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Chapter 17 Extraction and Isolation of Phenolic Compounds Celestino Santos-Buelga, Susana Gonzalez-Manzano, Montserrat Dueñas, and Ana M. Gonzalez-Paramas Abstract Phenolic compounds constitute a major class of plant secondary metabolites that are widely distributed in the plant kingdom and show a large structural diversity. These compounds occur as aglycones or glycosides, as monomers or constituting highly polymerized structures, or as free or matrix-bound compounds. Furthermore, they are not uniformly distributed in the plant and their stability varies significantly. This greatly complicates their extraction and isolation processes, which means that a single standardized procedure cannot be recommended for all phenolics and/or plant materials; procedures have to be optimized depending on the nature of the sample and the target analytes, and also on the object of the study. In this chapter, the main techniques for sample preparation, and extraction and isolation of phenolic compounds have been reviewed—from classical solvent extraction procedures to more modern approaches, such as the use of molecularly imprinted polymers or counter-current chromatography. Key words: Phenolic compounds, Sample preparation, Solvent extraction, Assisted extraction techniques, Solid-phase extraction, Column chromatography, Counter-current chromatography

1. Introduction Phenolic compounds constitute a major class of plant secondary metabolites that are widely distributed in the Plant Kingdom. Plant phenolics are biosynthesized through the shikimate/phenylpropanoid pathway leading to different compound classes that are summarized in Table 1. They may occur in their natural sources in free forms, as glycosylated or acylated derivatives, and as oligomeric and polymerized structures, such as hydrolyzable and condensed tannins, phlorotannins or lignins. They may also be found linked to plant matrix components like cell walls, carbohydrates or proteins. It should be indicated that although the terms “plant phenolics” and “polyphenols” are indistinguishably used by some

Satyajit D. Sarker and Lutfun Nahar (eds.), Natural Products Isolation, Methods in Molecular Biology, vol. 864, DOI 10.1007/978-1-61779-624-1_17, © Springer Science+Business Media, LLC 2012



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Table 1 Main classes of phenolic compounds Class Simple phenols (C6)

Phenolic acids Hydroxybenzoic acids (C6-C1)

Hydroxycinnamic acids C6-C3) and derivatives

Basic skeleton

Examples Phloroglucinol, catechol, resorcinol, vanillin, syringaldehyde

p-hydroxybenzoic acid, protoctechuic acid, vanillic acid, syringic acid, gallic acid, gentisic acid, salicylic acid Caffeic acid, coumaric acid, ferulic acid, sinapic acid, chlorogenic acid (5-caffeoylquinic acid)

Coumarins (C6-C3)

Scopoletin, umbelliferone, aesculetin

Naphtoquinones (C6-C4)

Juglone, pumblagin

Xanthones (C6-C1-C6)

Mangostin, mangiferin

Stilbenes (C6-C2-C6)

Resveratrol, piceid, e-viniferins

Anthraquinones (C6-C2-C6)

Emodin, physcion,

Flavonoids (C6-C3-C6) Flavan-3-ols

(Epi)catechin, (epi)gallocatecin



Extraction and Isolation of Phenolic Compounds


Table 1 (continued) Class

Basic skeleton



Apigenin, luteolin, chrysin, scutellarein, diosmetin, chrysoeriol


Quercetin, kaempferol, myricetin, galangin, fisetin, morin


Hesperidin, naringenin, taxifolin, eriodictyol, isosakuranetin


Cyanidin, delphinidin, malvidin, pelargonidin, petunidin, peonidin


Genistein, daidzein, glycitein, formononetin, biochanin A, puerarin


Phloretin, arbutin, butein, naringenin chalcone

Condensed tannins (proanthocyanidins) (C6-C3-C6)n

Procyanidins, prodelphidinins



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Table 1 (continued) Class

Basic skeleton


Hydrolysable tannins (gallotannins, ellagitannins)

