Box-Behnken Design-Based Optimization of Phytochemical Extraction from Diplazium esculentum (Retz.) Sw. Associated with Its Antioxidant and Anti-Alzheimer's Properties.

A previous study reported that the ethanolic extract of the edible fern, Diplazium esculentum (Retz.) Sw. (DE), obtained from a non-optimized extraction condition exhibited anti-Alzheimer's disease (AD) properties through the inhibition of a rate-limiting enzyme in amyloid peptide formation, ß-secretase-1 (BACE-1). Nevertheless, a non-optimized or suboptimal extraction may lead to several issues, such as a reduction in extraction efficiency and increased time and plant materials. In this study, extraction of the DE was optimized to obtain appropriate BACE-1 inhibition using a Box-Behnken design (BBD) and response surface methodology (RSM). Data revealed that the optimal extraction condition was 70% (v/v) aqueous ethanol, 50 min extraction time, 30 °C extraction temperature, and 1:30 g/mL solid/liquid ratio, giving BACE-1 inhibition at 56.33%. In addition, the extract also exhibited significant antioxidant activities compared to the non-optimized extraction. Metabolomic phytochemical profiles and targeted phytochemical analyses showed that kaempferol, quercetin, and their derivatives as well as rosmarinic acid were abundant in the extract. The optimized DE extract also acted synergistically with donepezil, an AD drug suppressing BACE-1 activities. Data received from Drosophila-expressing human amyloid precursor proteins (APPs) and BACE-1, representing the amyloid hypothesis, showed that the optimized DE extract penetrated the fly brains, suppressed BACE-1 activities, and improved locomotor functions. The extract quenched the expression of glutathione S transferase D1 (GSTD1), inositol-requiring enzyme (IRE-1), and molecular chaperone-binding immunoglobulin (Bip), while donepezil suppressed these genes and other genes involved in antioxidant and endoplasmic reticulum (ER) stress response, including superoxide dismutase type 1 (SOD1), activating transcription factor 6 (ATF-6), and protein kinase R-like endoplasmic reticulum kinase (PERK). To sum up, the optimized extraction condition reduced extraction time while resulting in higher phytochemicals, antioxidants, and BACE-1 inhibitors.

Change in the plasma proteome associated with canine cognitive dysfunction syndrome (CCDS) in Thailand.

BACKGROUND: Canine cognitive dysfunction syndrome (CCDS) is a progressive neurodegenerative disorder found in senior dogs. Due to the lack of biological markers, CCDS is commonly underdiagnosed. The aim of this study was to identify potential plasma biomarkers using proteomics techniques and to increase our understanding of the pathogenic mechanism of the disease. Plasma amyloid beta 42 (Aß42) has been seen to be a controversial biomarker for CCDS. Proteomics analysis was performed for protein identification and quantification. RESULTS: Within CCDS, ageing, and adult dogs, 87 proteins were identified specific to Canis spp. in the plasma samples. Of 87 proteins, 48 and 41 proteins were changed in the ageing and adult groups, respectively. Several distinctly expressed plasma proteins identified in CCDS were involved in complement and coagulation cascades and the apolipoprotein metabolism pathway. Plasma Aß42 levels considerably overlapped within the CCDS and ageing groups. In the adult group, the Aß42 level was low compared with that in the other groups. Nevertheless, plasma Aß42 did not show a correlation with the Canine Cognitive Dysfunction Rating scale (CCDR) score in the CCDS group (p = 0.131, R2 = 0.261). CONCLUSIONS: Our present findings suggest that plasma Aß42 does not show potential for use as a diagnostic biomarker in CCDS. The nano-LC-MS/MS data revealed that the predictive underlying mechanism of CCDS was the co-occurrence of inflammation-mediated acute phase response proteins and complement and coagulation cascades that partly functioned by apolipoproteins and lipid metabolism. Some of the differentially expressed proteins may serve as potential predictor biomarkers along with Aß42 in plasma for improved CCDS diagnosis. Further study in larger population-based cohort study is required in validation to define the correlation between protein expression and the pathogenesis of CCDS.

