Hydroxyester disaccharides from fruits of cape gooseberry (Physalis peruviana)

Article abstract of DOI:10.1016/S0031-9422(01)00467-8

The 3-O-β-D-glucopyranosyl-(1→6)-β-D -glucopyranoside of ethyl 3-hydroxyoctanoate and the diastereomeric 3-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosides of (3R) and (3S)-butyl 3-hydroxybutanoate, respectively, were isolated by chromatographic methods

Full text of DOI:10.1016/S0031-9422(01)00467-8

Phytochemistry 59 (2002) 439–445
www.elsevier.com/locate/phytochem
Hydroxyester disaccharides from fruits of cape
gooseberry (Physalis peruviana)
a
a,
b,1
b
Humberto Mayorga , Carmenza Duque *, Holger Knapp , Peter Winterhalter
aDepartamento de Quı´mica, Universidad Nacional de Colombia, AA 14490, Bogot a´ , Colombia
b
Institut fu¨r Lebensmittelchemie, TU Braunschweig, Schleinitzstrasse 20, D-38106, Braunschweig, Germany
Received in revised form 6 November 2001
Abstract
The 3-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranoside of ethyl 3-hydroxyoctanoate and the diastereomeric 3-O-a-l-arabino-
pyranosyl-(1!6)-b-d-glucopyranosides of (3R) and (3S)-butyl 3-hydroxybutanoate, respectively, were isolated by chromatographic
methods from fruits of cape gooseberry (Physalis peruviana) harvested in Colombia. Their structures were identified by ESI–MS/
MS and NMR spectroscopy. The three glycoconjugates can be considered as immediate precursors of ethyl 3-hydroxyoctanoate
and butyl 3-hydroxybutanoate, which are important aroma volatiles found in the fruit. # 2002 Elsevier Science Ltd. All rights
reserved.
Keywords: Physalis peruviana; Solanaceae; Uchuva; Cape gooseberry; Aroma precursors; Glycosides; Hydroxyester glycosides
1
. Introduction
in Colombia. Most importantly, during this study a
considerable number of a homologous series of 3- and 5-
hydroxyesters could be identified (Mayorga et al., 2001).
Our results were in line with previous reports on the
aroma of cape gooseberry fruits harvested in Germany
(Berger et al., 1989). Thus, to expand our knowledge on
flavour formation of Physalis peruviana fruit, we decided
to look for hydroxyester glycoconjugates which could
liberate volatile hydroxyesters as a mechanism of aroma
generation. The present paper describes the isolation and
characterization of three new hydroxyester glycosides: 3-
O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranoside of
ethyl 3-hydroxyoctanoate 1; 3-O-a-l-arabinopyranosyl-
(1!6) - b - d - glucopyranoside of butyl (3R) - hydroxy-
butanoate 2; and 3-O-a-l-arabinopyranosyl-(1!6)-b-d-
glucopyranoside of butyl (3S)-hydroxybutanoate 3.
During the last decade a considerable part of flavour
research has been directed not only to the free volatiles
as important contributors to the overall aroma of the
fruits but also to the examination of glycoconjugates. The
latter are known to act as volatile generators by enzymatic
or chemical pathways during fruit homogenization, e.g. in
the course of consumption, plant maturation, industrial
pretreatment or processing (Crouzet and Chassagne,
1
999). In the course of our studies on the aroma of
Colombian fruits, we have isolated several glycoconju-
gates and established their role as aroma progenitors in
fruits such as lulo (Solanum quitoense) (Wintoch et al.,
1
(
993; Osorio et al., 1999), pin
Parada and Duque, 1998), and melo
odorifera) (Parada et al., 2000).
˜
uela (Bromelia plumieri)
´
n de olor (Sicana
Recently we have examined the bound aroma com-
pounds of cape gooseberry (Physalis peruviana) cultivated
2. Results and discussion
2.1. Characterization of glycoside 1
*
Corresponding author. Tel.: +57-1-3165000x14472; fax: +57-1-
165220.
E-mail address: cduqueb@ciencias.ciencias.unal.edu.co (C. Duque).
Compound 1 was characterized in its acetylated form
a by ESI–MS/MS, as well as by H and 13C NMR
spectroscopy. The molecular mass of 1a was determined
3
1
1
1
Present address: Lebkuchenfabrik IfriSchuhmann, Kreuzburger
Str. 12, 90471 Nurnberg, Germany.
¨
to be 806 amu as the ESI mass spectrum revealed the
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00467-8

440
H. Mayorga et al. / Phytochemistry 59 (2002) 439–445
Fig. 1. Hydroxyester glycosides isolated from cape gooseberry (Physalis peruviana).

