Chicken egg is included among the main components of the human diet as an important source of nutrients, such as proteins, lipids, vitamins, minerals and carotenoids. The latter are terpenoid pigments present in egg yolks, providing their color and playing a vital role because of their significant bioactivities. The carotenoid content of egg yolk varies considerably since it is strongly influenced by the respective laying hens’ farming and feeding procedures, and there is therefore a need to establish an efficient method for their assessment. The absence of such a method prompted us to develop a novel procedure consisting of the extraction, saponification and quantitative assessment of contained carotenoids. For this purpose, the optimal conditions for the extraction of carotenoids from egg yolks were defined, along with the optimal saponification conditions of carotenoids, with respect to reaction duration and pH influence on the extract’s contents of lutein and zeaxanthin. The carotenoid content of extracts was determined using a novel, developed herein LC-MS/MS method that allows the accurate, fast and simultaneous quantitation of the 11 most abundant carotenoids in egg yolks. The method accuracy and reliability were validated for six different parameters determined for each analyte. The novel procedure was applied for the assessment of the carotenoid content of ten egg yolks of diverse origin, indicating the bioactive carotenoids lutein and retinol as the most abundant, while lesser amounts of the remaining natural and synthetic carotenoids were found and there was no trace of fucoxanthin or astaxanthin molecules. The results herein revealed a variation in the carotenoid content of chicken eggs that depended on the diet and farming method of egg-laying hens.

This study aimed to assess the effects of partial and complete replacement of synthetic vitamin E (Vit E) with grape pomace extract, rich in polyphenols, and wine lees derivatives, abundant in beta-glucans, on broiler performance, meat quality, health biomarkers, and intestinal integrity. There was a control group (CON) fed a basal diet with commercially recommended Vit E levels, while a second group (GP) was fed the same basal diet included zin-bacitracin as an antimicrobial growth promoter. The remaining three groups (W25, W50, W100) had 25%, 50%, and 100% of Vit E replaced by 25, 50, and 100 mg polyphenols as GAE/kg feed from grape pomace extract, respectively, supplemented also with 150 mg beta-glucans/kg feed. The W25 group tended to have lower body weight compared to the CON and GP groups (2565 g vs. 2762 g and 2727 g, respectively; p = 0.056), while feed intake was significantly higher in the CON group (p = 0.021). Malondialdehyde concentrations in breast and thigh muscles were elevated in the W100 group at 24- and 72-hours post-mortem (p = 0.039 and p = 0.030, respectively). Glutathione reductase levels in blood plasma were significantly reduced (p = 0.022) in the GP, W50, and W100 groups compared to CON. Additionally, CLDN1 expression in the jejunal mucosa was significantly increased in W100 compared to the CON group (p = 0.045). These findings suggest that partial substitution of Vit E with grape pomace and wine lees derivatives by 50% could be a feasible commercial dietary strategy since growth performance, oxidative status, and gene expression were not impaired.

Legumes, one of the first crops of humanity, inherently constitute a staple nutritional source for mankind, attracting significant research attention that has been afforded to the development of numerous cultivars. The study herein concerns the exploitation of the nutritional and bio-functional content of beans harvested from eleven Greek cultivars belonging to five different species, namely Cicer arietinum L., Pisum sativum L., Vicia faba L., Lens culinaris L., and Phaseolus vulgaris L. The final goal is to define their varietal identity and correlate their phytochemical content with their potential utilization as functional foods and/or feed of high nutritional value. In this respect, their extracts were screened against the presence of 27 fatty acids and 19 phenolic compounds, revealing the presence of 22 and 15 molecules, respectively. Specifically, numerous fatty acids were detected in significant amounts in all but C. arietinum extract, while significant polyphenolic content was confirmed only in P. vulgaris. Among individual compounds, linoleic acid was the major fatty acid detected in amounts averaging more than 150 mg/g, followed by oleic acid, which was present as a major compound in all extracts. Among the nine polyphenols detected in P. vulgaris, the molecules of genistein (3.88 mg/g) and coumestrol (0.82 mg/g) were the most abundant. Their antioxidant properties were evaluated through DPPH and FRAP assays, which were highlighted as most potent in both tests of the V. faba extract, while C. arietinum was determined as totally inactive, indicating a potential correlation between the phenolic content of the plant species and antioxidant activity. These results are indicative of the significant advances achieved for the cultivars investigated and reveal their important role as nutritional crops for human and animal consumption.

