Structure–mechanism relationship of antioxidant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by pH

The aims of this study were to assess bioactive properties (ACE inhibition and antioxidant capacity) from wheat gluten hydrolysate peptides fractionated by pH (4.0, 6.0 and 9.0), to determine peptide action mechanism, and to relate it to the secondary structure and functional groups of peptides. Gluten hydrolysate extracts (GHE) were enriched in peptides with medium hydrophobicity and molecular weight (≈ 60% MH and 5.5 kDa, respectively). Gluten peptides inhibited ACE I by uncompetitive mechanism and a direct relationship between α-helix structure and IC50% value was obtained (r = 0.9127). TEAC and cooper chelating activity from GHE 6.5 were the highest and directly correlated with MH peptides. GHE 9.0 had high carotene bleaching inhibition (47.5 ± 0.3%) and reducing power activity (163.1 ± 2.9 mg S₂O₃^2 − equivalent g^− 1 protein), which were directly related to disulfide bonds content of peptides (r = 0.9982 and 0.9216, respectively). pH was a good alternative to select bioactive peptides from wheat gluten hydrolysate.     

Antiangiogenic properties of carotenoids: A potential role of maize as functional food

Several human disorders are associated with neovascularization. The current in vivo study examines the effect in the blood vessel formation of a xanthophyll-rich seed extract of maize. The angiostatic effects of treatment with maize seed carotenoid extract (0.1–10 μM of zeaxanthin equivalent/implant) were evaluated in the chick yolk sac membrane (YSM) (12–50% inhibition, P < 0.05) as well as in the chorioallantoic membrane (CAM) assay (23–52% inhibition, P < 0.05) as compared to control group. A similar inhibitory action was found by treatments with the retinoic acid (10 μM/embryo, 49% and 57% inhibition) and commercial lutein (10 μM/embryo, 65% and 55% inhibition) (positive controls) performed in the YSM and CAM, respectively. Results point to maize seeds as interesting sources of xanthophylls which effectively inhibited the process of vessel formation, suggesting a potential role of such compounds in the prevention of diseases associated with vascular dysfunction.     

Enzymatic extraction of fucoxanthin from brown seaweeds

Brown seaweeds contain a number of bioactive compounds. The xanthophyll, fucoxanthin, has in vivo efficacy against disorders such as type 2 diabetes, obesity and cancer. Organic solvents are traditionally employed to extract fucoxanthin, but carry a toxic chemical and environmental burden. The aim of this study was to optimise a fucoxanthin extraction method using enzymes, water, low‐temperature dehydration and mechanical blending, to produce yields comparable to those achieved with an organic solvent (acetone). Response surface methodology was applied, using Fucus vesiculosus as a model species. A fucoxanthin yield of 0.657 mg g^?1 (dry mass) was obtained from F. vesiculosus blade using the enzymatic method, equivalent to 94% of the acetone‐extracted yield. Optimum extraction parameters were determined to be enzyme‐to‐water ratio 0.52%, seaweed‐to‐water ratio 5.37% and enzyme incubation time 3.05 h. These findings may be applied to the development of value‐added nutraceutical products from seaweed.     

Experimental study on the continuous production of velvet bean-based bioactive peptides in a membrane reactor and bioactivity mapping

