Developing patient-derived organoids to demonstrate JX24120 inhibits SAMe synthesis in endometrial cancer by targeting MAT2B.

Endometrial cancer (EC) is one of the most common gynecologic malignancies, which lacking effective drugs for intractable conditions or patients unsuitable for surgeries. Recently, the patient-derived organoids (PDOs) are found feasible for cancer research and drug discoveries. Here, we have successfully established a panel of PDOs from EC and conducted drug repurposing screening and mechanism analysis for cancer treatment. We confirmed that the regulatory ß subunit of methionine adenosyltransferase (MAT2B) is highly correlated with malignant progression in endometrial cancer. Through drug screening on PDOs, we identify JX24120, chlorpromazine derivative, as a specific inhibitor for MAT2B, which directly binds to MAT2B (Kd = 4.724 µM) and inhibits the viability of EC PDOs and canonical cell lines. Correspondingly, gene editing assessment demonstrates that JX24120 suppresses tumor growth depending on the presence of MAT2B in vivo and in vitro. Mechanistically, JX24120 induces inhibition of S-adenosylmethionine (SAMe) synthesis, leading to suppressed mTORC1 signaling, abnormal energy metabolism and protein synthesis, and eventually apoptosis. Taken together, our study offers a novel approach for drug discovery and efficacy assessment by using the PDOs models. These findings suggest that JX24120 may be a potent MAT2B inhibitor and will hopefully serve as a prospective compound for endometrial cancer therapy.

Novel simultaneous analysis of 18 types of glycosaminoglycan-derived disaccharides using 4-aminobenzoic acid ethyl ester derivatization by HPLC with fluorescence detection.

Glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), heparan sulfate (HS)/heparin (HP), and keratan sulfate (KS), play pivotal roles in living organisms. Generally, GAGs are analyzed after enzymatic digestion into unsaturated or saturated disaccharides. Due to high structural similarity between disaccharides, however, separation during analysis is challenging. Additionally, little is known about the structures of GAGs and their functional relationships. Elucidating the function of GAGs requires highly sensitive quantitative analytical methods. We developed a method for the simultaneous analysis of 18 types of disaccharides derived from HA (1 type), CS/DS (7 types), HS/HP (8 types), and KS (2 types) potentially detectable in analyses of human urine. The simple method involves HPLC separation with fluorescence detection following derivatization of GAG-derived disaccharides using 4-aminobenzoic acid ethyl ester (ABEE) as a pre-labeling agent and 2-picoline borane as a reductant. The ABEE derivatization reaction can be performed under aqueous conditions, and excess derivatization reagents can be easily, rapidly, and safely removed. This method enables highly sensitive simultaneous analysis of the 18 abovementioned types of GAG-derived disaccharides using HPLC with fluorescence detection in small amounts of urine (1 mL) in a single run. The versatile method described here could be applied to the analysis of GAGs in other biological samples.

The Influence of Emodin Succinyl Ethyl Ester on Non-alcoholic Steatohepatitis Induced by a Diet High in Fructose, Cholesterol, and Fat in Mice.

Nonalcoholic steatohepatitis (NASH) is a subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis and evidence of hepatocyte injury (ballooning) and inflammation, with or without liver fibrosis. In this study, after 12 weeks of induction, the mice were treated with emodin succinyl ethyl ester (ESEE) for four weeks at doses of 10/30/90 mg/kg/d. The blood analysis of experimental endpoints showed that ESEE exhibited significant therapeutic effects on the progression of disorders of glycolipid metabolism and the induced liver injury in the model animals. Histopathological diagnosis of the liver and total triglyceride measurements revealed that ESEE had a significant therapeutic effect on the histopathological features of nonalcoholic fatty liver disease/hepatitis, such as cellular steatosis and activation of intrahepatic inflammation. Additionally, ESEE was able to improve hepatocyte fat deposition, steatosis, and the course of intrahepatic inflammatory activity. Furthermore, it showed some inhibitory effect on liver fibrosis in the model animals. In summary, this study confirms the therapeutic effects of ESEE on the NAFLD/NASH model in C57BL/6J mice induced by a high-fat, high cholesterol, and fructose diet. These effects were observed through improvements in liver function, inhibition of fibrosis, and inflammatory responses. Changes in blood glucose levels, blood lipid metabolism, liver histopathological staining, liver fibrosis staining, and related pathological scores further supported the therapeutic effects of ESEE. Therefore, this study has important implications for the exploration of novel drugs for nonalcoholic fatty liver disease.