H. Mayorga et al. / Phytochemistry 59 (2002) 439–445
441
adduct ions m/z 845 [M+K]+ and 829 [M+Na]+.
Furthermore, the ion spectrum of m/z 829 showed the
fragment m/z 769 [M+Na–AcOH]+ confirming the above
mentioned molecular weight. The presence of the frag-
ment ion m/z 528 in the ESI–MS spectrum of 1a evi-
denced a peracetylated disaccharide (dihexose) as a
sugar moiety. The molecular mass of the aglycon moiety
was calculated to be 188 amu (difference between the
molecular weight of the glycoside and the weight of the
peracetylated disaccharide residue).
COSY with an oxymethylene group (ꢀH 4.13, ꢀC 60.5)
indicating a CH –CH –O– unit, which in turn is con-
nected to a carbonyl group (ꢀ 171.1) according to the
C
3
2
HMBC data (correlation between the signal at ꢀ 4.13
H
1
1
and ꢀC 171.1). On the other hand, the H– H COSY,
DEPT and HMBC experiments allowed us to assign the
partial structure CH –CH –CH –CH –CH –CH–O–
3
2
2
2
2
whose oxymethine proton (ꢀH 4.08–4.20, ꢀC 76.8) is
connected to the carbonyl function at ꢀC 171.1 via a
methylene group (ꢀH 2.41and 2.50, ꢀ 40.0), thus indi-
C
From H (Table 1) as well as 13C NMR spectral data
1
cating the structure of ethyl 3-hydroxyoctanoate for the
aglycon moiety. Finally, the HMBC correlation between
the anomeric proton (ꢀ 4.63, ꢀ 100.3) and the signal at
(
Table 2) the presence of a disaccharide moiety in com-
pound 1a was confirmed. The 1H NMR spectrum
exhibited two doublets at ꢀ 4.63 (J=8.0 Hz) (ꢀ 100.3)
and at ꢀH 4.64 (J=8.0 Hz) (ꢀ 100.8) for two anomeric
H
C
ꢀC 76.8 lead us to link the sugar unit to C-3 of the aglycon
moiety. Thus, compound 1a has been established to be
the heptaacetate of 3-O-b-d-glucopyranosyl-(1!6)-b-d-
glucopyranoside of ethyl 3-hydroxyoctanoate (Fig. 1).
The stereochemistry at C-3 remains unsolved.
Deacetylation of 1a and subsequent treatment with
Rohapect D5L (nonselective glycosidase) led to the lib-
eration of ethyl 3-hydroxyoctanoate 6. Ester 6 is an
important free aroma volatile in the fresh fruit exhibiting
fruity, green and sweet odor notes. The identity of 6 was
confirmed by HRGC and HRGC–MS analyses.
H
C
C
protons indicating the presence of two b-glycosidic lin-
1
1
kages. The H– H COSY evidenced two sequences of
hydrogen connectivities corresponding to the axial pro-
tons of two b-glucopyranoside units linked to each
1
3
other. Furthermore, the C NMR spectral data were in
good agreement with data published for the per-O-
acetylated b-d-gentiobioside residue (Straubinger et al.,
1
a methyl group (ꢀ 1.27, ꢀ 14.0) coupling in the H– H
998). In addition, the NMR spectroscopy data showed
1
1
H
C
Table 1
1H NMR (300 MHz, CDCl
3
) spectral data for compounds 1a–3a
Ha
1a
2a
2.42, 1H, dd (16.0/5.5)
3a
2
2
3
4
5
8
1
2
3
4
1
2
3
4
5
6
6
1
2
3
4
5
5
5
6
6
a
2.41, 1H, dd (16.0/5.0)
