Cholesterol Esterase Research Articles

Unmasking the efficacy of Skipjack Tuna (Katsuwonus Pelamis) dark muscle hydrolyzate in lipid regulation: a promising component for functional food

ABSTRACT To effectively utilize the by-products of skipjack tuna (Katsuwonus Pelamis) for the preparation of peptides with lipid-lowering activity, alkaline proteinase was selected from six commercial proteases to hydrolyze dark muscle. An L16 (54) orthogonal design was employed to optimize the hydrolysis conditions, resulting in the production of a high-peptide-content hydrolyzate named KPHs-AL. This hydrolyzate exhibited the ability to significantly reduce TG content. KPHs-AL also showed higher persuasive pancreatic lipase (PL) and cholesterol esterase (CEase) inhibitory potential than various molecular weight (Mw) fractions and other enzymatic hydrolyzates, with IC50 values of 5.14 and 8.51 mg/mL, respectively. Furthermore, KPHs-AL exerted a combined effect of regulating lipid accumulation in FAA-induced steatosis HepG2 cells, leading to decreased total cholesterol (TC) and total triglyceride (TG). Based on the high binding likelihood and interaction with specific binding sites predicted by Pepsite2, 38 peptide sequences were identified as potential PL and CEase inhibitors. Hence, KPHs-AL holds promise as a beneficial component of functional food and exhibits the potential for anti-obesity effects through the alleviation of lipid accumulation

SCD1 inhibition enhances the effector functions of CD8+ T cells via ACAT1-dependent reduction of esterified cholesterol.

We previously reported that the inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor function of CD8+ T cells indirectly via restoring production of DC recruiting chemokines by cancer cells and subsequent induction of antitumor CD8+ T cells. In this study, we investigated the molecular mechanism of direct enhancing effects of SCD1 inhibitors on CD8+ T cells. In vitro treatment of CD8+ T cells with SCD1 inhibitors enhanced IFN-γ production and cytotoxic activity of T cells along with decreased oleic acid and esterified cholesterol, which is generated by cholesterol esterase, acetyl-CoA acetyltransferase 1 (ACAT1), in CD8+ T cells. The addition of oleic acid or cholesteryl oleate reversed the enhanced functions of CD8+ T cells treated with SCD1 inhibitors. Systemic administration of SCD1 inhibitor to MCA205 tumor-bearing mice enhanced IFN-γ production of tumor-infiltrating CD8+ T cells, in which oleic acid and esterified cholesterol, but not cholesterol, were decreased. These results indicated that SCD1 suppressed effector functions of CD8+ T cells through the increased esterified cholesterol in an ACAT1-dependent manner, and SCD1 inhibition enhanced T cell activity directly through decreased esterified cholesterol. Finally, SCD1 inhibitors or ACAT1 inhibitors synergistically enhanced the antitumor effects of anti-PD-1 antibody therapy or CAR-T cell therapy in mouse tumor models. Therefore, the SCD1-ACAT1 axis is regulating effector functions of CD8+ T cells, and SCD1 inhibitors, and ACAT1 inhibitors are attractive drugs for cancer immunotherapy.

Biological Effects and Crystal X-Ray Study of Novel Schiff Base Containing Pentafluorophenyl Hydrazine: In Vitro and in Silico Studies.

A novel Schiff base namely 3,5-di-tert-butyl-6-((2-(perfluorophenyl)hydrazono)methyl)phenol was successfully synthesized and characterized using FT-IR and 1 H-NMR, 13 C-NMR, and 19 F-NMR. The crystal structure analysis of the Schiff base compound was also characterized with single crystal X-ray diffraction studies and supported the spectroscopic results. The cytotoxicity, anti-bacterial properties, and enzyme inhibition of the compound were also investigated. The molecular docking studies were performed in order to explain the interactions of the synthesized compound with target enzymes. The newly synthesized hydrazone derivative Schiff base compound showed high cellular toxicity on MCF-7 and PC-3 cells. Also, this compound caused low antibacterial effect on E. coli and S. aureus. Besides, the compound exhibited the inhibitory effect against pancreatic cholesterol esterase and carbonic anhydrase isoenzyme I, II with IC50 values 63, 99, and 188 μM, respectively. Consequently, it has been determined that the prepared Schiff base is an active compound in terms of cytotoxicity, enzyme inhibition, and anti-bacterial properties. These results provide preliminary information for some biological features of the compound and can play a major role in drug applications of the Schiff base compound.

