In vitro Accumulation of Polyphenols in Tea Callus Derived from Anther.

BACKGROUND: Tea is an economic important crop with high medicinal value due to rich polyphenols content. In the present research we studied the accumulation of polyphenols of in vitro regenerated callus from anthers. OBJECTIVE: Callus induction of tea anthers and in vitro accumulation of phenolic compounds from the anther-derived callus. MATERIALS AND METHODS: Standardization of callus induction for tea anthers. In vitro generated callus was screened for in vivo accumulation of catechins and its isomers were screened by FC reagent staining technique. The methanol extract of dry and green callus obtained were estimated qualitatively by Fourier transform infrared spectroscopy (FTIR)-alternative total reflection (ATR) and quantitatively by HPLC method. RESULTS: Anthers inoculated on half strength MS media fortified with 2,4-dichloro acetic acid (2 mg/L), Kn (1 mg/L), and BAP (1 mg/L) induced callus under photoperiod of 9:15 h light. The in vivo histochemical studies revealed the accumulation of polyphenols in the callus. The in vitro generated fresh and dry callus were used for extraction and screened for accumulated polyphenols [galic acid, (+)-catechin (C), (-)-epicatechin, (-)-epigallocatechin, (-)-epigallocatechin gallate, (-)-gallocatechins, (-)-epicatechin gallate] were estimated qualitatively by FTIR-ATR method and quantitatively by HPLC method. CONCLUSION: The FC staining technique used here helps in localization of polyphenol compounds accumulation in the tissues by instant microscopic studies. The study have scope in large-scale isolation of various medicinally important flavonol by using anther culture. Abbreviations used: HPLC: high pressure liquid chromatography; FTIR: Fourier transform infrared spectroscopy; 2,4-D: 2,4-dichloro acetic acid; BAP: N6-benzyl amino purine; kn: kinetin.

Combined electron transfer dissociation-collision-induced dissociation fragmentation in the mass spectrometric distinction of leucine, isoleucine, and hydroxyproline residues in Peptide natural products.

Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(•) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δm = 43 Da) or ethyl radicals (Δm = 29 Da), through collisional activation of z radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific z(•) ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS(n)) method has been successfully implemented in a liquid chromatography-MS(n) platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.