An Insight into the Thermal Degradation Pathway of γ-Oryzanol and the Effect on the Oxidative Stability of Oil.

Thermal stability and antioxidant ability of γ-oryzanol in oil have been widely studied. However, further research is needed to explore its thermal degradation products and degradation pathways. The thermal degradation products of γ-oryzanol in stripped soybean oil were identified and quantified by employing high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) during heating at 180 °C. The results revealed that γ-oryzanol undergoes ester bond cleavage to form trans-ferulic acid and free sterols, and trans-ferulic acid generated intermediate compound 4-vinylguaiacol, which ultimately generated vanillin. Analysis of kinetic and thermodynamic parameters revealed the thermal stability ranking of the four components of γ-oryzanol as follows: CampFA > CAFA > 24MCAFA > SitoFA. Furthermore, γ-oryzanol exhibited superior antioxidant activity at lower temperatures. The results of this study provide a theoretical basis for a better understanding of the thermal stability and antioxidant properties of γ-oryzanol in oil under thermal oxidation conditions.

Unsaturated bond recognition leads to biased signal in a fatty acid receptor.

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.

Chemical Synthesis Enables the Configurational Determination of Myristriol, a Highly Oxygenated Phenylpropanoid from Myristica fragrans Houtt.

A new phenylpropanoid, myristriol (1), along with 11 known ones were isolated from the seed kernel of Myristica fragrans Houtt. Their chemical structures were clearly elucidated by extensive spectroscopic analysis. In which, the relative configuration of 1 was finally determined as erythro-1 by comparison the NMR data of two synthetic erythro- and threo-diastereoisomers with that of natural 1.

Integration of physicochemical, molecular dynamics, and in vitro evaluation of electrosprayed γ-oryzanol-loaded gliadin nanoparticles.

Electrospraying is a technique to improve the application and stability of bioactive compounds in food. Here, electrospraying was applied to fabricate gliadin particles incorporated γ-oryzanol. The round particles were obtained, with an average diameter of 481.56 ± 283.74 nm, from scanning electron microscopy. Simulations demonstrated how γ-oryzanol-loaded gliadin particles were unfolded in acetic acid and culminated in their globular shape under an electric field. The results also revealed that γ-oryzanol was present in gliadin particles. Moreover, there was a successful formation of particles with a homogeneous distribution and an enhanced thermostabilization of γ-oryzanol. In food simulants, γ-oryzanol demonstrated an initial burst release, followed by a subsequent, slower release that occurred gradually. Finally, MTT assays showed concentration- and time-dependent inhibitions of γ-oryzanol-loaded gliadin particles on HT-29 cells, with IC50 values of 0.47 and 0.40 mg/mL for 24 and 48 h, respectively. This study described a protocol for developing γ-oryzanol-loaded gliadin particles with enhanced stability, valuable release-behavior, and decreased HT-29 proliferation.

Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs.

There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class Gammaproteobacteria. Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates. IMPORTANCE Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens.

Ketoprofen and Loxoprofen Platinum(IV) Complexes Displaying Antimetastatic Activities by Inducing DNA Damage, Inflammation Suppression, and Enhanced Immune Response.

Metastasis is a major contributor of death in cancer patients, and there is an urgent need for effective treatments of metastatic malignancies. Herein, ketoprofen (KP) and loxoprofen (LP) platinum(IV) complexes with antiproliferative and antimetastatic properties were designed and prepared by integrating chemotherapy and immunotherapy targeting cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and programmed death ligand 1 (PD-L1), besides DNA. A mono-KP platinum(IV) complex with a cisplatin core is screened out as a candidate possessing potent anti-proliferative and anti-metastasis activities both in vitro and in vivo. It induces serious DNA damage and further leads to high expression of γ-H2AX and p53. Moreover, it promotes apoptosis of tumor cells through mitochondrial apoptotic pathway Bcl-2/Bax/caspase3. Then, COX-2, MMP-9, NLRP3, and caspase1 as pivotal enzymes igniting inflammation and metastasis are obviously inhibited. Notably, it significantly improves immune response through restraining the expression of PD-L1 to increase CD3+ and CD8+ T infiltrating cells in tumor tissues.

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling.

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (1H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.

Rice varieties with a high endosperm lipid content have reduced starch digestibility and increased γ-oryzanol bioaccessibility.

