Chemoselective Preparation of New Families of Phenolic-Organoselenium Hybrids-A Biological Assessment.

Being aware of the enormous biological potential of organoselenium and polyphenolic compounds, we have accomplished the preparation of novel hybrids, combining both pharmacophores in order to obtain new antioxidant and antiproliferative agents. Three different families have been accessed in a straightforward and chemoselective fashion: carbohydrate-containing N-acylisoselenoureas, N-arylisoselenocarbamates and N-arylselenocarbamates. The nature of the organoselenium framework, number and position of phenolic hydroxyl groups and substituents on the aromatic scaffolds afforded valuable structure-activity relationships for the biological assays accomplished: antioxidant properties (antiradical activity, DNA-protective effects, Glutathione peroxidase (GPx) mimicry) and antiproliferative activity. Regarding the antioxidant activity, selenocarbamates 24-27 behaved as excellent mimetics of GPx in the substoichiometric elimination of H2O2 as a Reactive Oxygen Species (ROS) model. Isoselenocarbamates and particularly their selenocarbamate isomers exhibited potent antiproliferative activity against non-small lung cell lines (A549, SW1573) in the low micromolar range, with similar potency to that shown by the chemotherapeutic agent cisplatin (cis-diaminodichloroplatin, CDDP) and occasionally with more potency than etoposide (VP-16).

Decreased Human Platelet Activation and Mouse Pulmonary Thrombosis by Rutaecarpine and Comparison of the Relative Effectiveness with BAY11-7082: Crucial Signals of p38-NF-κB.

Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum, a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1-6 µM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.

Cascade Reactions by Nitric Oxide and Hydrogen Radical for Anti-Hypoxia Photodynamic Therapy Using an Activatable Photosensitizer.

Organelle-targeted activatable photosensitizers are attractive to improve the specificity and controllability of photodynamic therapy (PDT), however, they suffer from a big problem in the photoactivity under both normoxia and hypoxia due to the limited diversity of phototoxic species (mainly reactive oxygen species). Herein, by effectively photocaging a π-conjugated donor-acceptor (D-A) structure with an N-nitrosamine substituent, we established a unimolecular glutathione and light coactivatable photosensitizer, which achieved its high performance PDT effect by targeting mitochondria through both type I and type II (dual type) reactions as well as secondary radicals-participating reactions. Of peculiar interest, hydrogen radical (H•) was detected by electron spin resonance technique. The generation pathway of H• via reduction of proton and its role in type I reaction were discussed. We demonstrated that the synergistic effect of multiple reactive species originated from tandem cascade reactions comprising reduction of O2 by H• to form O2•-/HO2• and downstream reaction of O2•- with •NO to yield ONOO-. With a relatively large two-photon absorption cross section for photoexcitation in the near-infrared region (166 ± 22 GM at 800 nm) and fluorogenic property, the new photosensitizing system is very promising for broad biomedical applications, particularly low-light dose PDT, in both normoxic and hypoxic environments.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

Anthocyanins from Pomegranate (Punica granatum L.) and Their Role in Antioxidant Capacities in Vitro.

As phytochemicals, anthocyanins are not only responsible for the diverse colors in nature, but are associated with broad-spectrum health-promoting effects for human beings. Pomegranate is abundant in anthocyanins which possess high antioxidant capacities. However, the pomegranate anthocyanins profile and their contributions to antioxidant capacities are not fully depicted. The purpose of this article is to review anthocyanins from pomegranate as important antioxidants. Total anthocyanin content (TAC) and six major components vary greatly with intrinsic and extrinsic factors. In pomegranate, anthocyanins mainly acted as primary antioxidants, while their action as secondary antioxidants were not conclusive. The antioxidant potentials of anthocyanins were significantly affected by factors especially chemical structure and detection assays in vitro. The current knowledge may provide insights into potential applications for pomegranate anthocyanins based on their antioxidant activities.

Purification and Identification of Novel Antioxidant Peptides from Enzymatically Hydrolysed Samia ricini Pupae.

