Optimizing the Extraction of Bioactive Compounds from Porphyra linearis (Rhodophyta): Evaluating Alkaline and Enzymatic Hydrolysis for Nutraceutical Applications.

Porphyra sensu lato is one of the most economically significant and widely cultured and consumed algae in the world. Porphyra species present excellent nutraceutic properties due to their bioactive compounds (BACs). This research aimed to find the most efficient aqueous extraction method for BACs by examining alkaline and enzymatic hydrolysis. Alkaline hydrolysis with 2.5% sodium carbonate (SC) and at 80 °C proved optimal for extracting all BACs (phycobiliproteins, soluble proteins, polyphenols, and carbohydrates) except mycosporine-like amino acids (MAAs), which were best extracted with water only, and at 80 °C. Enzymatic hydrolysis, particularly with the 'Miura' enzymatic cocktail (cellulase, xylanase, glycoside hydrolase, and ß-glucanase), showed superior results in extracting phycoerythrin (PE), phycocyanin (PC), soluble proteins, and carbohydrates, with increases of approximately 195%, 510%, 890%, and 65%, respectively, compared to the best alkaline hydrolysis extraction (2.5% SC and 80 °C). Phenolic content analysis showed no significant difference between the 'Miura' cocktail and 2.5% SC treatments. Antioxidant activity was higher in samples from alkaline hydrolysis, while extraction of MAAs showed no significant difference between water-only and 'Miura' treatments. The study concludes that enzymatic hydrolysis improves the efficiency of BACs extraction in P. linearis, highlighting its potential for the nutraceutical industry, and especially with respect to MAAs for topical and oral UV-photoprotectors.

Development of Lipid Nanoparticles Containing Omega-3-Rich Extract of Microalga Nannochlorpsis gaditana.

Microalgae are described as a new source of a wide range of bioactive compounds with health-promoting properties, such as omega-3 lipids. This biomass product is gaining attention mainly due to its potential to accumulate different compounds depending on the species and environment, and it has been commonly recognized as a valuable nutraceutical alternative to fish and krill oils. In this work, we obtained the extract of the microalga Nannochloropsis gaditana, selected on the basis of its content of eicosapentaenoic acid (EPA) and glycolipids, which were determined using GC-MS and high-performance liquid chromatography (HPLC), respectively. To develop an oral formulation for the delivery of the extract, we used a 23 factorial design approach to obtain an optimal lipid nanoparticle formulation. The surfactant and solid lipid content were set as the independent variables, while the particle size, polydispersity index, and zeta potential were taken as the dependent variables of the design. To ensure the potential use of the optimum LN formulation to protect and modify the release of the loaded microalga extract, rheological and differential scanning calorimetry analyses were carried out. The developed formulations were found to be stable over 30 days, with an encapsulation efficiency over 60%.

Therapeutic Approaches for Age-Related Macular Degeneration.

Damage to the retinal pigment epithelium, Bruch's membrane and/or tissues underlying macula is known to increase the risk of age-related macular degeneration (AMD). AMD is commonly categorized in two distinct types, namely, the nonexudative (dry form) and the exudative (wet form). Currently, there is no ideal treatment available for AMD. Recommended standard treatments are based on the use of vascular endothelial growth factor (VEGF), with the disadvantage of requiring repeated intravitreal injections which hinder patient's compliance to the therapy. In recent years, several synthetic and natural active compounds have been proposed as innovative therapeutic strategies against this disease. There is a growing interest in the development of formulations based on nanotechnology because of its important role in the management of posterior eye segment disorders, without the use of intravitreal injections, and furthermore, with the potential to prolong drug release and thus reduce adverse effects. In the same way, 3D bioprinting constitutes an alternative to regeneration therapies for the human retina to restore its functions. The application of 3D bioprinting may change the current and future perspectives of the treatment of patients with AMD, especially those who do not respond to conventional treatment. To monitor the progress of AMD treatment and disease, retinal images are used. In this work, we revised the recent challenges encountered in the treatment of different forms of AMD, innovative nanoformulations, 3D bioprinting, and techniques to monitor the progress.

