Isolation and Structure Characterization of Flavonoids

Flavonoids are one of the most important classes of secondary metabolites from natural products due to their several applications in medicine, foods, diet industries, and so on. Even though a huge number has been reported from natural and synthetic sources, ­scientists­ are­ still­ interested­ in­ flavonoids­ and­ derivatives.­ The­ biggest­ challenge­ for­ working on secondary metabolites is related to the use of the predicted theoretical method­to­isolate­the­expected­compound­and­finally­analyse­the­spectroscopic­data­to­elucidate­and­fully­characterize­the­structure.­This­chapter­was­designed­to­document­useful­techniques­for­isolation­and­structure­characterization­of­flavonoids.­Besides­the­ well-known methods that have been used so far, we would also put together updated information about novel challenge techniques published in recent articles on isolation and­characterization­of­flavonoids.­Our­data­were­obtained­mainly­from­academic­library­ and from reported data online by using research links such as Google Scholar, Scopus, SciFinder,­Scirus,­PubMed,­and­so­on.­Our­field­experience­on­phytochemistry­of­isola - tion­and­characterization­of­flavonoids­was­also­used­in­this­chapter.


Introduction
Flavonoids are a large group of C-15 (C 6 -C 3 -C 6 ) secondary metabolites widespread in higher plants and are also detected in some lower plants such as algae. An important number has been reported from natural and synthetic sources due to their several applications in pharmaceutical and diet industries. Flavonoids occur in natural products specially blooming plantspecies,andcoloursofflowerscouldbeindicativefortheclassofcompounds.Flavonoids are mostly obtained as yellow pale, white, red, purple, blue, and so on from species of several plant families but are known to be widespread in the Fabaceae family. Flavonoids could be detected in natural products by using some analytical methods such as the Shinoda [1], sodium hydroxide [1] and p-dimethylaminocinnamaldehyde tests [2].

Sample preparation
Flavonoids especially those in plants could be extracted from several parts such as roots, barks, leaves, fruits, woods and flowers. Samples are more often dried and ground before the extraction process. This initial treatment of samples helps in facilitating the extraction yields as well as preserving constituents. In some cases, the extraction is carried out on fresh plantmaterials.Thedriedplantmaterialshavebeenreportedinseveralinvestigationstocontainmostflavonoidsthanfreshsamples [8][9][10].Obviously,thegroundsamplesalwaysgave higheryieldsofextraction,andthiscouldbejustifiedbythefactthatthesolventhascontact Flavonoids -From Biosynthesis to Human Health 46 with surface constituents when the powder has smaller particles. The extraction yields of flavonoidsfromnaturalproductsarealsoaffectedbysomefactorssuchastemperature,time and ratio of water in case of aqueous mixing solvents [11].
The extraction of powered seeds of Ziziphus mauritiana using different methods such as maceration, decoction, soxhlet extraction and sonication with 50 and 80% ethanol, and water (decoction) as solvents, was reported and the high totalflavonoidcontents was obtained from the sonication technique [19].
In the basic mechanism of the extraction techniques, the microwave-assisted extraction followsseveralstepswhencomparingtoconventionalextractions.Theseincludethepenetration of the solvent into the solid matrix, the solubilization and/or breakdown of constituents, the transportation of the solute outside of the solid matrix, the migration of the solute from the external surface of the solid into the solution, the movement of the extract with respect to the solid, and the separation and discharge of the extract and solid [20][21][22]. The main differencebetweenthemicrowave-assistedextractionandconventionalextractionsbeingthe directionsofheatandmassgradientsduringtheextraction:forthefirstprocess,bothmove from inside to outside while in the second case, the mass transfer goes from inside to outside when heat occurs from outside to inside of the subtract [20][21][22].
Following the traditional Indian medicinal preparations, Krishnan and Rajan recently reportedasuitableextractionofflavonoidsfromTerminalia bellerica Roxb., by the microwaveassisted solid-liquid method, an investigation conducted in view to study the influence of solvent-to-feed ratio and temperature on kinetics and thermodynamics of aqueous extraction [23].Totalflavonoidswithgoodyield(1.13%)obtainedunderoptimumconditions(ultrasonic power500 W,extractiontime20 min,materialsolventratio1:20,andethanol concentration 30%) using ultrasound-assisted extraction were reported from the corn silk (Zea mays L.), a Chinese medicinal herb, with a recommendation for this plant to be developed as food natural antioxidant reagents [24]. Ultrasonic extract of flowerfromLythrum salicaria L. was reported to possess good scavenging of hydrogen peroxide owing to the higher phenolic and flavonoidcontentswhenusingthreemethodsofextractionsuchaspercolation,ultrasonicassisted extraction and polyphenol fraction [25]. Allthesetechniquesallowtohaveflavonoidsinthecrudeextractwithgoodyieldbeforethe applicationofdifferentfractionationandpurificationproceduresfortheirisolation.