Exploring the use of fatty acid ethyl esters as a potential natural solution for the treatment of fish parasitic diseases.

Alternatives to conventional chemical treatments for parasitic diseases in fish are needed. Microalgal-sourced fatty acid ethyl esters (FAEEs) have shown an antiparasitic effect against Gyrodactylus turnbulli infection in guppies. Here, we tested a range of commercial FAEEs of various carbon chain lengths and unsaturation levels against two fish parasites. Guppies and barramundi infected with G. turnbulli and Trichodina sp., respectively, were used. The most effective FAEE, after excluding those toxic to fish, was ethyl laurate (12:0). For both parasites, the LD50 was 18.75 µM within 250 min of incubation. Ethyl eicosapentaenoate (20:5n3) was the next most effective FAEE against G. turnbulli, and dihomo-γ-linolenic acid ethyl ester (20:3n6) and ethyl α-linolenate (18:3n3) were the next most effective against Trichodina sp. In addition, FAEEs prepared from the microalga Phaeodactylum tricornutum residue, after fucoxanthin extraction, were examined against Trichodina sp. infection in barramundi for the first time. LD85 and LD100 was achieved at 2.5 and 5 µL mL-1 of the FAEE preparation, respectively. In vivo, immersion of infected barramundi in 1.25 µL mL-1 of this preparation for 24 h reduced infection prevalence from 100% to 53% and was non-toxic to fish.

Determination of N-Acetyl-L-cysteine Ethyl Ester (NACET) by Sequential Injection Analysis.

New sequential injection analysis (SIA) methods with optical sensing for the determination of N-acetyl-L-cysteine ethyl ester (NACET) have been developed and optimized. NACET is a potential drug and antioxidant with advantageous pharmacokinetics. The methods involve the reduction of Cu(II) in its complexes with neocuproine (NCN), bicinchoninic acid (BCA), and bathocuproine disulfonic acid (BCS) to the corresponding chromophoric Cu(I) complexes by the analyte. The absorbance of the Cu(I) complexes with NCN, BCA, and BCS was measured at their maximum absorbance wavelengths of 458, 562, and 483 nm, respectively. The sensing manifold parameters and experimental conditions were optimized for each of the Cu(II) complexes used. Under optimal conditions, the corresponding linear calibration ranges, limits of detection, and sampling rates were 8.0 × 10-6-2.0 × 10-4 mol L-1, 5.5 × 10-6 mol L-1, and 60 h-1 for NCN; 6.0 × 10-6-1.0 × 10-4 mol L-1, 5.2 × 10-6 mol L-1, and 60 h-1 for BCA; and 4.0 × 10-6-1.0 × 10-4 mol L-1, 2.6 × 10-6 mol L-1, and 78 h-1 for BCS. The Cu(II)-BCS complex was found to be best performing in terms of sensitivity and sampling rate. Usual excipients in pharmaceutical preparations did not interfere with NACET analysis.

Isolation, Identification, and Biological Activities of a New Chlorin e6 Derivative.