Nutraceuticals and Food-Grade Lipid Nanoparticles: From Natural Sources to a Circular Bioeconomy Approach.

Nutraceuticals have gained increasing attention over the last years due to their potential value as therapeutic compounds formulated from natural sources. For instance, there is a wide range of literature about the cardioprotective properties of omega-3 lipids and the antioxidant value of some phenolic compounds, which are related to antitumoral activity. However, the value of nutraceuticals can be limited by their instability under gastric pH and intestinal fluids, their low solubility and absorption. That is why encapsulation is a crucial step in nutraceutical design. In fact, pharmaceutical nanotechnology improves nutraceutical stability and bioavailability through the design and production of efficient nanoparticles (NPs). Lipid nanoparticles protect the bioactive compounds from light and external damage, including the gastric and intestinal conditions, providing a retarded delivery in the target area and guaranteeing the expected therapeutic effect of the nutraceutical. This review will focus on the key aspects of the encapsulation of bioactive compounds into lipid nanoparticles, exploring the pharmaceutical production methods available for the synthesis of NPs containing nutraceuticals. Moreover, the most common nutraceuticals will be discussed, considering the bioactive compounds, their natural source and the described biological properties.

Enzymatic modification to produce health-promoting lipids from fish oil, algae and other new omega-3 sources: A review.

Lipases are a versatile class of enzymes that have aroused great interest in the food and pharmaceutical industries due to their ability to modify and synthesize new lipids for functional foods. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have shown important biological functions promoting human health, especially in the development and maintenance of brain function and vision. Lipases allow selective production of functional lipids enriched in omega-3 PUFAs and are unique enzymatic tools to improve the natural composition of lipids and provide specific bioactivities. This review comprises recent research trends on the enzymatic production of bioactive, structured lipids with improved nutritional characteristics, using new enzymatic processing technologies in combination with novel raw materials, including microalgal lipids and new seed oils high in omega-3 fatty acids. An extensive number of lipase applications in the synthesis of health-promoting lipids enriched in omega-3 fatty acids by enzymatic modification is reviewed, considering the main advances in recent years for production of ethyl esters, 2-monoacylglycerols and structured triglycerides and phospholipids with omega-3 fatty acids, in order to achieve bioactive lipids as new foods and drugs.

Synthesis of omega-3 ethyl esters from chia oil catalyzed by polyethylene glycol-modified lipases with improved stability.

Enzymatic synthesis of fatty acid ethyl esters (FAEE) from chia (Salvia hispanica L.) oil has been performed with different modified derivatives and compared with commercial immobilized lipases to produce omega-3 rich FAEE. Therefore, the main objective was to synthesize omega-3 esters from chia oil catalysed by polyethylene glycol-modified lipases using a biocatalyst with higher stability than commercial derivatives. Lipase from Thermomyces lanuginosus (TLL) was immobilized by hydrophobic adsorption on Sepabeads C-18 followed by a physicochemical coating of lipase surface with a dense layer of PEG. Ethanolysis reactions were carried out using pressurized liquid extracted chia seed oil and with different lipase derivatives to compare the omega-3 FAEE yield and ratio of reaction products, which were analysed by HPLC-ELSD. Furthermore, reutilization of lipase derivatives was studied to evaluate the stability after several reaction cycles to minimize decreasing of catalytic activity and develop a feasible enzymatic process for food industrial applications.

Alternative oil extraction methods from Echium plantagineum L. seeds using advanced techniques and green solvents.