Wolbachia interferes with Zika virus replication by hijacking cholesterol metabolism in mosquito cells.

Zika virus is a member of the arbovirus Flaviviridae family transmitted by Aedes mosquitos and it is associated with microcephaly in infants born to infected mothers. Wolbachia is an intracellular gram-negative alpha-proteobacteria that infects many species of arthropods, including mosquitos. The presence of Wolbachia in mosquitos has been shown to control the vector population and suppress arbovirus transmission. One mechanism of Wolbachia-mediated interference with virus replication is competition over host resources between Wolbachia and the virus. We hypothesize that cholesterol metabolism is involved in Wolbachia-mediated virus suppression due to its important role in Zika virus replication. In this study, we determined that Wolbachia impacted virus replication by altering cholesterol biosynthesis in Aedes albopictus C6/36 cells, diverting resources from the host cell mevalonate (MVA) pathway to fulfill the needs of the bacteria. This resulted in a decrease of total cholesterol, increased Wolbachia loads, and decreased viral titers. Inhibition of the MVA pathway using fluvastatin decreased total cholesterol and viral titers, mimicking the effects of Wolbachia on the virus in Wolbachia-free cells. We also found that Wolbachia-infected cells had depleted lipid droplets, the main component of which is cholesterol esters. We confirmed that cholesterol esterases were upregulated in response to virus infection in C6/36 cells. Functional analysis showed that alteration of cholesterol metabolism simulated Wolbachia-mediated inhibition of virus infection in C6/36 cells. Our study provides a mechanism behind Wolbachia-induced interference of arbovirus replication and could help advance strategies to control arbovirus pathogens in insect vectors and human infections. IMPORTANCE Arthropod-borne viruses are emerging pathogens that are spread widely by mosquitos. Zika virus is an arbovirus that can infect humans and be transmitted from an infected mother to the fetus, potentially leading to microcephaly in infants. One promising strategy to prevent disease caused by arboviruses is to target the insect vector population. Recent field studies have shown that mosquito populations infected with Wolbachia bacteria suppress arbovirus replication and transmission. Here, we describe how intracellular bacteria redirect resources within their host cells and suppress Zika virus replication at the cellular level. Understanding the mechanism behind Wolbachia-induced interference of arbovirus replication could help advance strategies to control arbovirus pathogens in insect vectors and human populations.

Highly sensitive electrochemical detection of cholesterol based on Au–Pt NPs/PAMAM–ZIF-67 nanomaterials

A cholesterol biosensor was constructed by bimetallic (Au and Pt) and poly(amidoamine)-zeolite imidazole framework (PAMAM–ZIF-67). First, PAMAM–ZIF-67 nanomaterial was immobilized onto the electrode, and then Au and Pt were modified on the electrode by the electro-deposition method. Subsequently, cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) were fixed on the electrode. The stepwise modification procedures were recorded by impedance spectroscopy and voltammetry. The current response presented a linear relation to the logarithm of cholesterol content when content ranged between 0.00015 and 10.24 mM, and the minimum detection concentration reached 3 nM. The electrode was also used for the cholesterol assay in serum, which hinted at its potentially valuable in clinical diagnostics.Graphical abstractAn electrochemical biosensor based on gold nanoparticles, platinum nanoparticles, and polyamide–zeolitic imidazolate frameworks was developed for detection of cholesterol. First, polyamide–zeolitic imidazolate frameworks nanomaterial was fixed onto the electrode modified of mercaptopropionic acid by Au–S bond. Then, gold nanoparticles and platinum nanoparticles were electrodeposited on the above electrode. Subsequently, cholesterol oxidase and cholesterol esterase were co-immobilized on the surface of the modified electrode to fabricate the cholesterol biosensor. The biosensor has also been used for the measurement of cholesterol in human serum, which implied potential applications in biotechnology and clinical diagnostics.

