Extraction of plant material

Plant tissues are difficult to extract because of active lipases which hydrolyze rapidly phospholipids glycolipids and increase the amount of free fatty acids in the extract. Thus, a solvent frequently used to inhibit these enzymes is isopropanol.
Nichols’ method: Plant tissues are minced and macerated with 100 parts (w/w) of isopropanol. The mixture is filtered, the solid is extracted again with 200 parts of chloroform/isopropanol mixture (1/1, v/v). The combined filtrates are evaporated, dissolved in a small volume of chloroform/methanol (2/1, v/v) and, if necessary, washed according the Folch’s procedure. Optimal conditions of extraction of microalgae with aqueous isopropanol combined or not with cell rupture has been reported (Yao L et al., JAOCS 2013, 90, 571). The oil yield was largely increased after ultrasonic cell rupture after extraction with 88 or 95% isopropanol.

The extraction of algae is made with hot isopropanol (60°C) added to the cell suspension. It appears that unicellular algae (plankton) must be extracted rapidly with a minimum of preparative mechanical treatments (centrifugation, filtration). A preliminary small scale extraction is recommended to choose the procedure to be adopted.
A comparative evaluation of several extraction methods was done in three types of macroalgae (Kumari P et al., Anal Biochem 2011, 415, 134). Care should be taken while selecting the method for macroalgae, according to the group to which they belong, otherwise there would be a risk of obtaining erratic and inaccurate results.
The extraction of neutral lipids from microalgae has been efficiently done on lyophilized material (Fajardo AR et al., Eur J Lipid Sci Technol 2007, 109, 120). Briefly, first, 96% ethanol was used to extract the lipids from the dry biomass. Second, a biphasic system was formed by adding water and hexane to the extracted crude oil. Thus, the purified lipids were transferred to the hexane phase while most impurities remained in the aqueous phase.
An overview of advances made in technologies for extracting microalgae oil may be consulted before doing experiments (Mercer P et al., Eur J Lipid Sci Technol 2011, 113, 539). Solvent extraction technologies with extraction alternatives such as mechanical milling and pressing, enzymatic and supercritical fluid extraction are compared.
A comparative study of various extraction treatments have been reported using the microalgae Spirulina (Zheng G et al., JAOCS 2012, 89, 561). A purification was used to remove the pigments from the extracted lipids.

Efficient extraction of carotenoids from dry plant material (maize endosperm) has been described using mixtures of methanol/ethyl acetate (6/4, v/v) and methanol/tetrahydrofuran (1/1, v/v) (Rivera S et al., Molecules 2012, 17, 11255).


Extraction of plant sphingolipids

Because of their large hydrophilic polar head, solubilisation of glycosylated sphingolipids in usual organic solvents is inaccurate. When doing a phase partition in chloroform/methanol/water mixtures, these lipids remain insoluble for the most part or are recovered in the aqueous phase and interphase. A new extraction method to purify total plant sphingolipids has been developed (Buré C et al., Rapid Commun. Mass Spectrom 2013, 25, 3131). Briefly, plants cells were blended with cold 0.1 N aqueous acetic acid in a chilled Waring Blendor. The slurry was filtered under vacuum and the aqueous acetic acid filtrate was discarded. The residue was then re-extracted with hot 70% ethanol containing 0.1 N HCl. The filtrate was chilled and left at room temperature overnight. The precipitate was pelleted by centrifugation. The sphingolipid-enriched pellet was washed twice with cold acetone, and twice with cold diethyl ether to yield a whitish precipitate. Glycosylated sphingolipid contained in the precipitate were then dissolved in tetrahydrofuran (THF)/methanol/water (4/4/1, v/v/v) containing 0.1% formic acid by heating at 60°C, followed by gentle sonication. This solution was further used for mass spectrometry analyses (Cacas JL et al., Phytochemistry 2013, 96, 191).


Enzyme-assisted aqueous extraction (EAEP)

EAEP has been employed to extract different compounds from plants, and has been proved to be effective in improving the yield of various components. Improved lipid extraction was observed in many different oil-bearing plant materials including soybean (Freitas SP et al., Fett-Lipid 1997, 99, 333), sunflower seeds (Sineiro J et al., Food Chem 1998, 61, 467) and sesame (Latif S et al., Food Chem. 2011, 125, 679).14 In addition, EAEP will make it possible to extract and separate oil directly from algae in the natural aqueous environment of algae cultures, which avoids the collection and drying process of algae biomass.
As insoluble nonhydrolyzable biopolymers (algaenans) are present in cell wall of algae, the EAEP methods established for common terrestrial plants cannot be applied directly to the lipid extraction from microalgae. Improvement in lipid extraction of these vegetals has been done in using sonication combined with enzyme treatment (Liang K et al., J Agric Food Chem 2012, 60, 11771).

Oilseeds may be analyzed for oil content by an exhaustive extraction with petroleum ether. A comparison of five methods to measure the oil contents in oilseeds may be studied before choosing a specific procedure (Barthet VJ et al., J Oleo Sci 2002, 51, 589).

A one-tep extraction has been described to study the composition of triglycerides in small piece of seed and is suitable for a large number of tissue samples should be examined as in selecting new plant varieties. This easy and reliable method is based on an incubation of samples (20-50 mg) without shaking in a mixture containing heptane / 0.17 M NaCl in methanol (66.6/33.3, v/v), for 2 h at 80°C. After cooling, the upper phase containing mainly triglycerides was transferred to a new test tube for further analysis. Even under incomplete triglyceride extraction (80% maximum) the triglyceride ecomposition is representative of the total triglycerides found in the tissue (Ruiz-Lopez N et al., Anal Biochem 2003, 317, 247).

An efficient surfactant-based extraction of corn oil from corn germ has been proposed (Kadioglu SI et al., JAOCS 2011, 88, 863). Hexane and/or other organic solvents were avoided in the process. Greater than 80% of corn oil can be extracted with low surfactant and salt concentrations. It was concluded that aqueous-based surfactant microemulsion oilseed extraction is a promising alternative approach for oil extraction.

The extraction of xanthophyll was shown to be improved using cellulolytic enzymes and highly competitive when compared to the traditional process of pigment extraction (Navarrete-Bolanos JL et al., J Agric Food Chem 2004, 52, 3394). These data may foster the development of new extraction procedures in plants based on previous enzymatic hydrolysis of cell membranes.

The extraction of flour can be made with hexane at room temperature but the efficiency of phospholipid recovery is dependent upon the temperature and the moisture content (Snyder HE, Inform 2004, 15, 575).

The reading of the review of various techniques of preparing plant material by Romanik G et al. is suggested before any plant extraction (Romanik G et al., J Biochem Biophys Methods 2007, 70, 253).


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