The edible oil processing industry involves large losses of organic solvent into the atmosphere and long extraction times. In this work, fast and environmentally friendly alternatives for the production of echium oil using green solvents are proposed. Advanced extraction techniques such as Pressurized Liquid Extraction (PLE), Microwave Assisted Extraction (MAE) and Ultrasound Assisted Extraction (UAE) were evaluated to efficiently extract omega-3 rich oil from Echium plantagineum seeds. Extractions were performed with ethyl acetate, ethanol, water and ethanol:water to develop a hexane-free processing method. Optimal PLE conditions with ethanol at 150 °C during 10 min produced a very similar oil yield (31.2%) to Soxhlet using hexane for 8 h (31.3%). UAE optimized method with ethanol at mild conditions (55 °C) produced a high oil yield (29.1%). Consequently, advanced extraction techniques showed good lipid yields and furthermore, the produced echium oil had the same omega-3 fatty acid composition than traditionally extracted oil.

Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of Omega-3 Rich Oil from Chia Seeds (Salvia hispanica L.).

Chia (Salvia hispanica L.) seeds contain an important amount of edible oil rich in omega-3 fatty acids. Fast and alternative extraction techniques based on polar solvents, such as ethanol or water, have become relevant for oil extraction in recent years. However, chia seeds also contain a large amount of soluble fiber or mucilage, which makes difficult an oil extraction process with polar solvents. For that reason, the aim of this study was to develop a gentle extraction method for mucilage in order to extract chia oil with polar solvents using pressurized liquids and compare with organic solvent extraction. The proposed mucilage extraction method, using an ultrasonic probe and only water, was optimized at mild conditions (50 °C and sonication 3 min) to guarantee the omega-3 oil quality. Chia oil extraction was performed using pressurized liquid extraction (PLE) with different solvents and their mixtures at five different extraction temperatures (60, 90, 120, 150, and 200 °C). Optimal PLE conditions were achieved with ethyl acetate or hexane at 90 °C in only 10 min of static extraction time (chia oil yield up to 30.93%). In addition, chia oils extracted with nonpolar and polar solvents by PLE were analyzed by gas chromatography-mass spectrometry (GC-MS) to evaluate fatty acid composition at different extraction conditions. Chia oil contained ∼65% of α-linolenic acid regardless of mucilage extraction method, solvent, or temperature used. Furthermore, tocopherols and tocotrienols were also analyzed by HPLC in the extracted chia oils. The mucilage removal allowed the subsequent extraction of the chia oil with polar or nonpolar solvents by PLE producing chia oil with the same fatty acid and tocopherol composition as traditional extraction.

In vitro intestinal bioaccessibility of alkylglycerols versus triacylglycerols as vehicles of butyric acid.

Butyric acid has been the subject of much attention last years due to its bioactivity. However, the potential advantages of butyrate are limited by the problem to reach enough plasma concentrations; therefore, pro-drugs have been proposed as an alternative to natural butyrate. A comparative study on in vitro intestinal digestion of 2,3-dibutyroil-1-O-octadecyl glycerol (D-SCAKG) and tributyrin (TB), as potential pro-drugs of butyric acid, was performed. Aliquots were taken at different times of digestion for studying the extent and rate of hydrolysis of both substrates. The micellar phase (MP) and oily phase (OP) formed in the digestion media were separated and their composition in lipid products was analyzed. Initially, it was confirmed that the in vitro model reproduced physiological results by testing against olive oil as a standard lipid. The progress of in vitro intestinal digestion of D-SCAKG was slower than that of TB. TB hydrolyzed completely to butyric acid, whereas D-SCAKG mainly yielded 2-butyroil-1-O-octadecyl glycerol (M-SCAKG), followed by butyric acid and 1-O-octadecyl glycerol (AKG). The MP from both substrates mainly consisted of butyric acid. Minor levels of M-SCAKG and AKG were also found in the MP after hydrolysis of D-SCAKG, the M-SCAKG being mainly distributed in the OP. Therefore, D-SCAKG produced a stable form of esterified butyric acid as M-SCAKG after in vitro intestinal digestion, unlike TB. Additionally, such a product would integrate both bioactive compounds, butyric acid and alkylglycerol, within the same molecule. Free butyric acid and AKG would be also released, which are lipid products of interest as well.