The amount and distribution of rice endosperm lipids can influence starch digestibility and nutritional properties of white rice. However, this aspect has been poorly investigated thus far. We investigated the digestion properties of five rice varieties and common rice having different lipid contents (8.1-24.2 g kg-1) showing that the lipid content is positively correlated with the resistant starch content and negatively correlated with digestion extent (C∞) and estimated glycemic index (eGI). After non-starch lipid (NSL) removal from selected high-lipid mutants (ALK3 and RS4), C∞ was significantly enhanced compared to native samples when digested by α-amylase, while this phenomenon was not observed in low-lipid rice (GZ93). When pancreatin was used, starch digestion was only delayed; triglycerides were gradually hydrolyzed by pancreatic lipase and the lipids-starch complex became no longer resistant to hydrolysis by α-amylase. These results indicated that rice endosperm lipids inhibited starch digestion, by transforming part of the starch into a slowly digestible starch fraction. High-lipid mutants also had a higher total amount of, and more bioaccessible, γ-oryzanol than low-lipid varieties. This study indicates that high-lipid white rice has great potential in designing functional rice-based foods, combining a relatively lower eGI and a high γ-oryzanol content.

Crystal Structures of the Human Peroxisome Proliferator-Activated Receptor (PPAR)α Ligand-Binding Domain in Complexes with a Series of Phenylpropanoic Acid Derivatives Generated by a Ligand-Exchange Soaking Method.

Peroxisome proliferator-activated receptor (PPAR)α, a member of the nuclear receptor family, is a transcription factor that regulates the expression of genes related to lipid metabolism in a ligand-dependent manner, and has attracted attention as a target for hypolipidemic drugs. We have been developing phenylpropaonic acid derivatives as PPARα-targeted drug candidates for the treatment of metabolic diseases. Recently, we have developed the "ligand-exchange soaking method," which crystallizes the recombinant PPARα ligand-binding domain (LBD) as a complex with intrinsic fatty acids derived from an expression host Escherichia (E.) coli and thereafter replaces them with other higher-affinity ligands by soaking. Here we applied this method for preparation of cocrystals of PPARα LBD with its ligands that have not been obtained with the conventional cocrystallization method. We revealed the high-resolution structures of the cocrystals of PPARα LBD and the three synthetic phenylpropaonic acid derivatives: TIPP-703, APHM19, and YN4pai, the latter two of which are the first observations. The overall structures of cocrystals obtained from the two methods are identical and illustrate the close interaction between these ligands and the surrounding amino acid residues of PPARα LBD. This ligand-exchange soaking method could be applicable to high throughput preparations of co-crystals with another subtype PPARδ LBD for high resolution X-ray crystallography, because it also crystallizes in complex with intrinsic fatty acid(s) while not in the apo-form.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

Chemical Genetics Reveals a Role of Squalene Synthase in TGFß Signaling and Cardiomyogenesis.

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

Copper(II) complexes with non-steroidal anti-inflammatory drugs: Structural characterization, in vitro and in silico biological profile.

Six novel copper(II) complexes with the non-steroidal anti-inflammatory drugs ibuprofen, loxoprofen, fenoprofen and clonixin as ligands were synthesized and characterized by diverse techniques including single-crystal X-ray crystallography. The in vitro scavenging activity of the complexes against 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) free radicals and the ability to reduce H2O2 were studied in the context of the antioxidant activity studies. The complexes may interact with calf-thymus DNA via intercalation as revealed by the techniques employed. The affinity of the complexes for bovine and human serum albumins was evaluated by fluorescence emission spectroscopy and the corresponding binding constants were determined. Molecular docking simulations on the crystal structure of calf-thymus DNA, human and bovine serum albumins were also employed in order to study in silico the ability of the studied compounds to bind to these target biomacromolecules, in terms of impairment of DNA and transportation through serum albumins, to explain the observed in vitro activity and to establish a possible mechanism of action.

A Simple and Efficient Method for Synthesis and Extraction of Ethyl Ferulate from γ-Oryzanol.

Ethyl ferulate (EF) is a ferulic acid (FA) derivative with high commercial value. It is not found naturally and is mostly synthesized from FA via esterification with ethanol. The present work aimed to synthesize the EF from γ-oryzanol, a natural antioxidant from rice bran oil via acid-catalyzed transethylation at refluxing temperature of ethanol. The reaction was optimized by central composite design (CCD) under response surface methodology. Based on the CCD, the optimum condition for the synthesis of EF from 0.50 g of γ-oryzanol was as follows: γ-oryzanol to ethanol ratio of 0.50:2 (g/mL), 12.30% (v/v) H2SO4, and a reaction time of 9.37 h; these conditions correspond to a maximum EF yield of 87.11%. Moreover, the optimized transethylation condition was further validated using 12.50 g of γ-oryzanol. At the end of the reaction time, distilled water was added as antisolvent to selectively crystallize the co-products, phytosterol and unreacted γ-oryzanol, by adjusting the ethanol concentration to 49.95% (v/v). The recovery yield of 83.60% with a purity of 98% of EF was achieved. In addition, the DPPH and ABTS assays showed similar antioxidant activities between the prepared and commercial EF.

