Dietary Polyphenols Support Akkermansia muciniphila Growth via Mediation of the Gastrointestinal Redox Environment.

Obesity and metabolic dysfunction have been shown to be associated with overproduction of reactive oxygen species (ROS) in the gastrointestinal (GI) tract, which contributes to dysbiosis or imbalances in the gut microbiota. Recently, the reversal of dysbiosis has been observed as a result of dietary supplementation with antioxidative compounds including polyphenols. Likewise, dietary polyphenols have been associated with scavenging of GI ROS, leading to the hypothesis that radical scavenging in the GI tract is a potential mechanism for the reversal of dysbiosis. The objective of this study was to investigate the relationship between GI ROS, dietary antioxidants and beneficial gut bacterium Akkermansia muciniphila. The results of this study demonstrated A. muciniphila to be a discriminant microorganism between lean (n = 7) and obese (n = 7) mice. The relative abundance of A. muciniphila was also found to have a significant negative correlation with extracellular ROS in the GI tract as measured using fluorescent probe hydroindocyanine green. The ability of the dietary antioxidants ascorbic acid, ß-carotene and grape polyphenols to scavenge GI ROS was evaluated in tandem with their ability to support A. muciniphila bloom in lean mice (n = 20). While the relationship between GI ROS and relative abundance of A. muciniphila was conserved in lean mice, only grape polyphenols stimulated the bloom of A. muciniphila. Analysis of fecal antioxidant capacity and differences in the bioavailability of the antioxidants of interest suggested that the poor bioavailability of grape polyphenols contributes to their superior radical scavenging activity and support of A. muciniphila in comparison to the other compounds tested. These findings demonstrate the utility of the GI redox environment as a modifiable therapeutic target in the treatment of chronic inflammatory diseases like metabolic syndrome.

Elemental iron protects gut microbiota against oxygen-induced dysbiosis.

Gut dysbiosis induced by oxygen and reactive oxygen species may be related to the development of inflammation, resulting in metabolic syndrome and associated-conditions in the gut. Here we show that elemental iron can serve as an antioxidant and reverse the oxygen-induced dysbiosis. Fecal samples from three healthy donors were fermented with elemental iron and/or oxygen. 16S rRNA analysis revealed that elemental iron reversed the oxygen-induced disruption of Shannon index diversity of the gut microbiota.The bacteria lacking enzymatic antioxidant systems also increased after iron treatment. Inter-individual differences, which corresponded to iron oxidation patterns, were observed for the tested donors. Gut bacteria responding to oxygen and iron treatments were identified as guilds, among which, Escherichia-Shigella was promoted by oxygen and depressed by elemental iron, while changes in bacteria such as Bifidobacterium, Blautia, Eubacterium, Ruminococcaceae, Flavonifractor, Oscillibacter, and Lachnospiraceae were reversed by elemental iron after oxygen treatment. Short-chain fatty acid production was inhibited by oxygen and this effect was partially reversed by elemental iron. These results suggested that elemental iron can regulate the oxygen/ROS state and protect the gut microbiota from oxidative stress.

Isothiocyanate-rich moringa seed extract reduces skin inflammation in mouse ear edema model.

