required for penetration. Urolithins prevented ß-amyloid fibrillation in vitro and methyl-urolithin B (3-methoxy-6H-dibenzo[b,d]pyran-6-one), but not PE or its predominant ellagitannins, had a protective effect in Caenorhabditis elegans post induction of amyloid ß(1-42) induced neurotoxicity and paralysis. Therefore, urolithins are the possible brain absorbable compounds which contribute to pomegranate's anti-AD effects warranting further in vivo studies on these compounds.

New Antiglycative Compounds from Cumin (Cuminum cyminum) Spice.

Cumin (Cuminum cyminum L.), a widely consumed food spice, has been reported to have antiglycative effects in vitro and in vivo, but there is a paucity of data on its bioactive compounds. Herein, we report the isolation and structure elucidation (by NMR, HRESIMS, and CD) of 21 (1-21) compounds from a methanol extract of cumin seeds. The isolates included five new compounds: two sesquiterpenoids, two pairs of monoterpeneoid epimers, and a chalcone, named cuminoids A-E, respectively. The isolates were evaluated for antiglycative effects using the bovine serum albumin-fructose intrinsic fluorescence assay. At equivalent concentrations, several of the isolates, including cuminoids C-E, were more potent inhibitors than the positive control, aminoguanidine, a synthetic antiglycative agent (>50 vs 35%, respectively).