In vitro models to study insulin and glucocorticoids modulation of trimethyltin (TMT)-induced neuroinflammation and neurodegeneration, and in vivo validation in db/db mice.

Brain susceptibility to a neurotoxic insult may be increased in a compromised health status, such as metabolic syndrome. Both metabolic syndrome and exposure to trimethyltin (TMT) are known to promote neurodegeneration. In combination the two factors may elicit additive or compensatory/regulatory mechanisms. Combined effects of TMT exposure (0.5-1 µM) and mimicked metabolic syndrome-through modulation of insulin and glucocorticoid (GC) levels-were investigated in three models: tridimensional rat brain cell cultures for neuron-glia effects; murine microglial cell line BV-2 for a mechanistic analysis of microglial reactivity; and db/db mice as an in vivo model of metabolic syndrome. In 3D cultures, low insulin condition significantly exacerbated TMT's effect on GABAergic neurons and promoted TMT-induced neuroinflammation, with increased expression of cytokines and of the regulator of intracellular GC activity, 11ß-hydroxysteroid dehydrogenase 1 (11ß-Hsd1). Microglial reactivity increased upon TMT exposure in medium combining low insulin and high GC. These results were corroborated in BV-2 microglial cells where lack of insulin exacerbated the TMT-induced increase in 11ß-Hsd1 expression. Furthermore, TMT-induced microglial reactivity seems to depend on mineralocorticoid receptor activation. In diabetic BKS db mice, a discrete exacerbation of TMT neurotoxic effects on GABAergic neurons was observed, together with an increase of interleukin-6 (IL-6) and of basal 11ß-Hsd1 expression as compared to controls. These results suggest only minor additive effects of the two brain insults, neurotoxicant TMT exposure and metabolic syndrome conditions, where 11ß-Hsd1 appears to play a key role in the regulation of neuroinflammation and of its protective or neurodegenerative consequences.

Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells.

BACKGROUND: Activation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators. METHODS: BV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca(2+) ([Ca(2+)]i) were measured using luminescence and fluorescence assays. RESULTS: Treatment of BV-2 microglia with 100 µM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca(2+)]i, following Ca(2+) release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca(2+) chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca(2+)]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC. CONCLUSIONS: The results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca(2+)-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca(2+) release from the ER.

Enhancing Therapeutic Efficacy of Donepezil, an Alzheimer's Disease Drug, by Diplazium esculentum (Retz.) Sw. and Its Phytochemicals.

Alzheimer's disease (AD) is the most common type of dementia and a significant concern to global public health due to the prevalence of aging populations. Donepezil is one of only a few medications approved for use as an anti-AD agent but all have adverse side effects. Reducing the dosage of AD drugs with plant extracts (phytotherapy) while maintaining efficacy is one strategy to minimize adverse side effects. We previously reported the anti-AD properties of an edible fern, Diplazium esculentum (Retz.) Sw. (DE), which inhibited key enzymes involved in AD pathogenesis including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and ß-secretase 1 (BACE-1). This study aimed to determine whether DE exhibited a synergistic effect with donepezil. The enzyme inhibitory assay showed that DE extract and its bioactive compounds, kaempferol, and quercetin, slightly impeded AChE inhibition with donepezil, while DE extract and quercetin showed synergistic or additive effects with donepezil against BChE and BACE-1, respectively. DE extract combined with donepezil also improved eye phenotypes in a Drosophila model of AD by preventing ommatidia atrophia and bristle breakages. Furthermore, the DE extract exhibited no genotoxic activities, as determined by the Ames test. Our data revealed that DE extract showed promise when combined with donepezil during AD treatment by targeting BChE and BACE-1.

Multi-Endpoint Toxicological Assessment of Chrysin Loaded Oil-in-Water Emulsion System in Different Biological Models.

