Current procedures for extraction and purification of citrus flavonoides

In the food industry, antimicrobial agents are used for preserving the quality and safety of processed food. Flavonoids found in citrus extracts inhibit cell growth of a large group of infectious microorganisms, therefore, these compounds may be useful as antiviral, antifungal and antibacterial agents. Hesperetin, hesperidin, luteolin, naringenin, naringin, narirutin, neohesperidin, nobiletin and tangeretin are some of the main flavonoids found in various citrus fruits. The processes used in recent years to extract, purify and analyze typical flavonoids from citrus species are reviewed.


Introduction
Flavonoids are a group of polyphenols that are found in fruit, vegetables, nuts, seeds, stems and flowers as well as tea, wine, propolis and honey (Tham & Liew, 2014).Some flavonoids are responsible for fruit coloration.Most of flavonoids are structured basically by 3 rings, two of them are aromatic benzene rings (called rings A and B), connected by an oxygen pyrane ring (called ring C), as shown in figure 1, and there is the characteristic presence of hydroxyl groups in one or more R positions.
Especially abundant in citrus fruits, there are some flavonoids that can be found in almost all citrus fruits (table 1), like hesperidin, which consolidate a group called citrus flavonoids.Their concentration in peels is higher than in juice and seeds (Codoñer & Valls, 2010) as a result of flavonoids role for fruit coloration.
For nourishment purposes, research has concluded that consumers in theory are ready to accept food rich in flavonoids, by informing them about the scientific benefits (Zang et al., 2014;Zhang et al., 2014a;Jung et al., 2011;Lampila et al., 2009).In Europe, an average adult person spends up to €454.7 per year in flavonoids contained in cardiovascular drugs (Sanfelix et al., 2010).

Raw materials
Citrus fruits contain a range of key nutrients such as vitamin C, folate, dietary fiber, minerals and phytochemicals, which attributes to their health-promoting properties (Ledesma & Luque, 2014).It is believed that vitamin C is a major contributor to the anti-oxidant capacity of citrus.However, the major contribution of citrus anti-oxidant activity comes from the combination of phytochemicals and from their synergistic action with vitamin C. The major phytochemicals in citrus fruits are the terpenes and phenolic compounds, which possess anti-inflammatory and anti-carcinogenic activity (Wang et al., 2014;Natarajan et al., 2011;Codoñer & Valls, 2010).
The main citrus fruits in flavonoids research have been orange (C.sinensis), lemon (C.limon), grapefruit (C.paradisi) and tangerine (C.reticulata), for at least the past 30 years.Nowadays, native varieties are of special Table 1.Substitution in the general structure for some of the main citrus flavonoids (Barreca et al., 2011a;Gonzalez et al., 2010) interest.Tough Citrus species are harvested all around the world (Lorente et al., 2014), some species are better developed than others, depending on climate conditions, and its availability varies from one country to another or even regions within the same country (Roussos, 2011).This influences the research on specific Citrus species (table 2), where the main citrus producing countries have more studies on a wider variety of species, exploring even the wild varieties found in unique locations.

Raw material conditioning
Citrus have been collected and extracted in early springtime or late winter (Sandoval et al., 2012;Barreca et al., 2010), and about 2-5 months after the flowering period (Barreca et al., 2011a;Chen et al., 2011;Yoo et al., 2009).Even though most compounds are found in the peel, as indicated before, studies also have been made to obtain extracts rich in flavonoids from the juice and the seeds.In order to facilitate the extraction of the components, raw material must be ground to a small particle size, improving extract transport from the solid matrix towards the solvent phase.
Raw material can be used either fresh or dry (Ye et al., 2011), the use of fresh raw materials involves the presence of an aqueous phase in the extract, and a further separation, like decantation, must be carried out.
When dry raw material is used, it must be conditioned first, in order to permit cells to stretch back to their original size and shape, allowing the components to transfer through the cell's structure into solvent bulk.Juices, peels and other tissues can be separated manually (Barreca et al., 2011a) or using commercial extractors.

Preliminary Separation
Organic solvents are often used for the extraction of citrus compounds.There are two main operations to extract the compounds from the citrus matrix.One is simple maceration, with solvents extracting the compounds by diffusion from the citrus matrix (Yoo et al., 2009), where methanol is a frequently used solvent (table 3).The second one is centrifugation of the juices, eliminating aqueous phases (Barreca et al., 2011a).

