862
A. Bertrand et al. / Carbohydrate Research 341 (2006) 855–863
0
0
described above for luteolin solubility in different hydro-
organic solvents).
7.56 (d, 1H, J6 ,5 = 8.3 Hz, H-60), 6.97 (d, 1H, H-50),
6.60 (s, 1H, H-3), 6.50 (s, 1H, H-8), 6.21 (s, 1H, H-6)
and 5.50 (d, 1H, J1 ,2 = 3.7 Hz, H-100).
00 00
3.2.4. Glucansucrase activity. GS activity was deter-
mined by measuring the initial rate of fructose produc-
tion by using the 3,5-dinitrosalicylic acid assay.37,38
Thus, one unit of glucansucrase activity was defined as
the amount of enzyme that caused the consumption of
1 lmol of sucrose per minute at 30 ꢁC and, as a conse-
quence, the release of 1 lmol of fructose per minute.
Reactions were carried out at 30 ꢁC in sodium acetate
buffer (20 mM, pH 5.2), sucrose 100 g Lꢀ1 and various
solvent concentrations.
3.2.8. Structural characterisation of quercetin gluco-
sides. Quercetin-40-O-a-D-di-glucopyranoside
(P8):
LC/MS: Anal. Calcd for C27H30O17: 627.2 [MH]+.
Found: 627.3; LC/1H NMR (Me2SO-d6): d 7.63 (s,
1H, H-20), 7.61 (s, 1H, H-60), 7.29 (s, 1H, H-50), 6.46
(s, 1H, H-8), 6.21 (s, 1H, H-6), 5.53 (s, 1H, Glc-1, H-
100) and 5.21 (s, 1H, Glc-2, H-100).
Quercetin-40-O-a-D-glucopyranoside (P9): LC/MS:
Anal. Calcd for C21H20O12: 465.2 [MH]+. Found:
465.3; LC/1H NMR (Me2SO-d6): d 7.65 (s, 1H, H-20),
7.63 (s, 1H, H-60), 7.25 (s, 1H, H-50), 6.45 (s, 1H, H-
8), 6.20 (s, 1H, H-6) and 5.50 (s, 1H, H-100).
Quercetin-30-O-a-D-glucopyranoside (P10): LC/MS:
Anal. Calcd for C21H20O12: 465.2 [MH]+. Found:
465.3; LC/1H NMR (Me2SO-d6): d 8.00 (s, 1H, H-20),
7.65 (s, 1H, H-60), 6.97 (s, 1H, H-50), 6.47 (s, 1H, H-
8), 6.20 (s, 1H, H-6) and 5.45 (s, 1H, H-100).
3.2.5. Enzymatic transglucosylation. Transglucosyl-
ation reaction was carried out at 30 ꢁC in acetate buffer
(20 mM, pH 5.2) containing different concentrations of
water-miscible organic solvents, sucrose (120 mM), flav-
onic acceptor (9 mM) and glucansucrase (3 U mLꢀ1). At
fixed times an aliquot of the reaction mixture was heated
at 95 ꢁC for 5 min to stop the reaction. Then, the sam-
ples were centrifuged at 10,000 rpm for 10 min, and an
aliquot of the supernatant was diluted to the adequate
concentration with Me2SO or a mixture of water/aceto-
nitrile (20:80, v/v). HPLC analysis using a C18 column
or a NH2 column, respectively, allowed to determine
the amounts of substrates and products.
3.2.9. Structural characterisation of myricetin gluco-
sides. Myricetin-30-O-a-D-glucopyranoside (P11): LC/
MS: Anal. Calcd for C21H20O13: 481.2 [MH]+. Found:
481.3; LC/1H NMR (Me2SO-d6): d 7.58 (s, 1H, H-20),
7.37 (s, 1H, H-60), 6.45 (s, 1H, H-8), 6.20 (s, 1H, H-6)
and 5.42 (s, 1H, H-100).
3.2.6. Preparation of luteolin glucosides. A reaction
mixture (388 mL) containing luteolin (1 g, 3.5 mmol),
sucrose (16.1 g, 47 mmol), MEE (30%, v/v), dextransu-
crase B-512F (3 U mLꢀ1) and sodium acetate buffer
(20 mM, pH 5.2) was incubated at 30 ꢁC until total
sucrose consumption. Then, the reaction was stopped
after 7 h of reaction by heating the mixture at 95 ꢁC
for 5 min. The reaction mixture was extracted with 100
Myricetin-40-O-a-D-glucopyranoside (P12): LC/MS:
Anal. Calcd for C21H20O13: 481.2 [MH]+. Found:
481.3; LC/1H NMR (Me2SO-d6): d 7.20 (s, 1H, H-20),
7.20 (s, 1H, H-60), 6.45 (s, 1H, H-8), 6.20 (s, 1H, H-6)
and 5.20 (s, 1H, H-100).
Acknowledgements
mL (10 10 ml) of ethyl acetate and dried on MgSO .
*
4
The organic phase layer was evaporated until a yellow
powder was obtained, which was dried in a desiccator.
The mixture was fractionated by preparative chroma-
tography with a C18 column (PROCHROM LC-50
Phase SYMMETRY C18, 7.0 lm) using a gradient of
water/trifluoroacetic acid (99.90:0.10, v/v) and acetoni-
trile/trifluoroacetic acid (99.90:0.10, v/v) at a flow rate
of 60 mL minꢀ1: 0 min (85/15), 45 min (60/40). The frac-
tions containing the products were collected and lyophi-
lised. The structure of pure luteolin mono-glucoside P1
This work was supported by SERVIER Laboratories
´
(Orleans, France) (contract CRITT Bio-Industries
´ ´
Midi-Pyrenees, Toulouse, France) and by the Program
Proteomic and Engineering of Protein of the CNRS.
´ ˆ
We are grateful to Jerome Binet, Pierre Escalier and
Sandrine Moreau for their technical assistance.
References
1
was confirmed by H and 13C NMR analyses.
1. Harborne, J. B.; Williams, C. A. Phytochemistry 2000, 55,
481–504.
2. Havsteen, B. H. Pharmacol. Ther. 2002, 96, 67–202.
3. Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Free Radical
Biol. Med. 1996, 20, 933–956.
4. Brown, J. E.; Rice-Evans, C. A. Free Radic. Res. Sep.
1998, 29, 247–255.
5. Di Carlo, G.; Mascolo, N.; Izzo, A. A.; Capasso, F. Life
Sci. 1999, 65, 337–353.
6. Kimata, M.; Inagaki, N.; Nagai, H. Planta Med. 2000, 66,
25–29.
3.2.7. Structural characterisation of luteolin gluco-
sides. Luteolin-40-O-a-D-glucopyranoside (P1): LC/
MS: Anal. Calcd for C21H20O11: 449.2 [MH]+. Found:
449.3; LC/NMR: 1H and 13C NMR data given in Table 3.
Luteolin-30-O-a-D-glucopyranoside (P2): LC/MS:
Anal. Calcd for C21H20O11: 449.2 [MH]+. Found:
449.3; LC/1H NMR (Me2SO-d6): d 7.74 (s, 1H, H-20),