Background: Moringa (Moringa oleifera Lam.) seed extract (MSE) and its primary bioactive compound, moringa isothiocyanate-1(MIC-1), mitigate inflammation, oxidative stress, diabetes, and cancer in the in vivo rodent models following oral application. Purpose: To investigate the topical anti-inflammatory activity of MSE and purified MIC-1 in a TPA-induced mouse ear edema model. Study Design: The present study elucidates the topical anti-inflammatory effects and mechanisms of action of MSE, containing 38% of MIC-1 and purified MIC-1 using a mouse ear edema model utilizing 12-O-tetradecanoylphorbol-13-acetate (TPA), as the pro-inflammatory agent. Methods: A time-dependent and dose-dependent response was determined by pretreating CD-1 mice with various doses of MSE and MIC-1, positive control, dexamethasone, or vehicle control, followed by TPA, and the subsequent difference in ear thickness was measured using digital Vernier calipers. The effective doses of MSE and MIC-1were then selected to evaluate the change in weight of the ears using 6 mm biopsy punches and the results were confirmed by microscopy. Inflammatory markers were quantified with Luminex multiplex immunoassay. Results: MSE and MIC-1 were effective in a dose-dependent manner in a TPA-induced ear edema model, causing a reduction in ear thickness and a 48% and 49% decrease in ear punch weight, respectively. MSE and MIC-1 also caused a reduction in the levels of cytokine and chemokines, interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and keratinocyte chemoattractant (KC) in the ear tissue. MSE and MIC-1 reduced IL-6 expression by 84% and 78%, MCP1 by 74% and 73%, and KC by 56% and 43%, respectively. Additionally, the anti-inflammatory effect of MSE and MIC-1 was confirmed by hematoxylin and eosin (H&E) staining, used to assess the thickness of the ear swelling. MSE significantly reduced the thickness of the ears by 20% compared to TPA. Conclusion: These results reveal the topical anti-inflammatory properties of MSE, and MIC-1 likely transmitted via the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB) pathways as mentioned in previous studies. This work also suggests therapeutic uses of MSE and/or MIC-1 for skin inflammation.

Phytochemical and Agronomic Characterization of High-Flavonoid Lettuce Lines Grown under Field Conditions.

Flavonoids are antioxidant phytochemicals that confer a beneficial effect on human health. We have previously developed and characterized eight lettuce (Latuca sativa L.) lines that accumulated high levels of diverse flavonoids and their precursors in controlled environment conditions. Three Rutgers Scarlet lettuce (RSL) lines selected in tissue culture for deep-red color (RSL-NAR, RSL-NBR, RSL-NFR) accumulate anthocyanins and quercetin, three lines identified in a chemically mutagenized red lettuce population accumulate kaempferol (KfoA and KfoB) or naringenin chalcone (Nco), and two lines that were spontaneous green mutants derived from the red line RSL-NAR (GSL, GSL-DG) accumulate quercetin. These eight lines were field-grown in the Salinas Valley of California for four years together with seven control accessions of varying colors (light green, dark green, red, and dark red). At market maturity, a substantial variation in plant composition was observed, but the three RSL lines consistently accumulated high levels of cyanidin, GSL and GSL-DG accumulated the highest levels of quercetin, KfoA and KfoB accumulated kaempferol, and Nco amassed naringenin chalcone, confirming that these mutant lines produce high levels of beneficial phytochemicals under field conditions. Mutant lines and control accessions were also assessed for their biomass production (plant weight, height, and width), overall content of pigments (leaf chlorophyll and anthocyanins), resistance to diseases (downy mildew, lettuce drop, and Impatiens necrotic spot virus), postharvest quality of processed tissue (deterioration and enzymatic discoloration), and composition of 23 mineral elements. All but one mutant line had a fresh plant weight at harvest comparable to commercial leaf cultivars; only Nco plants were significantly (p < 0.05) smaller. Therefore, except for Nco, the new, flavonoid hyperaccumulating lines can be considered for field cultivation.

Moringa isothiocyanate-1 inhibits LPS-induced inflammation in mouse myoblasts and skeletal muscle.

This study aims to investigate the anti-inflammatory effects of moringa isothiocyanate-1 (MIC-1) extracted from seeds of Moringa oleifera Lam. in lipopolysaccharide (LPS)-induced inflammation models. MIC-1 decreased nitric oxide production and reduced the expression of pro-inflammatory markers (TNF-α, Ifn-α, IL-1ß, IL-6) in C2C12 myoblasts. The daily oral treatment of MIC-1 (80 mg/kg) for three days significantly reduced the expression of pro-inflammatory markers in gastrocnemius muscle tissue of LPS-treated C57BL/6 male mice. Transcriptomic analysis provided further insights into the inhibitory effects of MIC-1 on the LPS-induced inflammation, which suggested that MIC-1 affects inflammation and immunity-related genes in myoblasts and skeletal muscle tissue. MIC-1 inhibited the nuclear accumulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-treated myoblasts. Our data support the hypothesis that the MIC-1's effects in the muscle cells are mediated through the inhibition of the NF-κB translocation in the nucleus, which, in turn, results in immunomodulating and anti-inflammatory responses at the gene expression levels.

