´
Marquez Hernandez et al.
´
4726 J. Agric. Food Chem., Vol. 58, No. 8, 2010
samples 21 and 24 (Madruga), 34 (San Antonio), and 66 (Nueva Paz);
(mass range 40-800 amu) at 2.9 scans/s, with a multiplier voltage of 2000
V and an ionization energy of 70 eV.
Holguın, sample 60 (Baguano); Las Tunas, samples 2 (Puerto Padre) and
´ ´
€
11 (Jobabo); Matanzas, samples 44 and 46 (Jaguey Grande), 47 and
Identification. The structures identified were proposed on the basis of
their fragmentation patterns. The individual peaks were also compared
with the PMW-TOX, NIST 98, and Wiley 275 computer mass libraries. All
triterpenoids 1-14 were cochromatographed to confirm GC retention
times and mass spectra. Some minor components in the chromatograms
remained unidentified because of the lack of authentic samples and library
spectra of the corresponding compounds.
Comparative Study with Samples Previously Classified as
YCP (16). At the end of this study, the same analytical procedure was
applied to the 19 samples classified as YCP previously (16) and collected in
November 2004. These samples were compared with those used herein
(collected between October and December 2008 in the same zone and
having the same number). 1D NMR spectra and HPLC-PAD chromato-
grams showed no significant differences between the two different groups,
and also, the GC-MS profiles were similar enough both from qualitative
and quantitative points of view.
ꢀ
49 (Limonar), and 48 and 50 (Union de Reyes); Pinar del Rio, samples 39
ꢀ
(Candelaria), 41 (Bahia Honda), and 42 (Consolacion del Sur); Santiago
de Cuba, sample 8 (Guama); and Villa Clara, sample 28 (Remedios). Both
propolis samples and the dried methanol extracts were stored at 5 °C in the
dark until required for analysis.
NMR Analysis. A portion of each extract of YCP (about 100 mg) was
dissolved employing CDCl3 as the solvent (ca. 0.5 mL). A Bruker DRX-
600 spectrometer operating at 599.19 MHz for 1H and 150.858 for
13C, using the UXNMR software package, was used for the NMR experi-
ments.
HPLC-PDA Analysis. HPLC analysis of YCP extracts (5 mg/mL)
was performed on an Agilent 1100 series system consisting of a G-1312
binary pump, a G-1328A Rheodyne injector (20 μL loop), a G-1322A
degasser, and a G-1315A photodiode array detector(PDA), equipped with
a μ-Bondapack C-18 column (250 ꢀ 4.6 mm i.d., particle size 10 μm).
Analysis of YCP extracts was carried out as reported previously (16).
Triterpenoid Acetylation. Lanosterol acetate, β-amyrin acetate,
germanicol acetate, R-amyrin acetate, and lupeol acetate were prepared
by our group employing the standards mentioned above. Each triterpe-
noid (10 mg) was mixed with Ac2O (0.5 mL) and dry C5H5N (0.5 mL) and
heated in a water bath for 5 h. Acetyl derivatives were purified by
preparative TLC (mobile phase Hex/EtOAc 7:3, v/v), and their purity
was determined by differential scanning calorimetry (DSC) (Perkin-Elmer
Norwalk, CT). All acetyl derivatives were at least 98% pure.
Preparation of Extracts for Quantitative Analysis. Propolis sam-
ples (5 g) were extracted with methanol (25 mL ꢀ 5) for 3 h with occasional
stirring. The extracts were filtered and evaporated to dryness under
reduced pressure (40 °C). About 200 mg of the residues was dissolved in
MeOH (4 mL) in order to obtain a final concentration of 50 mg mL-1
(stock solutions of propolis). The quantification process was developed by
an internal standard method. An aliquot of stock solution of propolis
(100 μL) was mixed with 20 μL of a solution of internal standard
(cholesterol, 1 mg mL-1) and evaporated to dryness by a gentle stream
of nitrogen. One hundred microliters of MSTFA was added to the
propolis/cholesterol mixtures in a sealed glass tube for 15 min at 60 °C.