Chrysin is hypothesized to possess the ability to prevent different illnesses, such as diabetes, cancer, and neurodegenerative disorders. Nonetheless, chrysin has a low solubility under physiological conditions, resulting in limited bioavailability. In a previous study, we utilized an oil-in-water emulsion system (chrysin-ES or chrysin-NE) to encapsulate chrysin, thereby increasing its bioaccessibility and preserving its antioxidant and anti-Alzheimer's properties. To promote the chrysin-ES as a supplementary and functional food, it was obligatory to carry out a safety assessment. Cytotoxicity testing showed that chrysin-ES was harmless, with no killing effect on 3T3-L1 (adipocytes), RAW 264.7 (macrophages), HEK293 (kidney cells), and LX-2 (hepatic stellate cells). The acute toxicity evaluation demonstrated that the 50% lethal dose (LD50) for chrysin-ES was greater than 2000 mg/kg BW. Genotoxicity assessments found that chrysin-ES did not induce DNA mutations in vitro or in vivo. Furthermore, chrysin and chrysin-ES exhibited anti-mutagenic properties against PhIP-induced and IQ-induced mutagenesis in the Ames test, while they inhibited urethane-, ethyl methanesulfonate-, mitomycin C-, and N-nitrosomethylurea-mediated mutations in Drosophila. The present study illustrates the safety and anti-genotoxicity properties of chrysin-ES, allowing for the further development of chrysin-based food supplements and nutraceuticals.

Protein profiling and assessment of amyloid beta levels in plasma in canine refractory epilepsy.

Introduction: The relationship between epilepsy and cognitive dysfunction has been investigated in canines, and memory impairment was prevalent in dogs with epilepsy. Additionally, canines with epilepsy have greater amyloid-ß (Aß) accumulation and neuronal degeneration than healthy controls. The present study investigated plasma Aß42 levels and performed proteomic profiling in dogs with refractory epilepsy and healthy dogs. Methods: In total, eight dogs, including four healthy dogs and four dogs with epilepsy, were included in the study. Blood samples were collected to analyze Aß42 levels and perform proteomic profiling. Changes in the plasma proteomic profiles of dogs were determined by nano liquid chromatography tandem mass spectrometry. Results and discussion: The plasma Aß42 level was significantly higher in dogs with epilepsy (99 pg/mL) than in healthy dogs (5.9 pg/mL). In total, 155 proteins were identified, and of these, the expression of 40 proteins was altered in epilepsy. Among these proteins, which are linked to neurodegenerative diseases, 10 (25%) were downregulated in dogs with epilepsy, whereas 12 (30%) were upregulated. The expression of the acute phase proteins haptoglobin and α2-macroglobulin significantly differed between the groups. Complement factor H and ceruloplasmin were only detected in epilepsy dogs, suggesting that neuroinflammation plays a role in epileptic seizures. Gelsolin, which is involved in cellular processes and cytoskeletal organization, was only detected in healthy dogs. Gene Ontology annotation revealed that epilepsy can potentially interfere with biological processes, including cellular processes, localization, and responses to stimuli. Seizures compromised key molecular functions, including catalytic activity, molecular function regulation, and binding. Defense/immunity proteins were most significantly modified during the development of epilepsy. In Kyoto Encyclopedia of Genes and Genomes pathway analysis, complement and coagulation cascades were the most relevant signaling pathways affected by seizures. The findings suggested that haptoglobin, ceruloplasmin, α2-macroglobulin, complement factor H, and gelsolin play roles in canine epilepsy and Aß levels based on proteomic profiling. These proteins could represent diagnostic biomarkers that, after clinical validation, could be used in veterinary practice as well as proteins relevant to disease response pathways. To determine the precise mechanisms underlying these relationships and their implications in canine epilepsy, additional research is required.

Kaempferia parviflora Extracts Protect Neural Stem Cells from Amyloid Peptide-Mediated Inflammation in Co-Culture Model with Microglia.