This study investigates the continuous production of bioactive peptides from fermented velvet bean (Mucuna pruriens) using a cost-effective enzymatic membrane reactor. The optimal operating conditions for continuous hydrolysis were residence time of 9 h, enzyme-to-substrate ratio of 10%, and the use of a 5-kDa PES membrane for separation. The resulting permeate exhibited notable antioxidant activity with DPPH and FRAP assay values of 0.28 and 0.12 mg AEAC/mL, respectively, and an angiotensin-converting enzyme (ACE) inhibition of 83.28%. Further fractionation of the permeate with a 2-kDa membrane led to increased antioxidant activity and ACE inhibition, with resulting half maximal inhibitory concentration (IC₅₀) values of 7.6 and 0.6 μg protein/mL, respectively. Liquid chromatography-mass spectrometry was used to identify peptide sequences from the <2-kDa fraction, which were matched with the BIOPEP-UWM™ database. The results revealed that the fermented velvet bean peptides were predominantly dipeptidyl peptidase (DPP)-4 inhibitors and ACE inhibitors.Industrial relevanceThe combination of a stirred tank reactor and membrane separation has the potential to enable the continuous production of bioactive peptides from velvet bean through the use of a freely suspended enzyme system. This reactor design offers an alternative solution to the existing delay in commercializing bioactive peptides, by (i) simplifying the industrial process development for continuous bioactive peptide production, (ii) eliminating the need for enzyme immobilization, and (iii) eliminating batch-to-batch variability in product quality and unproductive time associated with batch-wise production.     

High hydrostatic pressure treatment as an alternative to pasteurization to maintain bioactive compound content and texture in red sweet pepper

Red sweet peppers (Capsicum annuum) are an excellent source of essential nutrients and bioactive compounds. High hydrostatic pressures (HHP) not only increase shelf-life but also maintain nutritional and organoleptic properties better in a number of food products. The aim of this work was to measure the effect of HHP and a thermal treatment, pasteurization (PA) in a water bath at 70 °C for 10 min, on some bioactive compounds (fibre, carotenoids and antioxidant activity) and on the texture (TPA; firmness and shear force) of red Lamuyo-type sweet peppers, in order to discover the relationship between treatment (HHP and PA), tissue microstructure and bioactive compound extractability. The results show that HHP at 500 MPa and PA treatments had less impact on the microstructure, bioactive compound content (fibre and antioxidant activity) and texture of red sweet peppers, than when low pressures were used. Consequently, new functional foods could be developed using red sweet pepper tissues treated with high pressures (500 MPa) and/or PA.Industrial relevanceToday’s consumers demand foods that are rich in bioactive compounds with beneficial health effects and safer, more natural, minimally-processed food products. Red sweet peppers (Capsicum annuum) are an excellent source of essential nutrients and bioactive compounds such as carotenoids and fibre. High hydrostatic pressure (HHP) processing is considered one of the most economically viable of the non-thermal technologies that helps to preserve red sweet peppers with high nutritional and quality parameters. Therefore, it would be interesting to study the microstructure of HHP-treated red sweet pepper tissues in order to discover whether this treatment promotes the extractability of bioactive compounds, and to compare the results with those obtained by pasteurizing the red sweet pepper. Thus, these enhanced red sweet peppers could be used as ingredients in the formulation of new functional foods.     

FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds

Phycocolloids present in three brown (Himanthalia elongata, Bifurcaria bifurcata, Saccharina latissima) and five red edible seaweeds (Mastocarpus stellatus, Gigartina pistillata, Chondracanthus acicularis, Nemalion helminthoides and Dumontia contorta) were studied by FTIR-ATR spectroscopy. Infrared spectra of polysaccharide standards (alginate, agar, iota-, kappa- and lambda-carrageenan) were obtained for comparison. The main polysaccharide found in brown seaweeds was alginate, a linear copolymer of mannuronic (M) and guluronic acid (G). Alginate M/G ratio was tentatively estimated from specific absorption bands (808/787 cm⁻¹ and 1030/1080 cm⁻¹) in infrared spectra, suggesting higher values of mannuronic than guluronic acid blocks (M/G > 1) for brown seaweeds. According to their infrared spectra, all the red seaweeds studied were mainly carrageenan producers. Thus, M. stellatus showed absorption bands at 929.0, 844.7 and 803.2 cm⁻¹ of a typical hybrid kappa/iota/mu/nu-carrageenan, meanwhile G. pistillata and C. acicularis, showed the characteristic broad band (830–820 cm⁻¹) of lambda-type carrageenan. Moreover, when the second-derivative was obtained to improve resolution of overlapped bands in the original FTIR spectra, this band was divided into several sharper signals, indicating the presence of lambda-/theta-/xi-carrageenans. Accordingly with their FTIR spectra, N. helminthoides contained sulphated polysaccharides, such as carrageenan or mannans, while D. contorta produced lambda- with lesser amounts of kappa-carrageenan. Therefore, FTIR-ATR spectroscopy is proposed as a useful tool for the food, pharmaceutical and cosmetics industry to check the phycocolloid quality of a raw seaweed material by a quick and non-destructive method.     

Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates

Microwave pretreatment and hydrolysis were applied to rainbow trout (Oncorhynchus mykiss) by-products to produce bioactive peptides with dual in vitro angiotensin-I converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV) inhibitory activities. Peptides were fractionated using the single step electrodialysis with ultrafiltration membrane (EDUF). Concentration of cationic peptides (CP) increased in the recovery solution, reaching 125 μg mL⁻¹ after a 4-h treatment with migration rate of 15.68 ± 2.98 g m⁻² h. CP fractions displayed ACE and DPP-IV I inhibitory properties, with IC₅₀ values of 0.0036 mg mL⁻¹ and 1.23 mg mL⁻¹ respectively. The bioactivity was attributed to the low molecular weight peptides (300–500 Da) recovered. CP exhibited non-competitive inhibition patterns for ACE and DPP-IV, which were dose dependent. These results showed that bioactive peptides can successfully be separated from complex hydrolysate mixtures by EDUF. The fractionated peptides can serve as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.     

Enzymatically hydrolysed asparagus (Asparagus officinalis L.) hard-stem exhibits the ability to inhibit angiotensin-converting enzyme (ACE)

Asparagus (Asparagus officinalis L.) hard-stem, woody bottom of the asparagus stalk, is a rich source of bioactive compounds and is discarded as an eco-friendly agricultural waste. Therefore, we intended to explore the antioxidant, amino acid composition, and antihypertensive potentials of enzymatically hydrolysed asparagus hard-stem. The treated samples exhibited higher total free amino acids (11437 mg 100 g⁻¹), total phenolics (1749 mg 100 g⁻¹), and ability to inhibit (78.38%) angiotensin-converting enzyme (ACE) compared to control (ACE inhibition of 46.88%). Hydrophobic amino acids (39%) and gallic acid (48%) were the highly eluted bioactive compounds. The ability to inhibit ACE had been positively correlated with hydrophobic amino acids (r = 0.959–0.987) and gallic acid (r = 0.966), indicating the role of amino acids and phenolics in controlling ACE reactions. Thus, asparagus hard-stem can be a potential source to develop natural health supplements for the management of hypertension and related health risks.     

Determination of reaction kinetics of hydrolysis of tilapia (Oreochromis niloticus) protein for manipulating production of bioactive peptides with antioxidant activity,angiotensin‐I‐converting enzyme inhibitory activity and Ca‐binding properties

To manipulate enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein for production of bioactive peptides, its reaction kinetics was intensively studied. The study showed that the production of peptides with different bioactive properties including antioxidant activity, angiotensin‐I‐converting enzyme (ACE) inhibition and Ca‐binding property and their kinetics were affected by the degree of hydrolysis and substrate concentration. A comparative study on reaction kinetics found that the kinetic parameters for the production of each bioactive peptide are unique, that is, the maximum initial velocity, V_max, for hydrolysis of protein was as high as 1.07 mg mL^?1 min^?1, but that for the production of peptides with antioxidant activity and Ca‐binding property were very low, range of 7.14–66.7 μg mL^?1 min^?1, and that for the production of peptides with ACE inhibitory activity was the lowest, at 2.57 μg mL^?1 min^?1. This knowledge of reaction kinetics of protein hydrolysis would be useful for manipulating and optimising the production of peptides with desired bioactive properties.     