2.50, 1H, dd (16.0/8.0)
4.08–4.20, 1Hb
2.36, 1H, dd (16.0/4.5)
2.54, 1H, dd (16.0/8.5)
4.14–4.26, 1H, m
b
2.66, 1H, dd (16.0/7.0)
4.25–4.37, 1H, m
1.20, 3H, d (6.5)
1.50–1.63, 2H, m
1.24–1.36, 6H, m
0.91, 3H, t (7.0)
1.30, 3H, d (6.5)
/6/7
0
0
0
0
4.13, 2H, q (7.0)
1.27, 3H, t (7.0)
4.08, 2H, t (6.5)
1.62, 2H, q (7.0)
3.99–4.14, 2H, m
1.61, 2H, q (7.0)
1.39, 2H, s (7.5)
0.94, 3H, t (7.5)
4.56, 1H, d (8.0)
1.38, 2H, s (7.5)
0.94, 3H, t (7.5)
4.62, 1H, d (8.0)
0
0
0
0
0
0
0
0
0
0
4.63, 1H, d (8.0)
4.88, 1H, dd (9.5/8.0)
5.15, 1H, dd (9.5/9.5)
4.89, 1H, dd (9.5/9.5)
3.62–3.71,1H, mc
4.88, 1H, dd (9.5/8.0)
5.18, 1H, dd (9.5/9.5)
4.92, 1H, dd (9.5/9.5)
3.63, 1H, br dd (9.5/7.0)
3.70, 1H, dd (11.0/7.0)
3.78, 1H, br d (11.0)
4.79, 1H, d (6.5)
4.88, 1H, dd (9.5/8.0)
5.17, 1H, dd (9.5/9.5)
4.92, 1H, dd (9.5/9.5)
3.68, 1H, ddd (9.5/7.0/2.0)
3.62, 1H, dd (11.0/7.0)
3.84, 1H, dd (11.0/2.0)
4.52, 1H, d (6.5)
0
0
0
3.62–3.71,1H, mc
a
0
b
3.82, 1H, br d (9.5)
4.64, 1H, d (8.0)
0
0
0
0
0
00
00
00
00
00
4.98, 1H, dd (9.5/8.0)
5.18, 1H, dd (9.5/9.5)
5.06, 1H, dd (9.5/9.5)
3.62–3.71, 1H, mc
5.14, 1H, dd (9.0/6.5)
5.21, 1H, dd (9.0/3.5)
5.24, 1H, ddd (3.5/3.5/2.0)
5.14, 1H, dd (9.0/6.5)
5.03, 1H, dd (9.0/3.5)
5.24, 1H, ddd (3.5/3.5/2.0)
0
00
0
00
0
a
3.80, 1H, br d (13.0)
4.00, 1H, dd (13.0/3.5)
3.60, 1H, dd (13.0/2.0)
4.02, 1H, dd (13.0/3.5)
00
b
0
a
4.12, 1H, dd (12.5/2.5)
4.27, 1H, dd (12.5/5.0)
1.98, 1.99, 2.08, 2.10, 12 H,
00
b
CH
3
CO
1.98, 2.01, 2.08, 2.13, 12 H,
4Âs, 2.02, 6H, s (6X)
1.97, 2.04, 2.07, 2.12, 12 H,
4Âs, 2.02, 6H, s (6X)
4Âs, 2.02, 9H, s (7X)
a
b
c
1
1
Assignments were based on H– H COSY, HMQC, HMBC and NOESY.
Obscured.
Signal overlapped.

442
H. Mayorga et al. / Phytochemistry 59 (2002) 439–445
Table 2
13C NMR (75 MHz, CDCl
3
) spectral data for compounds 1a–3a
Ca
1a
2a
3a
13C
DEPT
C
13C
13C
DEPT
C
1
2
3
4
5
6
7
8
171.1
40.0
76.8
35.4
24.6
31.8
22.5
14.2
60.5
14.0
171.0
42.0
71.9
19.6
170.8
42.1CH
74.2
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
2
CH
2
2
2
2
3
2
3
21.9
CH
3
0
0
0
0
1
64.4
30.6
64.4
30.7
19.2
13.6
101.1
71.5
73.0
69.2
73.3
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
2
2
3
2
3
19.1
13.7
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
1
2
3
4
5
6
100.3
71.5
73.0
69.3
73.5
68.2
100.8
71.2
73.0
68.5
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
98.8
71.6
73.0
69.0
74.2
2
67.167.5
100.4
69.6
2
100.4
69.1
69.9
67.4
CH
CH
CH
CH
CH
70.0
68.0
72.1CH
62.0
63.0
62.7
2
3
CH
CH
2
3
CH
3
CO
CO
20.5, 1C; 20.6, 5C;
2
20.6, 3C; 20.7, 1C;
20.8, 1C; 20.9, 1C
20.5, 1C; 20.6, 3C;
20.7, 1C; 20.9, 1C
(6X)
CH
C
0.7, 1C (7X)
(6X)
CH
3
169.2, 169.3, 169.4,
C
169.2, 169.3, 169.5,
170.1, 170.2, 170.3
(6X)
169.2, 169.3, 169.5,
170.0, 170.1, 170.2
(6X)
1
1
69.5, 170.1, 170.2,
70.6 (7X)
a
Assignments were based on DEPT, HMQC and HMBC experiments.