Prenylated Coumaric Acids from Artemisia scoparia Beneficially Modulate Adipogenesis.

Two new diprenylated coumaric acid isomers (1a and 1b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant's 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.

Elemental iron modifies the redox environment of the gastrointestinal tract: A novel therapeutic target and test for metabolic syndrome.

Metabolic syndrome (MetS, i.e., type 2 diabetes and obesity) is often associated with dysbiosis, inflammation, and leaky gut syndrome, which increase the content of oxygen and reactive oxygen species (ROS) in the gastrointestinal (GI) tract. Using near-infrared fluorescent, in situ imaging of ROS, we evaluated the effects of oral administration of elemental iron powder (Fe0) on luminal ROS in the GI tract and related these changes to glucose metabolism and the gut microbiome. C57Bl/6J mice fed low-fat or high-fat diets and gavaged with Fe0 (2.5 g per kg), in both single- and repeat-doses, demonstrated decreased levels of luminal ROS. Fourteen days of repeated Fe0 administration reduced hyperglycemia and improved glucose tolerance in the obese and hyperglycemic animals compared to the untreated obese controls and reduced the relative amount of iron oxides in the feces, which indicated an increased redox environment of the GI tract. We determined that Fe0 administration can also be used as a diagnostic assay to assess the GI microenvironment. Improved metabolic outcomes and decreased gastrointestinal ROS in Fe0-treated, high-fat diet-fed animals correlated with the increase in a co-abundance group of beneficial bacteria, including Lactobacillus, and the suppression of detrimental populations, including Oscillibacter, Peptococcus, and Intestinimonas. Daily Fe0 treatment also increased the relative abundance of amplicon sequence variants that lacked functional enzymatic antioxidant systems, which is consistent with the ability of Fe0 to scavenge ROS and oxygen in the GI, thus favoring the growth of oxygen-sensitive bacteria. These findings delineate a functional role for antioxidants in modification of the GI microenvironment and subsequent reversal of metabolic dysfunction.

Moringa isothiocyanate-1 regulates Nrf2 and NF-κB pathway in response to LPS-driven sepsis and inflammation.

This study aims to document the dual mode of pharmacological action of moringa isothiocyanate-1 (MIC-1) derived from seeds of Moringa oleifera Lam. Oral administration of chemically stable MIC-1 (80 mg/kg) significantly reduced the expression of inflammatory markers (Tnf-α, Ifn-α, IL-1ß, IL-6) in the liver, kidney, spleen, and colon and decreased spleen weight in the lipopolysaccharide (LPS)-induced sepsis / acute inflammation model in mice. Transcriptomic analysis of the effect of MIC-1 on the liver and in the LPS-induced RAW264.7 murine macrophage showed that MIC-1 decreases inflammation via inflammation, immunity, and oxidative stress pathways. These results are supported by the immunocytochemical observations that MIC-1 increased the nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor and decreased the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-induced macrophages. Transcriptional activation of antioxidant genes by MIC-1 translated into a reduction of reactive oxygen species (ROS) in the cytoplasm, decrease of mitochondrial superoxide content, and restoration of the mitochondrial membrane potential in the LPS-induced macrophages. Our data indicate that MIC-1 affects inflammation and oxidative stress, two key processes involved in the etiology of many chronic diseases. These effects involve upstream regulation of two key transcriptional factors regulating responses to these processes at a gene expression level.

Isolating an active and inactive CACTA transposon from lettuce color mutants and characterizing their family.

