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Acetylated Anthocyanidin 3-O-di-glycosides in Red-purple Flowers and Grayed-purple Leaves of Saintpaulia ‘Tomoko’

A new acetylated anthocyanin was extracted from the red-purple flowers of Saintpaulia ‘Tomoko’ with 5% HOAc-H2O or 5% formic acid-H2O, and determined to be peonidin 3-O-[6-O-(4-O-(acetyl)-αrhamnopyranosyl)-β-glucospyranoside] (1), by chemical and spectroscopic methods. In addition, two known acetylated cyanidin glycosides, cyanidin 3-O-[6-O-(4-O-(acetyl)-α-rhamnopyranosyl)-β-glucospyranoside] (2) and cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acetyl)-β-glucopyranoside] (3), were also identified in the redpurple flowers and grayed-purple leaves of S. ‘Tomoko’, respectively. These three anthocyanins have not been reported hitherto in plant tissues in the genus Saintpaulia.


Introduction
Saintpaulia cultivars (Gesneriaceae) are widely cultivated as one of the most popular ornamental indoor plants bred from some wild species native to Southern Africa, with white, pink, red, red-purple, purple, and violet-blue flowers. Recently, we reported the structural determination of malvidin, peonidin, and pelargonidin 3-acetyl-rutinoside-5-glucosides in the violet-blue, purple, and pink flowers of Saintpaulia 'Thamires' (Saintpaulia sp.) along with apigenin 4'-glucuronide as 5% HOAc-H 2 O extract (Tatsuzawa et al., 2012). However, there seem to be no previous reports on acylated peonidin and/or cyanidin 3-O-di-glycosides in the flowers and leaves of Saintpaulia, except some studies of non-acylated cyanidin 3-sambubioside in the leaves of Saintpaulia ionantha (Harborne, 1966). As part of our ongoing research on various flower colors in Saintpaulia cultivars, we studied the anthocyanin components of the red-purple flowers of S. 'Tomoko' along with grayed-purple leaves of S. 'Tomoko'. In this paper, we report the isolation and structural elucidation of a new acylated anthocyanin and two known ones from the flowers and/or leaves of S. 'Tomoko'.

Plant materials
The

Isolation and purification of pigments
Dried red-purple flowers (ca. 5 g) and grayed-purple leaves (ca. 50 g) of S. 'Tomoko' were immersed in 5% HOAc-H 2 O (5 L each) at room temperature for 12 h and then extracted. The extract was passed through a Diaion HP-20 (Nippon Rensui Co., Tokyo, Japan) column (90 × 150 mm), on which pigments were absorbed. Next the column was thoroughly washed with 5% HOAc-H 2 O (20 L) and eluted with 5% HOAc-MeOH (500 mL) to recover the pigments. After concentration, the pigments were separated and purified by paper chromatography using BAW. The separated pigments were further purified by preparative HPLC, which was performed on a Waters C18 (19 × 150 mm, Waters) column at 40°C with a flow rate of 4 mL/min and monitoring at 530 nm. The solvent used was as follows: a linear gradient elution for 15 min from 60% to 70% solvent B in solution A. The fraction was transferred to a Diaion HP-20 column, on which pigment was adsorbed. Pigments were eluted with 5% HOAc-MeOH (5:95, v/v) followed by the addition of excess Et 2 O and then dried. Purified pigment 1 (ca. 25 mg), pigment 2 (ca. 5 mg), and pigment 3 (ca. 45 mg) were obtained as dried dark-red powders.

Analyses of pigment
The identification of anthocyanin was performed by standard procedures involving deacylation with acid, and both alkaline and acid hydrolyses (Harborne, 1984).

Results and Discussion
Pigments from red-purple flowers and grayed-purple leaves of Saintpaulia HPLC analyses of 5% HOAc-H 2 O or 5% formic acid-H 2 O extracts from red-purple flowers and grayedpurple leaves of S. 'Tomoko' revealed that two major anthocyanins (pigments 1 and 2) (81.3% and 10.4%, of the total anthocyanin contents calculated from the HPLC peak area at 530 nm) and one major anthocyanin (pigment 3) (85.9%), respectively, were observed in extract at retention times of 28.1 min (pigment 1), 24.1 min (pigment 2), and 28.3 min (pigment 3), along with some other minor peaks.