Chlorin e6 is a well-known photosensitizer used in photodynamic diagnosis and therapy. A method for identifying and purifying a novel process-related impurity during the synthesis of chlorin e6 has been developed. Its structure was elucidated using NMR and HRMS. This new impurity is formed from chlorophyll b rather than chlorophyll a, which is the source of chlorin e6. The intermediates formed during chlorin e6 synthesis were monitored using HPLC-mass spectrometry. This new impurity was identified as rhodin g7 71-ethyl ester, the structure of which remains unknown to date. The cytotoxic effects of this novel compound in both dark and light conditions were studied against five cancer cell lines (HT29, MIA-PaCa-2, PANC-1, AsPC-1, and B16F10) and a normal cell line (RAW264.7) and compared to those of chlorin e6. Upon irradiation using a laser at 0.5 J/cm2, rhodin g7 71-ethyl ester demonstrated higher cytotoxicity (2-fold) compared to chlorin e6 in the majority of the cancer cell lines. Furthermore, this new compound exhibited higher dark cytotoxicity compared to chlorin e6. Studies on singlet oxygen generation, the accumulation in highly vascular liver tissue, and the production of reactive oxygen species in MIA-PaCa-2 cancer cells via rhodin g7 71-ethyl ester correspond to its higher cytotoxicity as a newly developed photosensitizer. Therefore, rhodin g7 71-ethyl ester could be employed as an alternative or complementary agent to chlorin e6 in the photodynamic therapy for treating cancer cells.

Lactobacillus plantarum Metabolites Elicit Anticancer Effects by Inhibiting Autophagy-Related Responses.

Lactobacillus plantarum (L. plantarum) is a probiotic that has emerged as novel therapeutic agents for managing various diseases, such as cancer, atopic dermatitis, inflammatory bowel disease, and infections. In this study, we investigated the potential mechanisms underlying the anticancer effect of the metabolites of L. plantarum. We cultured L. plantarum cells to obtain their metabolites, created several dilutions, and used these solutions to treat human colonic Caco-2 cells. Our results showed a 10% dilution of L. plantarum metabolites decreased cell viability and reduced the expression of autophagy-related proteins. Moreover, we found co-treatment with L. plantarum metabolites and chloroquine, a known autophagy inhibitor, had a synergistic effect on cytotoxicity and downregulation of autophagy-related protein expression. In conclusion, we showed the metabolites from the probiotic, L. plantarum, work synergistically with chloroquine in killing Caco-2 cells and downregulating the expression of autophagy-related proteins, suggesting the involvement of autophagy, rather than apoptosis, in their cytotoxic effect. Hence, this study provides new insights into new therapeutic methods via inhibiting autophagy.

Benefits of Icosapent Ethyl Across the Range of Kidney Function in Patients With Established Cardiovascular Disease or Diabetes: REDUCE-IT RENAL.

BACKGROUND: Chronic kidney disease is associated with adverse outcomes among patients with established cardiovascular disease (CVD) or diabetes. Commonly used medications to treat CVD are less effective among patients with reduced kidney function. METHODS: REDUCE-IT (Reduction of Cardiovascular Events with Icosapent Ethyl-Intervention Trial) was a multicenter, double-blind, placebo-controlled trial that randomly assigned statin-treated patients with elevated triglycerides (135-499 mg/dL) who had CVD or diabetes and 1 additional risk factor to treatment with icosapent ethyl (4 g daily) or placebo. Patients from REDUCE-IT were categorized by prespecified estimated glomerular filtration rate (eGFR) categories to analyze the effect of icosapent ethyl on the primary end point (composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina) and key secondary end point (a composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke). RESULTS: Among the 8179 REDUCE-IT patients, median baseline eGFR was 75 mL·min-1·1.73 m-2 (range, 17-123 mL·min-1·1.73 m-2). There were no meaningful changes in median eGFR for icosapent ethyl versus placebo across study visits. Treatment with icosapent ethyl led to consistent reduction in both the primary and key secondary composite end points across baseline eGFR categories. Patients with eGFR <60 mL·min-1·1.73 m-2 treated with icosapent ethyl had the largest absolute and similar relative risk reduction for the primary composite end point (icosapent ethyl versus placebo, 21.8% versus 28.9%; hazard ratio [HR], 0.71 [95% CI, 0.59-0.85]; P=0.0002) and key secondary composite end point (16.8% versus 22.5%; HR 0.71 [95% CI, 0.57-0.88]; P=0.001). The numeric reduction in cardiovascular death was greatest in the eGFR <60 mL·min-1·1.73 m-2 group (icosapent ethyl: 7.6%; placebo: 10.6%; HR, 0.70 [95% CI, 0.51-0.95]; P=0.02). Although patients with eGFR <60 mL·min-1·1.73 m-2 treated with icosapent ethyl had the highest numeric rates of atrial fibrillation/flutter (icosapent ethyl: 4.2%; placebo 3.0%; HR 1.42 [95% CI, 0.86-2.32]; P=0.17) and serious bleeding (icosapent ethyl: 5.4%; placebo 3.6%; HR, 1.40 [95% CI, 0.90-2.18]; P=0.13), HRs for atrial fibrillation/flutter and serious bleeding were similar across eGFR categories (P-interaction for atrial fibrillation/flutter=0.92; P-interaction for serious bleeding=0.76). CONCLUSIONS: In REDUCE-IT, icosapent ethyl reduced fatal and nonfatal ischemic events across the broad range of baseline eGFR categories. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01492361.

