Laura Jaime

Screening for Bioactive Compounds from Algae

In the present work, a comprehensive methodology to carry out the screening for novel natural functional compounds is presented. To do that, a new strategy has been developed including the use of unexplored natural sources (i.e., algae and microalgae) together with environmentally clean extraction techniques and advanced analytical tools. The developed procedure allows also estimating the functional activities of the different extracts obtained and even more important, to correlate these activities with their particular chemical composition. By applying this methodology it has been possible to carry out the screening for bioactive compounds in the algae Himanthalia elongata and the microalgae Synechocystis sp. Both algae produced active extracts in terms of both antioxidant and antimicrobial activity. The obtained pressurized liquid extracts were chemically characterized by GC-MS and HPLC-DAD. Different fatty acids and volatile compounds with antimicrobial activity were identified, such as phytol, fucosterol, neophytadiene or palmitic, palmitoleic and oleic acids. Based on the results obtained, ethanol was selected as the most appropriate solvent to extract this kind of compounds from the natural sources studied.

Chemical composition and antimicrobial activity of Rosmarinus officinalis L. Essential oil obtained via supercritical fluid extraction

The chemical composition and antimicrobial activity of essential oil-rich fractions obtained by supercritical CO2 extraction from Rosmarinus officinalis L. were investigated. Gas chromatography-mass spectroscopy analysis of these fractions resulted in the identification of 33 compounds of the essential oil. The main components of these fractions were alpha-pinene, 1,8-cineole, camphor, verbenone, and borneol, constituting ca. 80% of the total oil. The antimicrobial activity was investigated by the disc diffusion and broth dilution methods against six microbial species, including gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the essential oil-rich fractions obtained showed antimicrobial activity against all of the microorganisms tested, with inhibition zones and minimal bactericidal and fungicidal concentration values in the range of 17 to 33 mm and 2.25 to 0.25 mg/ml, respectively. The most active fraction was the one obtained in experiment 4 (4% ethanol as modifier; extraction pressure, 25 MPa; extraction temperature, 60 degrees C). S. aureus was found to be the most sensitive bacteria to the rosemary extracts, whereas the least susceptible was A. niger. alpha-Pinene, 1,8-cineole, camphor, verbenone, and borneol standards also showed antimicrobial activity against all the microorganisms tested, borneol being the most effective followed by camphor and verbenone. In that way, it was confirmed that essential oil from experiment 4, with the best antimicrobial activity, presented the highest quantity of camphor, borneol, and verbenone.

Subcritical water extraction and characterization of bioactive compounds from Haematococcus pluvialis microalga

In this work, extraction and characterization of compounds with antioxidant and antimicrobial activity from Haematococcus pluvialis microalga in red phase have been carried out. To do this, subcritical water extraction (SWE) has been combined with analytical techniques such as HPLC-DAD, HPLC-QqQ-MS and GC-MS and in vitro assays (i.e., for antioxidant and antimicrobial activity). The effect of the extraction temperature (50, 100, 150 and 200 degrees C) and solvent polarity has been studied in terms of yield and activity of the extracts. Results demonstrate that the extraction temperature has a positive influence in the extraction yield and antioxidant activity. Thus, the extraction yield achieved with this process was higher than 30% of dry weight at 200 degrees C as extraction temperature. Moreover, the extract obtained at 200 degrees C presented the highest antioxidant activity by far, while temperature does not seem to significatively affect the antimicrobial activity. Chemical composition was determined by HPLC-DAD, HPLC-QqQ-MS and GC-MS. Short chain fatty acids turned out to be responsible of the antimicrobial activity, whereas the antioxidant activity was correlated to vitamin E (present exclusively in the 200 degrees C extract), together with simple phenols, caramelization products and possible Maillard reaction products obtained during the extraction at high temperatures.

Pressurized Fluid Extraction of Bioactive Compounds from Phormidium Species

In the search for new functional ingredients with potential use in the food industry, extracts of unknown species of microalgae, such as Phormidium species have been studied. Three solvents of different polarities (i.e., hexane, ethanol, and water) have been used to obtain pressurized liquid extracts with different compositions. Moreover, extractions were performed at four different extraction temperatures (50, 100, 150, and 200 degrees C) with 20 min as extraction time. Antioxidant activity of the extracts has been measured by the TEAC assay. In general, hexane and ethanol extracts showed a higher antioxidant capacity that was mainly attributed to carotenoid compounds, as the TEAC value trend seems to be similar to the carotenoid content of the extracts. On the other hand, the high antioxidant activity of the 200 degrees C water extracts is likely related to the presence of Maillard reaction compounds produced by thermal degradation of the sample. beta-Carotene, lutein, violaxanthin, and neoxanthin were identified in 150 degrees C ethanol extracts. Four different microbial species ( Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger) were used to screen the potential antimicrobial activity of the Phormidium sp. extracts. The most sensitive microorganism was the yeast, C. albicans, whereas the fungus, A. niger, was the most resistant. In general, no drastic differences were found for solvents and temperatures tested, showing a very diverse nature of the compounds responsible for the antimicrobial activity of these microalgae. In ethanol extracts, antimicrobial activity could be mainly attributed to the presence of terpenes (i.e., beta-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) in the samples. Toxicity studies carried out with the extracts evaluated in the present work showed a cellular toxicity lower than those of other cyanobacteria such as Spirulina plantensis.

