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Apply protocols to see how the accumulation of metabolites changes


  • Phenolic compounds show large structural diversity. Naturally occurring phenolic compounds contain two distinctive frameworks; hydroxycinnamic acid and hydroxybenzoic acid structures.  


  • The basic skeleton remains same but the position of hydroxyl groups on the aromatic rings gives rise to variety of compounds.


  • In a given tissue of a particular plant system simple as well as complex phenolics could be present.


  • Flavonoids, an important class of secondary metabolites also show great structural diversity arising from the various hydroxylation, methylation, glycosylation and acylation patterns.


  • Most frequently encountered group of flavonoids aglycones includes flavones, flavonols, anthocyandins, isoflavones, flavanones, dihydroflavonols, biflavonoids, chalcones and aurones.


  • These compounds occur as aglycones or glycosides as monomers or constituting highly polymerized structures.


  • The objective in extracting phenolics from their plant source is to liberate these compounds from the vacuolar structures where they are found either through rupturing plant tissue or through a process of diffusion.


  • Solvent extraction methods can be used to separate soluble phenolic compounds by diffusion from a solid matrix (plant tissue) using a liquid matrix (solvent).


  • A single standardized procedure cannot be recommended for all phenolics and /or plant materials. The extraction and isolation procedures have to be optimized depending on the nature of the sample and the target analytes.


  • Phenolic extracts of plant materials are always a mixture of different classes of phenolics that are soluble in the solvent system used.  Aliphatic and chlorinated hydrocarbons, esters and lower alcohols are commonly used for extraction.They include analytical or HPLC grade n-hexane, petroleum ether, ethyl-acetate, n-butanol, ethanol and water.


  • Non-polar solvents solubilises mostly lipophilic compounds e.g. alkanes, fatty acids, pigments, waxes, sterols, some terpenoids, alkaloids and coumarins. Polar solvents are used for polar compounds e.g. flavonoid glycosides, tannins and some alkaloids.  






  • In deciding on the type of phenolic analysis it is important to determine whether target analytes are in their various conjugated forms or as aglycones. 


  • This strategy simplifies the composition of the sample, facilitating the compound extraction and increasing the possibilities of detection and quantification. 


  • Another important aspect to keep in mind before the extraction of flavonoids from any plant system is the information on the localization or distribution of the compounds in particular part of the plant i.e. (root, stem, leaf, flower and fruit).  


  • Flavonoids have been shown to be found in the cell wall, the cytoplasm, in oil bodies and associated with the nucleus and cell proteins as well in the vacuole. 


  • Therefore, it is beneficial to collect multiple plant parts or whole plant to ensure the extracts prepared are a representative of the range of secondary metabolites produced by the plants. 


  • There is variation in the specific secondary metabolites both quantitatively and qualitatively among closely related species or within a single species.



  • Tea leaf is characterized as containing catechin as well as caffeine. Catechins (flavan-3-ols) which are the most important secondary metabolites in the tea plant comprise maximum amount of tea polyphenols.


  • They are rich in young leaves and shoots of the tea plant. 


  • Catechins are divided into galloylated and non-galloylated compounds. Galloylated catechins, including (-) - epigallocatechin gallate (EGCG) and (-) - epicatechin gallate (ECG), esterified often with gallic acid in the 3-OH group of the flavan-3-ols units, are the major catechin compounds in the tea plant. 


  • Flavonols including quercetin, kaempferol and myricetin which are mostly present as aglycones represents a small fraction of the tea polyphenolic content.  


  • During the fermentation process, the catechins are converted to theaflavins and thearubigins. The majority of theaflavins formed undergoes further condensation reaction to form polymeric thearubigins. 


  • The polyphenols comprise one of the most distinguishing characteristics of the tea plant and have been more thoroughly investigated than any other class of compounds in tea. 


  • Due to the complexity among structures of different catechins problems arise in choosing the best and exact solvent system for the extraction of these compounds from different parts of the tea plant.


  • Although various solvents such as ethanol, methanol etc are used but water and methanol are considered as the best solvent for the extraction of catechin compounds from tea infusions.



  • Maize is one of the most important cereals whose anthocyanin pathway is well studied. In the maize plant, anthocyanins generally appear in the epidermal cells. 


  • They are a group of intensely colored pigments responsible for the orange, red, purple and blue colors of many fruits, vegetables, flowers, leaves, roots and other storage organisms of plants. 


  • They are found in the nature in the form of polyhydroxylated and or methoxylated heterosides which are derived from the flavylium ion or 2-phenylbenzopyrilium. Aglycon (anthocyanidin) is found united to one or various sugars, which in turn can be acylated with different organic acids. 


  • The presence of these hydroxyl groups on the rings, as well as one or several sugar molecule, makes these compounds quite soluble in water, ethanol and methanol. Anthocyanin stability increases with the number of methoxy’s in the B-ring and decreases as hydroxyl increases. 


  • So, acidic aqueous solvents have been used as extraction solvent in order to disrupt cell membranes and at the same time dissolve the water soluble pigments. 


  • Ethanol or methanol along with acid is generally used to prevent degradation of non-acylated compounds during extraction.



  • Petunia is another plant system where anthocyanins have been well studied. 


  • Anthocyanin hydroxylation patterns are of prime importance in flower color as they have a major effect on the color resulting from the pigment. Anthocyanins are usually present as the colored flavylium cation. 


  • The interaction of the anthocyanin structure and concentration and vacuolar pH determine final pigmentation.


  • Acidified methanol is considered as the suitable solvent for the extraction of anthocyanin from petunia flowers. Anthocyanins are highly soluble in water and alcoholic solutions. 


  • They are more stable at low pH. In aqueous solution they co- exist as four main equilibrium species; the flavylium cation, the quinonoidal base, the carbinol or the pseudobase and the chalcone. 


  • Depending on the pH of the solution and structure of particular anthocyanin the relative amounts of each equilibrium form vary.





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