1Note portant sur l'auteur1

2Note portant sur l'auteur2

3Note portant sur l'auteur3

4Note portant sur l'auteur4

5Note portant sur l'auteur5

6Note portant sur l'auteur6

Introduction

7The Ribes nigrum leaves are traditionally used for its diuretic and anti-inflammatory activities (Racz-Kotilla et al., 1977; Chanh et al., 1986; Declume, 1986; Pommier, 1990; Serrano et al., 1990; Mongold et al., 1993).

8Flavonoids are the main constituents of the leaf: quercetin, kaempferol, myricetin and isorhamnetin glycosides have been identified (Anton, 1999; Calamita et al., 1983; Gaspodinava et al., 1966). The flavonoids are used in the control of this drug: identification and dosage.

9Till now, the presence of acylated flavonoids has never been detected in this plant.

10In this work, we report for the first time, the isolation of kaempferol- 3-0-(6"-0-malonyl)-fi-D-glucopyranoside (KGM), in the leaves of black-currrant.

11An HPLC method for the qualitative and quantitative determination of isoquercitrin, astragalin and KGM in ribes leaves is described.

Material and methods

Plant material

12Ribes nigrum leaves were provided by the laboratory "La Tisane Provençale" (Aubagne, France).

Isolation and structure determination of KGM

13Powdered dried leaves (10 g) were extracted with 500 ml of 60 % aqueous methanol.

14The residual aqueous layer was successively extracted with methy- lethylketone.

15The methylethylketone extract (1.5 g) was subjected to polyamide column chromatography (50 g, polycarpolactam, 0.07 mm, Macherey-Nagel) and eluted with methanol (250 ml), ethanol (250 ml), acetone-water (8:2,v/v, 2.5 l).

16The acetone-water extract furnished 49 mg of pure KGM.

17The ¹H and ¹³C-NMR spectra were recorded on a Bruker AMX400 spectrometer in CD₃OD solution, internal reference was TMS. FAB- Mass spectra were obtained from Nermag R-10-10H mass spectrometer in the negative ion mode.

HPLC analysis

18Apparatus: Waters HPLC system (Milford, MA, USA):

• Software: Millenium³², v. 3.01
• Two solvent delivery systems 510
• Photodiode array detector 996
• Autosampler WISP 717

19Analytical conditions

• Analytical column Novapak C-18 (Waters), 4 m, 150 x 3.9 mm + Guard Pak Novapak insert
• Mobile phase: solvent A: H2O/H3PO4 (100:0.3, v/v) solvent B: CH3CN/H2O/H3PO4 (80:20:0.3, v/v) The solvent gradient profile was: 0-30 min: 17-25 % B; 30-35 min: 25-30 % B; 35-45 min: 30 % B.
• flow rate: 0.7 ml-min'¹
• UV detection: 340 nm
• Run time: 46 min
• Injection volume: 20 μl of filtered standards and samples.

20Samples

• Standard solutions: 1.0-8.0 mg of astragalin, isoquercetrin and KGM were dissolved in 100 ml 60 % aqueous methanol.
• Extract solutions: 0.45 g-3.60 g of powdered leaves were extracted under reflux with 90 ml of 60 % aqueous methanol for 30 min. The solutions were filtered and completed to 100 ml with the same solvent.

Results

Identification of KGM

21From the methylethylketone extract, the kaempferol-3-0-(6"-O-malonyl)-ß-D-glucopyranoside was isolated by column chromatography on polyamide. This malonated flavonol glycoside was identified by comparison of its ¹³C and ¹H-NMR spectra with published data (Wald et al., 1989; Veit et al., 1990).

22The FAB-Mass spectra of this compound showed a molecular ion peak at m/z 533 [MH]- corresponding to a molecular formula of C₂₄H₂₂O₁₄.

23This flavonoid was obtained for the first time from Ribes nigrum L. (figures 1 and 2)

Chromatography

24Under the chromatographic conditions described, very satisfactory results were obtained within 46 min for the identification of rutin, isoquercitrin, astragalin and KGM (figure 1).

Validation

25The validation data are presented in Table 1.

26The linearity of the calibration curve was achieved between 0.01-0.08 mg-ml^-1 for standards and between 0.45-3.0 g-100 ml^-1 for black currant leaves.

27Calibration curve were plotted by correlating peak areas against the corresponding concentrations. The assay was linear in the concentration range studied (r > 0.999).

28The precision of the method was tested by both intra-day (n=6) and inter-day (3 days, n=6) reproducibilities from the aqueous methanolic extract of Ribes nigrum leaves, at a concentration of 1.80.100 ml^-1 (Table 1).

29The CV values were less than 2.25 % for intra-day assays and 1.92 % for inter-day assays.

30The accuracy values showed very satisfactory results for precision: % of recovery ≈ 101 % (CV<2 %).

Dosage

31Three samples of Ribes nigrum leaves from different suppliers have been tested.

32Chromatic profiles and content of isoquercitrin, astragalin and KGM were determined and compared (Table 2). The chromatographic profiles obtained from the samples were similar.

33The comparison of the concentration of the quantified flavonoids showed that the content in kaempferol-3-glucomalonyl was eight times higher than the content in isoquercitrin and astragalin.

Conclusion

34The Ribes nigrum leaves are currently used in phytotherapy for its anti-inflammatory properties. Flavonoids are chosen for the drug identification and dosage.

35Phytochemical interest: we report, for the first time, the isolation and the identification of a malonated flavonol glycoside: kaempferol-3-0-(6"-0-malonyl)-(ß-D-glucopyranoside in the Ribes nigrum leaves.

36Analytical interest: for the first time an HPLC method for the dosage of these flavonoids in black currant is reported. This method allowed a complete separation and a simultaneous determination of isoquercitrin, astragalin and KGM. This highly selective HPLC method can be proposed for the qualitative and quantitative control of the drug, plant extracts or phytomedicines prepared from Ribes nigrum leaves.

Table 1. Validation data