Pigment 1
Pigment 1 was isolated from the 5% HOAc-H 2 O extract of dried petals, and purified using Diaion HP-20 column chromatography, preparative HPLC, and paper chromatography, according to the procedure described previously (Tatsuzawa et al., 2012). The chromatographic and spectroscopic properties of pigment 1 are shown in Materials and Methods.
Acid hydrolysis of pigment 1 yielded peonidin as its aglycone, glucose, rhamnose, and acetic acid. Alkaline hydrolysis of pigment 1 yielded a deacylanthocyanin and acetic acid. The deacylanthocyanin was identified as peonidin 3-rutinoside by HPLC, TLC, and UV-vis spectroscopy in comparison with authentic peonidin 3rutinoside.
The molecular ion [M] + of pigment 1 was observed at m/z 651 (C 30 H 35 O 16 ) using FABMS, indicating that pigment 1 is composed of peonidin with one molecule each of glucose, rhamnose, and acetic acid. The elemental components of pigment 1 were confirmed by high-resolution FABMS.
The structure of pigment 1 was further elucidated by investigation of its 1 H and 13 C NMR spectra, including 2D COSY, 2D NOESY, HMQC, and HMBC spectra (Fig. 1).
The chemical shifts of six aromatic protons of the peonidin moiety with their coupling constants were assigned on the basis of analysis of the 2D COSY spectrum. The signals of two anomeric protons of sugar moieties in pigment 1 appeared at δ 5.47 (d, J = 7.6 Hz, Glucose) and δ 4.62 (d, J = 1.5 Hz, Rhamnose), and the chemical shifts of other sugar protons were assigned by analysis of the 2D COSY spectrum with their coupling constants, indicating that the glucose residues of pigment 1 must be β-glucopyranose. In the rhamnose moiety, the doublet signal corresponds to an anomeric proton (δ 4.62, d, J = 1.5 Hz) and doublet signals of methyl protons (δ 0.91, d, J = 6.4 Hz) at C-5 suggested the presence of α-rhamnopyranose. The signal of the anomeric proton of glucose correlated with that of the C-3 carbon (δ 144.2) of peonidin in the HMBC spectrum and also to the signal of H-4 proton (δ 8.39) in the NOESY spectrum of peonidin. The characteristic feature revealed that the OH-3 position of peonidin is glycosylated by glucose. The signal of the anomeric proton of rhamnose correlated with that of the C-6 carbon (δ 66.4) in the HMBC spectrum and to the signal of H-6a and b protons (δ 3.57 and 3.91) in the NOESY spectrum of glucose. Therefore, rhamnose was bonded with glucose at OH-6 of glucose forming rutinose in the pigment. The proton signal of the H-4 of rhamnose (δ 4.75, t, J = 9.8 Hz) was shifted downfield, indicating that the OH-4 of rhamnose is acylated with acetic acid. This linkage was further confirmed by HMBC correlation (Fig. 1). Consequently, the structure of pigment 1 was elucidated to be peonidin 3-O-[6-O-(4-O-(acetyl)-αrhamnopyranosyl)-β-glucopyranoside] (Fig. 1), which is a new anthocyanin in plants ( Andersen and Jordheim, 2006;Harborne and Baxter, 1999;Grayer, 2008, 2011).

Pigment 2
Acid hydrolysis of pigment 2 yielded cyanidin as its aglycone, glucose, rhamnose, and acetic acid. Alkaline hydrolysis of pigment 2 yielded a deacylanthocyanin and acetic acid. The deacylanthocyanin was identified as cyanidin 3-rutinoside by HPLC, TLC, and UV-vis spectroscopy in comparison with authentic cyanidin 3rutinoside.
The molecular ion [M] + of pigment 2 was observed at m/z 637 (C 29 H 33 O 16 ) using FABMS, indicating that pigment 2 is composed of cyanidin with one molecule each of glucose, rhamnose, and acetic acid. The elemental components of pigment 2 were confirmed by high-resolution FABMS.
The structure of pigment 2 was further elucidated by investigation of its 1 H and 13 C NMR spectra, including 2D COSY, 2D NOESY, HMQC, and HMBC spectra (Fig. 1).