Purification
The mixtures obtained from extraction are quite complex, showing many species from the different tissues in the fruit.In order to obtain a higher concentration of some compounds, it's necessary to carry out a further purification.
Compound purification has been carried out by column chromatography, allowing high single concentration of compounds (Levaj et al., 2008).Purification through adsorption is versatile, simple and low cost, which makes it attractive for the selective recovery of a variety of phenolics and polyphenols.Adsorption shows other advantages like selectivity, environmental impact and toxicological effects.Studies on the characterization of the detailed interactions between resins and individual plant phenolics are needed for design (Soto et al., 2011).
High speed countercurrent chromatography (HSCC) is also used to extract and purify flavonoids using two-phase solvent systems, flowing simultaneously in opposite directions.In addition, HSCC also realizes multiple forms of the gradient elution process; thus it can be used not only to remove impurities from crude extract of raw materials but also to purify the final product.Moreover, some pure compounds can even be obtained through one step from crude extract without sample pretreatment (Duo et al., 2011).
Even tough, there is few available data for citrus flavonoids purification processes, i.e. the one used for purifying hesperidin, naringin, and narirutin with a Zorbax C18 column and a mobile phase of citric acid and ammonium acetate in water and methanol 60:40 (Levaj et al., 2008).Also, preparative high performance liquid chromatography (HPLC) using an instrument equipped with a UV-vis detector has not been employed widely in the isolation of flavonoid compounds.Most mobile phases consisted of a linear gradient of acetonitrile in H2O.Crude juice is diluted with DMF, flavonoids are collected in the HPLC course range time of 5-30 min.
The fractions collected are joined, evaporated to dryness in a rotary evaporator and redissolved to regenerate the original concentration of analytes in crude juice (Barreca et al., 2011d).

Analysis methods
Once the extract is obtained, it's important to analyze it, to know if the procedure was correct and the present species were separated as expected.

Qualitative methods
Thin layer chromatography (TLC) continues to be an important method for qualitative investigation of plant compounds because of its inherent advantages-many samples can be analyzed simultaneously and quickly and multiple separation techniques and detection procedures can be applied.TLC is one of the most powerful and general analytical tools used for qualitative purposes, indicating the presence of specific flavonoids in a simpler way than HPLC.It follows from numerous publications that, owing to large polarity differences, it is difficult to find a TLC system which separates similar structure molecules on a single chromatogram.
Most common stationary phase is silica gel, using a mobile phase of mixtures ethyl acetate -methanolformic acid (Mohammad et al., 2010).Conventional separation on silica gel with moderately polar mobile phases consisting of small amounts of methanol with less polar solvents has been successfully used for the polyphenolic compounds.The retention factor (Rf) values of the different compounds reflects their polarity, given by the number of -OH groups, wich displays much more affinity for the stationary phase.
Using the Folin-Ciocalteu reaction, the phenolic compounds form blue complexes with the phosphomolyb-dic-phosphotungstic reagent at high pH.The analysis is simple, highly reproducible under carefully controlled conditions, and, therefore, widely used.The Folin method represents a classic approach to estimate total phenolic compounds in a variety of matrices.Although the method is nonspecific, it is frequently applied as a measure of total phenolics in biochemical, animal, and clinical trials (Soto et al., 2012).Fluorescence detection of the flavonoids is also used to identify the effective separation of individual flavonoid compounds (Andreu et al., 2010).

Quantitative methods
High performance liquid chromatography (HPLC) is widely used to quantify the amount of flavonoid compounds in the obtained extracts, and there are several methods reported for HPLC sets (table 4).For every method, it must be considered the polarity of the species to be analyzed, so the correct column and mobile phase may be chosen.The hypothesis proposes that the difference in the orientation of the -OH could result in different affinities of the two isomers for the stationary phase and hence their separation.Good characterization of mobile-phase systems can be achieved by determination of relationships between retention and mobile-phase composition.Gas chromatography is also used, but due to its characteristics, volatile samples are required (Cheong et al., 2012).In addition, water samples are not allowed, only the species that are soluble in volatile solvent should be measured.
Liquid chromatography has been the most used technique to analyze the obtained extracts from citrus fruits (Jiang et al., 2011), performing tests at different pH levels and using a huge variety of detection methods.High performance liquid chromatography (HPLC) combined with ultraviolet (UV) detection and mass spectrometric (MS), electrospray ionization (ESI), and/or two mass spectrometer tandem (MS/MS) measurement provides the most useful techniques currently available to identify specific classes and structures of food phenolics (Barreca et al., 2013;He et al., 2011).The differences in ultraviolet spectra are an important tool in determining which wavelengths to monitor for detection and quantification by HPLC (Soto et al., 2012;González et al., 2010).

Conclusions
Since citrus fruits are original from Asia, most of the varieties on the current literature were found and studied in Far East countries.It doesn't mean that others countries are not interested in studying citrus flavonoids, only that they don't have so much of wild or endemic citrus species.Most of the studies used grounded dry raw material for extraction, from peels and whole fruit.Methanol mixtures are the main solvent used in the extraction of citrus flavonoids.
There's few literature found about purification of single flavonoids, since few details of purification behavior of single flavonoid compounds have been provided in most of the publications dealing with their isolation and structural elucidation, and, in some cases, inadequate information is supplied, there is an entire opportunity field for new research in purification techniques, and their efficiencies in flavonoids isolation.
High performance liquid chromatography has been used as the best analysis technique to quantify and identify structures of the obtained flavonoids and thin layer chromatography provides a quick method for qualitative identification of the compounds along the experimental process.

Table 2 .
Citrus species used in recent flavonoid research.

Table 3 .
Solvents used for flavonoids extraction in citrus species.

Table 4 .
HPLC sets for flavonoids in citrus species.