Direct and Rapid Profiling of Biophenols in Olive Pomace by UHPLC-DAD-MS

Olive mill by-products are effluents generated during olive oil production process. The two-phase centrifugation system produces a semi-solid olive pomace called “alperujo.” This by-product is a combination of liquid and solid wastes derived from the three-phase manufacturing process. A direct and fast analytical method by ultra-high-pressure liquid chromatography-DAD coupled with ESI/MS-MS has been developed for the profiling of phenolic compounds. Thirty-five metabolites belonging to phenyl alcohols, secoiridoids, flavonoids, and iridoids were identified as the main constituents of alperujo in 12 min, including p-coumaroyl aldarate and a verbascoside derivative found for the first time in alperujo and a new ligstroside derivative. Six quantitatively significant components were determined at concentrations ranging from 17.7 mg/L for p-coumaric acid to 370.7 mg/L for hydroxytyrosol. Our data confirm that alperujo is an interesting source of phenolic compounds that could be extracted for use as nutraceuticals.     

Transfer of phenolic compounds during olive oil extraction in relation to ripening stage of the fruit

The transfer of phenolic compounds of Olea europaea L. cv. Arbequina variety during olive oil extraction in relation to ripening stage was investigated. The parameters of oil extraction by the Abencor system are shown together with mass balances of the products and by products from the olive oil extraction in relation to olive paste. The phenolic compounds in olive paste, pomace, oil and wastewater were identified and measured by HPLC. Throughout the study, the concentrations of simple phenols, secoiridoids and flavonoids were higher in the olive paste and pomace phases than in oil and wastewater phases. High concentrations of 4‐(acetoxyethyl)‐1,2‐dihydroxybenzene (3,4‐DHPEA‐AC) and secoiridoid derivatives such as the dialdehydic form of elenolic acid linked to 3,4‐DHPEA (hydroxytyrosol) or p‐HPEA (tyrosol) (3,4‐DHPEA–EDA, p‐HPEA–EDA, where EDA is elenolic acid dialdehyde) and an isomer of oleuropein aglycone (3,4‐DHPEA–EA, where EA is elenolic acid aldehyde) were found in olive oil, together with lignan compounds. It was observed that 3,4‐DHPEA–EDA was the most abundant polyphenol present in the wastewater phase. This indicates that biotransformation occurred during olive extraction, especially in the crushing and malaxation operations, and reflects the possible chemical changes that lead to the formation of new compounds. Moreover, the distribution of compounds showed their affinities toward different phases. Copyright © 2005 Society of Chemical Industry     

Antioxidant activity of phenolic fractions in olive mill wastewater

Olive mill wastewater (OMW) contains a substantial amount of valuable antioxidant phenols that can be recovered for industrial application as food additives and pharmaceuticals. The present study was aimed at extracting different phenolic OMW fractions, and determining their antioxidant potential. Five different OMW fractions were obtained using fractionation techniques, their antioxidant potential determined by DPPH, ORAC and a β-carotene bleaching test. The total phenol level ranged between 115 and 170 mg/l. The phenolic compounds present in individual fractions were identified using the HPLC–PAD method, where the main compounds were hydroxytyrosol, tyrosol, caffeic acid, vanillic acid, verbascoside, oleuropein, ferulic acid, and p-coumaric acid. The five OMW fractions showed different antioxidant levels depending on the test used. DPPH test showed that the fraction of alkyl aromatic alcohols (AAAs) was the best with EC₅₀ of 20 mg/l and the pure hydroxytyrosol with 2 mg/l. ORAC test showed that AAA and semi hydrolysed total phenol (s-TP) fractions were significantly better than Trolox when compared to 20 mg/l of Trolox.     