7-Hydroxy-2-octenoic acid-ethyl ester mixture as an UV protectant secondary metabolite of an endolichenic fungus isolated from Menegazzia terebrata.

Enodolichenic fungi (ELF) are considered a promising bio-resource since they produce a variety of novel secondary metabolites with bioactivities. Ultraviolet (UV) radiation in sunlight containing UVA and UVB can cause acute and chronic skin diseases, and the demand for UV protectants in sunscreens has been increasing. Such situations evoke the strong interest of researchers in seeking effective UV protectants from natural products. In this study, we obtained partially purified 7-hydroxy-2-octenoic acid-ethyl ester (7E) from the secondary metabolites of ELF000548, which has UVA absorption activity. The antioxidant properties were performed by in vitro tests. The superoxide anion scavenging activity and inhibition of linoleic acid peroxidation of the 7E mixture were higher than ascorbic acid (ASA) and butyl hydroxyl anisole (BHA). Furthermore, the compound recovered the damage caused by UVB irradiation and inhibited melanin synthesis. Additionally, the 7E mixture exhibited no cytotoxicity toward the mouse melanoma cell lines, B16F1 and B16F10, except for the normal cell line, HaCaT. In general, these results are the first report about bioactivities of 7E, and those demonstrated that this compound might be a UV protectant to go further study.

Sustainable bio-based antimicrobials derived from fatty acids: Synthesis, safety, and efficacy.

Some conventional sanitizers and antibiotics used in food industry may be of concerns due to generation of toxic byproducts, impact on the environment, and the emergence of antibiotic resistance bacteria. Bio-based antimicrobials can be an alternative to conventional sanitizers since they are produced from renewable resources, and the bacterial resistance to these compounds is of less concern than those of currently used antibiotics. Among the bio-based antimicrobial compounds, those produced via either fermentation or chemical synthesis by covalently or electrovalently attaching specific moieties to the fatty acid have drawn attention in recent years. Disaccharide, arginine, vitamin B1, and phenolics are linked to fatty acids resulting in the production of sophorolipid, lauric arginate ethyl ester, thiamin dilauryl sulfate, and phenolic branched-chain fatty acid, respectively, all of which are reported to exhibit antimicrobial activity by targeting the cell membrane of the bacteria. Also, studies that applied these compounds as food preservatives by combining them with other compounds or treatments have been reviewed regarding extending the shelf life and inactivating foodborne pathogens of foods and food products. In addition, the phenolic branched-chain fatty acids, which are relatively new compounds compared to the others, are highlighted in this review.

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity.

Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe 3, GL-TyrOMe 6, GL-PheOEt 11, and GL-LysOMe 21 were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 µM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits 3, 6, 11, and 21 had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe 3 and GL-PheOEt 11 were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe 3 and GL-PheOEt 11 exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates 3 and 11 significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds 3 and 11 on DENV2 E-mediated attachment. Molecular docking data revealed that Compounds 3 and 11 have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds 3 and 11 were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure−activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.