Study of total fructan and fructooligosaccharide content in different onion tissues

The objective of this work was to determine the fructan and fructooligosaccharide (FOS) content of different onion tissues in order to evaluate the potential use of onion by-products from the food industry as a source of FOS and fructans. Assays with two methods were carried out to optimise the extraction procedure. The main FOS, namely kestose (GF2), nystose (GF3) and fructofuranosylnystose (GF4), were measured directly using standard sugars. The method for total fructans was based on enzymatic treatment (Novozym 230) of ethanolic/aqueous extract followed by determination of released fructose and glucose by HPLC. Data showed a clear predominance of GF2 in every onion tissue and no occurrence of highly polymerised fructans. The tissues richest in fructans were the fleshy layers, so that the outer two fleshy layers turn out to be the best onion by-product as a possible fructan source. © 2000 Society of Chemical Industry

Supercritical Carbon Dioxide Extraction of Compounds with Antimicrobial Activity from Origanum vulgare L.: Determination of Optimal Extraction Parameters

Oregano leaves were extracted using a pilot-scale supercritical fluid extraction plant under a wide range of extraction conditions, with the goal of determining the extraction and fractionation conditions to obtain extracts with optimal antimicrobial activity. In this investigation, the essential oil-rich fractions were selectively precipitated in the second separator, and their chemical composition and antimicrobial activity were investigated. Gas chromatography-mass spectrometry analysis of the various fractions resulted in the identification of 27 compounds of the essential oil. The main components of these fractions were carvacrol, trans-sabinene hydrate, cis-piperitol, borneol, terpinen-4-ol, and linalool. Antimicrobial activity was investigated by the disk diffusion and broth dilution methods against six different microbial species, including two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the supercritical fluid extraction fractions obtained showed antimicrobial activity against all of the microorganisms tested, although the most active fraction was the one obtained in experiment 5 (fraction was obtained with 7% ethanol at 150 bar and 40 degrees C). C. albicans was the most sensitive microorganism to the oregano extracts, whereas the least susceptible was A. niger. Carvacrol, sabinene hydrate, borneol, and linalool standards also showed antimicrobial activity against all of the microorganisms tested, with carvacrol being the most effective. Consequently, it was confirmed that essential oil from experiment 5, with the best antimicrobial activity, also presented the highest quantity of carvacrol.

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.

Extraction of functional ingredients from spinach (Spinacia oleracea L.) using liquid solvent and supercritical CO2 extraction

BACKGROUND In this work three different techniques were applied to extract dry leaves of spinach (Spinacia oleracea): solid-liquid extraction (SLE), pressurised liquid extraction (PLE) and supercritical fluid extraction (SFE) to investigate the influence of extraction solvent and technique on extracts composition and antioxidant activity. Moreover, the influence of carotenoids and phenolic compounds on the antioxidant and anti-inflammatory activities of spinach extracts was also studied. RESULTS The higher concentrations of carotenoids and the lower content of phenolic compounds were observed in the supercritical CO₂ extracts; whereas water and/or ethanol PLE extracts presented low amounts of carotenoids and the higher concentrations of phenolic compounds. PLE extract with the highest content of phenolic compounds showed the highest antioxidant activity, although SFE carotenoid rich extract also showed a high antioxidant activity. Moreover, both extracts presented an important anti-inflammatory activity. CONCLUSION PLE seems to be a good technique for the extraction of antioxidant and anti-inflammatory compounds from spinach leaves. Moreover, spinach phenolic compounds and carotenoids present a high antioxidant activity, whereas spinach carotenoids seem to show a higher anti-inflammatory activity than phenolic compounds. It is worth noting that of our knowledge this is the first time the anti-inflammatory activity of lipophilic extracts from spinach leaves is reported.

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.

Antiviral Properties of Supercritical CO2 Extracts from Oregano and Sage

The antiviral properties of supercritical CO2 extracts obtained from oregano and sage were evaluated against the herpes simplex virus type 1 at different stages during virus infection. All of the extracts tested presented a moderate extracellular direct virucidal activity, although a pre-treatment of Vero cells, with 10 μg/mL of sage extracts before virus addition, inhibited 70% of the virus infection. Moreover, supercritical extracts of sage and oregano were able to significantly inhibit the in vitro virus replication, showing IC50 values of 1.88 and 5.33 μg/mL, respectively. Carvacrol and thymol could be pointed out as the compounds responsible for the antiviral activity found in oregano supercritical extracts; meanwhile, borneol, camphor, and 1,8-cineole could be proposed as antiviral compounds in supercritical sage extracts. Results demonstrated that supercritical extraction was an appropriate technique to obtain antiviral extracts from oregano and sage.