Maximizing hypocholesterolemic peptides from an olive byproduct by enzymatic hydrolysis

Novel food proteases (Maxipro A, Maxipro B, Maxipro C, and Maxipro D) with broad specificity and different effective pH range (from 2.5 to 9.0) were evaluated to produce hypocholesterolemic peptides from olive seeds proteins. Box-Behnken experimental design was employed to evaluate the effect of the hydrolysis time, pH, temperature, and enzyme:substrate ratio on the amount of released peptides. The production of peptides, under optimal conditions, was higher with Maxipro C and lower with Maxipro B. The capability of extracts to reduce the absorption of exogenous cholesterol and the production of endogenous cholesterol was also evaluated. All hydrolysates showed a higher capacity to bind bile acids and inhibit cholesterol esterase enzyme than the obtained with usual Alcalase enzyme. Proteomic analysis using high resolution RP-HPLC-ESI-QTOF with database searching and de novo identification was employed to identify peptides in hydrolysates. Some sequences with hypocholesterolemic features were detected among the 45 peptides identified in Maxipro A hydrolysate, the 56 peptides in that of Maxipro C, and the 50 in the Maxipro B one. This work demonstrates that Maxipro A enzyme can hydrolyze olive seeds proteins, under acidic pH, releasing peptides with multifunctional and higher hypocholesterolemic capacity than the obtained with Alcalase, under alkaline conditions.

Triterpenoids from Protorhus longifolia Exhibit Hypocholesterolemic Potential via Regulation of Cholesterol Biosynthesis and Stimulation of Low-Density Lipoprotein Uptake in HepG2 Cells.

The increasing incidence of hypercholesterolemia-related diseases even in the presence of the currently available cholesterol-lowering drugs indicates a need to discover new therapeutic drugs. This study aimed to investigate the hypocholesterolemic potential of two triterpenoids isolated from Protorhus longifolia stem bark. In silico techniques and in vitro enzyme assays were used to evaluate the potential inhibition of cholesterol esterase and HMG-CoA reductase by the triterpenoids (ARM-2 and RA-5). The toxicity, modulation of low-density lipoprotein (LDL) uptake, and associated gene expression were determined in HepG2 hepatocytes. In silico molecular docking revealed that ARM-2 compared with RA-5 has a relatively stronger binding affinity for both enzymes. Both triterpenoids further demonstrated promising in silico drug-likeness properties and favorable ADMET profiles characterized by high intestinal absorption and lack of CYP450 enzyme inhibition. The compounds further showed, to varying degrees of efficacy, inhibition of cholesterol micellization as well as both cholesterol esterase and HMG-CoA reductase activities with IC50 values ranging from 16.4 to 41.1 μM. Moreover, enhanced hepatic cellular LDL uptake and the associated upregulation of the LDL-R and SREBP-2 gene expression were observed in the triterpenoid-treated HepG2 cells. It is evident that the triterpenoids, especially ARM-2, possess hypocholesterolemic properties, and these molecules can serve as leads or structural templates for the development of new hypocholesterolemic drugs.

Synthesis and Biodegradation Test of a New Polyether Polyurethane Foam Produced from PEG 400, L-Lysine Ethyl Ester Diisocyanate (L-LDI) and Bis-hydroxymethyl Furan (BHMF).

In this paper we produced a bio-based polyether-polyurethane foam PU1 through the prepolymer method. The prepolymer was obtained by the reaction of PEG 400 with L-Lysine ethyl ester diisocyanate (L-LDI). The freshly prepared prepolymer was extended with 2,5-bis(hydroxymethyl)furan (BHMF) to produce the final polyurethane. The renewable chemical BHMF was produced through the chemical reduction of HMF by sodium borohydride. HMF was produced by a previously reported procedure from fructose using choline chloride and ytterbium triflate. To evaluate the degradation rate of the foam PU1, we tested the chemical stability by soaking it in a 10% sodium hydroxide solution. The weight loss was only 12% after 30 days. After that, we proved that enzymatic hydrolysis after 30 days using cholesterol esterase was more favoured than hydrolysis with NaOH, with a weight loss of 24%, probably due to the hydrophobic character of the PU1 and a better adhesion of the enzyme on the surface with respect to water. BHMF was proved to be of crucial importance for the enzymatic degradation assay at 37 °C in phosphate buffer solution, because it represents the breaking point inside the polyurethane chain. Soil burial degradation test was monitored for three months to evaluate whether the joint activity of sunlight, climate changes and microorganisms, including bacteria and fungi, could further increase the biodegradation. The unexpected weight loss after soil burial degradation test was 45% after three months. This paper highlights the potential of using sustainable resources to produce new biodegradable materials.

The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis

Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor’s spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-β-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR’s ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-β-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.

