# Regarding the Nature of the Blue Pigment in Arils of Ravenala Madagascariensis Sonn. (Strelitziaceae)

## TL;DR

This study identifies a blue pigment in the arils of the Traveler’s tree as a copper-containing protein called phytocyanin.

## Contribution

The paper is the first to describe the structure of the blue pigment in Ravenala madagascariensis arils as a phytocyanin.

## Key findings

- The blue pigment in the arils is a 14 kDa protein identified as a phytocyanin via UHPLC/MS/MS analysis.
- The pigment's blue coloration is due to copper coordination with the protein.
- Optimal pigment extraction was achieved using 10% aqueous SDS at pH 5.

## Abstract

Ravenala madagascariensis, the Traveler’s
tree, is native to Madagascar but has adapted globally and is utilized
for medical and cosmetic purposes. Focusing on the brightly blue colored
arils of the plant’s seeds, this study aimed to examine their
morphological and physicochemical properties and to shed light upon
the previously undescribed structure of the pigment. Plant arils were
examined by energy-dispersive X-ray spectroscopy (EDS) and scanning
electron microscopy (SEM). Centesimal analyses indicated a lipid-rich
composition (81.45%), carbohydrates (7.03%), moisture (3.71%), ash
(0.78%), and proteins/peptides (7.1%). Analysis of the fatty acid
methyl esters obtained from the lipid fraction by GC–MS indicated
a composition of 41% palmitic, 14% stearic, 34% oleic and 7% linoleic
acids. A battery of qualitative tests employing hexane, ethanol, and
10% aqueous sodium dodecyl sulfate (SDS) extracts of R. madagascariensis arils for the presence the following
secondary metabolites was performed: alkaloids, triterpenes and phytosteroids,
coumarins, phenolics, flavonoids, anthroquiones, saponins and tannins.
Results for all classes were negative or, in one case, inconclusive.
Factorial optimization of extraction conditions for isolation of the
pigment was performed indicating 10% aqueous SDS at pH = 5 to be the
most efficient to remove the pigment from the arils, while spectrophotometer
scans indicated absorption peaks at 400 and 620 nm. Thin layer chromatography
(TLC) of a 6 M HCl digest of the arils tested positive for amino acids
when developed with ninhydrin. SDS-PAGE electrophoresis revealed a
14 kDa protein profile and the peptide fractions obtained after partial
degradation with trypsin were analyzed by (UHPLC/MS/MS), lending important
structural information that strongly suggests the presence of a phytocyanin
(Blue Copper Protein). Analysis of our data and by comparison to the
NCBI BLAST data bank indicated a phytocyanin domain-containing protein,
responsible for the blue coloration of Ravenala arils due to copper
coordination with the protein.