Pentagalloylglucose, vescalagin, castalagin

Lignans (C6-C2)2

Secoisolariciresinol, matairesinol, sesamin, pinoresinol, syringaresinol

Lignins (C6-C3)n

authors, they are not synonymous. The term “polyphenol” should be reserved for the phenylpropanoid-derived compounds featuring more than one phenolic ring, which leaves out the phenolic classes consisting of only one aromatic ring (e.g., most simple phenols and phenolic acids) whatever the number of substituting hydroxyl groups, as well as all the monophenolic structures. Further information on structural features, metabolic pathways and properties of plant phenolics can be found in a recent review by Quideau et al. (1). The structural diversity of phenolic compounds affects their physicochemical behavior, such as solubility and partitioning characteristics. The polarity of the compounds varies significantly with their structure, conjugation status, and association with sample matrix; bound forms and high molecular weight phenolics may be quite insoluble. Furthermore, phenolic compounds are not uniformly distributed in the plant and their stability varies significantly; some are relatively stable and others are volatile, thermolabile and/ or easily prone to oxidation. The task of recovery is further complicated as many foods and plants have high levels of enzyme activity. Hence, extreme care must be taken in the choice of correct extraction


Extraction and Isolation of Phenolic Compounds


protocols ensuring avoidance of any possible chemical modifications, which invariably result in artifacts through hydrolysis, oxidation, and/or isomerization (2). There is no standardized procedure that can be suitable for sample preparation and extraction of all phenolics or a specific class of phenolic substances in plant materials. The procedure has to be optimized depending on the (a) nature of the sample and the analytes (e.g., extraction of total phenolics, specific phenolic classes, individual compounds; bound or free phenolics; polymeric species); (b) object of the analysis (e.g., interest in structure elucidation or quantification); (c) availability of techniques. Because of the complexity of most of the matrices, i.e., food, drink, plant, and biological samples, the sample preparation procedure is a critical step of the entire process. Drying, grinding, homogenization, and filtration (or centrifugation) are common pretreatment steps prior to extraction. Also, in some cases, a hydrolysis step is included to release compounds from matrix structures and/or simplify extract composition (e.g., removal of the glycosidic moieties in view to the analysis of aglycones). Solvent extraction is commonly used for the preparation of the crude extracts, but assisted extraction methods, such as those using ultrasounds, microwaves, or pressurized or supercritical liquids, have also been highly employed. Phenolic extracts of plant materials are always a mixture of different classes of phenolics that are soluble in the solvent system used. Additional steps may be required for the removal of unwanted phenolics and nonphenolic substances, and an effective clean-up method is required. Solid-phase extraction (SPE), column chromatography (CC), and droplet countercurrent chromatography (DCCC) are usual techniques applied for the purification of extracts and/or isolation of phenolic compounds. More modern approaches, such as the use of molecularly imprinted polymers (MIPs), are also emerging as alternatives for selective compound extraction.

2. Materials Sample preparation may require various devices, solvents, and/or chemicals to perform drying, homogenization/grinding, sieving, extraction, preconcentration, derivatization, and hydrolysis. Enzymes, e.g., b-glycosidases and/or sulfatases, are required for enzymatic hydrolysis.


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In solvent extraction methanol (MeOH), ethanol (EtOH), acetone, and ethyl acetate (EtOAc) and combinations of them and with water are frequently used for the extraction of phenolics. Accelerated solvent extractor, Soxhlet, supercritical fluid extraction (SFE) device, or microwave-assisted extractors are also used (see Chapters 3–5). Ultrasound-assisted extractors (UAE) can be performed using ultrasonic baths or ultrasonic probes in either discrete or continuous mode. Most reported applications are of the static type and use an ultrasonic bath, cheaper and easier to operate than ultrasonic probes and that may even offer better results. Typically, a vessel containing the sample in the solvent is immersed in the transmitting liquid held in a bath. For the isolation of phenolic compounds, various chromatographic methods are applied, and usual chromatographs, e.g., CC, HPLC, mobile and stationary phases are required (see Chapters 7–12 for details).