The use of polyphenolic extract,purified hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid from olive mill wastewater for the stabilization of refined oils: a potential alternative to synthetic antioxidants

Ethyl acetate extracts of olive mill waste water (OMWW) were prepared, under in optimal conditions, using a continuous counter-current extraction unit. The antiradical and antioxidant activities of the OMWW extract as well as pure phenolic compounds identified in this extract were evaluated. Results showed that pure hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid had the highest radical-scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical and the highest antioxidant activities using the β-carotene linoleate model system.The effect of addition of individual phenolic compounds and OMWW extract to refined olive and husk oils was compared with that of control, BHA and BHT at 50 °C. Unexpectedly, 3,4-dihydroxyphenyl acetic acid had the highest protective effect against oil oxidation. Oils with added 3,4-dihydroxyphenyl acetic acid had lower PV than oils with added hydroxytyrosol, the most studied powerful antioxidant. Moreover, the addition of OMWW extract, at 500 ppm, resulted in lower PV values than BHA, p-hydroxyphenylacetic acid, tyrosol on the control.The results suggested that 3,4-dihydroxyphenyl acetic acid, hydroxytyrosol and OMWW extract possess useful antioxidant properties and may be used as alternatives in the search for natural replacement of synthetic antioxidant food additives.     

Phenolic and antioxidant potential of olive oil mill wastes

Olive oil mill waste was subjected to conventional liquid solvent extraction and supercritical fluid extraction using different solvents and carbon dioxide, respectively. The optimum solvent extraction conditions of phenols were 180 min using ethanol, at a solvent to sample ratio 5:1 v/w, and at pH 2. Solvent and SFE extracts were tested for their antioxidant activity by the DPPH radical scavenging method and by determination of peroxide value on virgin olive oil and sunflower oil. The ethanol extract exhibited the highest antiradical activity, and no correlation was found between antiradical activity and phenol content. The SFE extract exerted good antioxidant capacity although its phenolic yield was not quite high. Moreover, the ethanol extract appeared to be a stronger antioxidant than BHT, ascorbyl palmitate and vitamin E by the Rancimat method on sunflower oil. HPLC analysis of the extracts showed that the predominant phenolic compound was hydroxytyrosol. Various phenolic acids and flavonoids were also identified.     

Stability and antioxidant potential of purified olive mill wastewater extracts

A purified olive extract (POE) rich in phenolic and oleosidic compounds was prepared from olive mill wastewaters by adsorption onto an amphoteric polymer resin with the yield of 2.2% (w/v). In addition to complex and simple phenolic compounds found in the POE, the highly hydrophilic elenolic acid and DEDA (dialdehydic form of decarboxymethyl elenolic acid, a non-phenolic secoiridoids) were also present in high concentration (45%, w/w). Among the phenolic constituents, the major compounds are hydroxytyrosol and its glucoside, DEDA linked to hydroxytyrosol (Hy-DEDA), p-coumaric acid and caffeic acid, verbascoside and some flavonoids. The high content (23%, w/w in gallic acid equivalent) and structural diversity of these phenolic compounds confer superior antioxidant potential of POE with an IC₅₀ less than 8.5 μg/ml determined by DPPH assay. The stability of bioactive components during storage of the extract and changes in antioxidant properties were studied under different conditions. Determination of kinetics of degradation revealed that air/oxygen is the determinant factor which influences the stability of POE under low temperature storage conditions, while exposure to sunlight did not have significant effect on their stability. Whilst an increase in storage temperature decreased the total content of phenolic compounds (20-24% reduction) and non-phenolic secoiridoid aglycons, a high antioxidant capacity was still observed particularly in the nitrogen-protected POE samples (93-95% of the initial value). This was believed to be due to the transformation of hydroxytyrosol-containing complex phenols, especially the secoiridoid ester Hy-DEDA, into free phenolic monomer. This contributed significantly to the maintenance of a high antioxidant potency of the whole extract during storage. The high antioxidant, radical scavenging activity and stability of the extract suggest potential applications in the food and pharmaceutical industries.     

Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract

Phenolic compounds derived from the olive plant (Olea europaea L.), particularly hydroxytyrosol and oleuropein, have many beneficial effects in vitro. Olive leaves are the richest source of olive phenolic compounds, and olive leaf extract (OLE) is now a popular nutraceutical taken either as liquid or capsules. To quantify the bioavailability and metabolism of oleuropein and hydroxytyrosol when taken as OLE, nine volunteers (five males) aged 42.8 ± 7.4 years were randomized to receive either capsulated or liquid OLE as a single lower (51.1 mg oleuropein, 9.7 mg hydroxytyrosol) or higher (76.6 mg oleuropein, 14.5 mg hydroxytyrosol) dose, and then the opposite strength (but same formulation) a week later. Plasma and urine samples were collected at fixed intervals for 24 h post‐ingestion. Phenolic content was analyzed by LC‐ESI‐MS/MS. Conjugated metabolites of hydroxytyrosol were the primary metabolites recovered in plasma and urine after OLE ingestion. Peak oleuropein concentrations in plasma were greater following ingestion of liquid than capsule preparations (0.47 versus 2.74 ng/mL; p = 0.004), but no such effect was observed for peak concentrations of conjugated (sulfated and glucuronidated) hydroxytyrosol (p = 0.94). However, the latter peak was reached earlier with liquid preparation (93 versus 64 min; p = 0.031). There was a gender effect on the bioavailability of phenolic compounds, with males displaying greater plasma area under the curve for conjugated hydroxytyrosol (11 600 versus 2550 ng/mL; p = 0.048). All conjugated hydroxytyrosol metabolites were recovered in the urine within 8 h. There was wide inter‐individual variation. OLE effectively delivers oleuropein and hydroxytrosol metabolites to plasma in humans.     

Residues of grape (Vitis vinifera L.) seed oil production as a valuable source of phenolic antioxidants

Phenolic compounds of seven grape seed samples originating from mechanical seed oil extraction were identified and quantified by HPLC–DAD before (intact seeds) and after (press residue) the oil recovery process. Total amounts of all identified compounds ranged from 4.81 (‘Cabernet Mitos’) to 19.12 g/kg (‘Schwarzriesling’) of defatted dry matter (DM; ‘Schwarzriesling’) for integral grape seeds, whereas their content in the press residues ranged from 2.80 (‘Cabernet Mitos’) to 13.76 g/kg of defatted DM (‘Spätburgunder’). This is the first study presenting comprehensive data on the contents of individual phenolic compounds comprising all polyphenolic subclasses of press residues from grape seed oil production also covering the determination of the antioxidant activities of each subclass (Folin–Ciocalteu, FRAP and TEAC assays). Additionally, the effects of different solvents on the yields of phenolic compounds were determined. Maximum yields were obtained using methanol/0.1% HCl (v:v), water [75 °C] and a mixture of ethanol and water [3:1; v:v], respectively, whereas pure ethanol resulted in poor polyphenol extraction. The results of the present study confirm the press residues of grape seed oil production still to be a rich source of polyphenolics with strong antioxidant activity.     

Study of the inhibitory activity of phenolic compounds found in olive products and their degradation by Lactobacillus plantarum strains

Lactobacillus plantarum is the main species responsible for the spontaneous fermentation of Spanish-style green olives. Olives and virgin oil provide a rich source of phenolic compounds. This study was designed to evaluate inhibitory growth activities of nine olive phenolic compounds against four L. plantarum strains isolated from different sources, and to explore the L. plantarum metabolic activities against these phenolic compounds. None of the nine compounds assayed (oleuropein, hydroxytyrosol, tyrosol, as well as vanillic, p-hydroxybenzoic, sinapic, syringic, protocatechuic and cinnamic acids) inhibited L. plantarum growth at the concentration found in olive products. Oleuropein and tyrosol concentrations higher than 100 mM were needed to inhibit L. plantarum growth. On the other hand, sinapic and syringic acid showed the highest inhibitory activity since concentrations ranging from 12.5 to 50 mM inhibited L. plantarum growth in all the strains analyzed. Among the nine compounds assayed, only oleuropein and protocatechuic acid were metabolized by L. plantarum strains grown in the presence of these compounds. Oleuropein was metabolized mainly to hydroxytyrosol, while protocatechuic acid was decarboxylated to catechol. Metabolism of oleuropein was carried out by inducible enzymes since a cell-free extract from a culture grown in the absence of oleuropein was unable to metabolize it. Independent of their isolation source, the four L. plantarum strains analysed showed similar behaviour in relation to the inhibitory activity of phenolic compounds, as well as their ability to metabolize these compounds.     