The existence of neuroinflammation and oxidative stress surrounding amyloid beta (Aß) plaques, a hallmark of Alzheimer's disease (AD), has been demonstrated and may result in the activation of neuronal death and inhibition of neurogenesis. Therefore, dysregulation of neuroinflammation and oxidative stress is one possible therapeutic target for AD. Kaempferia parviflora Wall. ex Baker (KP), a member of the Zingiberaceae family, possesses health-promoting benefits including anti-oxidative stress and anti-inflammation in vitro and in vivo with a high level of safety; however, the role of KP in suppressing Aß-mediated neuroinflammation and neuronal differentiation has not yet been investigated. The neuroprotective effects of KP extract against Aß42 have been examined in both monoculture and co-culture systems of mouse neuroectodermal (NE-4C) stem cells and BV-2 microglia cells. Our results showed that fractions of KP extract containing 5,7-dimethoxyflavone, 5,7,4'-trimethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone protected neural stem cells (both undifferentiated and differentiated) and microglia activation from Aß42-induced neuroinflammation and oxidative stress in both monoculture and co-culture system of microglia and neuronal stem cells. Interestingly, KP extracts also prevented Aß42-suppressed neurogenesis, possibly due to the contained methoxyflavone derivatives. Our data indicated the promising role of KP in treating AD through the suppression of neuroinflammation and oxidative stress induced by Aß peptides.

Antidiabetic effects of Andrographis paniculata supplementation on biochemical parameters, inflammatory responses, and oxidative stress in canine diabetes.

Introduction: Diabetes mellitus is a common endocrine disorder that causes hyperglycemia in dogs. Persistent hyperglycemia can induce inflammation and oxidative stress. This study aimed to investigate the effects of A. paniculata (Burm.f.) Nees (Acanthaceae) (A. paniculata) on blood glucose, inflammation, and oxidative stress in canine diabetes. A total of 41 client-owned dogs (23 diabetic and 18 clinically healthy) were included in this double-blind, placebo-controlled trial. Methods: The diabetic dogs were further divided into two treatments protocols: group 1 received A. paniculata extract capsules (50 mg/kg/day; n = 6) or received placebo for 90 days (n = 7); and group 2 received A. paniculata extract capsules (100 mg/kg/day; n = 6) or received a placebo for 180 days (n = 4). Blood and urine samples were collected every month. No significant differences in fasting blood glucose, fructosamine, interleukin-6, tumor necrosis factor-alpha, superoxide dismutase, and malondialdehyde levels were observed between the treatment and placebo groups (p > 0.05). Results and Discussion: The levels of alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, and creatinine were stable in the treatment groups. The blood glucose levels and concentrations of inflammatory and oxidative stress markers in the client-owned diabetic dogs were not altered by A. paniculata supplementation. Furthermore, treatment with this extract did not have any adverse effects on the animals. Non-etheless, the effects of A. paniculata on canine diabetes must be appropriately evaluated using a proteomic approach and involving a wider variety of protein markers.

Prevalence and characterization of antimicrobial-resistant Escherichia coli isolated from veterinary staff, pets, and pet owners in Thailand.

BACKGROUND: Companion animals may act as antimicrobial resistance (AMR) reservoirs. This study investigated the prevalence and AMR patterns of Escherichia coli in pets and people in close contact with pets. METHODS: A total of 955 samples were collected from veterinary clinics across Thailand by rectal and skin or ear swabs from dogs and cats and fecal swabs from veterinarians, veterinary assistants, and pet owners. The minimum inhibitory concentrations (MICs) of the obtained isolates were investigated using Sensititre™ MIC plates against 21 different antimicrobial drugs. RESULTS: Escherichia coli from pets was frequently resistant to ampicillin (100%) and amoxicillin-clavulanic acid (100%), whereas E. coli from pet owners, veterinarians, and veterinary assistants was mostly resistant to tetracycline. The multiple antibiotic resistance index revealed that multidrug-resistant E. coli isolates were frequently found in dogs (34.92%), cats (62.12%), veterinarians (61.11%), veterinarian assistants (36.36%), and pet owners (47.62%). The most common AMR genes identified in this study were blaCTX-M, blaTEM, tetA, and tetB, which were associated with the antimicrobial susceptibility results. Additionally, extended-spectrum beta-lactamase (ESBL)-associated genes (i.e., blaCTX-M, blaTEM, and blaSHV) were found in 21.69%, 71.97%, 27.78%, and 21.43% of E. coli isolated from dogs, cats, veterinarians, and pet owners, respectively. CONCLUSIONS: Our findings demonstrated the presence of AMR genes, particularly ESBL-associated genes, in E. coli isolated from healthy pets and veterinarians. This implies that these sources of E. coli could potentially be reservoirs for antibiotic resistance, thereby increasing the risk of harm to both humans and animals. These findings highlight the importance of implementing effective AMR control measures in veterinary practices, as bacteria resistant to commonly used antimicrobials are present in humans and animals.

Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells.

BACKGROUND: Microglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB) pathway in murine BV-2 microglial cells were studied. METHODS: BV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11ß-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays. RESULTS: GR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11ß-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF-κB translocation Cay-10512. Moreover, an increased expression of TNFR2 was observed upon treatment with 11-dehydrocorticosterone and aldosterone, which was reversed by 11ß-HSD1 inhibitors and/or spironolactone and Cay-10512. CONCLUSIONS: A tightly coordinated GR and MR activity regulates the NF-κB pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11ß-HSD1, which is upregulated by pro-inflammatory mediators and may represent an important feedback mechanism involved in resolution of inflammation.

Corticosterone potentiates ochratoxin A-induced microglial activation.

Microglial activation in the central nervous system (CNS) has been associated with brain damage and neurodegenerative disorders. Ochratoxin A (OTA) is a mycotoxin that occurs naturally in food and feed and has been associated with neurotoxicity, while corticosteroids are CNS' physiological function modulators. This study examined how OTA affected microglia activation and how corticosteroids influenced microglial neuroinflammation. Murine microglial cells (BV-2) were stimulated by OTA, and the potentiation effects on OTA-induced inflammation were determined by corticosterone pre-treatment. Expressions of pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) were determined. Phosphorylation of mitogen-activated protein kinases (MAPKs) was analyzed by western blotting. OTA significantly increased the mRNA expression of IL-6, TNF-α, IL-1ß, and iNOS and also elevated IL-6 and NO levels. Corticosterone pre-treatment enhanced the neuroinflammatory response to OTA in a mineralocorticoid receptor (MR)-dependent mechanism, which is associated with increases in extracellular signal-regulated kinase (ERK) and p38 MAPK activation. In response to OTA, microglial cells produced pro-inflammatory cytokines and NO, while corticosterone increased OTA-induced ERK and p38 MAPK phosphorylation via MR. Findings indicated the direct role of OTA in microglia activation and neuroinflammatory response and suggested that low corticosterone concentrations in the brain exacerbated neurodegeneration.

Synergistic Antibacterial and Anti-inflammatory Activities of Ocimum tenuiflorum Ethanolic Extract against Major Bacterial Mastitis Pathogens.

Mastitis is the most prevalent global illness affecting dairy cows. This bacterial infection damages and inflames the udder tissues. Several plant extracts have demonstrated synergistic antibacterial activities with standard drugs in mastitis treatment. Scant information exists on Ocimum tenuiflorum L. This study evaluated the antibacterial activity of O. tenuiflorum extract and its interaction with antibacterial drugs against common mastitis pathogens including Staphylococcus aureus, coagulase-negative Staphylococci (CNS), Streptococcus agalactiae, and Escherichia coli. Anti-inflammatory activities in LPS-stimulated RAW264.7 macrophage cells were also studied. The O. tenuiflorum extract exhibited antibacterial activities against S. aureus, CNS, and S. agalactiae with minimum inhibitory concentration (MIC) ranging from 3.9 to 31.2 µg/mL and minimum bactericidal concentration (MBC) ranging from 15.6 to 500 µg/mL. Combinations of O. tenuiflorum with penicillin or amoxicillin-clavulanic acid showed synergistic effects against all tested strains but an additive effect with cefazolin and gentamicin. Pretreatment of the extract significantly decreased the expression of inflammatory molecules (IL-6, TNF-α, IL-1ß, iNOS, COX-2, and PGE2) generated by LPS in macrophages. Results suggested O. tenuiflorum effectiveness against various Gram-positive mastitis bacteria, with the potential to reduce antibacterial doses and combat inflammation.

Curcuminoid supplementation in canine diabetic mellitus and its complications using proteomic analysis.