The imperative need to produce safe foodstuffs using environmentally sustainable practices has highlighted the incorporation of legumes in human and animal diets as an emerging nutritional staple. Since legumes comprise a family of plants known to display an extensive agricultural diversity with significant bioactivities, we report herein the exploitation outcome of the nutritional and bio-functional content of hay, derived from the post-harvest aerial parts of eight novel-improved Greek cultivars belonging to the following six taxa: Larthyrus sativus L., Medicago sativa L., Cicer arietinum L., Pisum sativum L., Vicia faba L., and Phaseolus vulgaris L. In specific, the phytochemical content of their hay extracts was screened against the presence of 37 fatty acids and 67 polyphenols using, respectively, GC-FID and LC-MS/MS instruments, while the determination of their total phenolic and tannin contents was achieved with spectrophotometric methods. In this respect, the presence of 26 different fatty acids was detected of which the acids linoleic, linolenic and palmitic were traced in all extracts in concentrations exceeding the 10 mg/g, while the oleic acid was determined as the major component of C. arietinum (77.58 mg/g), V. faba (36.11 mg/g), and L. sativus (12.89 mg/g) extracts. In addition, 30 different phenolics were traced as extracts’ components with the most abundant the molecule of isoquercetin, which was present in six extracts and isoliquiritigenin, calycosin, and chlorogenic acid which were detected in five extracts. Finally, the antioxidant properties of the extracts were evaluated by performing both DPPH• and FRAP assays, which highlighted the V. faba extract as the most potent in both tests, followed by the extracts of P. sativum and P. vulgaris. Results herein are indicative of the significant advances achieved, for the improvement of investigated plant cultivars with respect to their utilization as nutritional crops, since these novel cultivars hays have been found to contain significant amounts of essential nutrients in the form of fatty acids along with bioactive ingredients in the form of polyphenols, while simultaneously they were determined as deprived of tannins that constitute essential anti-nutritional factors.

Oregano (Origanum vulgare, Lamiaceae plant family) is a well-known aromatic herb with great commercial value, thoroughly utilized by food and pharmaceutical industries. The present work regards the comparative assessment of in vitro propagated and commercially available oregano tissue natural products. This study includes their secondary metabolites’ biosynthesis, antioxidant properties, and anticancer activities. The optimization of callus induction from derived oregano leaf explants and excessive oxidative browning was performed using various plant growth regulators, light conditions, and antioxidant compounds. The determination of oregano callus volatiles against the respective molecules in maternal herbal material was performed using gas chromatography–mass spectrometry (GC/MS) analysis. In total, the presence of twenty-seven phytochemicals was revealed in both leaf and callus extracts, from which thirteen molecules were biosynthesized in both tissues studied, seven compounds were present only in callus extracts, and seven metabolites only in leaf extracts. Carvacrol and sabinene hydrate were the prevailing volatiles in all tissues exploited, along with alkanes octacosane and triacontane and the trimethylsilyl (TMS) derivative of carvacrol that were detected in significant amounts only in callus extracts. The MTT assay was employed to assess the in vitro cytotoxic properties of oregano extracts against the epithelial human breast cancer MDA-MB-231 and the human neuroblastoma SK-N-SH cell lines. The extracts displayed concentration and time-dependent responses in cell proliferation rates.

The intensive use of plant materials as a sustainable alternative for fish feed production, combined with their phytochemical content, which affects the growth and production characteristics of farmed fishes, necessitates their monitoring for the presence of raw materials of plant origin. This study reported herein concerns the development, validation and application of a workflow using high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) for the quantification of 67 natural phytoestrogens in plant-derived raw materials that were used to produce fish feeds. Specifically, we verified the presence of 8 phytoestrogens in rapeseed meal samples, 20 in soybean meal samples, 12 in sunflower meal samples and only 1 in wheat meal samples in quantities enabling their efficient incorporation into clusters. Among the various constituents, the soybean phytoestrogens daidzein, genistein, daidzin, glycitin, apigenin, calycosin and coumestrol, as well as the sunflower neochlorogenic, caffeic and chlorogenic phenolic acids, displayed the highest correlations with their origin descriptions. A hierarchical cluster analysis of the studied samples, based on their phytoestrogen contents, led to the efficient clustering of raw materials. The accuracy and efficiency of this clustering were tested through the incorporation of additional samples of soybean meal, wheat meal and maize meal, which verified the utilization of the phytoestrogen content as a valuable biomarker for the discrimination of raw materials used for fish feed production.