An integrated microfluidic-based biosensor using a magnetically controlled MNPs-enzyme microreactor to determine cholesterol in serum with fluorometric detection

This work provides a microfluidic-based biosensor to determine total cholesterol in serum based on integrating the reaction/detection zone of a microfluidic chip of a magnetically retained enzyme microreactor (MREµR) coupled with the remote fluorometric detection through a bifurcated fiber-optic bundle (BFOB) connected with a conventional spectrofluorometer. The method is based on developing the enzymatic hydrolysis and oxidation of cholesterol at microscale size using both enzymes (cholesterol esterase (ChE) and cholesterol oxidase (ChOx)) immobilized on magnetic nanoparticles (MNPs). The biocatalyst reactions were followed by monitoring the fluorescence decreasing by the naphtofluorescein (NF) oxidation in the presence of the previous H2O2 formed. This microfluidic biosensor supposes the physical integration of a minimal MREµR as a bioactive enzyme area and the focused BFOB connected with the spectrofluorometer detector. The MREµR was formed by a 1 mm length of magnetic retained 2:1 ChE-MNP/ChOx-MNP mixture. The dynamic range of the calibration graph was 0.005–10 mmol L−1, expressed as total cholesterol concentration with a detection limit of 1.1 µmol L−1 (r2 = 0.9999, sy/x = 0.03, n = 10, r = 3). The precision expressed as the relative standard deviation (RSD%) was between 1.3 and 2.1%. The microfluidic-based biosensors showed a sampling frequency estimated at 30 h−1. The method was applied to determine cholesterol in serum samples with recovery values between 94.8 and 102%. The results of the cholesterol determination in serum were also tested by correlation with those obtained using the other two previous methods.Graphical abstract

Inhibitory effect of procyanidin B2 and tannin acid on cholesterol esterase and their synergistic effect with orlistat

This study was aimed to analyze the effect of procyanidin B2 (PC) and tannin acid (TA) on the activities of cholesterol esterase (CEase) and the inhibitory mechanisms of enzymatic activity. The interaction mechanisms were investigated by enzymatic kinetics, multi-spectroscopy methods, thermodynamics analysis, molecular docking, and dynamic simulations. PC and TA could bind with CEase and inhibit the activity of enzyme in a mixed-competitive manner and non-competitive manner, which was verified by molecular docking simulations and dynamics simulations. Also, PC and TA showed the synergistic inhibition with orlistat. Fluorescence, UV-vis and the thermodynamic analysis revealed that the complexes were formed from CEase and inhibitors by noncovalent interaction. As revealed by the circular dichroism results, both PC and TA decreased enzymatic activities by altering the conformations of CEase. The inhibition of PC and TA on CEase might be one mechanism for its cholesterol-lowering effect.

Measurement of cholesterol levels of lipoprotein subclasses in human serum using anion-exchange high-performance liquid chromatography with a linear concentration gradient of sodium perchlorate.

Relationships between the subclasses of high-density lipoprotein (HDL) or low-density lipoprotein (LDL) and the risk of atherosclerotic cardiovascular disease have been studied, and using various methods, such as ultracentrifugation, electrophoresis, and nuclear magnetic resonance, for analysing lipoprotein subclasses. We established a method for HDL and LDL subclasses using anion-exchange high-performance liquid chromatography (AEX-HPLC) with a linear concentration gradient of sodium perchlorate (NaClO4). In the AEX-HPLC, the subclasses of HDL and LDL were separated, and detected using a post-column reactor with an enzymatic cholesterol reagent, that contained cholesterol esterase, cholesterol oxidase, and peroxidase as major ingredients. LDL subclasses were divided based on the absolute value of first-derivative chromatogram. Three HDL subclasses, HDL-P1, HDL-P2, and HDL-P3, and three LDL subclasses, LDL-P1, LDL-P2, and LDL-P3, were separated by AEX-HPLC, and detected in order. The major components of HDL-P2 and HDL-P3 were HDL3 and HDL2, respectively. The linearity was determined for each lipoprotein subclass. The coefficients of variation of cholesterol concentration of the subclasses for within-day assay (n = 10) and between-day assay (n = 10) ranged between 3.08-8.94% and 4.52-9.97%, respectively. Cholesterol levels in HDL-P1 of diabetic patients were positively correlated with oxidized LDL levels (r = 0.409, p = 0.002). Moreover, cholesterol levels in LDL-P2 and LDL-P3 were positively correlated with oxidized LDL levels (r = 0.393, p = 0.004 and r = 0.561, p < 0.001, respectively). AEX-HPLC may be highly suitable as an assay to clinically assess lipoprotein subclasses.