Improving the nutritional profile of culinary products: oleogel-based bouillon cubes.

Structured fat phases are the basis of many consumer relevant properties of fat-containing foods. To realise a nutritional improvement - less saturated, more unsaturated fatty acids - edible oleogels could be remedy. The feasibility of traditional fat phases structured by oleogel in culinary products has been evaluated in this study. In this contribution the oleogel application in bouillon cubes as model system for culinary products is discussed. Three different gelators (sunflower wax (SFW), a mixture of ß-Sitosterol and γ-Oryzanol (SO) and ethylcellulose (EC)), at two concentration levels (5% and 10% (w/w)) each, were evaluated with respect to their physical properties, in the food matrix and application. The application of pure and structured canola oil (CO) was benchmarked against the reference, palm fat (PO). The assessment of the prototypes covered attempts to correlate the physicochemical analyses and sensory data. Organoleptic and analytical studies covered storage stability (up to 6 months) monitoring texture, color and fat oxidation. The results indicate that the substitution of palm fat by oleogel is essentially possible. The characteristics of the bouillon cubes are tuneable by gelator choice and inclusion level. Most importantly, the data show that the anticipated risk of intolerable effects of oxidation during shelf life is limited if antioxidants are used.

Prevention of UV-induced skin cancer in mice by gamma oryzanol-loaded nanoethosomes.

AIMS: Skin cancer is the most widespread cancer worldwide, mainly caused by exposure to ultraviolet radiation (UV) in sunlight. Utilizing topical preventive agents in routinely daily used cosmetics may prevent UV-related skin damages and skin cancers. γ-Oryzanol (GO) is a natural component derived from rice bran oil, with potential antioxidant and skin anti-aging properties. MAIN METHODS: We biologically thorough studied the antioxidant and anticancer effects of GO in vitro to found the effective signaling pathways, then evaluated the sun protection factor of prepared formulation, and finally investigated the long-term preventive effects of GO-loaded nanoethosomes (GO-NEs) against UVB-induced skin cancer in mice. KEY FINDINGS: GO-NEs could effectively prevent UVB-induced skin cancer. SIGNIFICANCE: Our results suggest that GO-NEs could be utilized as an innovative ingredient in cosmetics.

Supercritical CO2 extraction of lycopene from pink grapefruit (Citrus paradise Macfad) and its degradation studies during storage.

Lycopene was extracted from pink grapefruit using SC-CO2 and rice bran oil as co-solvent. Response surface methodology was employed to evaluate the individual and interactive effects of three process parameters varied at five levels i.e. pressure (250, 300, 375, 450 & 500 bar), temperature (55, 60, 70, 80 & 85 °C), and extraction time (60, 90, 135, 180 & 210 min). Single optimum point for multiple response variables was achieved at 325 bar, 64 °C, and 143 min with overall desirability of 0.92 at which 70.52 ± 3.65% (lycopene extraction efficiency) and 11154 ± 148 ppm (γ-oryzanol) were predicted. Extraction temperatures of more than 80 °C and time beyond 180 min led to the isomerization of lycopene. Lycopene storage at 3 °C, 10 °C, & 25 °C showed average k and half-life values as 0.018, 0.030, & 0.075 and 40, 23, & 9 days, respectively for first-order degradation kinetics; depicting faster degradation at higher storage temperatures.

Design and biological evaluation of cinnamic and phenylpropionic amide derivatives as novel dual inhibitors of HIV-1 protease and reverse transcriptase.