The emergence of excessive free radicals leads to the destruction of various systems within the body. These free radicals also affect nutritional values, color, taste, and emit an odor akin to rancid food. Most food industries use synthetic antioxidants, such as BHT (butylated hydroxytoluene) or BHA (butylated hydroxy anisole). However, high doses of these can be harmful to our health. Therefore, an antioxidant compounds, such as bioactive peptides from edible animals or plants, have emerged to be a very promising alternative as they reduce potential side effects. This study focused on the purification and identification of antioxidant peptides from protein hydrolysates of wild silkworm pupae (Samia ricini). Antioxidant peptides were purified from the hydrolysate by ultrafiltration and RP-HPLC. The results showed that protein hydrolysate from S. ricini pupae by trypsin with a molecular weight lower than 3 kDa and highly hydrophobic property, exhibited strong DPPH radical scavenging activity and chelating activity. Further identification of peptides from the fraction with the highest antioxidant activity was carried out using LC-MS/MS. Three novel peptides, i.e., Met-Ley-Ile-Ile-Ile-Met-Arg, Leu-Asn-Lys-Asp-Leu-Met-Arg, and Glu-Asn-Ile-Ile-Leu-Phe-Arg, were identified. The results of this study indicated that the protein hydrolysate from S. ricini pupae possessed potent biological activity, and the novel antioxidant peptides could be utilized to develop health-related antioxidants in food industry.

Minimalistic Principles for Designing Small Molecules with Multiple Reactivities against Pathological Factors in Dementia.

Multiple pathogenic elements, including reactive oxygen species, amyloidogenic proteins, and metal ions, are associated with the development of neurodegenerative disorders. We report minimalistic redox-based principles for preparing compact aromatic compounds by derivatizing the phenylene moiety with various functional groups. These molecular agents display enhanced reactivities against multiple targets such as free radicals, metal-free amyloid-ß (Aß), and metal-bound Aß that are implicated in the most common form of dementia, Alzheimer's disease (AD). Mechanistic studies reveal that the redox properties of these reagents are essential for their function. Specifically, they engage in oxidative reactions with metal-free and metal-bound Aß, leading to chemical modifications of the Aß peptides to form covalent adducts that alter the aggregation of Aß. Moreover, the administration of the most promising candidate significantly attenuates the amyloid pathology in the brains of AD transgenic mice and improves their cognitive defects. Our studies demonstrate an efficient and effective redox-based strategy for incorporating multiple functions into simple molecular reagents.

Tackle the free radicals damage in COVID-19.

COVID-19 is a severe pandemic which has caused a devastating amount of loss in lives around the world, and yet we still don't know how to appropriately treat this disease. We know very little about the pathogenesis of SARS-CoV-2, the virus which induces the COVID-19. However, COVID-19 does share many similar symptoms with SARS and influenza. Previous scientific discoveries learned from lab animal models and clinical practices shed light on possible pathogenic mechanisms in COVID-19. In the past decades, accumulated scientific findings confirmed the pathogenic role of free radicals damage in respiratory virus infection. Astonishingly very few medical professionals mention the crucial role of free radical damage in COVID-19. This hypothesis aims to summarize the crucial pathogenic role of free radical damage in respiratory virus induced pneumonia and suggest an antioxidative therapeutic strategy for COVID-19.

Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: example of avarol.

The liposomal integration method, in conjunction with electron paramagnetic resonance (EPR) spectroscopy, has been presented for the investigation of antioxidant activity of selected water-insoluble compound towards biologically relevant free radicals. This method was applied to avarol, a sesquiterpenoid hydroquinone isolated from the marine sponge Dysidea avara. The antioxidant activity of water-insoluble avarol towards •OH, O2•- and NO• radicals was attained by its incorporation into the DPPC liposomes bilayer, and towards ascorbyl radicals in the organic solvent. Avarol's activity towards •OH, O2•-, NO• and ascorbyl radicals was 86.2%, 50.9%, 23.6% and 61.8%, respectively, showing its significant radical scavenging potential.

Crystal Guava (Psidium guajava L. "Crystal"): Evaluation of In Vitro Antioxidant Capacities and Phytochemical Content.