An intelligent and novel electrochemical biosensor for simultaneous enzymatic biosensing of cholesterol and glucose in the presence of uric acid based on first-and second-order calibration methods

In this work, for the first time, a novel electrochemical biosensor was fabricated based on modification of a glassy carbon electrode with multi-walled carbon nanotubes-ionic liquid (MWCNTs-IL) which was used as a platform to the synthesis of molecularly imprinted polymers having glucose (GL), glucose oxidase (GO), cholesterol (CL), cholesterol oxidase (CO), cholesterol esterase (CE), horse radish peroxidase (HRP), and uric acid (UA) as template molecules. For simultaneous biosensing of GL and CL, the biosensor (MIPs/MWCNTs-IL/GCE) was incubated with the enzymes and then, it was individually incubated with GL and CL. Response of the biosensor was based on the reduction of the hydrogen peroxide produced from enzymatic reactions. Individual incubation of the biosensor with GL and CL generated two very similar peaks while its incubation with both GL and CL generated a single peak which needed to be assisted by first- and second-order calibration methods to choose the best method. Second-order hydrodynamic differential pulse voltammetric data were generated and modeled by MCR-ALS, PARASIAS, and PARAFAC2 to simultaneous determination of them in the presence of UA as uncalibrated interference based on exploiting second-order advantage. Amperometric responses of the biosensor to GL and CL were also calibrated and assisted by first-order calibration methods for simultaneous determination of GL and CL in the presence of UA based on exploiting first-order advantage. The result confirmed that the biosensor assisted by second-order data modeled by MCR-ALS showed the best performance for simultaneous determination of CL and GL in the presence of UA as uncalibrated interference in both synthetic samples and human serum samples which was comparable with HPLC-UV as the reference method. It should be noted the biosensor was able to simultaneous determination of CL and GL in the ranges of 0.5–15 pM and 1–18 pM with limits of detection of 0.81 pM and 0.23 pM, respectively, and response of the biosensor was stable, selective, ultrasensitive, repeatable, and reproducible. This work as the first report chemometric assisted enzymatic biosensing of GL and CL can be continued to develop more projects for biomedical purposes.

Chemical composition and anti-cholesterol activity of tea (Camellia sinensis) flowers from albino cultivars.

Albino tea cultivars are mutant tea plants with altered metabolisms. Current studies focus on the leaves while little is known about the flowers. To evaluate tea flowers from different albino cultivars, the chemical composition and anti-cholesterol activity of tea flowers from three albino cultivars (i.e., Baiye No.1, Huangjinya, and Yujinxiang) were compared. According to the results, tea flowers from Yujinxiang had more amino acids but less polyphenols than tea flowers from the other two albino cultivars. A reduced content of procyanidins and a high chakasaponins/floratheasaponins ratio were characteristics of tea flowers from Yujinxiang. In vitro anti-cholesterol activity assays revealed that tea flowers from Yujinxiang exhibited stronger activity in decreasing the micellar cholesterol solubility, but not in cholesterol esterase inhibition and bile salt binding. It was noteworthy that there were no specific differences on the chemical composition and anti-cholesterol activity between tea flowers from albino cultivars and from Jiukeng (a non-albino cultivar). These results increase our knowledges on tea flowers from different albino cultivars and help food manufacturers in the cultivar selection of tea flowers for use.

Novel Plant-Protein (Quinoa) Derived Bioactive Peptides with Potential Anti-Hypercholesterolemic Activities: Identification, Characterization and Molecular Docking of Bioactive Peptides