Upon the basis of both possible ligand-binding site interactions and the uniformity of key residues in active sites, a novel class of HIV-1 PR/RT dual inhibitors was designed and evaluated. Cinnamic acids or phenylpropionic acids with more flexible chain and smaller steric hindrance were introduced into the inhibitors, giving rise to significant improvement in HIV-1 RT inhibitory activity by one or two orders of magnitude, with comparable or even improved potency against PR at the same time, compared with coumarin anologues in our previous studies. Among these inhibitors, 38d displayed a 19-fold improvement in anti-PR activity with IC50 value of 0.081 nM compared to the control DRV. In addition, inhibitor 38c exhibited an excellent anti-RT IC50 value of 0.43 µM, only a 4.7-fold less potent activity than the control EFV. More significantly, the disparate ratio between HIV-1 PR and RT inhibition became more reasonable with ratio of 1: 10.4, just as 37b. Furthermore, the assays on HIV-1 late stage and early stage supported the rationality of designing dual inhibitors. The SAR data as well as molecular modeling studies provided new insight for further optimization of more potent HIV-1 PR/RT dual inhibitors.

Mechanism of action of various terpenes and phenylpropanoids against Escherichia coli and Staphylococcus aureus.

Foodborne, disease-causing microorganisms are increasingly resistant to commercial antibiotics. Thus, there is a need for the development of new agents capable of acting efficiently in the control of these pathogens. Terpenoids and phenylpropanoids stand out for having high biological activity and a broad spectrum of action. The objectives of this study were to evaluate the antibacterial potential of limonene, ß-citronellol, carvone, carvacrol, eugenol and trans-cinnamaldehyde and to investigate the mechanism of activity of these compounds against the bacteria Escherichia coli and Staphylococcus aureus. The terpene and phenylpropanoid compoundswere purchased and their antibacterial potential was assessed by macrodilution. The mechanism of action was verified by tests of potassium ion efflux, salt tolerance, extravasation of cellular contents, absorption of crystal violet and morphological changes analyzed by electron microscopy. Bacteriostatic and bactericidal effects caused by the compounds carvone, carvacrol, eugenol and transcinemaldehyde were observed in both species; antibacterial activity against only S. aureus was observed for ß-citronelol and limonene. Reduced salt tolerance was found for strains of E. coli treated with carvacrol and S. aureus treated with ß-citronelol. There was extravasation of cellular materials induced by treatments with carvone, carvacrol, eugenol and trans-cinnamaldehyde in both microorganisms. The absorption of crystal violet increased for E. coli after incubation with each treatment. Deleterious effects and morphological changes were observed. Therefore, the monoterpenes and phenylpropanoids under study are potentially applicable for antimicrobial use against E. coli and S. aureus, and the mechanism of action involves changes in membrane permeability without cell lysis.

Syringin: A Phenylpropanoid Glycoside Compound in Cirsium brevicaule A. GRAY Root Modulates Adipogenesis.

Cirsium brevicaule A. GRAY is a wild perennial herb, and its roots (CbR) have traditionally been used as both food and medicine on the Japanese islands of Okinawa and Amami. The present study evaluated the antiadipogenic effect of CbR using mouse embryonic fibroblast cell line 3T3-L1 from JCRB cell bank. Dried CbR powder was serially extracted with solvents of various polarities, and these crude extracts were tested for antiadipogenic activity. Treatment with the methanol extract of CbR showed a significant suppression of lipid accumulation in 3T3-L1 cells. Methanol extract of CbR was then fractionated and subjected to further activity analyses. The phenylpropanoid glycosidic molecule syringin was identified as an active compound. Syringin dose dependently suppressed lipid accumulation of 3T3-L1 cells without cytotoxicity, and significantly reduced the expressions of peroxisome proliferator-activated receptor gamma, the master regulator of adipogenesis, and other differentiation markers. It was demonstrated that syringin effectively enhanced the phosphorylation of the AMP-activated protein kinase and acetyl-CoA carboxylase. These results indicate that syringin attenuates adipocyte differentiation, adipogenesis, and promotes lipid metabolism; thus, syringin may potentially serve as a therapeutic candidate for treatment of obesity.

Interactions between α-tocopherol and γ-oryzanol in oil-in-water emulsions.

The interaction between antioxidants is affected by many factors, such as concentration, ratio and system. In this study, different concentrations of α-tocopherol and γ-oryzanol showed antagonistic effect in the oil-in-water emulsion, and the distribution of α-tocopherol increased in aqueous phase after combined with γ-oryzanol. The concentration could affect the degree of antagonism. According to fluorescence quenching, cyclic voltammetry measurements and the oxidative decomposition of antioxidants during storage, the inhibitory effect of γ-oryzanol on the regeneration of α-tocopherol was proposed to be responsible for the antagonism. This work can provide suggestions for studying the mechanism of antioxidant interaction in emulsion system.