n
Compound 1 is, to our best knowledge, reported here
for the first time. In contrast, the aglycon is a known
bound aroma compound of cape gooseberry (Berger et
with HR–FABMS measurements. In ESI–MS experi-
ments, product ion spectra of m/z 729, 669 and 609 (see
Experimental), respectively, confirmed the molecular
weight. These data also indicated the presence of a
terminal triacetylated pentosyl unit as part of the struc-
ture of compound 2a due to the presence of a char-
˜
al., 1989; Mayorga et al., 2001), and pinuela (Bromelia
plumieri) (Parada and Duque, 1998). The aglycon has
also been reported as free volatile in fruits such as
mountain papaya (Carica pubescens) (Idstein et al.,
acteristic fragment at m/z 239 [pentose(Ac) +Na-
3
1
985), pineapple (Ananas comosus) (Umano et al., 1992),
AcOH]+ in the product ion spectrum of m/z 609. Fur-
ther, the latter spectrum reveals ions at m/z 507
caja fruit (Spondias lutea) (Allegrone and Barbeni, 1992),
bachang (Mangifera foetida), kuini (Mangifera odorata)
+
[M+Na–3AcOH–CH =C=O] , 407 [pentose(Ac)
2
3
+
(
Wong and Ong, 1993), apple (Malus silvestris) (Beuerle
hexose(Ac) +Na–2AcOH–CH =C=O–H O] and 347
3 2 2
+
et al., 1996), pear (Pyrus communis) (Beuerle and
Schwab, 1997), and yellow passion fruit (Passiflora edulis
flavicarpa) (Werkhoff et al., 1998).
[407–AcOH] ; which is in accordance with the presence
of a disaccharide unit as sugar moiety. Additionally, the
+
fragment ion at m/z 142 [aglycon–H O] suggested a
2
weight of 160 amu for the aglycone moiety and the
+
fragment ion at m/z 551[M+Na–2AcOH–C H –H]
2
.2. Structure elucidation of glycosides 2 and 3
4
9
provided a hint for the presence of a C H alkyl chain in
9
4
Glycosides 2 and 3 were similarly characterized in
the aglycone structure.
their peracetylated forms 2a and 3a. A molecular for-
mula of C H O for 2a could be deduced from HR–
FABMS. The ESI–MS spectrum of compound 2a
The presence of two doublets at ꢀH 4.56 (J=8.0 Hz)
(ꢀC 98.8) and at ꢀH 4.79 (J=6.5 Hz) (ꢀC 100.4) in the
NMR spectra indicated a disaccharide as sugar moiety
containing one b- and one a-glycosidic linkage. In both
the H and C NMR spectra the characteristic signals
for a terminal a-l-arabinopyranosyl moiety linked to a
3
1
46 18
+
showed strong adduct ions at m/z 766 [M+AcOH] ,
7
45 [M+K]+ and 729 [M+Na]+ from which a mole-
cular mass of 706 amu could be calculated in agreement
1
13

H. Mayorga et al. / Phytochemistry 59 (2002) 439–445
443
b-d-glucopyranosyl unit was observed (Straubinger et
al., 1999). A correlation between the protons at C-6 of
the hexose and the C-1in the pentose moiety revealed a
Enzymatic hydrolysis (Rohapect D5L) of the glyco-
sides 2 and 3 liberated butyl 3-hydroxybutanoate 7
which was unambiguously identified by HRGC and
HRGC–MS. By using HRGC–O, the aroma of 7 was
determined as fruity, estery, sour and gooseberry-like.
To the best of our knowledge this is the first time that
these diastereoisomeric glycosides have been found in
nature. The aglycones, however, are known constituents
of several fruits e.g. hog plum (Spondias mombins L.)
(Adedeji et al., 1991), mango (Mangifera indiga L.)
(Adedeji et al., 1992), mountain papaya (Krajewski et al.,
1997), caja fruit (Allegrone and Barbeni, 1992), kuini
(Wong and Ong, 1993), and banbangan (Mangifera
panjang Kostermans) (Wong and Siew, 1994).
1
!6 linkage. In addition, the NMR spectral data
showed one carbonyl (ꢀC 171.0), one oxymethine (ꢀH
.25-4.37, ꢀ 71.9) and one low-field shifted methylene
4
C
1
1
group (ꢀH 2.42 and 2.66, ꢀ 42.0). In the H– H COSY
C
spectrum, the methylene group displayed a coupling to
the oxymethine proton indicating a ÀCHO–CH –COÀ
2
unit, which in turn is connected to a methyl group (ꢀH
1
.20, ꢀC 19.6). A methylene group at ꢀH 4.08 (ꢀC 64.4)
correlating in the HMBC spectrum to the above men-
tioned carbonyl group indicates an ester structure in the
aglycon. Since there are three additional carbon signals
left, butanol can be assumed as the alcohol moiety of the
ester. This assumption is in accordance with the observed
loss of a C H -chain in the mass spectrum and with the
Finally, it has to be stressed that glycoconjugates 1–3
could be considered as immediate precursors of ethyl 3-
hydroxyoctanoate and butyl 3-hydroxybutanoates which
are important aroma contributors in cape gooseberry
fruits.