Metabolomic approach with LC-QTOF to study the effect of a nutraceutical treatment on urine of diabetic rats.

The rat treated with streptozotocin has been proposed as the most appropriate model of systemic oxidative stress for studying antioxidant therapies. In that sense, rosemary extracts have long been recognized as having antioxidant properties, and folic acid may be able to improve endothelial progenitor cell function. A mixture containing both has been tested as a possible nutraceutical to improve health complications in diabetes. We have developed the methodology to evaluate metabolic changes in the urine of streptozotocin-induced diabetic rats after supplementing their diet with rosemary extract obtained with supercritical fluids (SFE) containing 10% folic acid in an acute but short-term study. It has been done with a metabolomics approach using LC-QTOF as an analytical tool. About 20 endogenous metabolites have been identified by databases and MS/MS showing statistically significant changes. Among them, several amino acids and their metabolites point to changes due to the effect of the gut microbiota. In addition, the comparison between control and streptozotocin-diabetic rats has permitted the showing of some metabolic coincidences between type 1 diabetes and other (possible) autoimmune diseases such as autism and/or Crohn's disease, and the nutraceutical intervention has succeeded in inducing changes in such biomarkers.

Pressurized liquid extraction as an alternative process to obtain antiviral agents from the edible microalga Chlorella vulgaris.

The antiviral properties of pressurized liquid extracts (PLE) (acetone, ethanol, and water) obtained from the edible microalga Chlorella vulgaris were evaluated against herpes simplex virus type 1 (HSV-1). None of the extracts tested showed extracellular direct virucidal activity against the virus, although a pretreatment of Vero cells with 75 microg/mL of water and ethanol extracts before virus addition inhibited 70% of the virus infection. Moreover, water and ethanol extracts were able to significantly inhibit the in vitro virus replication, showing IC(50%) values of 61.05 and 80.23 microg/mL respectively. To identify the type of compounds responsible for the antiviral activity found in the water extract, the polysaccharide fraction was isolated. This activity was found to correlate with polysaccharides, because the polysaccharide-rich fraction (46% concentrated) showed higher antiviral activity than the complete water extract. A concentration of 75 microg/mL of this fraction inhibited 90% virus infection when added as a pretreatment and showed an IC(50%) value of 33.93 microg/mL for intracellular virus replication. GC-MS characterization of the ethanol extract showed that the antiviral activity of this extract could be partially related with the presence of phytol, although other compounds could be involved in this activity.

Acute and repeated dose (28 days) oral safety studies of an alkoxyglycerol extract from shark liver oil in rats.

Shark liver oil has been used for over 50 years as both a therapeutic and preventive agent. The active ingredients in shark liver oil have been found to be a group of ether-linked glycerols known as alkoxyglycerols. Despite its popularity, there is little published toxicology data on alkoxyglycerols. The toxicity of a supercritical fluid extract of shark liver oil (AKG-1 extract) has been evaluated in acute and repeated dose (28 days) oral toxicity studies in rats at doses of 200 and 100 times the maximum recommended dose by supplement manufacturers in humans, respectively. The AKG-1 extract administered in a single oral gavage dose of 2000 mg kg(-1) of body weight resulted in no adverse events or mortality. The AKG-1 extract administered as a daily dose of 1000 mg kg(-1) of body weight for 28 days by gavage resulted in no adverse effects or mortality. For both studies, no abnormal clinical signs, behavioral changes, body weight changes, or change in food and water consumption occurred. There were no changes in hematological and serum chemistry values, organ weights, or gross or histological characteristics. It is concluded that the AKG-1 extract is well tolerated in rats at an acute dose of 2000 mg kg(-1) and at a subchronic (28 days) dose of 1000 mg kg(-1).

Dunaliella salina extract effect on diabetic rats: metabolic fingerprinting and target metabolite analysis.