Effect of Soy Flour and Maize Flour Addition on Phase Separation in Moi-Moi from Soaked Cowpea (Vigna unguiculata) and Cowpea Flour from Different Cowpea Varieties

Two improved varieties of cowpea (IT89KO and IT90K-76) and one local variety (lsiocha) were used to investigate the effects of added soyflour and maize flour on the phase separation in moi-moi made from soaked cowpea and cowpea flour. Different levels (5%, 10%, 15%, 20% and 25%) of maize flour, soyflour and a 1:1 blend of soyflour and maize flour (soy/maize flour) were separately added. The moi-moi from the different combinations was evaluated for phase separation and per cent height of the upper layer calculated. Soaking of cowpea reduced the size of the upper layer in moi-moi compared with the use of cowpea flour. The addition of maize flour or the soy/maize flour reduced the upper layer compared with when either flour was added alone. The upper layer of moi-moi made from cowpea flour with added soy flour (21.20%) was significantly higher (p < 0.05) than moi-moi from soaked cowpea with added soy flour (15.2%). Moi-moi with added maize flour made from cowpea flour produced a significantly (p < 0.05) higher mean per cent upper layer (19.72%) compared with that from soaked cowpea (8.95%). Addition of soyflour produced the greatest upper layer for all varieties used. There were no significant (p > 0.05) differences in the per cent upper layers for moi-moi from all cowpea varieties when maize flour was added. Increasing the proportion of added flours increased the size of the upper layer. Complete prevention of the occurrence of phase separation in moi-moi by addition of soy flour and maize flour alone is not feasible.     

Terpenoids,DEET and short chain fatty acids as toxicants and repellents for Rhyzopertha dominica (coleoptera: Bostrichidae) and Lasioderma serricorne (Coleoptera: Ptinidae)

Plant-based products and common repellents have been suggested as promising alternatives for management of stored product insects. In this study, contact toxicity and repellent activity of the safe natural products carvacrol, citronella oil, geraniol, nootkatone, ocimene and R-(+)-pulegone, and the synthetic commercial repellents, N,N-Diethyl-3-methylbenzamide (DEET), and the fatty acid mixture of octanoic, nonanoic, and decanoic acids (C8910) were evaluated against the lesser grain borer, Rhyzopertha dominica and the cigarette beetle, Lasioderma serricorne under laboratory conditions. In contact toxicity assays the compounds were tested at concentrations ranging between 0.008 and 0.4 mg/cm². Carvacrol and R-(+)-pulegone exhibited the highest contact toxicity with LC₅₀ values of 0.019 and 0.023 mg/cm² against L. serricorne and LC₅₀ values of 0.012 and 0.019 mg/cm² against R. dominica, respectively. Similarly, C8910, geraniol and citronella oil showed toxic effect against both insects. The repellent activity of compounds was tested using the preference method assay at concentrations ranging between 3.125 and 50.0 μg/cm². The highest repellency percentage (RP) was achieved by C8910 against L. serricorne with a RP value of 76.0% at the lowest concentration (6.25 μg/cm²), while carvacrol showed the highest repellent activity against R. dominica with RP value of 88.0% at 3.125 μg/cm² within 3 h of insect exposure. The tested compounds caused higher repellent activity to R. dominica than L. serricorne. In vitro inhibition studies of acetylcholinesterase (AChE) in adults of both species showed that R-(+) pulegone strongly inhibited AChE activity of R. dominica and L. serricorne with 69.0% and 88.0% inhibition at 40 mM, respectively. Carvacrol caused 41.8% inhibition in AChE activity of R. dominica compared to 66.7% of L. serricorne at 40 mM. The results indicated that the tested natural compounds may be useful alternatives for controlling R. dominica and L. serricorne.     