3. Methods 3.1. Sample Preparation

Sampling and sample preparation are the initial steps in the analytical process. Sample preparation is required to (a) improve sample stability; (b) enhance the efficiency of the extraction process; (c) eliminate or reduce potential interferences; (d) enrich analytes or to transform them into derivatives that can be more easily detected or quantified. Sample preparation may consist of multiple steps, such as sample drying, homogenization, sieving, extraction, preconcentration, derivatization, and hydrolysis. Although the importance of the sample preparation process has long been recognized, it has received relatively less attention than separation and detection, even though it is crucial to ensure the quality and consistency of the obtained results. It has been estimated that approximately 60% of analysis time and around 30% of analytical errors stem from the sample preparation step (3).

3.1.1. Physical Treatments

Phenolic content and composition considerably vary depending on the part of the plant, i.e., leaves, roots, bark, flowers, or fruits. Within fruits, different distribution pattern usually exists in flesh, peel, and seeds. Samples collected must be representative of the plant material to be analyzed, e.g., the entire plant or the selected part of it. On the other hand, samples must be adequately conserved up to the analysis so that no changes in their chemical composition


Extraction and Isolation of Phenolic Compounds


are produced. However, in most papers no sufficient attention is usually paid to these steps and sample collection and storage are overlooked or not adequately documented. A significant decrease in the levels of phenolic compounds in the source material may occur during the time between sample collection and analysis depending on the storage time and conditions. Samples can be stored in fresh, frozen, or dried form. Fresh samples can be kept under refrigeration, although they are quite unstable and a rapid decrease in the content of phenolic compounds usually occurs. Similar losses in the levels of total glucosinolates (71–80%), total flavonoids (59–62%), sinapic acid derivatives (44–51%), and caffeoylquinic acid derivatives (73–74%) were found by Vallejo et al. (4) during the storage of broccoli for 3 days at 15ºC or 7 days at 1ºC. In these circumstances, it is essential to inactivate all enzymatic, metabolic, and chemical reactions in order to maintain accurate sample identity and demonstrate the effectiveness of such procedures in any report (3). Sample blanching and/or storage in an inert atmosphere may help to improve compound stability. Freezing and drying are the preferred options for long storage. Freezing of the sample facilitates further extraction, since the increase of sample volume and the formation of ice crystals lead to tissue disruption facilitating the release of compounds. Enzymatic and chemical reactions may, however, take place during thawing of the samples leading to changes in the phenolic composition. The thawing method (refrigerator, room temperature, or microwave) shows differential effects on the levels of phenolics. Microwave thawing usually produces the most reliable results and is also the most practical approach for routine analyses (2). Most usually solid samples are air or freeze-dried. Generally, freeze-drying retains higher levels of phenolics content in plant samples than air-drying (5). Oven drying is less recommended as it may induce degradation of thermolabile compounds. The influence of different sample storage conditions (in fresh, freezing, air and oven-drying and freeze-drying) in the concentration and profile of phenolic compounds of birch leaves was assessed by Keinanen and Julkunen-Tiitto (6). The results suggested that for quantitative analysis, the samples should be analyzed immediately or alternatively stored fresh frozen for a few days. For long storage, freeze-drying of leaves frozen at −18ºC was the preferred treatment; no relevant differences were found when previous freezing was made in liquid N2. Oven drying at 40 and 80ºC might cause thermal damage and did not show advantages over air-drying at ambient temperature. When samples have to be dried at ambient or low temperature, a method of enzyme inactivation, such as a short initial drying period at higher temperature or microwaving, should be tested (6).


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After drying, solid samples are normally submitted to milling or grinding before homogenization in the extraction solvent. Liquid samples like juices, tea, or biological fluids require minimum preparation and are just filtered or centrifuged, concentrated or diluted, before extraction or direct analysis. Mechanical processes, such as grinding, blending, and sieving, are used to reduce the particle size in solid samples. Gravity-based methods, such as centrifugation and sedimentation, are often used for the fractionation of heterogeneous samples and the separation of liquid layers in liquid–liquid extraction. Size-exclusion filtration through porous membranes can also be used to separate molecules according to their molecular size. A filter with a certain size of pores allows smaller molecules to pass through freely while larger molecules are retained. Filtration can be operated simply by gravity, under pressure or with assistance of vacuum or centrifugation. Solvent-induced protein precipitation is the sample preparation technique most usually employed in the case of biological fluids like plasma or urine. Miscible organic solvents (acetonitrile, methanol) are normally used for these purposes. Samples are usually filtered and/or centrifuged before and especially after solvent treatment in order to separate the resultant protein precipitates. As the procedure does not involve actual extraction the chance of workup losses during sample preparation is reduced. However, the extracts (supernatants) are relatively unclean as they might still contain a significant amount of unprecipitated sample components. Therefore, selectivity is usually low and further purification by SPE may be required (7). 3.1.2. Hydrolysis