Common vetch (Vicia sativa L.) is one of the most cultivated feed crops with extensive agricultural diversity and numerous cultivars. This study concerns the first-time investigation of the dry plant biomass and grains of six vetch cultivars to define the detailed fingerprint of their phenolic and fatty acid content, along with their respective antioxidant potencies. The results revealed a substantial variation in the feed quality traits among the tested Vicia sativa varieties, highlighting the crucial role and influence the genotype plays in the achievement of high-quality livestock nutrition. Among the six varieties tested, Istros and M-6900 displayed a particularly intriguing phytochemical profile characterized by elevated phenolic content, significant antioxidant potency and remarkably high fatty acid indices. These findings are indicative of the great potential of these varieties to function as suitable candidates for incorporation into farm animal diets either in the form of dry biomass (hay) or as a grain feed additive.

The significant beneficial effects of phytoestrogens on human and animal health have emerged the necessity to develop a sensitive, accurate and rapid method for their efficient quantitation in human and animal foods. Study herein concerns the development of a novel analytical method using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) that allows the simultaneous quantitation of 67 natural phytoestrogens. The novel method was fully validated through the determination of the following parameters: selectivity, sensibility, recovery, precision, accuracy of detection and quantification limits for each analyte. Specifically, the novel method proved accurate displaying linear response with R2 values >= 0.9991, quantitation limits from 5.5 to 331.8 ng/mL for 98.5% of analytes, recovery values ranging between 64.8% and 150.3%, while the precision and accuracy scores remained below 20% for most analytes. The method was successfully applied on the following plant samples used in human and/or animal diet belonging to Fabaceae plant family: Trifolium repens, Vicia sativa, Pisum sativum and Glycine max (soybean meal). Furthermore, the novel method was applied for the determination of the phytoestrogenic content of two animal feeds (for livestock and fishes) and two foodstuffs (chicken meat and fish fillet).

The global demand for increased meat production has brought to the surface several obstacles concerning environmental impacts, animals' welfare, and quality features, revealing the need to produce safe foodstuffs with an environmentally acceptable procedure. In this regard, the incorporation of legumes into animal diets constitutes a sustainable way out that prevents these apprehensions. Legumes are plant crops belonging to the Fabaceae family and are known for their rich content of secondary metabolites., displaying significant antioxidant properties and a series of health and environmental benefits. The study herein aims to investigate the chemical composition and antioxidant activities of indigenous and cultivated legume plants used for food and feed. The respective results indicate that the methanolic extract of Lathyrus laxiflorus (Desf.) Kuntze displayed the highest phenolic (64.8 mg gallic acid equivalents/g extract) and tannin (419.6 mg catechin equivalents/g extract) content, while the dichloromethane extract of Astragalus glycyphyllos L., Trifolium physodes Steven ex M.Bieb. and Bituminaria bituminosa (L.) C.H.Stirt. plant samples exhibited the richest content in carotenoids lutein (0.0431 mg/g A. glycyphyllos extract and 0.0546 mg/g B. bituminosa extract), α-carotene (0.0431 mg/g T. physodes extract) and β-carotene (0.090 mg/g T. physodes extract and 0.3705 mg/g B. bituminosa extract) establishing their potential role as vitamin A precursor sources. Results presented herein verify the great potential of Fabaceae family plants for utilization as pasture plants and/or dietary ingredients, since their cultivation has a positive impact on the environment, and they were found to contain essential nutrients capable to improve health, welfare, and safety.