Dietary flavonoids play an important role in human nutrition and health. Flavonoid biosynthesis genes have recently been identified in lettuce (Lactuca sativa); however, few mutants have been characterized. We now report the causative mutations in Green Super Lettuce (GSL), a natural light green mutant derived from red cultivar NAR; and GSL-Dark Green (GSL-DG), an olive-green natural derivative of GSL. GSL harbors CACTA 1 (LsC1), a 3.9-kb active nonautonomous CACTA superfamily transposon inserted in the 5' untranslated region of anthocyanidin synthase (ANS), a gene coding for a key enzyme in anthocyanin biosynthesis. Both terminal inverted repeats (TIRs) of this transposon were intact, enabling somatic excision of the mobile element, which led to the restoration of ANS expression and the accumulation of red anthocyanins in sectors on otherwise green leaves. GSL-DG harbors CACTA 2 (LsC2), a 1.1-kb truncated copy of LsC1 that lacks one of the TIRs, rendering the transposon inactive. RNA-sequencing and reverse transcription quantitative PCR of NAR, GSL, and GSL-DG indicated the relative expression level of ANS was strongly influenced by the transposon insertions. Analysis of flavonoid content indicated leaf cyanidin levels correlated positively with ANS expression. Bioinformatic analysis of the cv Salinas lettuce reference genome led to the discovery and characterization of an LsC1 transposon family with a putative transposon copy number greater than 1,700. Homologs of tnpA and tnpD, the genes encoding two proteins necessary for activation of transposition of CACTA elements, were also identified in the lettuce genome.

Designing a Clinical Study With Dietary Supplements: It's All in the Details.

A successful randomized clinical trial of the effect of dietary supplements on a chosen endpoint begins with developing supporting data in preclinical studies while paying attention to easily overlooked details when planning the related clinical trial. In this perspective, we draw on our experience studying the effect of an ethanolic extract from Artemisia dracunculus L. (termed PMI-5011) on glucose homeostasis as a potential therapeutic option in providing resilience to metabolic syndrome (MetS). Decisions on experimental design related to issues ranging from choice of mouse model to dosing levels and route of administration in the preclinical studies will be discussed in terms of translation to the eventual human studies. The more complex considerations in planning the clinical studies present different challenges as these studies progress from testing the safety of the dietary supplement to assessing the effect of the dietary supplement on a predetermined clinical outcome. From the vantage point of hindsight, we will outline potential pitfalls when translating preclinical studies to clinical studies and point out details to address when designing clinical studies of dietary supplements.

Metabolomic differences between invasive alien plants from native and invaded habitats.

Globalization facilitated the spread of invasive alien species (IAS), undermining the stability of the world's ecosystems. We investigated the metabolomic profiles of three IAS species: Chromolaena odorata (Asteraceae) Datura stramonium (Solanaceae), and Xanthium strumarium (Asteraceae), comparing metabolites of individual plants in their native habitats (USA), to their invasive counterparts growing in and around Kruger National Park (South Africa, ZA). Metabolomic samples were collected using RApid Metabolome Extraction and Storage (RAMES) technology, which immobilizes phytochemicals on glass fiber disks, reducing compound degradation, allowing long-term, storage and simplifying biochemical analysis. Metabolomic differences were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) of samples eluted from RAMES disks. Partial Least Squares-Discriminant Analysis (PLS-DA) of metabolomes of individual plants allowed statistical separation of species, native and invasive populations of each species, and some populations on the same continent. Invasive populations of all species were more phytochemically diverse than their native counterparts, and their metabolomic profiles were statistically distinguishable from their native relatives. These data may elucidate the mechanisms of successful invasion and rapid adaptive evolution of IAS. Moreover, RAMES technology combined with PLS-DA statistical analysis may allow taxonomic identification of species and, possibly, populations within each species.

Proanthocyanidin-Rich Grape Seed Extract Reduces Inflammation and Oxidative Stress and Restores Tight Junction Barrier Function in Caco-2 Colon Cells.