An important decision when dealing with phenolic analysis needs to be taken as to whether to determine the target analytes in their various conjugated forms or as the aglycones. Thus, prior to extraction, samples can be submitted to hydrolysis so that only aglycones are further analyzed (see Note 1). This strategy simplifies the composition of the sample, facilitating compound extraction and increasing the possibilities of detection and quantification. Furthermore, hydrolyses can be used as an aid to structural elucidation and characterization of phenolic conjugates (e.g., glycosylated and/ or acylated derivatives), and are also required to release insoluble phenolic compounds bound to sample matrix components that are not directly extractable by organic solvents. However, this is a decision that needs to be carefully considered since hydrolysis techniques are not always efficient and may destroy some compounds. Nevertheless, hydrolysis may still be undertaken if the aglycones are the target analytes. Alkaline and acid hydrolyses are the most commonly used hydrolysis techniques, although enzymatic treatment with, e.g., proteases, pectinases, cellulases, or amylases may also help release matrix-bound phenolics (8–10). Hydrolysis with b-glycosidases and/ or sulfatases has also been employed for selective cleavage of func-


Extraction and Isolation of Phenolic Compounds


tional groups to aid flavonoid identification (7). Alkaline hydrolysis is used to release matrix-bound phenolics. It is performed with NaOH or KOH 2N to 10N at variable incubation times from a few minutes to several hours, usually under an inert atmosphere and protecting from light. Successive hydrolyses in alkaline and acid conditions may improve the release of bound phenolics (11, 12), although this strategy does not prove to be efficient in all cases (13). Alkaline hydrolysis has also been used analytically to assist in the identification of acylated flavonoids; ester linkages are cleaved and the released products (acyl residues and base flavonoid) can be further identified by HPLC or other suitable techniques (14). Acid hydrolysis has been the traditional approach for the measurement of aglycones and phenolic acids from flavonoid glycosides and phenolic acid esters, respectively (2). It is usually carried out in a concentrated hydrochloric acid medium (e.g., 1N to 6N HCl in methanol) under boiling or reflux conditions using reaction times from a few minutes to 1 h. In the case of O-glycosylated phenolics, as most naturally occurring flavonoids, acid treatment leads to the separation of the constituting sugars and aglycones, which can be used for their identification and analysis. The reaction also allows distinguishing between O-glycosides and C-glycosides, as the latter are not cleaved. The use of mild hydrolysis conditions with organic acids, such as 10% acetic acid, might offer some structural information, based on the identification of intermediate products from the partial cleavage of the conjugating moieties (7). Differential pH’s treatment has been applied for extraction of free, esterified, and insoluble-bound phenolic acids. Free phenolic acids were recovered from an acidified acetone extract of the plant material with diethyl ether/ethyl acetate. Alkaline hydrolysis of the extracts with 4M NaOH under N2 was used to liberate esterified phenolic acids that were then extracted with diethyl ether/ethyl acetate upon acidification. Insoluble bound phenolic acids were released by the treatment of the plant residue remaining after acetone extraction with 4M NaOH and further extracted with ethyl ether/ ethyl acetate (15, 16). Hydrolyses have to be performed with care (see Note 1). Gallic acid has been shown to be unstable during alkaline hydrolysis, and chlorogenic and other caffeoylquinic acids have been found to be rapidly hydrolyzed to caffeic acid which may decompose further (11). Relevant losses of cinnamic acids and derivatives were also reported during alkaline hydrolysis of rapeseed, cereal, and potato flours that were particularly high for caffeic and sinapic acids (15, 16). The loss of o-diphenols via oxidation to the corresponding quinones is also a concern under alkaline conditions (2). The use of inert atmospheres and addition of antioxidants like as ascorbic acid and EDTA have been used as a routine precaution to improve