Mechanism of fungal inhibition activity of Nα-lauroyl-L-arginine ethyl ester (LAE) and potential in control of Penicillium expansum on postharvest citrus 'Benimadonna' (Citrus reticulata × Citrus sinensis).

BACKGROUND: Citrus 'Benimadonna' (Citrus reticulata × Citrus sinensis) is a high-value perishable fruit; thus there is an urgent need for a preservation technology with high effectiveness and low safety risk from industries. Nα-Lauroyl-l-arginine ethyl ester hydrochloride (LAE) was applied to enhance preservability by compounding with natamycin, and a possible fungal inhibition mechanism based on the hypothesis of an impact on the cell membrane by surfactant was investigated. RESULTS: In vitro testing showed that the minimum inhibitory concentration of LAE against Penicillium expansum (PE), isolated as the predominant spoilage-inducing fungus, was 32 mg L-1 and it was partially synergistic with natamycin. Subsequent in vivo testing proved the inhibition capacity. During 90 days' refrigerated preservation, spoilage rate was significantly decreased by preharvest spraying versus control without extra taste loss, and LAE showed an alleviating benefit on total pectin loss. Subsequently, electron microscopic imaging and intracellular protein levels of PE exposed to LAE indicated that LAE stress led to increased permeability and decreased cell integrity. Moreover, peroxidase, superoxide dismutase and catalase revealed that LAE enhanced oxidative stress, while pectinase was antagonized. CONCLUSION: The present investigation first introduced LAE as a candidate active ingredient for citrus preservative. A theoretical basis was provided for the development of preservation technology for high-value perishable fruit. According to the authors' knowledge this study is the first report on the inhibition mechanism of LAE in terms of oxidative stress. © 2022 Society of Chemical Industry.

FAEE exerts a protective effect against osteoporosis by regulating the MAPK signalling pathway.

CONTEXT: Ferulic acid ethyl ester (FAEE) is abundant in Ligusticum chuanxiong Hort. (Apiaceae) and grains, and possesses diverse biological activities; but the effects of FAEE on osteoporosis has not been reported. OBJECTIVE: This study investigated whether FAEE can attenuate osteoclastogenesis and relieve ovariectomy-induced osteoporosis via attenuating mitogen-activated protein kinase (MAPK). MATERIALS AND METHODS: We stimulated RAW 264.7 cells with receptor activator of NF-κB ligand (RANKL) followed by FAEE. The roles of FAEE in osteoclast production and osteogenic resorption of mature osteoclasts were evaluated by tartrate resistant acid phosphatase (TRAP) staining, expression of osteoclast-specific genes, proteins and MAPK. Ovariectomized (OVX) female Sprague-Dawley rats were administered FAEE (20 mg/kg/day) for 12 weeks to explore its potential in vivo, and then histology was undertaken in combination with cytokines analyses. RESULTS: FAEE suppressed RANKL-induced osteoclast formation (96 ± 0.88 vs. 15 ± 1.68) by suppressing the expression of osteoclast-specific genes, proteins and MAPK signalling pathway related proteins (p-ERK/ERK, p-JNK/JNK and p-P38/P38) in vitro. In addition, OVX rats exposed to FAEE maintained their normal calcium (Ca) (2.72 ± 0.02 vs. 2.63 ± 0.03, p < 0.05) balance, increased oestradiol levels (498.3 ± 9.43 vs. 398.7 ± 22.06, p < 0.05), simultaneously reduced levels of bone mineral density (BMD) (0.159 ± 0.0016 vs. 0.153 ± 0.0025, p < 0.05) and bone mineral content (BMC) (0.8 ± 0.0158 vs. 0.68 ± 0.0291, p < 0.01). DISCUSSION AND CONCLUSIONS: These findings suggested that FAEE could be used to ameliorate osteoporosis by the MAPK signalling pathway, suggesting that FAEE could be a potential therapeutic candidate for osteoporosis.