4
9
observed molecular mass of 706 amu. Thus, the structure
of compound 2a could be assigned as the peracetate of 3-
O-a-l-arabinopyranosyl-(1!6)-b-d-glucopyranoside of
butyl 3-hydroxybutanoate (Fig. 1). The stereochemistry
of carbon 3 could be deduced as R by comparison of the
NMR spectroscopy data with those published by Kra-
jewski et al. (1997) for a similar glycoside containing glu-
cose instead of arabinose–glucose as the sugar moiety. The
two diastereoisomers differ in the coupling constants of the
diastereotopic protons of C-2 and in the H and 13C
NMR chemical shifts of positions 2, 3 and 4 as well as of
the anomeric signal of the glucose moiety (cf. Table 3).
HR–FABMS measurements of 3a indicated a mole-
cular formula of C H O . The ESI–MS/MS data of
3. Experimental
3.1. General
MLCCC was performed in the head to tail mode at a
rotational speed of 800 rpm, using a 75 m  2.6 mm i.d.
1
PTFE tubing (total volume=400 ml) and CHCl –
3
1
13
MeOH–H O (7:13:8) as solvent system. H– C NMR
2
spectra were recorded in CDCl at 300/75 or 500/125
3
MHz, respectively. HR–FABMS was taken in a glycerol
matrix. ESI–MS/MS was employed in the positive mode
by direct introduction of samples with acetonitrile–H O
3
1
46 18
3
a was identical to that of compound 2a suggesting an
C
isomeric structure for both compounds. 1H and 13
2
NMR chemical shifts of glycoside 3a were also very
similar to those of 2a (cf. Tables 2 and 3) except for the
signals of C-2, C-3 and C-4, as well as for the anomeric
signals of the disaccharide. The values clearly indicated
S configuration at C-3 when compared with the syn-
thetic glucosides described by Krajewski et al. (1997).
On the basis of the overall results for compound 3a the
structure of 3-O-a-l-arabinopyranosyl-(1!6)-b-d-glu-
copyranoside of butyl (3S)-3-hydroxybutanoate (Fig. 1)
could be assigned to compound 3.
(9:1) at a flow rate of 4 ml/min. Electrospray parameters:
nebulizing gas, 10 psi; drying gas, 4 l/min (both nitrogen);
ꢀ
drying temperature 320 C. Voltages: end plate, À3000 V;
capillary, À3500 V; skimmer 1,+50 V; skimmer 2, +10
V; capillary exit, +150 V; multiplier, 1500 V; detection
mode: positive. Acquisition: accumulation cut-off, 40 m/z;
scan range, 50–1500 m/z; summation, 8 spectra. Pres-
À5
sure: about 1Â10 mbar in the ion trap. For flash
chromatography, Merck silica gel 60 (0.063–0.200 mm)
was used. HRGC of the aglycones was carried out with
Table 3
a
a
3
Selected NMR spectral data of compounds 2a, 3a (300 MHz, CDCl ) and 4 and 5
Position
2a (3R)
3a (3S)
4 (3R)
5 (3S)
2.35 (16.0/4.5)
H-2a
H-2b
H-4
2.42 (16.0/5.5)
2.66 (16.0/7.0)
1.20 (6.5)
4.56 (8.0)
42.0
2.36 (16.0/4.5)
2.54 (16.0/8.5)
1.30 (6.5)
4.62 (8.0)
42.142.4
74.2
2.40 (15.7/7.3)
2.76 (15.7/5.8)
1.20 (6.3)
4.58 (8.1)
42.1
2.54 (16.0/8.5)
1.28 (6.3)
4.62 (8.1)
0
0
H-1
C-2
C-3
C-4
71.9
19.6
73.4
20.2
74.4
21.6
21.9
101.1
0
0
C-1
98.8
99.9
101.1
a
Compounds 4 and 5: synthetic 3-O-(tetra-O-acetyl-b-d-glucopyranoside) of butyl 3-hydroxybutanoates (400 MHz, CDCl
3
) (Krajewski et al., 1997).

444
H. Mayorga et al. / Phytochemistry 59 (2002) 439–445
a fused-silica WCOT column (30 mÂ0.25 mm i.d., 0.25
mm film thickness) coated with DB-Wax. The column
temperature was programmed 4 min isothermally at 50 C,
subsequent purification to preparative HPLC using first
an Eurospher Si 100-5 column (5 mm, 250Â16 mm) with
TBME as mobile phase at a flow rate of 3 ml/min, fol-
lowed by chromatography on a Eurospher 100 C18
ꢀ
ꢀ
ꢀ
ꢀ
then raised to 130 C at 4 C/min, from 130 to 190 C at
ꢀ
ꢀ
ꢀ
1 C/min and then raised again from 190 to 220 C at 4 C/
min, and finally kept at this temperature for 20 min.
column (5 mm, 250Â16 mm) with MeOH–H O (7:3) at a
2
flow rate of 5 ml/min. The fraction obtained from the
latter column in a range of 50–55 min was finally pur-
ified on a LichroCart RP-18 column (5 mm, 250 mmÂ4
Injector and detector temperatures were maintained at
ꢀ
2
20 C. The carrier gas flow was 1.0 ml/min He.
Volumes of 1 ml were injected with a split ratio of 1:10.