Although carotenoids generally possess antimicrobial and antioxidant properties, the in vivo synergistic action of carotenoid blends derived from plant-based by-products has not been thoroughly studied. Therefore, the carotenoid characterization and antimicrobial potential of Citrus reticulata extract as well as the impact of this carotenoid-rich extract (CCE) dietary supplementation on the performance, meat quality, and immune-oxidative status of broiler chickens were determined. One hundred and twenty one-day-old hatched chicks (Ross 308) were allocated to two dietary groups, with four replicate pens of 15 birds each. Birds were fed either a basal diet (CON) or the basal diet supplemented with 0.1% CCE (25 mg carotenoid extract included in 1 g of soluble starch) for 42 d. β-Cryptoxanthin, β-Carotene, Zeaxanthin, and Lutein were the prevailing carotenoid compounds in the Citrus reticulata extract. The CCE feed additive exerted inhibitory properties against both Gram-positive (Staphylococcus aureus) and negative (Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium) bacteria. Both the broiler performance and meat quality did not substantially differ, while the breast muscle malondialdehyde (MDA) concentration tended to decrease (p = 0.070) in the CCE-fed broilers. The inclusion of CCE decreased the alanine aminotransferase and MDA concentration, and the activity of glutathione peroxidase, while the activity of superoxide dismutase was increased in the blood. Catalase and NADPH oxidase 2 relative transcript levels were significantly downregulated in the livers of the CCE-fed broilers. Additionally, Interleukin 1β and tumor necrosis factor (TNF) relative transcript levels were downregulated in the livers of the CCE- fed broilers, while TNF and interferon γ (IFNG) tended to decrease in the spleens and bursa of Fabricius, respectively. The present study provided new insights regarding the beneficial properties of carotenoids contained in Citrus reticulata in broilers' immune-oxidative status. These promising outcomes could be the basis for further research under field conditions.

Citrus fruits Cold Press Essential Oils (CPEOs) constitute a low-cost by-product produced during the juice making process that are composed by a volatile and a non-volatile fraction. Their non-volatile fractions are rich in valuable secondary metabolites, such as carotenoids, coumarins, psoralens and flavonoids [1,2]. Study herein concerns the development of a quantitative method for their carotenoids analyses using the LC-MS/MS and LC-DAD methodology. • CPEOs carotenoids content was characterized quantitatively. • Natural carotenoids β-cryptoxanthin, lutein and zeaxanthin were determined as the most abundant high added value molecules.

Cold Press Essential Oil (CPEO) of Citrus fruits, a co-product of juice-making industry, is investigated herein as a sustainable source of high added value industrial products exploring for first time its potentials for simultaneous carotenoid and fine chemicals isolation. Main obstacle towards this target is the physical and chemical properties differentiation of the main CPEO ingredients. To overcome it three different separation approaches were tested on orange, lemon, and tangerine CPEOs. The results indicated as most potent the novel procedure of negative pressure azeotropic condensation, which achieved yields of carotenoid crude extract consistently above 3% in all CPEOs, (lemon: 3.2%, orange 3.9%, tangerine: 5.2%). In addition, their total carotenoid content was found to excel in recovery rates after the volatiles removal, achieving 68% in tangerine, 85% in orange, and 38% in lemon. Tangerine CPEO was further processed, concluding to the pilot scale isolation of β-cryptoxanthin, which is presented herein for first time. In specific, the application of this process yielded 1.07% of pure all-trans-β-cryptoxanthin and 95.08% D-limonene, proving the feasibility and sustainability of the proposed protocol. This paradigm of circular economy regards the transformation of a low value industrial co-product into two high added value fine chemicals. In specific, the added value of the crude tangerine CPEO, with a commercial price 7 euros per litre, after the separation and recovery of limonene and β-cryptoxanthin is significantly increased as suggested by the cumulative price of 14 270 euros per litre for both isolates (limonene: 270 euros, β-cryptoxanthin:14 000 euros).

Vinification by-products display great potential for utilization as feed additives rich in antioxidant compounds. Thus, the effect of dietary ground grape pomace (GGP), wine lees extract rich in yeast cell walls (WYC), and grape stem extracts (PE) on the relative expression of several genes involved in liver oxidative mechanisms and the oxidative status of the blood and breast muscle of broiler chickens was investigated. In total, 240 one-day-old as hatched chicks (Ross 308) were assigned to four treatments, with four replicate pens and 15 birds in each pen. Birds were fed either a basal diet (CON) or a basal diet supplemented with 25 g/kg GGP, or 2 g/kg WYC, or 1 g starch including 100 mg pure stem extract/kg (PE) for 42 days. The polyphenolic content of vinification by-products was determined using an LC-MS/MS library indicating as prevailing compounds procyanidin B1 and B2, gallic acid, caftaric acid, (+)-catechin, quercetin, and trans-resveratrol. Body weight and feed consumption were not significantly affected. The relative transcript level of GPX1 and SOD1 tended to increase in the liver of WYC-fed broilers, while NOX2 tended to decrease in the PE group. SOD activity in blood plasma was significantly increased in WYC and PE compared to the CON group. The total antioxidant capacity measured with FRAP assay showed significantly higher values in the breast muscle of PE-fed broilers, while the malondialdehyde concentration was significantly decreased in both WYC- and PE-fed broilers compared to the CON group. The exploitation of vinification by-products as feed additives appears to be a promising strategy to improve waste valorization and supply animals with bioactive molecules capable of improving animals’ oxidative status and products’ oxidative stability.

