Two diglucosylated anthocyanins from Combretum paniculatum flowers

From Combretum paniculatum flowers, two diglucosylated derivatives from cyanidin and pelargonidin were identified using chromatographic (TLC), chemical (degradation by hydrolysis, tests of revelations) and spectral [UV-visible, 1 H-NMR ( 1 H and 13 C, TOCSY-1D, DQF-COSY, NOESY-2D)] methods. These pigments were found to con-sist of cyanidin 3,5-O- β -D-diglu-copyranoside and pelargonidin 3,5-O- β -D-diglucopyranoside


INTRODUCTION
The plant kingdom contains a wide range of natural substances such as polyphenols, carotenoids, phytosterols, phyto-estrogens, glucosinolates, etc. [1]. Polyphenols which are found in all parts of higher plants (roots, stems, leaves, flowers, pollen, fruits, seeds and wood), are secondary metabolites characterized by the presence of an aromatic ring bearing free hydroxyl groups or with a carbohydrate [2,3]. It is known that Aqueous extracts of inflorescences of Combretum paniculatum studied in this work are known to have an anti-tumor activity of carcinoma of the lung [4].
In this paper, we report for the first time the identification of 3,5-β-O-diglucosides of cyanidin and pelargonidin in red petals of C. paniculatum flowers.

METHODS AND MATERIALS
Plant material: belonging to the family of Combretaceae, C. paniculatum is a robust liana reaching at least 15 m high. Loose terminal panicles are formed of dense spikes of flower petals and Red fillets usually appearing during the dry season during defoliation.
Extraction, purification and isolation of the petals of flowers of C. paniculatum: freshly harvested, the red petals of C. paniculatum flowers are immediately freezedried. 100 g of powder obtained by crushing the petals are extracted by maceration at 5˚C successively with 1000 mL and twice with 200 mL of methanol-hydrochloric acid (99:1) system [5]. The filtrate were pooled and concentrated almost dry vacuum at 30˚C. 50 mL of HCl-H 2 O (0.5:99) system/MeOH (7:3 mL) are added. The solution was filtered and concentrated at 25 mL. This solution was filtered and fixed on a nonionic polymeric adsorbent (Amberlite XAD-7, Aldrich Chemical Co., Milwaukee, WI) column (length 300 mm, i.d. 20 mm) which was prewashed with 0.5% HCl/H 2 O; the pigments were then eluted with MeOH/H 2 O/HCl, 70:30:0.3). The eluate was concentrated, filtered through a Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden) column (length 300 mm, i.d. 20 mm). The final purification was achieved by preparative thin-layer chromatography (TLC) on silica gel 60 F254 (Merck-Clevenot Corp.) using EtOAc/HCO 2 H/AcOH/H 2 O, 100:11:11:26 (EFAW), as solvent system. The two main bands from this chromatography are numbered from bottom to top. Then to the band which had the small frontal reference is assigned 1 and another 2. The isolated bands of adsorbent were eluted with 0.5% TFA/MeOH, and the solutions were concentrated and filtered through a RP-18 column using (0.5% TFA/H 2 O)/MeOH (6:4). The eluates were finally concentrated and freeze-dried to give 1 (5 mg) and 2 (10 mg) as TFA salts.
Chemical analysis: Acidic hydrolysis was performed by dissolving 1 mg of each pure compound in 4 mL in hydrochloric acid 1 N (in vials) and placed in a water-bath at 100˚C. Successive samplings carried out between 0 and 60 min were cooled and analyzed by TLC with the solvent system: BAW [Butanol acetic acid/water 4:1:5; upper phase] on a plate of cellulose. Samples at 60 min were cooled and extracted twice by 0.5 mL of 3-methylbutan-2-ol from 1 and 2. These samples at 60 min were analyzed by TLC on cellulose plate with the Forestal [acetic acid/HCl concentrate water (30:3:10)] as mobile phase together with cyanidin and pelargonidin as anthocyanidins standards available.
Electronic spectroscopy: (UV-visible) spectra were obtained with DES 190 double energy system spectro-meter from SAFAS-MONACO, using MeOH-HCl 0.01 N as solution system. About three drops of aluminium chloride (AlCl 3 ) were added to the previous solution to highlight the eventual vicinal hydroxyl free groups.

RESULTS AND DISCUSSION
UV-visible spectra of 1 and 2 ( Table 1) were showed absorption in UV area at 280 nm corresponding to nonacylated anthocyanins. Furthermore, in visible area 1 and 2 were absorbed respectively at 506 and 530 nm. 1 would be a derivative of cyanidin and 2 is probably a derivative of pelargonidin. By the addition of AlCl 3 , 1 has given red shift effect indicating the presence of vicinal hydroxyl groups on the B ring. But 2 has caused no red shift confirming that it is a derivative of the pelargonidin. The ratio A 440 /A max.vis. (respectively 28% and 22%; Table 1) suggested that the 5 position of these compounds was not free [5][6][7]. The TLC of the products of acidic hydrolysis for each of the two compounds in the Forestal with standards has showed two spots corresponding to cyanidin (1) and the pelargonidin (2). Controlled hydrolyses of compounds 1 and 2 have given each an intermediate spot between the aglycon and anthocyanin indicating that they were diglycosylated.
All sugars protons appear between 3 and 5.5 ppm. However, two doublets corresponding to anomeric protons of glucoses A and B were clearly observed at 5.31 and 5.18 ppm for 1 (5.31 and 5.19 ppm for 2) respectively, are characteristic of the protons on 3 and 5 positions respectively [5,6] (Table 2). Their β-D configurations of each are confirmed by the high values of their coupling constants (J ≈ 7 -8 Hz) [6]. TOCSY spectra obtained by selective excitation protons H1" of the glucose allowed to confirm the membership of each methylenic proton of glucoses A and B of 1 and 2. To determine connection positions of sugars, correlation NO-ESY 2-D experiments were performed. The correlation between H1"A (5.31 ppm) and the aglycon H4 indicated that glucose A is bound in 3 position. Similarly, irradiation of H1"B signal (5.19 ppm) reveals a proximity with the aglycon H6 (downfield shift of 1H NMR signal of H-6 protons in both anthocyanins), confirming that glucose B is attached in 5 position.
Spectra DQF-COSY proton coupling constants allowed assignments of protons of glucoses A and B. Indeed, anomeric proton 5.31 ppm (H1''A) is nearby of a proton to 3.72 ppm (H2''A) which is linked to H3''A appearing at 3.65 ppm. This triplet (with a coupling constant about 9.6 Hz) is connected to an another triplet H4''A appearing at 3.43 ppm. The last one is connected to H5''A at 3.56 ppm. Signal at 4.01 ppm with a wide coupling con-  3.56 ppm. Signal at 4.01 ppm with a wide coupling constant (12 Hz) reveals a geminal coupling between two methylenic protons on 6'' position. It characterized H6''bA; the other proton (H6''aA) also appears at 3.72 ppm. By a similar reasoning based on spectra DQF-COSY 2, the glucoses A and B into 2 are identical to those detected in 1. Thus, isolated compounds from C. paniculatum flowers are identified as respectively as cyanidin and pelargonidin 3,5-O-β-D-diglucopyranoside (Figure 1).