One microliter of the mixtures was directly analyzed by GC-MS. Initial
qualitative analyses were realized in the same manner. Stock solutions of
triterpenoids (1-14) and cholesterol (internal standard, IS) were prepared
by dissolving appropriate amounts of the compounds in MeOH to achieve
concentrations of 1 mg mL-1. Appropriate dilutions of the stock solution
of triterpenoids were made with MeOH to prepare three calibration
solutions containing 100, 200, and 300 μg mL-1of each triterpenoid,
respectively. Aliquots of IS solution were added in order to obtain the
same final concentrations for cholesterol (50 μg mL-1). Successively,
100 μL of calibration solutions, containing IS, were derivatized with
MSTFA, and 1 μL was set as injection volume in all cases. All areas were
measured and referenced to the area of IS by the data system. Triterpe-
noids were quantified by GC/EI-MS-SIM using base peaks as quantifiers:
lanosterol (1, m/z 393), β-amyrone (2, m/z 218), β-amyrin (3, m/z 218),
germanicol (4, m/z 204), R-amyrone (5, m/z 218), R-amyrin (6, m/z 218),
lupeol (7, m/z 189), cycloartenol (8, m/z 393), lanosterol acetate (9, m/z
393), β-amyrin acetate (10, m/z 218), germanicol acetate (11, m/z 204),
24-methylene-9,19-ciclolanostan-3β-ol (12, m/z 422), R-amyrin acetate
(13, m/z 218), and lupeol acetate (14, m/z 189). The results represent the
mean ( SD of three determinations.
RESULTS AND DISCUSSION
The analyzed crude extracts were obtained by maceration with
methanol since the plant exudates’ fraction of propolis, which
usually contains the bioactive components, is separated from the
wax by extraction with this solvent.
1D NMR Analysis. All 19 yellow-type Cuban propolis (YCP)
samples showed yellowish extracts and similar spectroscopic
characteristics, related to the presence of aliphatic compounds
(Figure 1). 1H NMR spectra showed the main signals almost
exclusively in the aliphatic region (δ 0.75-2.1) and downfield
signals of variable intensities between 3.0 and 6.7 ppm were
present in all YCP samples. Moreover, in many YCP samples,
two strong signals at ca. 80 and 176 ppm, in the 13C NMR spectra,
revealed the presence of carbon atoms bearing oxygen; these
chemical shifts can be associated with the C-3 and the acetyl
1
carbonyl group present in triterpenoids. All H NMR spectra
exhibited strong shielded signals as singlets between 0.79 and
0.95 ppm due to the presence of methyl groups attached to sp3
carbons. Both 1H NMR and 13C NMR data allowed us to group
them into two principal groups named YCP type A (samples 2,
11, 20, 41, 42, 46, 48, 50, and 66) and YCP type B (samples 8, 21,
24, 28, 34, 39, 44, 47, 49, and 60).
YCP Type A. In the 1H NMR spectra, a strong aliphatic
region was appreciated between 0.75 and 1.64 ppm. This aliphatic
part was divided into two signal groups (δ 0.75-0.96 and
1.57-1.64) by a strong signal at 1.25 ppm. Downfield signals
between 3.22 and 6.18 ppm exhibited variable intensities. Two
downfield signals as broad singlets (δ 5.32 and 6.18) were
observed in all of the 1H NMR spectra of this group only, while
signals with less intensity were observable in the aromatic region
over 7.0 ppm. These data indicated the presence of aliphatic
compounds as major constituents and suggested the presence of
aromatic compounds as minor components.
In the 13C NMR spectra, a very complex zone attributed to sp3
carbon atoms was observed between 13.9 and 55.1 ppm; however,
some signals belonging to sp2 carbon atoms (between 100 and
150 ppm) were observed, and these could be assigned to sp2
carbon atoms of alkenes substituted mainly by alkyl groups. The
simultaneous comparison of the 1H and 13C NMR spectral data
suggested the presence of aliphatic compounds (terpenoids and
sterols) as the main constituents of this yellow Cuban propolis
variety.
Analytical Conditions and GC-MS Analysis. A Hewlett-Packard
(HP) Model GC 6890 Series gas chromatograph coupled with an HP 5973
series mass-selective detector quadrupole mass spectrometer was em-
ployed for all analyses. Samples were separated on a 25 m ꢀ 0.20 mm
i.d., 0.33 μm film thickness, HP-Ultra 2 capillary column (Agilent
Technology, Palo Alto, CA). The column temperature was initially held
at 80 °C for 1 min, and then the temperature was raised to 310 °C at a rate
of 5 °C min-1, followed by an isothermal period of 20 min. The total run
time was 67 min. Ultrahigh-purity helium with an inline oxygen trap was
used as carrier gas at a flow rate of 0.8 mL/min. The injector was heated to
280 °C and was on split mode with a split ratio of 1:10, and the injection
volume was 1.0 μL. MS source and MS quad temperatures were 230 and
150 °C, respectively. The MSD was acquiring data in the full scan mode
1
YCP Type B. Also in the H NMR spectra of samples of
yellow propolis type B, a strong aliphatic region was appreciated
with proton signals occurred between 0.8 and 2.1 ppm. The NMR
spectra of YCP type B were very similar to that of type A except
for the presence, in all 10 samples, of singlets at δH ca. 2.0,