Free radicals can cause many diseases, such as cancer. Antioxidant is a compound that could scavenge free radicals. One of the natural antioxidants is guava. The goals of this research were to investigate the antioxidant activity of leaves and fruit of crystal guava by determining the value of the Antioxidant Activity Index (AAI) using DPPH, CUPRAC, and FRAP; evaluate the total phenolic content (TPC) and total flavonoid content (TFC); analyse the correlation between the TPC and TFC with AAI DPPH, CUPRAC, and FRAP, and analyse the correlation between the 3 methods. Extraction was performed by the reflux method using n-hexane, ethyl acetate, and ethanol. Determination of AAI DPPH, CUPRAC, FRAP, the TPC, and the TFC was performed by UV-visible spectrophotometry. The correlation of the TPC and TFC with AAI DPPH, CUPRAC, and FRAP and, also, the correlation of the 3 methods were investigated by Pearson's method. The antioxidant activity of leaves and fruit extracts of crystal guava showed AAI DPPH in the range of 0.33-56.46, CUPRAC 0.20-7.31, and FRAP 1.65-59.89. The highest TPC was given by ethanol leaf extracts (49.55 ± 1.45 g GAE/100 g), while the highest TFC was for n-hexane leaf extracts (9.68 ± 0.210 g QE/100 g). The TPC of leaves extract had a significantly positive correlation with AAI DPPH, CUPRAC, and FRAP. AAI DPPH, AAI CUPRAC, and AAI FRAP of leaves and fruit extract of crystal guava showed a significantly positive correlation. In general, leaves extract had strong antioxidant activity by the three methods. For the highest antioxidant activity, ethanol was the best solvent for extraction leaves and ethyl acetate for extraction fruit of crystal guava. The TPC in leaves extract contributed to the antioxidant activity by DPPH, CUPRAC, and FRAP methods. The Antioxidant activity of leaves and fruit extracts of crystal guava was linear by the three methods.

Antioxidant Content of Frozen, Convective Air-Dried, Freeze-Dried, and Swell-Dried Chokecherries (Prunus virginiana L.).

Chokecherry (Prunus virginiana L.) is rich in bioactive molecules as phenolics, which can act as antioxidants, anti-inflammatory, anticancer, among others; however, due to its high perishability, most of this fruit is wasted. Freezing and sun drying have been the most adopted techniques to avoid its postharvest deterioration. Nevertheless, both processes have presented some drawbacks as high storage costs and losses of bioactive molecules. Therefore, to preserve these molecules, this study compared the impact of convective airflow drying (CAD), freezing (FR), freeze drying (FD), and swell drying (SD). Total phenolics content (TPC), total flavonoids content (TFC), kuromanin concentration (KC), and antioxidant activity (antiradical activity (ARA) and Trolox equivalent antioxidant capacity assay (TEAC)) of chokecherries were measured. "Swell drying" is a drying process coupling convective airflow drying to the Instant Controlled Pressure Drop (DIC) expansion. A central composite rotatable design was applied to optimize the DIC variables and responses. Results showed that both freezing and swell drying effectively preserve the TPC, TFC, KC, and ARA. Moreover, SD samples also presented the highest TEAC. Contrary, in the case of CAD, it caused the highest losses of both antioxidant content and activity. Swell drying remedies the shrinkage and collapsing of dried food structure, which results in a better antioxidants extraction.

Participation of Free-Radical Processes in Structural and Metabolic Disturbances in the Lung Tissues Caused by Exposure to Coal-Rock Dust and their Adaptogenic Correction.

Adaptive correction of structural and metabolic disturbances in the lungs caused by longterm exposure to coal-rock dust were studied in experiments on rats. It was shown that the complex antioxidant preparation containing dihydroquercetin compensated disturbances in the redox balance in the lung tissue, prevented the formation of dust granulomas, and reduced the severity of degenerative changes in the bronchopulmonary system.

New Phenolic Derivatives of Thiazolidine-2,4-dione with Antioxidant and Antiradical Properties: Synthesis, Characterization, In Vitro Evaluation, and Quantum Studies.

Oxidative stress has been incriminated in the physiopathology of many diseases, such as diabetes, cancer, atherosclerosis, and cardiovascular and neurodegenerative diseases. There is a great interest in developing new antioxidants that could be useful for preventing and treating conditions for which oxidative stress is suggested as the root cause. The thiazolidine-2,4-dione derivatives have been reported to possess various pharmacological activities and the phenol moiety is known as a pharmacophore in many naturally occurring and synthetic antioxidants. Twelve new phenolic derivatives of thiazolidine-2,4-dione were synthesized and physicochemically characterized. The antioxidant capacity of the synthesized compounds was assessed through several in vitro antiradical, electron transfer, and Fe2+ chelation assays. The top polyphenolic compounds 5f and 5l acted as potent antiradical and electron donors, with activity comparable to the reference antioxidants used. The ferrous ion chelation capacity of the newly synthesized compounds was modest. Several quantum descriptors were calculated in order to evaluate their influence on the antioxidant and antiradical properties of the compounds and the chemoselectivity of the radical generation reactions has been evaluated. The correlation with the energetic level of the frontier orbitals partially explained the antioxidant activity, whereas a better correlation was found while evaluating the O-H bond dissociation energy of the phenolic groups.