Grape polyphenols have previously been shown to improve gut health and attenuate the symptoms of metabolic syndrome; however, the mechanism of these beneficial effects is still debated. In this study, we investigated the protective effect of proanthocyanidin-rich grape seed extract (GSE) on bacterial lipopolysaccharide (LPS)-induced oxidative stress, inflammation, and barrier integrity of human Caco-2 colon cells. GSE significantly reduced the LPS-induced intracellular reactive oxygen species (ROS) production and mitochondrial superoxide production, and upregulated the expression of antioxidant enzyme genes. GSE also restored the LPS-damaged mitochondrial function by increasing mitochondrial membrane potential. In addition, GSE increased the expression of tight junction proteins in the LPS-treated Caco-2 cells, increased the expression of anti-inflammatory cytokines, and decreased pro-inflammatory cytokine gene expression. Our findings suggest that GSE exerts its beneficial effects on metabolic syndrome by scavenging intestinal ROS, thus reducing oxidative stress, increasing epithelial barrier integrity, and decreasing intestinal inflammation.

The DESIGNER Approach Helps Decipher the Hypoglycemic Bioactive Principles of Artemisia dracunculus (Russian Tarragon).

Complementing classical drug discovery, phytochemicals act on multiple pharmacological targets, especially in botanical extracts, where they form complex bioactive mixtures. The reductionist approach used in bioactivity-guided fractionation to identify single bioactive phytochemicals is inadequate for capturing the full therapeutic potential of the (bio)chemical interactions present in such complex mixtures. This study used a DESIGNER (Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources) approach to selectively remove the known bioactives, 4'-O-methyldavidigenin (1; 4,2'-dihydroxy-4'-methoxydihydrochalcone, syn. DMC-1) and its isomer 4-O-methyldavidigenin (2; syn. DMC-2), from the mixture of phytochemicals in an ethanol extract from Artemisia dracunculus to determine to what degree the more abundant 2 accounts for the established antidiabetic effect of the A. dracunculus extract. Using an otherwise chemically intact "knock-out extract" depleted in 2 and its regioisomer, 1, in vitro and in vivo outcomes confirmed that 2 (and likely 1) acts as major bioactive(s) that enhance(s) insulin signaling in skeletal muscle, but also revealed that 2 does not account for the breadth of detectable biological activity of the extract. This is the first report of generating, at a sufficiently large preparative scale, a "knock-out extract" used as a pharmacological tool for in vitro and in vivo studies to dissect the biological impact of a designated bioactive in a complex phytochemical mixture.

Epigenome and transcriptome study of moringa isothiocyanate in mouse kidney mesangial cells induced by high glucose, a potential model for diabetic-induced nephropathy.

Moringa isothiocyanate (MIC-1) is a bioactive constituent found abundantly in Moringa oleifera which possesses antioxidant and anti-inflammation properties. However, epigenome and transcriptome effects of MIC-1 in kidney mesangial cells challenged with high glucose (HG), a pre-condition for diabetic nephropathy (DN) remain unknown. Herein, we examined the transcriptome gene expression and epigenome DNA methylation in mouse kidney mesangial cells (MES13) using next-generation sequencing (NGS) technology. After HG treatment, epigenome and transcriptome were significantly altered. More importantly, MIC-1 exposure reversed some of the changes caused by HG. Integrative analysis of RNA-Seq data identified 20 canonical pathways showing inverse correlations between HG and MIC-1. These pathways included GNRH signaling, P2Y purigenic receptor signaling pathway, calcium signaling, LPS/IL-1-mediated inhibition of RXR function, and oxidative ethanol degradation III. In terms of alteration of DNA methylation patterns, 173 differentially methylation regions (DMRs) between the HG group and low glucose (LG) group and 149 DMRs between the MIC-1 group and the HG group were found. Several HG related DMRs could be reversed by MIC-1 treatment. Integrative analysis of RNA-Seq and Methyl-Seq data yielded a subset of genes associated with HG and MIC-1, and the gene expression changes may be driven by promoter CpG status. These genes include Col4a2, Tceal3, Ret, and Agt. In summary, our study provides novel insights related to transcriptomic and epigenomic/CpG methylomic alterations in MES13 upon challenged by HG but importantly, MIC-1 treatment reverses some of the transcriptome and epigenome/CpG methylome. These results may provide potential molecular targets and therapeutic strategies for DN.