The valorization of vinification byproducts portrays a promising bioprocess for the enrichment of animals’ diet with bioactive compounds, such as polyphenols, which could regulate the immune response. Therefore, the impact of dietary grounded grape pomace (GGP), wine lees extract (WYC), and grape stem extract (PE) on the relative transcript level of immune related genes of broiler chickens were examined. Two hundred forty, one-day-old as hatched (male/female) chicks (Ross 308) were allocated to four dietary groups, with four replicate pens each with 15 birds. Birds were fed either a basal diet (CON) or the basal diet supplemented with 2.5% GGP, or 0.2% WYC, or 0.1% PE for 42 d. The relative expression of immune-related genes was investigated using a real-time PCR platform. The mRNA levels of Toll-like Receptor 4 (TLR4) were downregulated (p = 0.039) in the liver of broilers fed the GGP-containing diet compared to the CON, while in the spleen of PE-fed broilers, TLR4 was significantly upregulated (p = 0.043). The mRNA levels of interleukin 8 (IL8) tended to upregulate (p = 0.099) in the bursa of Fabricius and were significantly increased (p = 0.036) in the spleen of broilers fed the PE diet. Vinification byproducts depict a promising sustainable source of polyphenols for the poultry feed industry, but more research is needed under field conditions.

Cold pressed essential oil (CPEO) of mandarin (Citrus reticulata Blanco), a by-product of the juice-making industrial process known to contain large amounts of polymethoxyflavones, was exploited for its content in high added value natural coumarins. The study herein afforded a method referring to the evaporation of CPEO volatile fraction under mild conditions (reduced pressure and temperature below 35 °C) as azeotrope with isopropanol. This allowed the isolation of high added value coumarins from the non-volatile fragment using preparative High Performance Liquid Chromatography (HPLC). Pilot-scale application of this procedure afforded for each kg of CPEO processed the following natural bioactive coumarins in chemically pure forms: heraclenol (38–55 mg), 8-gerayloxypsoralen (35–51 mg), auraptene (22–33 mg), and bergamottin (14–19 mg). The structures of coumarins were verified by Nuclear Magnetic Resonance (NMR) spectroscopy and HPLC co-injection with authentic standards. Thus, the low market value mandarin CPEO with current value of 17 to 22 EUR/kg can be valorized through the production of four highly bioactive natural compounds worth 3479 to 5057 EUR/kg, indicating the great potentials of this methodology in the terms of the circular economy.

The well-established, health-benefitting effects of grapevines and derivatives (wines and vinification byproducts) are attributed to their antioxidant phenolic content. The dearth of an efficient method for the simultaneous quantitation of antioxidant phenolics prompted us to develop a novel method utilizing triple quadrupole LC–MS/MS for the accurate, fast, simultaneous quantitation of the 32 most abundant grapevine phenolics. The fully validated, novel method is capable to simultaneously record the quantitative presence of 12 phenolic acids, 19 polyphenols and coniferyl aldehyde (a phenolic compound extracted from cork stoppers into wines) and is applicable for the determination of antioxidant phenolics content of grape berries, pomace, stems and wines. Its utility was demonstrated for three native Greek grapevine varieties, two red (Mandilaria and Aidani mavro) and one white (Monemvassia). Results herein highlighted the stems of the Monemvassia white variety as particularly rich in antioxidant phenolics such as the flavonol monomer (+)-catechin (387 mg/kg) and the dimer procyanidin B1 (400 mg/kg) along with stilbene phytoalexin trans-resveratrol (24 mg/kg). These results are in line with the TPC, TFC and TTC content of stems and the determined antioxidant capacities, highlighting the stems of this Vitis vinifera variety as potentially exploitable source of antioxidant phenolics.