Preliminary Study: Comparison of Antioxidant Activity of Cannabidiol (CBD) and α-Tocopherol Added to Refined Olive and Sunflower Oils.

This study evaluates the antioxidant activity of cannabidiol (CBD), added to model systems of refined olive (ROO) and sunflower (SO) oils, by measuring the peroxide value, oxidative stability index (OSI), electron spin resonance (ESR) forced oxidation, and DPPH• assays. Free acidity, a parameter of hydrolytic rancidity, was also examined. CBD was compared using the same analytical scheme with α-tocopherol. CBD, compared to α-tocopherol, showed a higher scavenging capacity, measured by DPPH• assay, but not better oxidative stability (OSI) of the oily systems considered. In particular, α-tocopherol (0.5%) showed an antioxidant activity only in SO, registered by an increase of more than 30% of the OSI (from 4.15 ± 0.07 to 6.28 ± 0.11 h). By ESR-forced oxidation assay, the concentration of free radicals (µM) in ROO decreased from 83.33 ± 4.56 to 11.23 ± 0.28 and in SO from 19.21 ± 1.39 to 6.90 ± 0.53 by adding 0.5% α-tocopherol. On the contrary, the addition of 0.5% CBD caused a worsening of the oxidative stability of ROO (from 23.58 ± 0.32 to 17.28 ± 0.18 h) and SO (from 4.93 ± 0.04 to 3.98 ± 0.04 h). Furthermore, 0.5% of CBD did not lower dramatically the concentration of free radicals (µM) as for α-tocopherol, which passed from 76.94 ± 9.04 to 72.25 ± 4.13 in ROO and from 17.91 ± 0.95 to 16.84 ± 0.25 in SO.

Dose-dependency of multiple free radical-scavenging activity of edaravone.

BACKGROUND: Edaravone is a powerful free radical scavenger that is in clinical use. However, data concerning its dose-response relationship against multiple free radicals remain sparse. The purpose of the present study was to demonstrate the dose-dependency of direct scavenging activity of edaravone against multiple free radical species. MATERIALS AND METHODS: Free radical-scavenging activities of edaravone against six free radical species were evaluated by electron spin resonance spectroscopy using spin-trapping method. RESULTS: Edaravone scavenged the following free radicals in dose-dependent manners with reaction rate constants (kedaravone) or 50% inhibitory concentration (IC50) as indicated: hydroxyl radical (kedaravone = 5.2 × 1010 M-1 s-1), superoxide anion (kedaravone/kG-CYPMPO = 0.63), tert-butyl peroxyl radical (kedaravone/kG-CYPMPO = 8.8), ascorbyl free radical (IC50 = 0.17 ± 0.06 mM), 2,2-diphenyl-1-picrylhydrazyl (DPPH, IC50 = 4.7 ± 0.3 µM), and nitric oxide (kedaravone = 7.0 × 103 M-1 s-1). CONCLUSIONS: The dose-dependent scavenging activities of edaravone against multiple free radical species were clearly illustrated. It is speculated that edaravone acts as antioxidant by dose-dependently scavenging multiple free radical species along the chain reactions of oxidative stress in surgery.

Synthesis and biological evaluation of novel heteroring-annulated pyrrolino-tetrahydroberberine analogues as antioxidant agents.

Tetrahydroberberine (THB), otherwise known as canadine, is a natural alkaloid showing significant pharmacological properties and antioxidant protection against oxidative damage. Herein, we synthetized structurally complex THB analogues, namely pyrrolino-tetrahydroberberines (PTHBs) 4a-g, containing the pyrrolino[2,3-b]pyridine system, by means of the reactions of 1,2-diaza-1,3-dienes and 7,8-dihydroberberine. Aim of the study was to explore the in vitro antioxidant properties of PTHBs in comparison to THB thus to identify the most effective against free radical-induced oxidative injury, by using three different antioxidant tests: the ORAC method, the DNA nicking assay, and the DCFH-DA cellular assay. As a result, PTHB 4d emerged among the other THB analogues by exhibiting the best antioxidant properties. First, it was the only compound having an ORAC value completely comparable to that of THB, indicating the same ability to neutralize peroxyl radicals. Secondly, 4d showed an even better antioxidant capacity than THB in protecting DNA against ferrous ion-induced strand breaks. These observations were also confirmed in NCTC-2544 human keratinocytes exposed to hydrogen peroxide. Indeed, 4d protected cells against oxidation more efficiently than THB both in the short (1 and 3 h) and long (24 h) period of incubation, possibly suggesting increased cell membrane permeability and/or intracellular stability of 4d as compared to THB.