CpG methyl-seq and RNA-seq epigenomic and transcriptomic studies on the preventive effects of Moringa isothiocyanate in mouse epidermal JB6 cells induced by the tumor promoter TPA.

Epigenetic mechanisms play an important role in the early stages of carcinogenesis. Moringa isothiocyanate (MIC-1) is a major bioactive component derived from Moringa oleifera that has considerable antioxidant and anti-inflammatory effects. However, how MIC-1 influences epigenomic alterations in TPA-mediated JB6 cell carcinogenic transformation has not been evaluated. In this study, DNA and RNA isolated from TPA-induced JB6 cells in the presence or absence of MIC-1 were subjected to DNA Methyl-seq and RNA-seq to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs), respectively. When JB6 cells were challenged with TPA alone, there was a significant alteration of DEGs and DMRs; importantly, MIC-1 treatment reversed the patterns of some of the DEGs and DMRs. Transcriptome and CpG methylome profiling was performed in Ingenuity® Pathway Analysis (IPA) software to analyze the altered signaling pathways. Several anti-inflammatory responses, antioxidative stress-related pathways, and anticancer-related pathways were identified to be affected by MIC-1. These pathways included NF-kB, IL-1, LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, p53, and PTEN signaling pathways. Examination of correlations between transcriptomic and CpG methylome profiles yielded a small subset of genes, including the cancer-related genes Tmpt, Tubb3, and Muc2; the GTPases Gchfr and Igtp; and the cell cycle-related gene Cdc7. Taken together, our results show the potential contributions of epigenomic changes in DNA CpG methylation to gene expression to molecular pathways active in TPA-induced JB6 cells and demonstrate that MIC-1 can reverse these changes, supporting the potential preventive/treatment effects of MIC-1 against skin carcinogenesis.

Distinct Fractions of an Artemisia scoparia Extract Contain Compounds With Novel Adipogenic Bioactivity.

Adipocytes are important players in metabolic health and disease, and disruption of adipocyte development or function contributes to metabolic dysregulation. Hence, adipocytes are significant targets for therapeutic intervention in obesity and metabolic syndrome. Plants have long been sources for bioactive compounds and drugs. In previous studies, we screened botanical extracts for effects on adipogenesis in vitro and discovered that an ethanolic extract of Artemisia scoparia (SCO) could promote adipocyte differentiation. To follow up on these studies, we have used various separation methods to identify the compound(s) responsible for SCO's adipogenic properties. Fractions and subfractions of SCO were tested for effects on lipid accumulation and adipogenic gene expression in differentiating 3T3-L1 adipocytes. Fractions were also analyzed by Ultra Performance Liquid Chromatography- Mass Spectrometry (UPLC-MS), and resulting peaks were putatively identified through high resolution, high mass accuracy mass spectrometry, literature data, and available natural products databases. The inactive fractions contained mostly quercetin derivatives and chlorogenates, including chlorogenic acid and 3,5-dicaffeoylquinic acid, which had no effects on adipogenesis when tested individually, thus ruling them out as pro-adipogenic bioactives in SCO. Based on these studies we have putatively identified the principal constituents in SCO fractions and subfractions that promoted adipocyte development and fat cell gene expression as prenylated coumaric acids, coumarin monoterpene ethers, 6-demethoxycapillarisin and two polymethoxyflavones.

Genetic and Phytochemical Characterization of Lettuce Flavonoid Biosynthesis Mutants.