Addition of enzymes complex during olive oil extraction improves oil recovery and bioactivity of Western Australian Frantoio olive oil

Olive oil consumption has increased as many studies revealed the health benefits of regular consumption of olive oil. There is a need to find effective oil extraction techniques capable of increasing oil recovery without compromising its quality. This study investigated the impact of adding enzymes complex Viscozymes during olive oil extraction on oil recovery, total phenolic compounds, antiradical activity and the standard quality parameters. It was found that at a concentration of 0.30 g mL^?1, Viscozymes could significantly improve the oil recovery from 49 to 69% (P < 0.001) when compared to the Control sample. The concentration of total phenolic compounds was also significantly improved from 110 to 266 mg kg^?1 oil (P < 0.01) and the antiradical activity increased from 31 to 48% inhibition of 2,2‐diphenyl‐1‐picrylhydrazil radical (P < 0.001). Addition of Viscozymes therefore represents an effective extraction technique that increases oil recovery without compromising the concentration of total phenolic compounds and antiradical activity.     

Bioprocess intensification: an aqueous two-phase process for the purification of C-phycocyanin from dry Spirulina platensis

The dry Spirulina powders are rich in nutritional compounds especially including C-phycocyanin (C-PC) have been principal raw material in the food processing. But the purity of C-PC in the dry powders was not up to the food grade standard. In this study, C-PC was recovered and purified from the dry algae powders using aqueous two-phase system (ATPS). The optimal conditions were proved in polyethylene glycol (PEG) 1000 and sodium phosphate, system pH of 5.8, the tie-line length of 28.50 % (w/w) and the volume ratio (Vr) of 0.16 to increase the purity from the initial purity of 0.42 to 1.31 after the first extraction. The recovery yield was 89.52 %. After the third ATPS extraction, the purity and the purification factor were achieving up 2.11 and 5.01. It was successfully decreased the viscosity of the system and extraction time by application of PEG 1000. It facilitated the feasibility of the scaling-up in industry.     

Characterization of antioxidant olive oil biophenols by spectroscopic methods

BACKGROUND: Olive oil contains numerous phenolic components with well‐recognized health‐beneficial activity. The major phenolic compounds present in olives and virgin olive oil—hydroxytyrosol, oleuropein and the oleuropein aglycones 3,4‐DHPEA‐EA and 3,4‐DHPEA‐EDA—as well as some of their metabolites were studied in the present work, regarding their main structural preferences. Vibrational spectroscopy (Raman) coupled to theoretical methods were used, aiming at fully characterizing the systems and therefore enabling their quick and reliable identification in food samples. RESULTS: The Raman data, assisted by the theoretical simulations, allowed us to obtain the main geometrical and spectroscopic features of the olive oil constituents under study, which determine their known antioxidant and chemoprotective properties. In fact, it was verified that the spectra comprise distinctive bands for each compound, allowing their ready detection and differentiation. CONCLUSION: This is the first reported study on the structural behaviour of olive oil phenolic compounds, and it established Raman spectroscopy as a rapid, non‐destructive and reliable analytical technique for identifying these bioactive components in dietary extracts. It can surpass other analytical methods currently used, once it allows the concomitant identification of several olive oil components in a particular sample. Copyright © 2010 Society of Chemical Industry     

Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers

Hydroxytyrosol and hydroxytyrosyl acetate are two well-known phenolic compounds with antioxidant properties that are present in virgin olive oil. Since the in vivo biological activity of polyphenols is dependent on their intestinal absorption and metabolism, the absorption of hydroxytyrosol and hydroxytyrosyl acetate and the extent to which they are conjugated and metabolised during transfer across intestinal Caco-2/TC7 cell monolayers, was investigated. LC-DAD and LC-MS were used for the quantification and identification of metabolites. Further evidence was obtained by observing metabolite susceptibility to β-glucuronidase treatment and by comparison of products of in vitro conjugation reactions of authentic phenolics with those produced by the CaCo-2 cells. Homovanillyl alcohol was the only conjugate detected as a result of hydroxytyrosol metabolism, and accounted for 20% of the total metabolites detected in the basolateral compartment after 2 h of incubation. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol (38.4%) and subsequently metabolised into homovanillyl alcohol (6.7%). In addition, hydroxytyrosyl acetate glucuronide (17.4%) together with non-metabolised hydroxytyrosyl acetate (37.5%) were also detected. Both hydroxytyrosyl acetate and hydroxytyrosol were transferred across human Caco-2/TC7 cell monolayers, but the acetylated compound exhibited an apparent permeability (Papp_AP→BL/Papp _BL→AP) 2.1-fold higher than free hydroxytyrosol. For both compounds, all conjugates were preferentially transported to the basolateral side. These results show that the acetylation of hydroxytyrosol significantly increases its transport across the small intestinal epithelial cell barrier, and supports further research into hydroxytyrosyl acetate as a hydroxytyrosol prodrug offering enhanced bioavailability.     

Emerging Technologies for Recovery of Value-Added Components from Olive Leaves and Their Applications in Food/Feed Industries

Olive leaves are the most abundant agricultural waste source rich in polyphenolics. Due to the numerous health benefits associated with these compounds, the interest in recovering polyphenols from olive leaves has increased in the scientific community over the last decade. Recent studies have focused on improved extraction techniques and processing methods that are most suited for agro-biological industries involved in the development of nutraceutical and functional products. The major problems in olive leaves processing include bitter taste and the low stability of various phenolic compounds. Oleuropein and hydroxytyrosol are the most important phenolic compounds extracted from olive leaves. The present review highlights the importance of olive leaves, their composition, preparation methods, major phenolic compounds, and commercial applications. This review article focuses on integrating studies on olive leaf extract (OLE) pertinent to nutrition, health, and beauty. The different board categories of delivery systems available for the encapsulation of OLE are given. These novel delivery systems could improve fortification, supplementation, and dietary diversification in food and pharmaceutical products.     

Ultrasound-assisted enzyme catalyzed hydrolysis of olive waste and recovery of antioxidant phenolic compounds

The ultrasound-assisted enzyme hydrolysis (UAEH) procedure for extraction of phenolics from olive waste (OW) was established. The optimized conditions obtained with response surface methodology (RSM) were pH 5.75, treatment 40 min and temperature 55 °C, the yield obtained reached to 4.0%. The extraction kinetics model was fitted well to the ln (C_t/C₀) = kt, and the rate constants (k) increased with the increased in the extraction temperature. Moreover, the values of k were higher and the activation energy was lower than ultrasound treatment alone. The thermodynamic parameters indicated the extraction process was easier under the UAEH. Compared with the petroleum ether and n-butyl alcohol, ethyl acetate extract fraction had high phenolic content (64.4 mg/g), especially hydroxytyrosol and tyrosol (17.2 mg/g and 14.1 mg/g), and exhibited excellent antioxidant activities in DPPH·, ABTS⁺·and FRAP assay. These finding revealed that OW could be used for a enrich source of natural phenolic compounds for development of food industry.Industrial relevanceOlive waste (OW) has been widely paid attention as sources of high-added value compounds, such as phenolic which can be obtained by the several methods. And phenolic extract as a food additive can not only improve the sensory quality of food, but also can enhance antioxidant capacity in fatty food matrices. Moreover, the use of this extract has better economic benefits than synthetic additives usually used in food industry. In this study, a novel green, simple and efficient technology was employed to extract the phenolic compounds, which was suitable for large-scale industrial use. The results indicated that ultrasound-assisted enzyme hydrolysis (UAEH) has significant potential in the extraction process of phenolics from OW exhibiting higher phenolic yield in shorter extraction times.     