Hypercholesterolemia remains a serious global public health concern. Previously, synthetic anti-hypercholesterolemic drugs were used for ameliorating this condition; however, long-term usage presented several side-effects. In this regard, natural products as an adjunct therapy has emerged in recent times. This study aimed to produce novel bioactive peptides with anti-hypercholesterolemic activity (cholesterol esterase (CEase) and pancreatic lipase (PL)) from quinoa protein hydrolysates (QPHs) using three enzymatic hydrolysis methods (chymotrypsin, protease and bromelain) at 2-h hydrolysis intervals (2, 4, and 6 h). Chymotrypsin-generated hydrolysates showed higher CEase (IC50: 0.51 mg/mL at 2 h) and PL (IC50: 0.78 mg/mL at 6 h) inhibitory potential in comparison to other derived hydrolysates and intact quinoa proteins. Peptide profiling by LC-MS QTOF and in silico interaction with target enzymes showed that only four derived bioactive peptides from QPHs could bind in the active site of CEase, whereas twelve peptides could bind in the active site of PL. Peptides QHPHGLGALCAAPPST, HVQGHPALPGVPAHW, and ASNLDNPSPEGTVM were identified to be potential CEase inhibitors, and FSAGGLP, QHPHGLGALCAAPPST, KIVLDSDDPLFGGF, MFVPVPH, and HVQGHPALPGVPAHW were identified as potential PL inhibitors on the basis of the maximum number of reactive residues in these bioactive peptides. In conclusion, QPHs can be considered as an alternative therapy for the treatment of hypercholesterolemia.

In vitro antidiabetic and antihypercholesterolemic activities of camel milk protein hydrolysates derived upon simulated gastrointestinal digestion of milk from different camel breeds

Milk protein hydrolysates derived from 4 camel breeds (Pakistani, Saheli, Hozami, and Omani) were evaluated for in vitro inhibition of antidiabetic enzymatic markers (dipeptidyl peptidase IV and α-amylase) and antihypercholesterolemic enzymatic markers (pancreatic lipase and cholesterol esterase). Milk samples were subjected to in vitro simulated gastric (SGD) and gastrointestinal digestion (SGID) conditions. In comparison with intact milk proteins, the SGD-derived milk protein hydrolysates showed enhanced inhibition of α-amylase, dipeptidyl peptidase IV, pancreatic lipase, and cholesterol esterase as reflected by lower half-maximal inhibitory concentration values. Overall, milk protein hydrolysates derived from the milk of Hozami and Omani camel breeds displayed higher inhibition of different enzymatic markers compared with milk protein hydrolysates from Pakistani and Saheli breeds. In vitro SGD and SGID processes significantly increased the bioactive properties of milk from all camel breeds. Milk protein hydrolysates from different camel breeds showed significant variations for inhibition of antidiabetic and antihypercholesterolemic enzymatic markers, suggesting the importance of breed selection for production of bioactive peptides. However, further studies on identifying the peptides generated upon SGD and SGID of milk from different camel breeds are needed.

Multiple Bioactivities of Peptides from Hydrolyzed Misgurnus anguillicaudatus

Misgurnus anguillicaudatus (loach) is a widely distributed benthic fish in Asia. In this study, the alkaline protease was used to hydrolyze loach, and the hydrolysate products of different molecular weights were obtained by membrane separation. In vitro antioxidant assays showed that the <3 kDa fraction (SLH-1) exhibited the strongest antioxidant activity (DPPH, hydroxyl radical and superoxide radical scavenging ability, and reducing power), while SLH-1 was purified by gel filtration chromatography, and peptide sequences were identified by LC-MS/MS. A total of six peptides with antioxidant activity were identified, namely SERDPSNIKWGDAGAQ (D-1), TVDGPSGKLWR (D-2), NDHFVKL (D-3), AFRVPTP (D-4), DAGAGIAL (D-5), and VSVVDLTVR (D-6). In vitro angiotensin-converting enzyme (ACE) inhibition assay and pancreatic cholesterol esterase (CE) inhibition assay, peptide D-4 (IC50 95.07 μg/mL, 0.12 mM) and D-2 inhibited ACE, and peptide D-2 (IC50 3.19 mg/mL, 2.62 mM), D-3, and D-6 acted as pancreatic CE inhibitors. The inhibitory mechanisms of these peptides were investigated by molecular docking. The results showed that the peptides acted by binding to the key amino acids of the catalytic domain of enzymes. These results could provide the basis for the nutritional value and promote the type of healthy products from hydrolyzed loach.