Introduction: Inflammation and oxidative stress contribute to diabetes pathogenesis and consequences. Therapeutic approaches for canine diabetes remain a challenge. Curcumin has anti-inflammatory and anti-oxidative effects and is beneficial for humans with diabetes mellitus (DM); however, data on its impact on canine diabetes is limited. This study aimed to evaluate the potential for causing adverse effects, anti-inflammatory effects, anti-oxidative effects and proteomic patterns of curcuminoid supplementation on canine DM. Methods: Altogether, 18 dogs were divided into two groups: DM (n = 6) and healthy (n = 12). Curcuminoid 250 mg was given to the DM group orally daily for 180 days. Blood and urine sample collection for hematological parameters, blood biochemistry, urinalysis, oxidative stress parameters, inflammatory markers and proteomics were performed every 6 weeks. Results and discussion: Curcuminoid supplementation with standard therapy significantly decreased oxidative stress with the increased glutathione/oxidized glutathione ratio, but cytokine levels were unaffected. According to the proteomic analysis, curcuminoid altered the expression of alpha-2-HS-glycoprotein, transthyretin, apolipoprotein A-I and apolipoprotein A-IV, suggesting that curcuminoid improves insulin sensitivity and reduces cardiovascular complications. No negative impact on clinical symptoms, kidneys or liver markers was identified. This study proposed that curcuminoids might be used as a targeted antioxidant strategy as an adjunctive treatment to minimize diabetes complications in dogs.

Phikud Navakot extract attenuates lipopolysaccharide-induced inflammatory responses through inhibition of ERK1/2 phosphorylation in a coculture system of microglia and neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Phikud Navakot (PN), a mixture of nine herbal plants, is an ancient Thai traditional medicine used for relieving circulatory disorders and dizziness. PN has also shown anti-inflammatory effects in rats with acute myocardial infarction. Moreover, phytochemical-inhibiting neuroinflammation, including gallic acid, vanillic acid, ferulic acid, and rutin were detected in PN extract; however, the anti-neuroinflammatory activity of PN extract and its components in a coculture system of microglia and neuronal cells is limited. OBJECTIVE: To investigate the anti-neuroinflammatory activities of PN on lipopolysaccharide (LPS)-induced inflammation in a coculture system of microglia and neuronal cells. METHODS: ELISA and qRT-PCR were used to assess cytokine expression. The phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by Western blotting. Microglia-mediated neuroinflammation was evaluated using a BV-2 microglia-N2a neuron transwell co-culture. RESULTS: PN extract and its component, gallic acid, decreased LPS-induced the mRNA expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as well as IL-6 protein levels in both microglial monoculture and coculture systems. This was accompanied by a reduction in neurodegeneration triggered by microglia in N2a neurons with increased neuronal integrity markers (ßIII tubulin and tyrosine hydroxylase (TH)). These effects were caused by the ability of PN extract to inhibit extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. CONCLUSION: This is the first study to show that PN extract inhibits neurodegeneration in LPS-activated BV-2 microglia by targeting ERK signaling activity.

Ethanolic Fruit Extract of Emblica officinalis Suppresses Neuroinflammation in Microglia and Promotes Neurite Outgrowth in Neuro2a Cells.

Inhibiting neuroinflammation and modulating neurite outgrowth could be a promising strategy to prevent neurological disorders. Emblica officinalis (EO) may be a potent agent against them. Although EO extract reportedly has anti-inflammatory properties in macrophages, there is limited knowledge about its neuroprotective activity by suppressing microglia-mediated proinflammatory cytokine production and inducing neurite outgrowth. The present study aimed to elucidate the effect of EO fruit extract on the lipopolysaccharide- (LPS-) induced neuroinflammation using microglial (BV2) and neuroblastoma (Neuro2a) cells. The results demonstrated that, in LPS-treated BV2 cells, EO fruit extract reduced nitric oxide, interleukin-6, and tumor necrotic factor-α production. It also enhanced the neurite length of Neuro2a cells, which was linked to the upregulation of TuJ1 and MAP2 expressions. In conclusion, these findings indicate that the ethanolic extract of EO fruits has promising neuroprotective potential to exhibit antineuroinflammation activity and accelerative effect on neurite outgrowth in vitro. Therefore, EO fruit extract can be considered a novel herbal medicine candidate for managing neuroinflammatory diseases.