Pigment 3
Acid hydrolysis of pigment 3 yielded cyanidin as its aglycone, glucose, xylose, and acetic acid. Alkaline hydrolysis of pigment 3 yielded a deacylanthocyanin and acetic acid. The deacylanthocyanin was identified as cyanidin 3-sambubioside by HPLC, TLC, and UV-vis spectroscopy in comparison with authentic cyanidin 3sambubioside.
The molecular ion [M] + of pigment 3 was observed at m/z 623 (C 37 H 47 O 22 ) using FABMS, indicating that pigment 3 is composed of cyanidin with one molecule each of glucose, xylose, and acetic acid. The elemental components of pigment 3 were confirmed by highresolution FABMS.
The structure of pigment 3 was further elucidated by investigation of its 1 H and 13 C NMR spectra, including 2D COSY, 2D NOESY, HMQC, and HMBC spectra (Fig. 1).
The chemical shifts of six aromatic protons of the cyanidin moiety with their coupling constants were assigned on the basis of analysis of the 2D COSY spectrum. The signals of two anomeric protons of sugar moieties in pigment 3 appeared at δ 5.66 (d, J = 7.7 Hz, Glucose) and δ 4.71 (d, J = 7.6 Hz, Xylose), and the chemical shifts of other sugar protons were assigned by analysis of the 2D COSY spectrum with their coupling constants, indicating that those glucose and xylose residues of pigment 3 must be β-pyranose forms. The signal of the anomeric proton of glucose A correlated with that of the C-3 carbon (δ 143.7) of cyanidin in the HMBC spectrum and also to the signal of H-4 proton (δ 8.79) in the NOESY spectrum of cyanidin. The characteristic feature revealed that the OH-3 position of cyanidin is glycosylated by glucoses. The signal of the anomeric proton of xylose correlated with that of the C-2 carbon (δ 80.8) in the HMBC spectrum and to the signal of H-2 proton (δ 3.93) in the NOESY spectrum of glucose. Therefore, xylose was bonded with glucose at OH-2 of glucose forming sambubiose in the pigment. On the basis of the result of analysis of its COSY spectra, two characteristic methylene proton signals (δ 4.05 and 4.43) being shifted to lower magnetic fields were assigned to H-6a and b protons of glucose. This result indicated that the OH-6 of glucose must be esterified 80 with acetic acid. This linkage was further confirmed by HMBC correlation (Fig. 1). Consequently, the structure of pigment 3 was elucidated to be cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acetyl)-β-glucopyranoside] (Fig. 1), which was found in Saintpaulia for the first time (Andersen and Jordheim, 2006;Harborne and Baxter, 1999;Grayer, 2008, 2011), although this pigment has been found in the flowers of Camellia 'Dalicha' (Li et al., 2008).
The distributions of 3-acetyl-rutinoside-5-glucosides of malvidin, pelargonidin, and peonidin have already been reported in the violet-blue, purple, and pink flowers of S. 'Thamires' as their main anthocyanins (Sato et al., 2011;Tatsuzawa et al., 2012). However, in this study, it was revealed that the red-purple flowers of S. 'Tomoko' contain peonidin 3-acetyl-rutinoside and cyanidin 3-acetyl-rutinoside as the main anthocyanins. Therefore, it is presumed that S. 'Tomoko' has two characteristic biosynthetic features different from S. 'Thamires' (Sato et al., 2011;Tatsuzawa et al., 2012) for producing the red-purple flowers as follows: (1) it is devoid of flavonoid 3',5'-hydroxylase activity and (2) 5-OH of anthocyanidin is free from glucosylation. On the other hand, anthocyanin in the grayed-purple colored leaves of S. 'Tomoko' contained cyanidin 3-acetylsambubioside as a main anthocyanin. Therefore, it was thought that the biosynthesis of anthocyanin 3glucoside moiety for the leaf anthocyanin depended on another biosynthesis system, not the flower one.