The involvement of complement activation in various forms of cardiovascular disease renders it an important factor for disease progression and therapeutic intervention. The protective effect of resveratrol against cardiovascular disease via moderate red wine consumption has been established but the exact mechanisms are still under investigation. The current study utilised human coronary artery endothelial cells (HCAECs) in order to assess the extent to which the protective effect of resveratrol, at concentrations present in red wine, can be attributed to the upregulation of complement regulatory proteins through heme-oxygenase (HO)-1 induction. Resveratrol at concentrations as low as 0.001 μΜ increased HO-1 expression as well as membrane cofactor protein (MCP, CD46) and decay-accelerating factor (DAF, CD55) expression with no-effect on CD59. Silencing of HO-1 expression by HO-1 siRNAs abrogated both DAF and MCP protein expression with no effect on CD59. Resveratrol-mediated induction of DAF and MCP reduced C3b deposition following incubation of HCAECs with 10% normal human serum or normal rat serum as a source of complement. Incubation of HCAECs, with either a DAF blocking antibody or following transfection with HO-1 siRNAs, in the presence of 10% normal rat serum increased C3b deposition, indicating that both DAF and HO-1 are required for C3b reduction. These observations support a novel mechanism for the protective effect of resveratrol against cardiovascular disease and confirm the important role of HO-1 in the regulation of the complement cascade.

The aim of the present study was the investigation of the antioxidant activity of plant extracts from Rosa canina, Rosa sempervivens and Pyrocantha coccinea. The results showed that the bioactive compounds found at higher concentrations were in the R. canina extract: hyperoside, astragalin, rutin, (+)-catechin and (-)-epicatechin; in the R. sempervirens extract: quinic acid, (+)-catechin, (−)-epicatechin, astragalin and hyperoside; and in the P. coccinea extract: hyperoside, rutin, (−)-epicatechin, (+)-catechin, astragalin, vanillin, syringic acid and chlorogenic acid. The total polyphenolic content was 290.00, 267.67 and 226.93 mg Gallic Acid Equivalent (GAE)/g dw, and the total flavonoid content 118.56, 65.78 and 99.16 mg Catechin Equivalent (CE)/g dw for R. caninna, R. sempervirens and P. coccinea extracts, respectively. The extracts exhibited radical scavenging activity in DPPH and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)•+ assays and protection from ROO•-induced DNA damage in the following potency order: R. canina > R. sempervirens > P. coccinea. Finally, treatment with R. canina and P. coccinea extract significantly increased the levels of the antioxidant molecule glutathione, while R. canina extract significantly decreased Reactive Oxygen Species (ROS) in endothelial cells. The results herein indicated that the R. canina extract in particular may be used for developing food supplements or biofunctional foods for the prevention of oxidative stress-induced pathological conditions of endothelium.

It has been proposed that exercise-induced oxidative stress and adaptations are dependent on training status. In this study, we examined the effects of training background on free radical generation and adaptations after eccentric exercise. Forty volunteers were divided into two groups (trained and untrained) and were asked to perform eccentric exercise. Then, their blood samples were collected pre, 24, 48, and 72 hours postexercise. Biomarkers indicating oxidative damage and the antioxidant profiles of the participants were measured in plasma and erythrocyte lysate both spectrophotometrically and chromatographically. The results revealed that the untrained group depicted more severe oxidative damage (protein carbonyls, malondialdehyde), weaker antioxidant status (reduced glutathione, static and capacity oxidation-reduction potential), and weaker radical-scavenging activity (superoxide radical scavenging and reducing power) compared to the trained participants. Our findings show that trained individuals are less susceptible to oxidative damage and suggest that generalized nutritional recommendations regarding recovery after exercise should be avoided.

Present essay explores the potentials of Citrus juice industry's by-products as alternative bioactive natural products resources. Four crude Cold Pressed Essential Oils (CPEOs), derived from orange, lemon, grapefruit, and mandarin, were studied. All CPEOs were subjected to water distillation, in order to obtain the volatile fragment, which was further fractionated with respect to distillation period in two parts, concluding to eight samples. These samples along with the four original CPEOs were assessed in relation to their phytochemical content and their repellent and larvicidal properties against Asian Tiger Mosquito. The volatiles recovery rates ranged from 74% to 88% of the CPEO. Limonene presented a significant increase in all samples ranging from 8% to 52% of the respective CPEO's content and peaked in mandarin's 2nd volatile fragment which comprised 97% of the essential oil. The refinement process presented clear impacts on both bioassays: a significant increase in larvicidal potency was observed, annotated best by the improvement by 1100% and 1300% of the grapefruit volatile fractions; repellence testing provided only one significant result, the decrease of landings by 50% as a response to mandarin's second volatile fraction. The applied methodology thus may be considered for the improvement of Citrus juice industry's by-products chemistry and bioactivity.