The influence of TAS2R38 bitter taste gene polymorphisms on obesity risk in three racially diverse groups.

OBJECTIVES: Bitter taste perception affects food preference, eating behavior, and nutrient intake. The purpose of this study was to investigate the contribution of bitter taste gene polymorphisms to body fatness as measured by percentage of body fat. METHOD: Three common single nucleotide polymorphisms (SNPs) of the TAS2R38 gene which result in amino acid changes in the protein (A49P, V262A, and I296V), were studied in three racially diverse groups: European Americans n = 313, African Americans n = 109, and Asians n = 234. RESULTS: The allele frequencies of the three SNPs were similar to previous studies. The rare haplotypes, AAI and AAV, were found in high prevalence in the African American subgroup (22.94%) and European American subgroup (6.07%). The PROP non taster; AVI/AVI diplotype was associated with a higher risk of obesity in European American and Asian but not African American subjects after age adjustment. CONCLUSIONS: TAS2R38 polymorphisms could be associated with obesity development. In addition to taste perception, nutrient sensing and energy metabolism should be studied in relation to bitter taste receptors to confirm the association between genetic polymorphisms and body fatness. Genetic polymorphisms, race, gender, and environmental factors such as dietary patterns could all contribute to body fat.

Diplazium esculentum (Retz.) Sw. reduces BACE-1 activities and amyloid peptides accumulation in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD), one type of dementia, is a complex disease affecting people globally with limited drug treatment. Thus, natural products are currently of interest as promising candidates because of their cost-effectiveness and multi-target abilities. Diplazium esculentum (Retz.) Sw., an edible fern, inhibited acetylcholinesterase in vitro, inferring that it might be a promising candidate for AD treatment by supporting cholinergic neurons. However, evidence demonstrating anti-AD properties of this edible plant via inhibiting of neurotoxic peptides production, amyloid beta (Aß), both in vitro and in vivo is lacking. Thus, the anti-AD properties of D. esculentum extract both in vitro and in Drosophila models of Aß-mediated toxicity were elucidated. Findings showed that an ethanolic extract exhibited high phenolics and flavonoids, contributing to antioxidant and inhibitory activities against AD-related enzymes. Notably, the extract acted as a BACE-1 blocker and reduced amyloid beta 42 (Aß42) peptides in Drosophila models, resulting in improved locomotor behaviors. Information gained from this study suggested that D. esculentum showed potential for AD amelioration and prevention. Further investigations in vertebrates or humans are required to determine the effective doses of D. esculentum against AD, particularly via amyloidogenic pathway.

Development of Chrysin Loaded Oil-in-Water Nanoemulsion for Improving Bioaccessibility.

Chrysin (5,7-dihydroxyflavone) is a remarkable flavonoid exhibiting many health-promoting activities, such as antioxidant, anti-inflammatory, and anti-Alzheimer's disease (AD). Nevertheless, chrysin has been addressed regarding its limited applications, due to low bioaccessibility. Therefore, to improve chrysin bioaccessibility, a colloidal delivery system involving nanoemulsion was developed as chrysin nanoemulsion (chrysin-NE) using an oil-in-water system. Our results show that chrysin can be loaded by approximately 174.21 µg/g nanoemulsion (100.29 ± 0.53% w/w) when medium chain triglyceride (MCT) oil was used as an oil phase. The nanocolloidal size, polydispersity index, and surface charge of chrysin-NE were approximately 161 nm, 0.21, and -32 mV, respectively. These properties were stable for at least five weeks at room temperature. Furthermore, in vitro chrysin bioactivities regarding antioxidant and anti-AD were maintained as pure chrysin, suggesting that multistep formulation could not affect chrysin properties. Interestingly, the developed chrysin-NE was more tolerant of gastrointestinal digestion and significantly absorbed by the human intestinal cells (Caco-2) than pure chrysin. These findings demonstrate that the encapsulation of chrysin using oil-in-water nanoemulsion could enhance the bioaccessibility of chrysin, which might be subsequently applied to food and nutraceutical industries.