Retention indices were calculated using a mixture of
normal paraffins as standard. HRGC–MS of the agly-
cones was employed with the same type of column and
temperature conditions as mentioned above for HRGC.
Mass spectra were scanned at 70 eV in the range of 30–
mm) by a gradient solvent system MeOH–H O (80:20 to
2
90:10) during 20 min at a flow rate of 0.5 ml/min to
yield pure compound 1a (2.5 mg).
The fraction eluted from the above mentioned Euro-
spher 100 C18 preparative column in a range of 20–25
min was finally purified by HPLC using this time an
analytical column Eurospher Si 100-5 (5 mm, 250Â4 mm)
with hexane–TBME (1:1) at a flow rate of 2 ml/min as
well as a chiral column LichroCart (R,R)-Whelk-01(5
mm, 250Â4 mm) with hexane–iso-PrOH (7:3) as mobile
phase at 1ml/min. Crystallization in hexane– iso-PrOH
3
00 amu. Results of qualitative analyses (mass spectral
data studies) were verified by comparing the retention
indices and mass spectral data with those of authentic
reference substances. Optical rotations were measured on
a Perkin-Elmer 241Polarimeter.
(7:3) of the two peaks which eluted from the chiral column
yielded pure 2a (6 mg) and 3a (9 mg).
3
.2. Plant material
Fresh cape gooseberry fruits (Physalis peruviana L.)
were obtained from a local market in Bogota, Colombia
´
3.4. Enzymatic hydrolysis
in 1997. Fruits fully healthy were carefully selected
according to the degree of ripeness measured by fruit color
Three mg of each MLCCC fractions were dissolved in
25 ml of 0.2 M citrate–phosphate buffer (pH 5.0) and a
nonselective glycosidase (300 ml of Rohapect D5L,
(
brilliant orange) and pH=3.6 of its pulp.
¨
Rohm, Darmstadt, Germany) was added together with
phenyl b-d-glucopyranoside as internal standard (105
mg). Then, the mixture was incubated at 37 C over 36 h
and the liberated aglycones were extracted with diethyl
3
.3. Extraction and isolation of compounds 1–3
ꢀ
The fruits (15 kg) were homogenized in 15 l of water
adjusted to pH 7.0) and centrifuged (5000 g for 30 min).
(
ether. The organic phase was dried over anhydrous
ꢀ
The supernatant divided in ten portions was subjected to
LC on Amberlite-XAD-2 (glass column, 40Â700 mm, 10
ml/min) (Gunata et al., 1985). After rinsing the column
with 3 l of distilled water, the glycosides were eluted with
sodium sulfate, concentrated (Vigreux column, 38 C)
to 0.2 ml, and subjected to HRGC and HRGC–MS
analyses. For enzymatic hydrolyses of pure compounds,
1mg of each compound was used.
1
.5 l of methanol. The combined methanol eluates were
concentrated to dryness under reduced pressure, extracted
with diethyl ether to eliminate remaining volatiles and
lyophilized to afford 25.2 g of crude glycosides. Portions
of ꢁ1.8 g of the glycosidic extract were subjected to
MLCCC for separation of glycosides. Sixty-six fractions
3.5. Data for compounds 1a–3a, 6 and 7
2
2
ꢀ
Heptaacetate 1a. Colorless gum, ½ꢁꢂ À4.6 (MeOH, c
D
0.0867). For H NMR and C NMR spectra (see Tables 1
1
13
+
and 2). HR–FAB–MS: m/z 829.3077 [M+Na] (C H
56
3
6
(
each 5 ml) were collected. Each fraction was con-
O Na requires 829.3105). ESI–MS/MS m/z (rel. int.): 845
20
centrated to dryness, lyophilized, and an aliquot was
enzymatically hydrolyzed (Rohapect D5L). The released
volatiles (aglycons) were analyzed by HRGC–MS.