We previously developed red lettuce (Lactuca sativa L.) cultivars with high flavonoid and phenolic acid content and demonstrated their anti-diabetic effect. Here we report on developing three fertile and true-breeding lettuce lines enriched with flavonoids with reported beneficial health effects. These lines were identified in a segregating population of EMS-mutagenized red lettuce and characterized biochemically and genetically. Change in red coloration was used as a visual indicator of a mutation in a flavonoid pathway gene, leading to accumulation of flavonoid precursors of red anthocyanins. Pink-green kaempferol overproducing kfoA and kfoB mutants accumulated kaempferol to 0.6-1% of their dry weight, higher than in any vegetable reported. The yellow-green naringenin chalcone overproducing mutant (nco) accumulated naringenin chalcone, not previously reported in lettuce, to 1% dry weight, a level only observed in tomato peel. Kfo plants carried a mutation in the FLAVONOID-3' HYDROXYLASE (F3'H) gene, nco in CHALCONE ISOMERASE (CHI). This work demonstrates how non-GMO approaches can transform a common crop plant into a functional food with possible health benefits.

Moringa Isothiocyanate Activates Nrf2: Potential Role in Diabetic Nephropathy.

Moringa isothiocyanate (MIC-1) is the main active isothiocyanate found in Moringa oleifera, a plant consumed as diet and traditional herbal medicine. Compared to sulforaphane (SFN), MICs are less studied and most work have focused on its anti-inflammatory activity. The purpose of this study is to better understand the Nrf2-ARE antioxidant activity of MIC-1 and its potential in diabetic nephropathy. MIC-1 showed little toxicity from 1.25-5 µM. MIC-1 activated Nrf2-ARE at similar levels to SFN. MIC-1 also increased gene expression of downstream Nrf2 genes NQO1, HO-1, and GCLC. Protein expression of HO-1 and GCLC was elevated in MIC-1-treated cells versus control. MIC-1 suppressed pro-inflammatory cytokines in LPS-stimulated macrophages. MIC-1 reduced levels of reactive oxygen species in high glucose (HG)-treated human renal proximal tubule HK-2 cells. RNA-seq was performed to examine the transcriptome in HK-2 cells exposed to HG with or without MIC-1. Ingenuity Pathway Analysis (IPA) of RNA-seq on HK-2 cells exposed to HG identified TGFß1 and NQO1 regulation as potentially impacted and treatment of HG-exposed HK-2 cells with MIC-1 reversed the gene expression of these two pathways. Results implicate that the transcriptional regulator TGFß1 signaling is activated by HG and that MIC-1 can inhibit HG-stimulated TGFß1 activation. In summary, MIC-1 activates Nrf2-ARE signaling, increases expression of Nrf2 target genes, and suppresses inflammation, while also reducing oxidative stress and possibly TGFß1 signaling in high glucose induced renal cells. Taken together, it appears that one potential therapeutic strategy for managing DN and is currently under development in clinic is Nrf2 activation.

Tricin levels and expression of flavonoid biosynthetic genes in developing grains of purple and brown pericarp rice.

The methylated flavone tricin has been associated with numerous health benefits, including reductions in intestinal and colon cancers in animal models. Tricin is found in a wide range of plant species and in many different tissues. However, whole cereal grains, such as rice, barley, oats, and wheat, are the only food sources of tricin, which is located in the bran portion of the grain. Variation in tricin levels was found in bran from rice genotypes with light brown, brown, red, and purple pericarp color, with the purple pericarp genotypes having the highest levels of tricin. Here, we analyzed tricin and tricin derivative levels in developing pericarp and embryo samples of a purple pericarp genotype, IAC600, that had high tricin and tricin derivative levels in the bran, and a light brown pericarp genotype, Cocodrie, that had no detectable tricin or tricin derivatives in the bran. Tricin and tricin derivatives were detected in both the pericarp and embryo of IAC600 but only in the embryo of Cocodrie. The purple pericarp rice had higher total levels of free tricin plus tricin derivatives than the light brown pericarp rice. When expressed on a per grain basis, most of the tricin component of IAC600 was in the pericarp. In contrast, Cocodrie had no detectable tricin in the pericarp samples but did have detectable chrysoeriol, a precursor of tricin, in the pericarp samples. We also used RNA-Seq analysis of developing pericarp and embryo samples of the two cultivars to compare the expression of genes involved in the flavonoid biosynthetic pathway. The results presented here suggest that understanding the basis of tricin accumulation in rice pericarp may lead to an approach to increasing tricin levels in whole grain rice. From analysis of gene expression levels in the pericarp samples it appears that regulation of the flavone specific genes is independent of regulation of the anthocyanin biosynthetic genes. It therefore may be feasible to develop brown pericarp rice cultivars that accumulate tricin in the pericarp.