Angiotensin I-converting enzyme inhibitory activity of chickpea and pea protein hydrolysates

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of chickpea (kabuli and desi) and yellow pea (Golden) using in vitro gastrointestinal simulation, alcalase/flavourzyme, and papain. Protein/peptide profiles studied by SDS–PAGE and SE-HPLC, showed a rich composition of the hydrolysates in small peptides having MWs under 4 kDa. Papain hydrolysed yellow pea proteins showed the highest ACE inhibitory activity. In addition, chickpea desi proteins hydrolysed by in vitro gastrointestinal simulation showed higher ACE inhibition (IC₅₀ of 140 ± 1 μg/ml) compared to its digests obtained by alcalase/flavourzyme (IC₅₀ of 228 ± 3 μg/ml) or papain (IC₅₀ of 180 ± 1 μg/ml) and to chickpea kabuli hydrolysed by gastrointestinal simulation (IC₅₀ of 229 ± 1 μg/ml). The results demonstrate that enzymatic hydrolysates of chickpea and pea proteins contain bioactive ACE inhibitory peptides; furthermore, the type of enzyme used for hydrolysis affects the ACE inhibitory activity.     

Changes in phenolics, γ-aminobutyric acid content and antioxidant,antihypertensive and hypoglycaemic properties during ale white sorghum (Sorghum bicolor (L.) Moench) brewing process

Effects of white sorghum brewing process on free amino-acids, γ-aminobutyric acid (GABA), phenolics and bioactivity, including antioxidant (by ABTS⁺ and reducing power, RP, methods), antihypertensive (angiotensin converting enzyme-I, ACE-I inhibition assay), and hypoglycaemic activity (α-glucosidase inhibition assay) were evaluated. From the wort to the beer, free amino acids decreased, but GABA and phenolics increased significantly, positively modifying the bioactive potential. ABTS and α-glucosidase inhibition activity correlated positively with at least one of the phenolic acids evaluated. Ale white sorghum beer presented high content of GABA (7.8 mg L⁻¹), phenolics (40.7 mg total phenolic acids L⁻¹), antioxidant activity (9.14 mmol Trolox equivalent L⁻¹, and 48.8 mmol ascorbic acid equivalent L⁻¹, for ABTS⁺ and RP, respectively), and exhibit ACE-I inhibition (1.0 μg captopril equivalent L⁻¹) and α-glucosidase inhibition (34.5 mg acarbose equivalent L⁻¹) activities. The level of bioactive compounds and its low ethanol content (2.3%), make beer obtained from malted white sorghum a potential functional beverage.     

Rolliniastatin-1, a bis-tetrahydrofuran acetogenin: The major compound of Annona mucosa Jacq. (Annonaceae) has potent grain-protective properties

The widespread use of synthetic insecticides results in insecticide-resistant populations of maize weevil, Sitophilus zeamais Motsch. (Coleoptera: Curculionidae), a primary pest species of stored corn worldwide. Thus, new active ingredients with different modes of action are needed for integrated pest management (IPM) of stored grains. Thus, toxicological bioassays (using S. zeamais as bioindicator) associated to chromatographic techniques were performed to isolate insecticidal compounds from the grain-protective ethanolic extract of Annona mucosa Jacq. (Annonaceae) seeds. The acetogenin bis-tetrahydrofuran rolliniastatin-1 was identified as major bioactive constituent from A. mucosa seeds based on bioassay-guided fractionation, along with rolliniastatin-2, deethylrollinastatin-1, jimenezin and fractions containing triglycerides. This compound produced 51.1% of mortality of adult weevils when applied at 57.66 mg kg⁻¹, drastically reducing F₁ progeny and their damage to corn grains. Although the acute toxicity level was lower than that with the formulation based on diatomaceous earth at a concentration 17.3 times higher (Insecto®^, at recommended rate) used as a positive control, rolliniastatin-1 protected grain statistically similar to that of the positive control. Furthermore, bioassays indicate that compounds of different chemical natures have a synergistic effect on the overall biological activity of seed derivatives of A. mucosa. Efficacy and technical viability of the process to obtain rolliniastatin-1 from the seeds of A. mucosa should allow the production of a botanical insecticide to control populations of S. zeamais at corn warehouses.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates

Angiotensin I-converting enzyme (ACE) inhibitory activity was investigated for small red bean (Phaseolus vulgaris) protein hydrolysate produced by sequential digestion of Alcalase, papain followed by in vitro gastrointestinal simulation. The hydrolysate had ACE inhibitory activity with IC₅₀ of 67.2 ± 1.8 μg protein/mL. Peptides responsible for potent ACE inhibitory activity were isolated by a three-step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). The fraction obtained after RP-HPLC fractionation with the highest activity yielded an IC₅₀ of 19.3 ± 1.4 μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with K_i of 11.6 ± 1.7 μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC₅₀ value of 206.7 ± 3.9 μM. The results expand the knowledge base of ACE inhibitory properties of small red bean protein hydrolysate and should be useful in further identification of specific ACE inhibitory peptides in beans.     

Inhibition of the angiotensin-converting enzyme by grape seed and skin proanthocyanidins extracted from Vitis vinífera L. cv. País

The influence of two extracts of grape skin and seeds from Vitis vinífera L. cv. País (Chilean black grapes), rich in proanthocyanidins (PAs), was evaluated on the inhibition of the angiotensin I-converting enzyme (ACE), and the inhibition was related to the type and number of subunits of the polymeric PAs chain. Size exclusion chromatography was used to purify the extract and its characterization was made by acid catalysis depolymerization followed by HPLC. ACE activity was measured by quantitative HPLC, measuring the hyppuric acid (HA) produced from the hydrolysis of hippuryl-l-histidyl-l-leucine (HHL) by ACE. Structural compositions differed significantly between both extracts: the skin extracts do not exhibit epicatechin (EC) and epicatechin gallate (ECG), and the seed extracts did not present epigallocatechin (EGC). Skin extracts have a higher mean degree of polymerization (mDP) than seed extracts and a higher inhibition power (IC₅₀ = 0.14 ± 0.03 μM and IC₅₀ = 0.480 ± 0.03 μmol/L), respectively. The catechin (IC₅₀ = 1495 ± 90 μmol/L) and epicatechin (IC₅₀ = 1772 ± 121 μmol/L) monomers exerted lower inhibition than the either grape extract. The structural differences between grape skin and seed PAs could influence the ACE inhibition capacity. The larger inhibitory power of skin extract was associated to greater OH availability, higher mDP and the presence of EGC.     

Grain-protectant compounds from Duguetia lanceolata (Annonaceae) derivatives: Bioassay-guided searching and toxicity against the maize weevil

In order to obtain bioactive allelochemicals from Neotropical Annonaceae, bioguided chromatographic fractionations with derivatives prepared from Duguetia lanceolata St.-Hil. leaves were performed using the maize weevil, Sitophilus zeamais Mots. (Coleoptera: Curculionidae), as bioindicator, which is a primary insect pest of stored cereals in tropical conditions. For this end, four phases (hexane, dichloromethane, ethyl acetate and hydroalcohol) were obtained firstly by liquid-liquid partition and tested through a residual contact bioassay (at 1500 mg L⁻¹) in order to verify their bioactivity on adults of the maize weevil. Only the hexanic fraction caused significant mortality (74%) after the tenth day of insect exposure on treated samples of grains corn. Thus, thisfraction was separated into seven new subfractions by means of vacuum column chromatography and solvents of increasing polarities. Subfraction 2 promoted 90% of mortality whereas subfraction 6 promoted 30% of mortality of maize weevils. The aromatic compound 2,4,5-trimethoxystyrene and a mixture of steroids [campesterol (8.44%) + stigmasterol (12.37%) + sitosterol (79.19%)] were isolated from subfraction 2 and promoted promising grain protective activity, in a manner comparable to a diatomaceous-earth based formulation (our positive control). Furthermore, the results reported in the present study demonstrate the potential application of derivatives of D. lanceolata leaves in integrated pest management (IPM) of stored grains, mainly in the framework of maize weevil management.     