Combined MLCCC fractions (35–46, 580 mg) (containing
hydroxyester precursors, according to the results of
enzymatic hydrolysis) were acetylated overnight with 5
ml of Ac O-pyridine. The peracetylated glycosides thus
2
obtained were subjected to flash chromatography (Still
et al., 1978), using a silica gel column and the following
[M+K]+ (19), 829 [M+Na]+ (100), 528 [hexose(Ac)
4
+
hexose(Ac) +Na—AcO–AcOCH +H] (5). Daughter
3
2
+
ion spectrum of m/z 829: 769 [M+Na–AcOH] (100),
709 [769-AcOH]+ (38), 667 [709-CH CO] (11), 607
+
2
[667-AcOH]+ (9), 547 [607-AcOH]+ (4). Daughter ion
+
spectrum of m/z 769: 769 [M+Na–AcOH] (100), 709
+
[769-AcOH]+ (98), 649 [709-AcOH] (9), 607 [709-
+
+
AcOH–CH =C=O] , 547 [607-AcOH] (7).
2
ꢀ
22
D
Hexaacetate 2a. White needles, mp 104–106 C. ½ꢁꢂ
ꢀ
1
13
2
1
00 ml pentane–Et O discontinuous gradients (8:2, 6:4,
2
:1, 4:6, 2:8). One hundred fractions (each ꢁ10 ml) were
À2.1 (MeOH, c 0.2867). For H NMR and C NMR
spectra (see Tables 1and 2). HR–FABMS: m/z
729.2572 [M+Na]+ (C H O Na requires 729.2581).
collected. Combined fractions 72–85 (227 mg) were
concentrated in vacuum to dryness and subjected for
3
1
46 18
ESI–MS/MS m/z (rel. int.): 766 [M+AcOH]+ (15), 745

H. Mayorga et al. / Phytochemistry 59 (2002) 439–445
445
[
trum of m/z 729: 669 [M+Na–AcOH] (100), 609 [669-
M+K]+ (17), 729 [M+Na]+ (100). Daughter ion spec-
+
Allegrone, G., Barbeni, M., 1992. Identification of volatile compo-
nents of caja fruit (Spondias lutea L.) and chiral analysis of 3-
hydroxy aliphatic esters. Flav. Fragr. J. 7, 337–342.
AcOH]+ (27), 507 [609-AcOH–CH CO]+ (4). Daughter
2
Berger, R.G., Drawert, F., Kollmannsberger, H., 1989. The flavour of
cape gooseberry (Physalis peruviana L.). Z. Lebensm. Unters.
Forsch. 188, 122–126.
+
ion spectrum of m/z 669: 669 [M+Na–AcOH] (1), 609
669-AcOH]+ (100), 507 [M+Na-3AcOH–CH CO]
+
[
2
(
7). Daughter ion spectrum of m/z 609: 609 [M+Na–
Beuerle, T., Schreier, P., Brunerie, P., Bicchi, C., Schwab, W., 1996.
Absolute configuration of octanol derivatives in apple fruits. Phy-
tochemistry 43, 145–149.
AcOH]+ (100), 551 [609-C H –H] (49), 507 [609-
+
2
AcOH–CH CO] (61), 407 [pentose(Ac) hexose(Ac) +
Na-2AcOH–CH CO–H O]
2
(
se(Ac) +Na–AcOH] (21), 142 [aglycon–H O] (2).
4 9
+
2
3
3
Beuerle, T., Schwab, W., 1997. Octane-1,3-diol and its derivatives
from pear fruits. Z. Lebensm. Unters. Forsch. 205, 215–217.
Crouzet, J., Chassagne, D., 1999. Glycosidically bound volatiles in
plants. In: Ikan, R. (Ed.), Natural Occurring Glycosides. John
Wiley & Sons. New York, pp. 225–274.
+
+
(27), 347 [407-AcOH]
+
2
20), 293 [hexose(Ac) +Na–2H O] (10), 239 [pento-
3 2
+
+
3
2
ꢀ
20
D
Hexaacetate 3a. White crystals, mp 101–103 C. ½ꢁꢂ
ꢀ
1
13
Gunata, Y.Z., Bayonove, C.L., Baumes, R.L., Cordonnier, R.E.,
1
+
1.8 (MeOH, c 0.2267). For H NMR and C NMR
spectra (see Tables 1and 2). HR–FABMS: m/z
29.2604 [M+Na]+ (C H O Na requires 729.2581).
985. The aroma of grapes. I. Extraction and determination of free
and glycosidically bound fraction of some grape aroma compo-
nents. J. Chromatogr. 331, 83–90.
7
3
1
46 18
+
ESI–MS/MS m/z (rel. int.): 766 [M+AcOH] (15), 745
M+K]+ (17), 729 [M+Na]+ (100). Daughter ion spec-
+
Idstein, H., Keller, T., Schreier, P., 1985. Volatile constituents of
mountain papaya (Carica candamarcensis, syn. C. pubescens Lenne
et Koch) fruit. J. Agric. Food Chem. 33, 663–666.