Potencial terapéutico de plantas utilizadas en herboristería por el Gallego Otero / Therapeutic potential of plants used in herbal medicine by Gallego Otero

Introducción: la estrecha relación entre los grupos humanos y las plantas ha permitido la aparición y el desarrollo de la herboristería tradicional. En Cuba, la vasta experiencia del científico popular Enrique Otero, conocido popularmente como "Gallego Otero" en el uso de herboristería medicinal para la sanación de la población ha tenido un alcance nacional e internacional, por lo que resulta muy necesario evaluar técnicamente algunas propiedades de las especies más comunes utilizadas en sus prácticas habituales. Objetivo: investigar el potencial antioxidante y antifúngico de especies de plantas recomendadas como medicinales, a partir de los saberes populares del Gallego Otero. Métodos: se aplicaron los procedimientos establecidos para la elaboración de los extractos vegetales y los análisis antioxidantes y antifúngicos. Los resultados de cada ensayo fueron puntuados cualitativamente de 0 a 3, indicando "0" la ausencia de actividad visible en comparación con el control negativo y representando "3" el nivel de actividad más alto. Finalmente se compararon los resultados obtenidos para cada parte de la planta analizada. Resultados: el 51% de las especies analizadas demostró actividad antifúngica y el 100% antioxidante. A nivel de hojas y tallos, un 88 y 98% respectivamente revelaron respuesta antioxidante, mientras el 47 y 37% demostraron efecto antifúngico. Conclusiones: los "Ensayos en la naturaleza", utilizados para detectar actividades bioquímicas de interés en plantas identificaron actividad antioxidante y antifúngica en hojas y tallos de 51 especies entre las plantas seleccionadas en el "Complejo Integral de Investigación y Desarrollo de plantas medicinales "Gallego Otero".

Introduction: from the very origins of humankind, the close relationship between humans and plants led to the emergence and development of traditional herbal medicine. In Cuba, Enrique Otero, the botanical healer commonly known as "Gallego Otero", has become renowned domestically and internationally for his vast knowledge and experience in herbal medicine. For this reason, there is a pressing need for the technical evaluation of the properties of the species deployed most often as part of his treatments. Objective: to investigate the antioxidant and antifungal potential of plant species recommended for medicinal treatment by Gallego Otero. Methods: established procedures were applied for the preparation of plant extracts and for the antioxidant and antifungal assays. The results of each test were qualitatively scored from 0 to 3, with "0" indicating the absence of visible activity compared to the negative control, and "3" denoting the highest level of activity. Finally, the results were compared for all different plant parts tested. Results: 51% of the species tested displayed antifungal activity, while 100% resulted in antioxidant activity. At the leaf and stem level, 88% and 98% respectively showed antioxidant activity, whereas 47% and 37% indicated antifungal activity. Conclusions: "Screens-to-Nature" technology, used for the primary detection of biochemical activities of interest in traditional herbal medicine plants, identified antioxidant and antifungal activity in leaves and stems of 51 plant species out of all the plants selected for testing at the "Gallego Otero" Medicinal Plant Research and Development Center.