Pressurized green liquid extraction of betalains and phenolic compounds from Opuntia stricta var. Dillenii whole fruit: Process optimization and biological activities of green extracts

The Opuntia Stricta var. Dillenii's prickly pears are an underutilized fruits, which provide a great source of betalains (mainly, betacyanins) and phenolic compounds (phenolic acids and flavonoids) that could play an important role in health-promotion. This study focuses on the optimization of process for the green extraction of betalains and phenolic compounds from Opuntia stricta var. Dillenii's whole fruits by Pressurized liquid extraction (PLE), using a response surface methodology (RSM) by a central composite design (CCD) in order to obtain rich extracts in betalains and phenolic compounds with the similar profile of the original one found in O. dillenii fruits (avoiding any degradation of these compounds during the extraction process) with proven biological activities. For PLE optimization, the ethanol volume in water (0–100%, v/v) and the temperature (25–65°C) were selected as independent variables. The identification and quantification of the individual bioactive compounds of the obtained green extracts were done by HPLC-DAD-ESI/MS and HPLC-DAD-ESI/QTOF and their biological activities were determined by in vitro tests, as: the antioxidant activity by the ORAC method and the anti-inflammatory activity by the hyaluronidase inhibition method. Ethanol volume in water (%, v/v) was the variable with most significant (p ≤ 0.05) effect in the target responses (bioactive content and biological activities). The best results were obtained at 50% ethanol in water (v/v) and 25°C temperature (run 10) obtaining extracts with betalains as 2.34 ± 0.18 mg of betanin/g dry weight, 2.51 ± 0.04 mg of 5′´-O-E-sinapoyl-2′-apyosil-phyllocactin/g dry weight, 2.32 ± 0.19 mg of neobetanin/g dry weight and phenolic compounds, 2.08 ± 0.07 mg of piscicid acid/g dry weight and 0.25 ± 0.02 mg of isorhamnetin glucoxyl-rhamnosyl-pentoside (IG2) /g dry weight. The betalain and phenolic profile of the PLE extracts was quite similar to the profile from the conventional extraction, but PLE extraction process enhanced the extraction of some bioactives as neobetanin (39%) andpiscidic acid (124%). All employed PLE process CCD combinations significantly upgrade the in vitro biological activities (antioxidant and anti-inflammatory) of the Opuntia stricta var. Dillenii's PLE green extracts.     

Generation of phenolic-rich extracts from brewers' spent grain and characterisation of their in vitro and in vivo activities

Brewers' spent grain (BSG) is a co-product of the brewing industry, which is rich in phenolic acids. This study compared the in vitro bioactive properties of aqueous, aqueous pH-shift and enzyme-aided phenolic extracts from pale (unroasted) wet BSG. Overall, the pH-shift extracts showed higher oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant capacity (TEAC) and ferric-reducing antioxidant power (FRAP) values compared with the enzyme-aided extracts. A selected enzyme-aided extract showed highest in vitro ACE inhibitory activity (ACE IC₅₀: 0.133 ± 0.032 mg mL⁻¹) and mediated significant (p < 0.05) reductions in blood pressure markers in vivo following ingestion by spontaneously hypertensive rats (systolic blood pressure; −35.35 ± 9.04 and diastolic blood pressure; −28.55 ± 4.22 mmHg). The results demonstrated that the observed in vitro activity of phenolic-rich extracts may be translated into an in vivo hypotensive effect. Enzyme-assisted extraction has potential as an alternative solvent-free approach for generating phenolic-rich extracts from BSG that may find use in the management of hypertension.