REDUCE-IT USA: Results From the 3146 Patients Randomized in the United States.

BACKGROUND: Some trials have found that patients from the United States derive less benefit than patients enrolled outside the United States. This prespecified REDUCE-IT (Reduction of Cardiovascular Events with Icosapent Ethyl - Intervention Trial) subgroup analysis was conducted to determine the degree of benefit of icosapent ethyl in the United States. METHODS: REDUCE-IT randomized 8179 statin-treated patients with qualifying triglycerides ≥135 and <500 mg/dL and low-density lipoprotein cholesterol >40 and ≤100 mg/dL and a history of atherosclerosis or diabetes mellitus to icosapent ethyl 4 g/d or placebo. The primary composite end point was cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or hospitalization for unstable angina. The key secondary composite end point was cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. A hierarchy was prespecified for examination of individual and composite end points. RESULTS: A total of 3146 US patients (38.5% of the trial) were randomized and followed for a median of 4.9 years; 32.3% were women and 9.7% were Hispanic. The primary composite end point occurred in 24.7% of placebo-treated patients versus 18.2% of icosapent ethyl-treated patients (hazard ratio [HR], 0.69 [95% CI, 0.59-0.80]; P=0.000001); the key secondary composite end point occurred in 16.6% versus 12.1% (HR, 0.69 [95% CI, 0.57-0.83]; P=0.00008). All prespecified hierarchical end points were meaningfully and significantly reduced, including cardiovascular death (6.7% to 4.7%; HR, 0.66 [95% CI, 0.49-0.90]; P=0.007), myocardial infarction (8.8% to 6.7%; HR, 0.72 [95% CI, 0.56-0.93]; P=0.01), stroke (4.1% to 2.6%; HR, 0.63 [95% CI, 0.43-0.93]; P=0.02), and all-cause mortality (9.8% to 7.2%; HR, 0.70 [95% CI, 0.55-0.90]; P=0.004); for all-cause mortality in the US versus non-US patients, Pinteraction=0.02. Safety and tolerability findings were consistent with the full study cohort. CONCLUSIONS: Whereas the non-US subgroup showed significant reductions in the primary and key secondary end points, the US subgroup demonstrated particularly robust risk reductions across a variety of individual and composite end points, including all-cause mortality. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01492361.

Formation of taste-active pyroglutamyl peptide ethyl esters in sake by rice koji peptidases.

Formation of taste-active pyroglutamyl (pGlu) peptide ethyl esters in sake was investigated: 2 enzymes (A and B) responsible for the esterification were purified from a rice koji extract. MADLI-TOF/TOF analysis after deglycosylation identified enzyme (A) as peptidase S28 (GenBank accession number OOO13707.1) and enzyme (B) as serine-type carboxypeptidase (accession number AO090010000534). Both enzymes hydrolyzed pGlu peptides and formed ethyl esters under sake mash conditions: acidic pH (3-4) and in ethanol (5%-20% v/v) aqueous solutions. Enzyme (A) formed pGlu penta-peptide ethyl esters from pGlu undeca-peptides by a prolyl endo-type reaction. Enzyme (B) formed (pGlu) deca-peptide and its ethyl esters from pGlu undeca-peptides in an exo-type reaction. We are the first to report the enzymatic ethyl esterification reaction in the formation of pGlu peptides by rice koji peptidases.

Superior Properties of N-Acetylcysteine Ethyl Ester over N-Acetyl Cysteine to Prevent Retinal Pigment Epithelial Cells Oxidative Damage.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats' eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

Determination of N-Acetyl-l-cysteine Ethyl Ester (NACET) by Flow Injection Analysis and Spectrophotometric Detection Using Different Thiol-Sensitive Ligands.

