1354 Journal of Natural Products, 2009, Vol. 72, No. 7
Notes
were acquired on a PerkinElmer Spectrum One FT-IR instrument. LC-
MS/UV chromatograms were obtained using a Fractionlynx system
from Waters (Milford, MA) working in analytical mode and equipped
with a ZMD mass spectrometer and a 486 UV detector. A monolithic
column ONYX-C18 (100 × 3 mm) from Phenomenex (Torrance, CA)
was used for the run. Purification of compounds 1 and 2 from
Castagnaro bergamot samples was performed with the same system,
working in the semipreparative mode (21 mL/min) and using an AXIA
synergy fusion column (21.2 × 100 mm) from Phenomenex. The UV
on a Bruker Avance 500 MHz (1H: 500.13 MHz, 13C: 125.77 MHz)
instrument (Rheinstetten, Germany), dissolving purified samples in
CD3OD, except where otherwise stated. HRMS were acquired on a
Q-star pulsar-i (MDS Sciex Applied Biosystems, Toronto, Canada),
equipped with an ion-spray source on samples collected from the
analytical chromatographic runs. The MS/MS data were obtained at
15 and -40 eV collision energy voltages in the positive and negative
mode, respectively, using N2 as collision gas.
Basic hydrolyses were performed by dissolving 1 mg of purified
samples in 1 mL of saturated Na2CO3 water solution. The reaction lasted
20 h, and the formation of products was followed by HPLC UV/MS
until the disappearance of the reactant. NMR data (DMSO-d6) of the
3-hydroxymethylglutaric acid obtained after hydrolysis: CH3-3, δH 1.22
(s), δC 28.0 (q), CH2-2,4, δH 2.46 (m), δC 46.5 (q), 1,5-COOH, δH
8.21 (s), δC 173.0 (s), OH-3, δH not detected, δC 69.0 (s).
Enzymatic hydrolyses were performed as follows: 1 mg of purified
product and 1 mg of hesperidinase were dissolved in 1 mL of
McIlvaine’s buffer at pH 3.5. The reaction temperature was maintained
at 37 °C. The reaction lasted until the appearance of the aglycon moiety
as the major product. The product formation was followed by HPLC
UV/MS until the aglycon was evident.
powdered compounds. The semipreparative steps were repeated until
an appropriate amount was recovered for each experiment.
Hesperetin 7-[2′′-r-rhamnosyl-6′′-(3′′′′-hydroxy-3′′′-methylglu-
taryl)-ꢀ-glucoside] (brutieridin) (1): pale yellow powder; UV (MeOH)
λ
max (log ꢀ) nm 285 (3.80), 324 (3.12); IR (KBr) νmax 3304, 1720, 1641,
1574, 1515, 1440, 1295, 1274, 1202, 1129, 1085 cm-1 1H NMR
;
(CD3OD, 500 MHz) see Table 1; 13C NMR (CD3OD, 125 MHz) see
Table 1; (+) HRESIMS m/z 755.2387 [M + H]+, calcd for C34H43O19,
755.2404; (-) HRESIMS/MS m/z 691.2277 [(M - CO2 - H2O) -
H]-, calcd for C33H39O16, 691.2243; m/z 651.1890 [(M - C4H6O3) -
H]-, calcd for C30H35O16, 651.1930; m/z 609.1836 [(M - C6H8O4) -
H]-, calcd for C28H33O15, 609.1824; m/z 489.1357 [(M - C10H16O8)
- H]- or [0,2X0]-, calcd for C24H25O11, 489.1397; m/z 301.0715 [(M
- Rha - Glc - HMG) - H]-, calcd for C16H13O6, 301.0717; HPLC
(-) ESIMS tR 10.05 min, m/z 753 [M - H]-.
1
detector was set at 280 nm. H NMR spectra were recorded at 25 °C
Naringenin 7-[2′′-r-rhamnosyl-6′′-(3′′′′-hydroxy-3′′′-methylglu-
taryl)-ꢀ-glucoside] (melitidin) (2): pale yellow powder; UV (MeOH)
λmax (log ꢀ) 283 (3.80), 327 (3.23); IR (KBr) νmax 3348, 1717, 1642,
1520, 1177, 1087, 834 cm-1; 1H NMR (CD3OD, 500 MHz) see Table
1; 13C NMR (CD3OD, 125 MHz) see Table 1; (+) HRESIMS m/z
725.2290 [M + H]+, calcd for C33H41O18, 725.2287; (-) HRESIMS/
MS m/z 661.2114 [(M - CO2 - H2O) - H]-, calcd for C32H37O15,
661.2137; m/z 621.1864 [(M - C4H6O3) - H]-, calcd for C29H33O15,
621.1824; m/z 579.1764 [(M - C6H8O4) - H]-, calcd for C27H31O14,
579.1719; m/z 459.1311 [(M - C10H16O8) - H]- or [0,2X0]-, calcd for
C23H23O10, 459.1291; m/z 271.0601 [(M - Rha - Glc - HMG) -
H]-, calcd for C15H11O5, 271.0611; HPLC (-) ESIMS tR 9.85 min,
m/z 723 [M - H]-.
Acknowledgment. This work is part of the Italian National Project,
MIUR-FIRB RBIP06XMR_004.
Plant Material. Bergamot fruits (Citrus bergamia) of three different
cultivars (Castagnaro, Fantastico, and Femminello) were harvested in
different periods (October, November, and December 2007) by Union-
berg Association, located at Condofuri Marina (RC, Italy). Fruit samples
were classified by Furfari nursery as number 02004450801/814/06/
ZP.
Chemicals. HPLC grade solvents were purchased from Carlo Erba
(Milan, Italy). Standard flavonoids were purchased from Extrasynthese
(Genay, France). Other chemicals were purchased from Sigma-Aldrich
(St. Louis, MO).
Supporting Information Available: ESIMS/MS of compounds 1
and 2; 1H and 13C 1D and 2D NMR spectra of compounds 1 and 2; LC
MS chromatograms of basic and enzymatic hydrolysis of compounds
1 and 2; ESIMS/MS of product from enzymatic reaction on compound
1. This material is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
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Extraction and Isolation. The flavonoid extract of bergamot was
obtained by chopping the fruit in a ternary mixture of methanol, ethanol,
and chloroform, 65:30:5 (v/v/v); subsequently, it was filtered on a
Buchner funnel, concentrated, and passed through a C18 cartridge
(Supelclean LC-18, 60 mL, 10 g; Supelco, St. Louis, MO), previously
activated with MeOH and washed with water. The loaded material was
washed with 15 mL of water and then eluted with 2 × 15 mL of MeOH.
The eluate was evaporated under vacuum to dryness. For analytical
chromatography a solution of 1000 ppm was injected into the HPLC.
The run time was 30 min, the flow rate was 1.5 mL/min, and the
gradient was built using 0.1% HCOOH in H2O (solvent A) and CH3CN
(solvent B) as eluting phase. The solvent run was composed by the
following steps: linear gradient from 95% A to 5% A in 20 min; linear
gradient from 5% A to 95% A in 5 min; equilibration of the column
for 5 min. For semipreparative purposes, 500 mg of the dried residue
were dissolved in 1 mL of H2O-CH3OH (1:1) and submitted to HPLC
purification. The semipreparative run was performed at a 21 mL/min
flow rate, using 0.1% HCOOH in H2O (solvent A) and CH3CN (solvent
B) in two steps. The first step was performed to partially purify each
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mass signal of each product were mixed and the solvent was evaporated
under vacuum. Finally, the residual water was lyophilized to yield the
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