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the stability of phenolic acids during alkaline hydrolysis (12, 13). Flavanones are easily converted to isomeric chalcones in alkaline media (or vice versa in acidic media) provided that there is a hydroxyl substituent at position 2¢ (or 6¢) of the chalcone (17). 3.2. Extraction 3.2.1. Solvent Extraction

Solvent extraction is still the most common procedure for the extraction of phenolic compounds from plant materials. MeOH, EtOH, acetone, and EtOAc and combinations of them and with water are frequently used for the extraction of phenolics. EtOAc has the advantages of not being water miscible and having a relatively low boiling point, which make it easily removable. More polar phenolics (e.g., benzoic and cinnamic acids, or highly glycosylated flavonoids) could not be completely extracted with pure organic solvents, for that reason mixtures with water are quite usually employed, e.g., 70% aqueous MeOH or acetone (18). Less polar solvents (e.g., EtOAc, diethyl ether) are, however, suitable for the extraction of flavonoid aglycones. Soxhlet extraction, a general and well-established technique, is often used to isolate flavonoids from solid samples, which are usually first homogenized following (freeze-) drying or freezing with liquid N2. Soxhlet extraction surpasses in performance other conventional extraction techniques except for the extraction of thermolabile compounds. Other drawbacks of this technique are that is time-consuming and require relatively large quantities of solvents (19). Combination of Soxhlet with other auxiliary extraction techniques like microwaves may overcome some of these limitations. Different matrices have distinct composition, and different phenolics also have different solubility characteristics; it is, therefore, imperative to characterize solvent extraction efficiencies for the compounds of interest and the matrix to be extracted. It is not always possible to extract all the target compounds with a unique solvent and multiple solvents may be required to extract forms of varying polarities in compound mixtures. Acidification of the solvent increases the ability to extract phenolics, especially when protic polar solvents, e.g., MeOH and EtOH, are used. By acidifying the medium, the phenol-phenolate equilibrium shifts toward the less polar phenyl form, thus facilitating extraction with organic solvents. Acidification is even necessary for the extraction of anthocyanins that are structurally dependent on the pH of the medium, which modifies their characteristics of solubility and affects their stability. Soft acidic conditions must be used to prevent hydrolysis of conjugating residues during extraction. Losses of compounds (e.g., flavonoids containing labile acyl and sugar residues) may, however, occur during further solvent evaporation, especially when acidified solvents have been employed for extraction. The use of weak organic acids, such as formic, acetic, or trifluoroacetic acid (TFA) rather than inorganic acids for acidification of the solvents, and addition of water prior to concentration might minimize these losses (18). The formation of artifacts due to acylation of


Extraction and Isolation of Phenolic Compounds


the sugar moieties of flavonoid glycosides by formic or acetic acid used as solvent modifiers is another reported detrimental effect (20). During extraction oxidation, thermal degradation, isomerization, or enzymatic degradation/modification may also take place. The extraction conditions (e.g., pH, temperature, particle size, sample-to-solvent volume ratio, number of cycles, pressure) have to be carefully optimized to ensure that the target compounds are efficiently extracted without causing significant sample degradation (3). After solvent extraction, additional steps may be required. Centrifugation is usually used to separate the solid plant residue. Filtration is less advisable since the retention of certain phenolic compounds in the filtration membrane may occur. A short gentle heating in warm water (e.g., 55ºC, 15 min) prior to centrifugation has produced good results for the extraction of flavones and flavanones, still, it is unadvisable for other temperature sensitive flavonoids, such as proanthocyanidins or anthocyanins (18). Crude extracts often contain unwanted phenolic and nonphenolic substances, such as sugars, fats, terpenes, waxes, or pigments, which can interfere with later analysis. Consequently, a purification step is necessary. Washing the aqueous extract obtained after vacuum evaporation of the organic phase of the extraction solvent with a nonpolar solvent (petroleum ether, chloroform, or hexane) can be used for the removal of lipophilic side compounds. Whichever the method used, the final extracts are usually concentrated. To achieve this, vacuum evaporation at low temperature (