A new flow injection spectrophotometric method for the determination of N-acetyl-l-cysteine ethyl ester (NACET) was developed and validated. The method is based on the reduction of Cu(II)-ligand complexes to chromophoric Cu(I)-ligand complexes with the analyte. The studied ligands were neocuproine (NCN), bicinchoninic acid (BCA) and bathocuproine disulfonic acid (BCS). The absorbance of the Cu(I)-ligand complex was measured at 458, 562 and 483 nm for the reactions of NACET with NCN, BCA and BCS, respectively. The method was validated in terms of linear dynamic range, limit of detection and quantitation, accuracy, selectivity, and precision. Experimental conditions were optimized by a univariate method, yielding linear calibration curves in a concentration range from 2.0 × 10-6 mol L-1 to 2.0 × 10-4 mol L-1 using NCN; 2.0 × 10-6 mol L-1 to 1.0 × 10-4 mol L-1 using BCA and 6.0 × 10-7 mol L-1 to 1.2 × 10-4 mol L-1 using BCS. The achieved analytical frequency was 90 h-1 for all three ligands. The method was successfully employed for NACET determination in pharmaceutical preparations, indicating that this FIA method fulfilled all the essential demands for the determination of NACET in quality control laboratories, as it combined low instrument and reagent costs with a high sampling rate.

Natural flavor biosynthesis by lipase in fermented milk using in situ produced ethanol.

ABSTRACT: Many flavoring agents on the market are extracted from natural sources or synthesized chemically. Due to the disadvantages of both methods, biotechnology is becoming a promising alternative. In this study, short chain ethyl esters with fruity notes were biosynthesized in UHT whole milk via coupling ethanolic fermentation with lipase (Palatase®) transesterification. Kluyveromyces marxianus, Lactobacillus fermentum and Lb. paracasei were used for fermentation. Milk fat was esterified with in situ produced ethanol by adding lipase at 0, 8 and 24 h of fermentation. Viable cell counts and pH were monitored during 48 h fermentation period. Flavor active ethyl esters, ethanol and free fatty acids were analyzed using headspace SPME-GC. Free fatty acid levels were lower in K. marxianus samples than lactobacilli. K. marxianus produced higher amounts of ethanol and esters than lactic acid bacteria. Viable cell counts decreased after lipase application at 0 and 8 h, possibly due to fatty acid production. Addition of lipase at 24 h resulted in improved cell counts as well as ethanol and ester production in the case of K. marxianus. This study demonstrated that fermenting milk with alcohol producing cultures in conjunction with lipase application can be an alternative to artificial flavorings in fermented milks.

A self-emulsifying Omega-3 ethyl ester formulation (AquaCelle) significantly improves eicosapentaenoic and docosahexaenoic acid bioavailability in healthy adults.

PURPOSE: Application of intelligent formulation design has the ability to address the poor bioavailability and improve the fasted state bioavailability of fish oils. In this study we assessed the ability of a self-emulsifying drug delivery system (SEDDS), AquaCelle®, as an additive to enhance the oral absorption of Omega-3 ethyl esters (EE) in healthy subjects under low-fat diet conditions. METHODS: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) EE were formulated with AquaCelle®. A single dose (680 mg dose of oil containing 272 mg of EPA EE and 204 mg of DHA EE), randomized, double-blind, study measured uptake of EPA and DHA over 24 h in healthy adults. Participants were randomized into two groups, receiving either the SEDDS AquaCelle® fish oil formulation or the unformulated fish oil EE as control. RESULTS: The AquaCelle® fish oil EE formulation demonstrated instant and complete emulsification on addition to water to produce an emulsion with an average diameter of 43 µm, compared to the oil alone which did not emulsify. The study revealed a significant difference in absorption (Cmax and AUC0-24h) between the AquaCelle® group and the control group. The AquaCelle® group was capable of increasing maximum plasma concentrations and absorption (AUC0-24h) of total Omega-3 (EPA + DHA) 3.7- and 7.1-fold, respectively, compared to the control. CONCLUSION: Formulating Omega-3 EE with a SEDSS concentrate (AquaCelle®) demonstrated a significant improvement in the oral absorption of Omega-3 fatty acids without requiring a high-fat meal.

Production of melatonin and other tryptophan derivatives by Oenococcus oeni under winery and laboratory scale.

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.