Spray drying of a phenolic‐rich membrane filtration fraction of olive mill wastewater: optimisation and dried product quality

Olive mill wastewater (OMWW) from 3‐ and 2‐phase mills was subjected to a two‐step membrane filtration process. The obtained reverse osmosis retentate (RO‐R) is a phenolic‐rich coproduct stream, and the reverse osmosis permeate is a near‐pure water stream. A pilot‐scale spray dryer was used for the production of RO‐R powder. The optimum temperature/feed pump conditions were obtained for 3‐phase (150°C/4.4 Hz) and for 2‐phase RO‐R (175°C/3.8 Hz). The powders obtained by spray drying under these conditions were analysed for physicochemical properties, including identification and quantification of phenolic compounds. Protocatechuic acid, 3‐hydroxytyrosol, tyrosol, vanillic acid and p‐coumaric acid were quantified in both RO‐R powders, which show the possibility to obtain a phenolic‐rich powder from an OMWW filtration fraction. This will lead towards better usage of by‐products from olive oil production.     

Stability of encapsulated olive oil in the presence of caffeic acid

The aim of this study was to investigate the influence of microencapsulation and addition of the phenolic antioxidant caffeic acid (CA) on the storage stability of olive oil. Olive oil in the absence or presence of 300 ppm CA was encapsulated in 1.5% w/w sodium alginate shells. Encapsulated oil (with/without added CA) and unencapsulated oil were stored at 20 or 37 °C for 30 days and then subjected to stability and quality evaluation based on peroxide value (PV), p-anisidine value (p-AV), Totox value, free fatty acid (FFA), total extractable phenolic content (TEPC), and fatty acid composition. The CA addition increased the stability and TPC of the final oil product. Oxidation changes were generally slower in the encapsulated oil samples. Both encapsulation and addition of CA preserved unsaturated fatty acids (UFAs) including C18:1 (omega-9 FA), C18:2 (omega-6 FA) and C18:3 (omega-3 FA). We conclude that the current oil encapsulation method using alginate microspheres could be a feasible approach to increasing olive oil stability. The addition of CA to olive oil not only provides additional protection to the oil, but also improves the nutritional values of the final oil product in terms of elevated TEPC and desired UFAs.     

Contribution of olive seed to the phenolic profile and related quality parameters of virgin olive oil

BACKGROUND: Conflicting results have been reported about the effect of fruit de‐stoning on the virgin olive oil (VOO) phenolic profile. The aim of the present study was to determine whether olive seed plays any role in the synthesis of this oil phenolic fraction. RESULTS: Increases of around 25% of total phenolic compounds were observed in oils obtained from de‐stoned olive fruits in three main Spanish cultivars. To investigate the involvement of olive seed in determining the phenolic profile of VOO, whole intact olive fruits were added with up to 400% olive stones. Excellent regression coefficients were found in general for the decrease of total phenolic compounds and, particularly, of o‐diphenolics in the resulting oils. On the other hand, it was found that olive seed contains a high level of peroxidase (POX) activity (72.4 U g^?1 FW), accounting for more than 98% of total POX activity in the whole fruit. This activity is able to modify VOO phenolics in vitro, similar to the effect of adding stones during VOO extraction. CONCLUSION: Olive seed plays an important role in determining VOO phenolic profile during the process to obtain an oil that seems to be associated with a high level of POX activity. Copyright © 2007 Society of Chemical Industry     

Biological properties of olive oil phytochemicals

Olive oil is the principal source of fat in the Mediterranean diet, which has been associated with a lower incidence of coronary heart disease and certain cancers. Extra-virgin olive oil contains a considerable amount of phenolic compounds, for example, hydroxytyrosol and oleuropein, that are responsible for its peculiar taste and for its high stability. Evidence is accumulating to demonstrate that olive oil phenolics are powerful antioxidants, both in vitro and in vivo; also, they exert other potent biological activities that could partially account for the observed healthful effects of the Mediterranean diet.     

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.