Antiviral compounds obtained from microalgae commonly used as carotenoid sources

The final publication is available at Springer via http://dx.doi.org/10.1007/s10811-011-9692-1Pressurized liquid extraction (PLE), an environmentally friendly technique, has been used to obtain antiviral compounds from microalgae commonly used as carotenoids sources: Haematococcus pluvialis and Dunaliella salina. The antiviral properties of PLE extracts (hexane, ethanol and water) were evaluated against herpes simplex virus type 1 (HSV-1) at different stages during viral infection. Pre-treatment of Vero cells with 75 µg mL-1 of H. pluvialis ethanol extract inhibited virus infection by approx. 85%, whereas the same concentration of water and hexane extracts reduced the virus infectivity 75% and 50% respectively. D. salina extracts were less effective than H. pluvialis extracts and presented a different behaviour, since water and ethanol extracts produced a similar virus inhibition (65%). Moreover, H. pluvialis ethanol extract was also the most effective against HSV-1 intracellular replication. The antiviral activity of water PLE extracts was found to correlate with polysaccharides, since the polysaccharide-rich fraction isolated from these extracts showed higher antiviral activity than the original water extracts. A GC-MS characterization of the H. pluvialis ethanol extract showed the antiviral activity of this extract could be partially related with the presence of short chain fatty acids, although other compounds could be involved in this activity; meanwhile, in the case of D. salina ethanol extract other compounds seemed to be implied, such as: β-ionone, neophytadiene, phytol, palmitic acid and α-linolenic acid. Results demonstrated the use of PLE allows obtaining antiviral compounds from microalgae used as carotenoids sources, which gives both microalgae biomass an added value.This work has been financed by the Spanish Ministry of Education and Science (AGL2005-06726-C04), the programme CONSOLIDER-INGENIO 2010 (CDS2007-00063) and the regional programme ALIBIRD-CM S-0505/AGR-0153 from the Comunidad de Madrid, Spain

Purification of Natural Products by Selective Precipitation Using Supercritical/Gas Antisolvent Techniques (SAS/GAS)

Supercritical fluids offer considerable advantages for the isolation of natural products. Supercritical fluids can be used as antisolvents to precipitate selectively target compounds from a mixture and to remove impurities by the Supercritical Antisolvent technique (SAS). The present decade has experienced a considerable increase in the number of publications that apply this technique to natural extracts, especially for the isolation of polyphenols and carotenoids from plants and microalgae. However, the lack of a clear terminology and purpose adds confusion to the topic. The proposed review aims at defining a research field that consists in applying SAS to natural extracts for the purification of target compounds. To do so, we trace back the origin of the field, discuss the different terminology used to refer to such processes, and suggest appropriate terms for the process and for reported results. This work explores the scope of the topic by compiling all works published to date from a scattered literature, using all possible process terminologies for the search. The information given aims to highlight the most promising applications explored so far and possibly inspire further research

In situ quantification of minor compounds in pressurized carbon dioxide using Raman spectroscopy

In this work, we present an optical approach that allows quantification of compounds dissolved down to a sub 10 ppm (molar) level in supercritical carbon dioxide (scCO(2)) by in situ Raman spectroscopy. The quantification strategy is based on the consideration of Raman signal intensity ratios and of weak Raman signals of CO2. The first measure makes the quantification strategy independent of the refractive index, laser power fluctuations and changes in alignment, and is thus robust against variations of the operation conditions. The second measure circumvents the classical challenges related to the dynamic range limit of detectors, which is inherent to the quantification of minor compounds. Example solubilities of the wax alkyl ketene dimer (AKD) in CO2 were quantified at 333 K and a range of pressures from 10 MPa to 18 MPa. The solubilities are between 0.1 mg g(-1) and 1.5 mg g(-1) corresponding to similar to 9 ppm and similar to 132 ppm (molar), respectively. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved

Determination of carotenoids in microalgae using supercritical fluid extraction and chromatography

A method was developed based on supercritical fluid chromatography for quantitative determination of carotenoids in extracts of Scenedesmus sp. By utilizing the low backpressure in supercritical fluid chromatography, a C18 column and a 2-ethyl pyridine column were coupled in series. It was concluded that even minor changes in temperature had a substantial effect on selectivity. A standard mixture of 8 carotenoids and microalgae extracts obtained through supercritical fluid extraction with and without 10% ethanol as a co-solvent were successfully separated. All of the carotenoids were separated within 10 min, while the total analysis time was 20 min. The method was validated and the carotenoids of microalgae extracts were quantified. Furthermore, the method should be seen as a more rapid and environmentally sustainable alternative to traditional high-performance liquid chromatography methods utilizing organic solvents

Supercritical fluid extraction of lipids from linseed with on-line evaporative light scattering detection.