[
trum of m/z 729: 669 [M+Na–AcOH] (100), 609 [669-
Krajewski, D., Duque, C., Schreier, P., 1997. Aliphatic ß-d-gluco-
sides from fruits of Carica pubescens. Phytochemistry 45, 1627–
AcOH]+ (27), 507 [609-AcOH–CH CO]+ (4). Daughter
2
ion spectrum of m/z 669: 669 [M+Na–AcOH]+ (1), 609
1
631.
669-AcOH]+ (100), 507 [M+Na-3AcOH–CH CO]
+
Mayorga, H., Knapp, H., Winterhalter, P., Duque, C., 2001. Glycosi-
dically bound flavor compounds of cape gooseberry (Physalis per-
uviana L.). J. Agric. Food Chem. 49, 1904–1908.
[
2
(
7). Daughter ion spectrum of m/z 609: 609 [M+Na–
AcOH]+ (100), 551 [609-C H –H] (49), 507 [609-
+
2
AcOH–CH CO] (61), 407 [pentose(Ac) hexose(Ac) +
Na-2AcOH–CH CO–H O]
2
(
(
4 9
Osorio, C., Duque, C., Fujimoto, Y., 1999. C13-Norisoprenoid gluco-
conjugates from lulo (Solanum quitoense L.) leaves. J. Agric. Food
Chem. 47, 1641–1645.
+
2
3
3
+
+
(27), 347 [407-AcOH]
+
2
20), 293 [hexose(Ac) +Na–2H O] (10), 239 [pentose
3
Ac) +Na–AcOH]+ (21), 142 [aglycon–H O] (2).
˜
Parada, F., Duque, C., 1998. Studies on the aroma of pinuela fruit
2
+
pulp (Bromelia plumieri): free and bound volatile composition and
characterization of some glucoconjugates as aroma precursors. J.
High Res. Chromatogr. 21, 577–581.
3
2
Ethyl 3-hydroxyoctanoate 6. C H O , M 188. R
I
1
0
20
3
r
(
6
(
DB-Wax) 1880. EIMS m/z (rel. int.): 43 (73), 55 (31),
0 (17), 71 (44), 83 (13), 88 (31), 89 (28), 117 (100), 125
11), 170 (1), 187 (<1).
Parada, F., Duque, C., Fujimoto, Y., 2000. Free and bound volatile
composition and characterization of some glucoconjugates as
aroma precursors in melon de olor fruit pulp (Sicana odorifera). J.
´
Agric. Food Chem. 48, 6200–6204.
Butyl 3-hydroxybutanoate 7. C H O , M 160. R
I
8
16
3
r
Still, W.C., Kahn, M., Mitra, A., 1978. Rapid chromatographic tech-
nique for preparative separations with moderate resolution. J. Org.
Chem. 43, 2923–2925.
(
4
1
DB-Wax) 1684. EIMS m/z (rel. int.): 41(59), 43 ( 10 0),
5 (73), 56 (49), 57 (33), 60 (28), 71( 10 ), 87 (88), 89 (38),
05 (6), 145 (10), 159 (<1).
Straubinger, M., Bau, B., Eckstein, S., Fink, M., Winterhalter, P.,
1
998. Identification of novel glycosidic aroma precursors in saffron
Crocus sativus L.). J. Agric. Food Chem. 46, 3238–3243.
(
Acknowledgements
Straubinger, M., Knapp, H., Watanabe, N., Oka, N., Washio, H.,
Winterhalter, P., 1999. Three novel eugenol glycosides from rose
flowers, Rosa damascena Mill. Nat. Prod. Lett. 13, 5–10.
This work was supported by Colciencias and IPICS
Uppsala University, Sweden). The authors are indebted
Umano, K., Hagi, Y., Nakahara, K., Shoji, A., Shibamoto, T., 1992.
Volatile constituents of green and ripened pineapple (Ananas como-
sus [L.] Merr.). J. Agric. Food Chem. 40, 599–603.
(
to Katherine Jaramillo and Fabiola Espejo of the Instituto
de Inmunologıa de San Juan de Dios for the HMBC,
´
Werkhoff, P., Gu
998. Vacuum headspace method in aroma research: flavor
chemistry of yellow passion fruits. J. Agric. Food Chem. 46,
076–1093.
Wintoch, H., Morales, A., Duque, C., Schreier, P., 1993. (R)-(À)-(E)-
,6-Dimethyl-3,7-octadiene-2,6-diol 6-O-ß-d-glucopyranoside: nat-
¨
ntert, M., Krammer, G., Sommer, H., Kaulen, J.,
HMQC and NOESY measurements. We are also grateful
to Professor Yoshinori Fujimoto (Tokyo Institute of
Technology) for help with recording HR–FABMS spectra.
1
1
2
ural precursor of hotrienol from lulo fruit (Solanum vestissimun D.)
peelings. J. Agric. Food Chem. 41, 1311–1314.
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