Purified Stigmasterol Related Novel Phytosterol from Chrysopogon zizanioides (L.) Roberty Root Extract Exhibits Strong Cholesterol Esterase (CEase) and Diabetic Foot Ulcer (DFU) Causing Bacterial Pathogens Inhibitory Properties

Background: Chrysopogon zizanioides Roberty. root extracts have documented amylase and α-glucosidase inhibition, hepatoprotection, antioxidant, antimicrobial, antihyperglycemic, antidepressant, and antitubercular properties, but lack ample reports on health care applications of purified phytoconstituents. Objective: To study the inhibitory properties of phytosterol eluate against cholesterol esterase (CEase) and diabetic foot ulcer (DFU) causing pathogens (clinical isolates). Methods: The shake flask method was adopted to extract the phytoconstituents and standard spectrophotometric assays were carried out to quantify phytosterol constituent and CEase inhibitory activity. Central composite design (CCD) based response surface methodology (RSM) was adopted to observe the improvement of phytosterol constituent and CEase inhibitory activity. Two dimensional preparative thin layer chromatography (2D PTLC) technique was executed to purify the phytosterol content and HRLC based APCI – Q-TOF/ MS2 analysis was done to predict the phytosterol structures. Standard disc assay was used to explore the inhibitory effect of extract and purified phytosterol content against DFU causing pathogens. Results: The crude extracts exhibited a moderate CEase inhibitory activity (Ethylacetate: 67 ± 3.2%) and appreciable phytosterol content (Acetone:11.24 ± 0.52 mg/g tissue). CCD based RSM has improved the CEase inhibitory activity (Ethylacetate: 96.56 ± 1.1%) as well as the phytosterol content (6.45 ± 0.5 mg/g tissue). The purified phytosterol fractionate (PTLC eluate) was found to contain stigmasterol related novel phytosterol and revealed a significant CEase inhibitory activity (81 ± 2.5 %; Ki: 54.89 µg) with mixed type inhibition. Conclusion: Both the extract and the PTLC eluate have recorded a substantial control over DFU causing bacterial pathogens.

Co-extraction of soy protein and polysaccharide with lipid-lowering activity: Characterization of functional property, nutritional property and colonic fermentation property through a metabolomics approach

Defatted soy meal is a candidate for preparing lipid-lowering active ingredient. The study aimed to optimally co-extract soy protein and polysaccharide with lipid-lowering activity and improved functional and nutritional properties using non-conventional extraction techniques, i.e. thermal-, high pressure homogenization- and enzyme-assisted extraction. Non-conventional extraction enhanced the yields of soy protein and soluble saccharides and inactivated anti-nutrients. Soy extract (SE) prepared by pectinase-assisted extraction possessed not only strong in vitro lipid-lowering activity (i.e. cholesterol micellar inhibition activity, bile salts binding capacity, pancreatic lipase inhibition activity and cholesterol esterase inhibition activity), but also excellent functional (i.e. instant solubility of protein, low turbidity and acceptable color) and nutritional properties (i.e. none agglutinin activity). Resistant-digested fragment of 7S/11S played key role in lipid-lowering activity of SE. Moreover, SE beneficially altered colonic fermentation property of soy protein through promoting the release of bioactive peptides and organic acids, accelerating tryptophan and linoleic acid metabolism to produce indole acrylic acid, indole lactic acid and linoleic acid, and inhibiting putrefactive compounds (i.e. piperidine and tyramine). Oligosaccharide and polysaccharide in SE improved functional and nutritional properties of soy protein without compromising its lipid-lowering activity.

Purification, identification and hypolipidemic activities of three novel hypolipidemic peptides from tea protein

In this study, hypolipidemic peptides were obtained from tea protein by enzymatic hydrolysis, ultrafiltration and high-performance liquid chromatography. Subsequently, the hypolipidemic peptides were identified by mass spectrometry and screened through molecular docking technology, and the hypolipidemic activities and mechanisms of the active peptides were explored. The results showed that the hydrolysate of hypolipidemic peptides obtained by pepsin hydrolysis for 3 h had good bile salt binding ability. After purification, identification and molecular docking screening, three novel hypolipidemic peptides FLF, IYF and QIF were obtained. FLF, IYF and QIF can interact with the receptor proteins 1LPB and 1F6W through hydrogen bonds, π-π bonds, hydrophobic interactions and van der Waals forces, thus exerting hypolipidemic activities. Activity studies showed that, compared with the positive controls, FLF, IYF and QIF had excellent sodium taurocholate binding abilities, pancreatic lipase inhibitory activities and cholesterol esterase inhibitory activities. Moreover, FLF, IYF and QIF can effectively inhibit lipogenic differentiation of 3T3-L1 preadipocytes, reduce intracellular lipid and low-density lipoprotein content and increase high-density lipoprotein content. These results indicated that the three novel hypolipidemic peptides screened in this study had excellent hypolipidemic activities and were expected to be used as natural-derived hypolipidemic active ingredients for the development and application in functional foods.