Supercritical fluid extraction (SFE) is a green alternative method of extraction for neutral lipids in seeds compared to conventional methods utilizing organic solvents. In this work, a novel method where SFE is hyphenated with an evaporative light scattering detector is presented. The method was subsequently applied to determine lipid content in crushed linseed. The new method enables rapid quantification of extracted lipids as well as be ability to continuously monitor the extraction rate in real-time, thus being able to determine the time point of completed extraction. Both the detector and the method was validated. The results show that any of several tested oils can be used to calibrate the detection method for the determination of lipids extraction from linseed. The overall method repeatability and intermediate precision was 2.6% relative standard deviations. The extracted amount was significantly less than that obtained using the standard method of Soxhlet with petroleum ether, 26.0±0.4% (95% CI, n=9) compared to 32.3±1.3% (95% CI, n=3) of extracted amounts. It was found that channeling effects were present, and by either performing sequential repeated extractions with decompression in-between or by using a relatively large vessel a more complete extraction could be obtained. Interestingly, a substantially higher extracted amount (approximately 50%) was obtained compared to both a single extraction by SFE and the Soxhlet method. Therefore, it is recommended that an additional extraction including a rapid decompression in-between should be included in the validation of a method using supercritical fluid extraction, in order to either rule out channeling effects or to acquire a full recovery

In situ Raman spectroscopy for the evaluation of solubility in supercritical carbon dioxide mixtures

Solubility phenomena in supercritical carbon dioxide (SC-CO2) mixtures have been studied in situ using Raman spectroscopy. A Raman spectroscopy instrumentation has been linked to a high-pressure optical cell. The set-up was tested in the system SC-CO2/alkyl ketene dimer (AKD) under pressures up to 300 bars and temperatures up to 80 degrees C. As a proof of concept, the possibility to study solubility of AKD in SC-CO2 as well as solubility of SC-CO2 in AKD by studying the Raman signal of SC-CO2 has been presented. Statistically tested results show how these competitive phenomena depend on the density region of the SC-CO2. Raman spectra of AKD in the system AKD/SC-CO2 have also been acquired, demonstrating the prospect of quantitative analysis for solubility studies in SC-CO2. Furthermore, this versatile set-up provides information about thermal expansion of the vessel and evidences the need for corrected equations in this commonly used region of pressures and temperatures. (c) 2012 Elsevier B.V. All rights reserved

Pressurised hot water extraction with on-line particle formation by supercritical fluid technology

In this work, an on-line process for pressurised hot water extraction (PHWE) of antioxidants from plants as well as drying of the extract in one step by particle formation based on the use of supercritical carbon dioxide (SC-CO2) has been developed. This process has been called WEPO (R), water extraction and particle formation on-line. With this process, dried extracts from onion with the same composition of quercetin derivatives as non-dried extracts have been obtained as a fine powder with spherical particles from 250 nm to 4 mu m in diameter. The major compounds present in the extract were quercetin-3,4'-diglucoside, quercetin-4'-glucoside and quercetin. An auxiliary inert gas (hot N-2) was used to enhance the drying process. Parameters such as temperature (120 degrees C), SC-CO2 and N-2 pressures (80 and 12.5 bar, respectively) and flow rate of SC-CO2 (10 ml/min), have been settled by trial-and-error in order to achieve a fine and constant spray formation. Water content, size and morphology, antioxidant capacity and quercetin content of the particles were studied to evaluate the efficiency of the WEPO process. Results were compared with the ones from extracts obtained by continuous flow PHWE followed by freeze-drying. Results showed that both processes gave similar results in terms of antioxidant capacity, concentration of quercetin derivatives and water content, while only WEPO was able to produce defined spherical particles smaller than 4 mu m. (C) 2012 Elsevier Ltd. All rights reserved

Extraction with Water-in-Carbon Dioxide Microemulsions : A Case Study on Steviol Glycosides

This work explores the use of water-in-supercritical carbon dioxide (scCO2) microemulsions for the extraction of polar metabolites from plants. Stevia rebaudiana Bertoni leaves and polyethylene glycol trimethylnonyl ether (TMN) surfactants were selected for a case study. A CO2-water-TMN 10 mixture at 35 °C and 30.0 MPa extracted 7 mg target analyte/g dry leaves. The extraction was proven to occur due to a water-surfactant liquid solution rather than a water-in-CO2 microemulsion. Using a modified extraction setup, the microemulsion was created prior to extraction. TMN 6 was able to dissolve enough water in CO2 to extract steviol glycosides, in detectable but very small amounts

Dynamic pH determination at high pressure of aqueous additive mixtures in contact with dense CO2

A system consisting of a high-pressure tolerant microfluidic glass chip, high-speed absorbance imaging, and image processing has been developed to study rapid dynamic events like pH change in a multiphase flow. The system gives both kinetic and quantitative equilibrated information. By tracking the interactions of aqueous additive mixtures and liquid CO2, at 80 bar and 24 °C, under flow, measurement at a given P, T condition is done in 0.25 s. The acidification rate to steady state was found to be mass transport limited, occurring in less than 1 s. For 30 mM of the additives ammonium acetate and ammonium formate, equilibrium pH of 4.5 and 4.1, respectively, was seen. These additives are of key importance in common mobile phases used in SFC

Phase-behavior of alkyl ketene dimmer (AKD) in supercritical carbon dioxide. The implications of using different solubility measurement methods

In this work, three different methods were used to determine the phase behavior of alkyl ketene dimer (AKD) in supercritical carbon dioxide (SC-CO2). Rough values for the total solubility of AKD in SC-CO2 were obtained. The solubility ranges from 1 to 14 mg AKD/g CO2 at temperatures from 40 to 80 degrees C and pressures from 10 to 30 MPa. A significant increase in solubility was observed in the region above 50 degrees C and 20 MPa. A region of improved solubility was found, without the presence of a cross-over pressure point. The fact that different solubility methods gave different results was discussed and the importance of data interpretation was highlighted. H-1 NMR and MALDI-TOF MS analyses were utilized to chemically characterize the AKD wax. Its special behavior and the influence of data interpretation on the design of a RESS process were discussed. (C) 2011 Elsevier B.V. All rights reserved