- Determination of individual catechin contents by HPLC is expensive and time consuming .In the study, Chlorophyll has been demonstrated to be important factors in the regulation of catechin biosynthesis which might be associated with the sink- source balance in leaves. Therefore, Chlorophyll content might be utilized as potential indicators to predict catechin composition in Tea production, as the determination of individual catechin contents by HPLC is expensive and time consuming (Kang Wei et al; 2012).
- The isocratic HPLC system sucessfully seperated the tea catechins in the tea flower extract. Some flavonoids, such as quercetin, rutin, quercetin glycosides, apigenin, kamempferol and others could not be resolved by this system .Therefore, another gradient elution HPLC system has been developed for these flavonoids. Two solvent systems, namely, boiling water and 75% ethanol were used to extract the total catechins and caffeine from the tea flower. The results indicated that more catechins and caffeine were extracted by 75% ethanol system (Yung-Shenglin etal; 2003).
- By developing long LC gradient of HPLC not only Proanthocyanidin but also wide range of glycosylated and non- glycosylated flavonoids and Isoflavones has been resolved that often occur in crude plant extracts. This technique also shifts the emphasis away from laborious sample preparation in the laboratory to utilising the combinations of advanced instrumentation available today (Karl Fraser, Scott J. Harrison et al; 2012).
- The above mentioned protocols used for purification shows the presence of different types of complex compounds in various parts of maize plant. Maize is one of the cereals whose anthocyanin composition is well defined.
- The first study revealed the extraction and purification of anthocyanins from corn seeds of purple variety using preparative HPLC column.
- The next study showed the purification of phenolic compound such as maysin from kernels and cob tissues of maize.
- The third study reports the identification and purification of a derivative of ferulic acid from maize bran by using silica gel chromatography.
- The final study reports the accumulation of phenolic compounds in leaf and stem of maize which were purified using solid phase extraction method. All the discussed protocols have shown to have phenolic compound accumulation in maize. Although all the purified compounds were more or less similar in different parts of the maize plant, but different purification methods were employed in those studies. This reveals that the compounds present in the various tissues are complex in nature and depends on the solubility and polarity of the solvent used for extraction.
- Petunia is considered as the model system to understand the biosynthetic pathway of anthocyanin. The aglycone forms of anthocyanins i.e. anthocyanidins are generally accumulate as major compounds in petunia flowers.
- First study reveals the recovery of purified anthocyanidins from petal tissue by using HPLC.
- Next study showed the implementation of Amberlite-XAD resin along with preparative HPLC performed in gradient and elution mode to purify the acylated and glycosidic derivatives of anthocyanins from Petunia flowers.
- In the third study, Petunia leaves were used to characterize and purify flavonol glycosides using polyamide column and preparative HPLC. Final study reports the purification of acylated anthocyanins from Petunia flowers using Sephadex LH-20 along with preparative HPLC.
A crude natural product extract is literally a cocktail of compounds. It is difficult to apply a single technique for isolation and purification of individual compounds from the crude mixture. From the above methods used for purification, it is clear that these plant species contain various classes of compounds with structural diversity. Purification is the critical part of the recovery process of bioactive compounds from crude plant extracts. It isemployed to remove fractions and obtain given class of compounds in pure state from mixture of compounds. Moreover it is also important to obtain pure extracts to ensure the identity and safety of the antioxidant activity exhibited by those compounds to be used as food additives in industries. The purified compounds obtained from these plant extract species has been reported to have good radical scavenging and antimicrobial activities.