Chromatography as a main tool for isolation of flavonoids
The isolation of flavonoids from natural sources is conducted by repeated and successive chromatography techniques such as open column chromatography (CC), preparative thin-layerchromatography(prep.TLC),centrifugalpreparativethin-layerchromatography (CPTLC), high-speed counter-current chromatography (HSCCC), medium-pressure liquid chromatography (MPLC), high-pressure preparative liquid chromatography (prep. HPLC), and so on.
In column chromatography method, stationary phases could be normal or reverse phase silicagels,Sephadex(LH-20,G-10,G-25andG-50).Inviewtohaveflavonoids-richfractions, it is recommended to use some preliminary liquid-liquid extraction methods or polymeric resins such as Diaion HP-20, Amberlites (XAD-2, XAD-7) from the crude extract. These polymeric resins are very useful when the absorption of extracts is eluted in the open column chromatography with an increasing gradient of methanol in water.
Theopencolumnchromatography(CC)isstillthemostusefulandeasyisolationtechnique fornaturalproductsisolationandbymeansthatofpurificationofflavonoids.Thechoiceof the good solvents system for mobile phase is important and should be made from the check-upTLConthe crudeortheflavonoids-richfraction.Combinationandpolarity of solvents shouldbeuseddependingontheclassofflavonoidstargeted.Aftertheflash column,some majorfractionscoulddirectlybesubjectedtoSephadexLH-20orprep.TLCiftheydonot contain complex mixture of flavonoids. During the separation process, constituents from theflavonoid-richfractionscouldhavecloserretentionfactors(Rf)basedontheirpolarities. The change in phase of the adsorbent in some cases is useful to have good separation in eithersmallopenCCpurificationorprep.TLC.Severalinvestigationsreportedthe isolation of new flavonoids using CC. This included two dihydrochalcones, rare natural resources secondary metabolites, from Eriosema glaumerata [26],twopolyhydroxylated flavones having antioxidant activity from E. robustum [3], one isoflavanol from Kotschya strigosa [27], two Flavonoids -From Biosynthesis to Human Health glucoside isoflavonesfromIris kashmiriana [28], four dimeric chalcone derivatives from Uvaria siamensis [29], five flavonoids from Millettia griffithii [6], one pterocarpan, three isoflavones from the root, stem bark and leaves of Erythrina schliebenii [30],fourflavonoidC-glycosides with anti-inflammatory properties from the leaves of Piper aduncum [31]. The number of recent published articles using CC is indicative for the useful and convenience of this method. Nevertheless, the prep. HPLC technique has been widely used for isolating commonly polyphenolsandmorespecificallyflavonoids.Thesuitabilityofthismethodforthisclassof secondary metabolites is associated with its high absorption in UV that is used as detector during the isolation.
Several works on isolation of flavonoids from natural products using prep. HPLC have been published so far, and some of these compounds, recently reported, are documented in Table 1 as well as their sources, column characteristics and mobile phases used ( Table 1 and Figure 2).

Themassspectrometrytechniqueisveryhelpfulinthestructureelucidationofflavonoids.
It is used in the determination of the molecular weight for establishing the distribution of substituentsbetweentheA-andB-ringsandinthedeterminationofthenatureandsiteof attachmentofthesugar(s)inflavonoidC-andO-glucosides.Themolecularweightofthebasic , and so on] to one of the basic molecular weights above.Thelossofsomeion-fragmentsfromthemolecularorpseudo-molecularion isverycharacteristicinthemassspectraofflavonoids.Peaks obtained during this fragmentation process represent accurately the corresponding ion-fragments that are expressed as mass-to-charge ratio (m/z).Theexactmolecularweightforeachfragmentmaybemeasured tothenearest0.0001massunitifthemassspectrometerisoperatinginhighresolution.This information enables calculation of precise molecular formula from the molecular ion peak and ion-fragments [54].Aprerequisiteforsuccessfulmassspectrometryisthattheflavonoid shouldbesufficientlyvolatileinthehighvacuumwithinthemass spectrometer.Mostagly-conesaresufficientlyvolatileatprobetemperatureof100-230°C,highertemperaturesbeing required for the more polar polyhydroxyflavonesandflavonols.Glycosides,anthocyanidins andbiflavonoids,however,arenotsufficientlyvolatileandshouldthereforebederivatized to improve their volatility. Some standard methods used for derivatization of compounds are permethylation or perdeuteromethylation and trimethylsilylation [54].
Naturalproductsingeneralorflavonoidsinparticularremainanimportantsourcefordrug discovery. Determination of their absolute configurations is one of the most challenging tasks in the structure elucidation of chiral flavonoids. It has been proven that the change in absolute configuration of secondary metabolites consequently affected the difference in pharmacological activity of both stereo-compounds. Methods such as chiroptical approaches, chemical synthesis, analytical chemistry, chiral derivatization and X-ray crystallography could be used to determine the absolute configuration of flavonoids.An important investigation wasreportedonthedeterminationofabsoluteconfigurationofnaturalproductsandsome flavonoidsusingexperimentalandcalculatedelectroniccirculardichroism(ECD)data [56].

Conclusion
The extraction, isolation and characterization of flavonoids from natural products have been carried out successfully by natural product chemists and phytochemists using relevant techniques and new methods. Some of these techniques and methods have been documented inthischapterwithillustrationsowingtosomeflavonoidsrecentlyreported.Itisclearthat the HPLC and its combination with other available techniques of isolation are being often usedtoobtainflavonoidsfromnaturalsourcesespeciallyfromplantspecies.Thecharacterizationofflavonoidsremainsbasicallyfocusedontheanalysisoftheirspectroscopic,mass and UV data and some chemical investigations depending on the nature of the structure under elucidation. Theneedofflavonoidsinagriculture,foodanddrugindustriesstillone of the worldwide up-to-date research interests. Natural resources and especially medicinal plantsarestillavailabletodiscovernovelorefficientantioxidantflavonoidsthatcouldbe used as drugs to fight against degenerative diseases one of the issues the global health is facing today.
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