Potential antioxidant activity of Morita-Baylis-Hillman adducts.

The wide variety of potent biological activities of Morita-Baylis-Hillman adducts (MBH) encouraged us to synthesize new series of products belonging to this class of compounds, possessing different functionalities and exhibiting potential antioxidant activity. As part of our on-going program on targeting molecules with antioxidant activity, we describe herein different DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activities of MBH alcohols and their derivatives including acetates, phosphonates and hydrazonophosphonates. The obtained results showed that the strongest DPPH radical scavenging activity was observed in the case of hydrazonophosphonates in comparison to the other MBH derivatives.

Conventional and microwave prompted synthesis, antioxidant, anticholinesterase activity screening and molecular docking studies of new quinolone-triazole hybrids.

The synthesis of ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylates (4, 5) was performed via the reaction of corresponding anilines with diethyl ethoxymethylenemalonate under conventional and also microwave promoted conditions. The treatment of 4 and 5 afforded the corresponding hydrazides (6 and 7). These hydrazides were converted to the corresponding carbo(thio)amides (9a-f and 10a-e) which were then subjected to an intramolecular cyclisation leading to the formation of quinolone-triazole hybrids (11a-f and 12a-e). The newly synthesized compounds were screened for their biological activities such as antioxidant capacity (AC) and acetylcholinesterase Activity. Inhibition of cholinesterases is an effective method to curb Alzheimer's disease, a progressive and fatal neurological disorder. A series of some novel quinolonederivatives were designed, synthesized, and their inhibitory effects on AChE were evaluated. We obtained our compounds and determined their anticholinesterase activities according to the Ellman's method. 9b and 10c showed the best AChE inhibition with 0.48 ±â€¯0.02 and 0.52 ±â€¯0.07, respectively. Docking studies were performed for the most active compounds (9b, 10c) and interaction modes with enzyme active sites were determined. As a result of these studies, a strong interaction between these compounds and the active sites of AChE enzyme was revealed.

New bio-sensitive and biologically active single crystal of pyrimidine scaffold ligand and its gold and platinum complexes: DFT, antimicrobial, antioxidant, DNA interaction, molecular docking with DNA/BSA and anticancer studies.

Novel gold and platinum complexes [AuL2]·Cl, 1 and [PtL2]·2Cl, 2 with ligand, 2-methoxy-6-((2-(4-(trifluoromethyl)pyrimidin-2-yl)hydrazono)methyl)phenol (HL) have been synthesized and screened for their antimicrobial, antioxidant, DNA binding and anticancer (in vitro) activities. The single crystal of ligand HL was obtained by slow evaporation technique. The molecular structure of HL was confirmed from single crystal X-ray technique. Density functional theory calculations have been performed to gain insights into the electronic structure of these metal complexes. Antimicrobial result shows that, HL and complexes (1 and 2) have good antimicrobial agents against E. coli (bacteria) and C. albicans (fungi) than others bacterial and fungal strains. Antioxidant assay results suggest that, HL and complexes (1 and 2) possess good radical scavenging activity against diverse free radicals (DPPH, SOD, NO and H2O2). The intercalative interactions of HL and complexes (1 and 2) with CT-DNA were confirmed from spectroscopic titrations and viscometric measurements. Furthermore, the interactions of prepared compounds with DNA were confirmed by molecular docking analysis. In order to understand the nature of interactions between these metal complexes and BSA protein results clearly shows that complex 1 binds better than that of complex 2. The antitumor activities of prepared products were tested against single normal and different tumor cell lines by MTT assay. These results reveal that prepared complexes (1 and 2) have significant cytotoxic effect against tumor cell lines.

Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate.

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and prostaglandin-E2 (PGE2 ) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.