Antioxidant therapy has been proposed to improve the oxidative stress status of diabetic patients. Natural products are a source of substances such as carotenoids, with known antioxidant properties with possible benefits on diabetes. Among them, Dunaliella salina is a microalga with high content in carotenoids that can be extracted via an environmentally clean process such as supercritical fluid extraction with CO2. Five doses of D. salina extract with in vitro antioxidant properties were intragastrically administrated to adult male streptozotocin (STZ) diabetic rats. Urine fingerprints of control and diabetic rats, both with and without treatment, were obtained by capillary electrophoresis with two different modes (normal polarity and MEKC and reverse polarity and CZE). When the profiles were submitted together to pattern recognition techniques they showed the effects of D. salina extract on this acute and short-term treatment animal model in a rapid, simple and cost-effective way without identifying a single marker. In order to have a further biochemical knowledge of the effect, after treatment, rats were sacrificed and blood and liver glutathione, as well as plasma glucose, triglycerides, cholesterol, total protein, urea, acetoacetate, 3-hydroxybutyrate, lactate, pyruvate and urate, TBARS and urine 8-isoprostane were analysed. Vitamin E in plasma and liver was also measured. Twenty-seven parameters were individually assessed, and both univariate statistics (mean comparison after 1W-ANOVA) and multivariate data analysis were performed. D. salina extract induced changes showed up by the multivariate analysis. Results of the treatment from most of the parameters can be considered beneficial for diabetic animals; although an increase in hyperglycemia and 8-isoprostane excretion when STZ treated animals received the extract was observed.

Meat-based functional foods for dietary equilibrium omega-6/omega-3.

Nutritionists encourage improving the diet by combining meat products with fish or other sea-related foods, in order to equilibrate the omega-6/omega-3 ratio. Strong scientific evidence supports the beneficial health effects of a balanced omega-6/omega-3 PUFA (poly unsaturated fatty acids) diets. In the present work, the scientific bases of new functional meat products with both a balanced omega-6/omega-3 ratio and a synergic combination of antioxidants are discussed. The aim is to contribute to the dietary equilibrium omega-6/omega-3 and to increase the antioxidant intake. Conventional meat products supplemented with a specific fatty acids and antioxidants combination led to functional foods with healthier nutritional parameters.

Acute oral safety study of rosemary extracts in rats.

Increasing interest in rosemary plants is due to their antioxidant and health-enhancing properties. The aim of this study was to evaluate the potential acute toxicity of two supercritical fluid extracts of rosemary. An acute safety study of rosemary extracts was conducted in Wistar rats at a single oral gavage dosage of 2,000 mg/kg of body weight. Rosemary extracts were well tolerated; no adverse effects or mortality were observed during the 2-week observation period. No abnormal signs, behavioral changes, body weight changes, or change in food and water consumption occurred. Two weeks after a single oral rosemary extract dose of 2,000 mg/kg of body weight, there were no changes in hematological and serum chemistry values, organ weights, or gross or histological characteristics. Rosemary extracts appear to have low acute toxicity, and the oral lethal doses (LD50) for male and female rats are greater than 2,000 mg/kg of body weight.

Supercritical carbon dioxide fractionation of nonesterified alkoxyglycerols obtained from shark liver oil.

Ethanolysis of shark liver oil was carried out to generate a product enriched in nonesterified alkoxyglycerols and fatty acid ethyl esters. For the present study, the original oil contained very low amounts of squalene, and thus, unsaponifiable matter was mainly constituted by nonesterified alkoxyglycerols (NEAKG). A small percentage of monoesterified alkoxyglycerols (MEAKG) was also detected. Supercritical fluid extraction was employed to fractionate the mixture, achieving a complete elimination of esters and concentrating the alkoxyglycerol compounds in the raffinate product. Extractions were carried out in a countercurrent packed column, using extraction pressures in the range of 140-180 bar, temperatures from 45 to 65 degrees C, and a solvent-to-feed ratio of 15. NEAKG + MEAKG purity obtained in the raffinate at the best extraction conditions was around 78% w/w, and satisfactory yield (>60%) was also achieved. Therefore, the raffinate product can be re-esterified to design highly valuable ether lipid compounds.

Beta-carotene isomer composition of sub- and supercritical carbon dioxide extracts. Antioxidant activity measurement.

In the present work sub- and supercritical extraction conditions using carbon dioxide were studied in order to obtain extracts with different compositions from the green microalgae Dunaliella salina. Different compositions of beta-carotene isomers were identified in the extracts by using HPLC-DAD. Also, antioxidant activity of the extracts was measured using a TEAC assay. An experimental design was applied considering two factors, extraction pressure and temperature, in a wide range of values, trying to maximize the extraction yield. Higher yields were obtained at high pressures and low temperatures, that is, at higher CO2 densities. Attempts were made to correlate the antioxidant activity of the extracts with their chemical composition by means of principal component analysis. A certain relationship was found between their antioxidant activity and the isomeric composition of beta-carotenes. As a result, an original equation is proposed to predict the antioxidant activity of extracts from D. salina in terms of the ratio 9-cis-beta-carotene/all-trans-beta-carotene, the concentration of alpha-carotene, and, especially, the concentration of 9-cis-beta-carotene.

Separation of rosemary antioxidant compounds by supercritical fluid chromatography on coated packed capillary columns.

Antioxidant compounds in rosemary extracts obtained by supercritical fluid extraction (SFE) were separated by supercritical fluid chromatography (SFC) on packed capillary columns. The columns contained silica particles coated with SE-54 (5% phenyl, 95% methyl silicone) and Carbowax 20 M [poly(ethylene glycol)]. The use of coated packed capillary columns allowed the separation of polar compounds by SFC with neat CO2. The SFC conditions were selected on the basis of previous work. High pressures (up to 370 atm; 1 atm = 10,325 Pa) and moderate temperatures (up to 100 degrees C) were used to separate the compounds responsible for the antioxidant activity such as carnosic acid and camosol while lower pressures were sufficient to separate the compounds of the essential oil.

Countercurrent supercritical fluid extraction and fractionation of high-added-value compounds from a hexane extract of olive leaves.

Countercurrent supercritical fluid extraction (CC-SFE) at a pilot scale plant was used for fractionation of high-added-value products from a raw extract of olive leaves in hexane. Compounds found in the raw extract were waxes, hydrocarbons, squalene, beta-carotene, triglycerides, alpha-tocopherol, beta-sitosterol, and alcohols. The CC-SFE extraction process was investigated according to a 2(3) full factorial experimental design using the following variables and ranges: extraction pressure, 75-200 bar; extraction temperature, 35-50 degrees C; and ethanol as modifier, 0-10%. Data were analyzed in terms of extraction yield, enrichment, recovery, and selectivity. Higher extraction yields were attained at 200 bar. For most of the compounds analyzed enrichment was attained at the same conditions, that is, 75 bar, 35 degrees C, and 10% ethanol. Hydrocarbons were usually recovered in the separators, whereas waxes and alpha-tocopherol remain in the raffinate. Selectivity data reveal that alpha-tocopherol is the most easily separable compound. The influence of the experimental factors on the recovery of all the compounds was studied by means of regression models. The best fitted model was attained for beta-sitosterol, with R2 = 99.25%.

Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction, using micellar electrokinetic chromatography and reverse-phase liquid chromatography.

Antioxidants from orange juice were determined by the combined use of countercurrent supercritical fluid extraction (CC-SFE) prior to reverse-phase liquic chromatography (RP-LC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published LC procedure, both using diode array detection. The characterization of the different antioxidants was further done by LC-mass spectrometry. Advantages and drawbacks of LC and MEKC for analyzing the antioxidants found in the different orange extracts are discussed. Although LC yields higher peak area and slightly better reproducibility than MEKC, the latter technique provides information about the CC-SFE extracts in analysis times 7 times faster than by LC. This analysis advantage can be used for the quick adjustment of CC-SFE conditions, thus providing a fast way to obtain orange fractions of specific composition.