Triterpene phytoalexins from nectarine fruits

Article abstract of DOI:10.1016/S0031-9422(99)00316-7

Seven triterpenoid phytoalexins were isolated from peel of unripe fruits of nectarine (Prunus persica cv Fantasia) wounded and inoculated with Colletotrichum musae. Two were new triterpenoids, identified as 1β,2α,3α,24-tetrahydroxyurs-12-en-28-oic acid and 1β,2α,3α,24- tetrahydroxyolean-12-en-28-oic acid. 2α,3α,24-Trihydroxyolean-12-en-28-oic acid, 2α,3α,24-trihydroxyurs-12-en-28-oic acid, 2α,3β,24-trihydroxyolean- 12-en-28-oic acid, 2α,3α-dihydroxyolean-12-en-28-oic acid, and 2α,3α- dihydroxyurs-12-en-28-oic acid were previously reported as constitutive natural products from other plants, but were never described as phytoalexins. All showed antifungal activity against the fungus mentioned.

Full text of DOI:10.1016/S0031-9422(99)00316-7

Phytochemistry 52 (1999) 623±629
Triterpene phytoalexins from nectarine fruits
a, b
Hassane El Lahlou , Nobuhiro Hirai *, Mitsuya Tsuda , Hajime Ohigashi
a, b,
c
b
a
CREST, Japan Science and Technology Corporation (JST), Japan
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan
Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan
b
c
Received 28 September 1998; received in revised form 28 April 1999
Abstract
Seven triterpenoid phytoalexins were isolated from peel of unripe fruits of nectarine (Prunus persica cv Fantasia) wounded and
inoculated with Colletotrichum musae. Two were new triterpenoids, identi®ed as 1b,2a,3a,24-tetrahydroxyurs-12-en-28-oic acid
and 1b,2a,3a,24-tetrahydroxyolean-12-en-28-oic acid. 2a,3a,24-Trihydroxyolean-12-en-28-oic acid, 2a,3a,24-trihydroxyurs-12-en-
2
8-oic acid, 2a,3b,24-trihydroxyolean-12-en-28-oic acid, 2a,3a-dihydroxyolean-12-en-28-oic acid, and 2a,3a-dihydroxyurs-12-en-
2
8-oic acid were previously reported as constitutive natural products from other plants, but were never described as
phytoalexins. All showed antifungal activity against the fungus mentioned. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Prunus persica; Nectarine; Colletotrichum musae; Phytoalexin; Triterpenoid; Antimicrobial activity
1. Introduction
lation and characterisation of seven phytoalexins from
unripe fruits of nectarine.
Resistance in many plant-pathogen interactions is
accompanied by the rapid deployment of a multicom-
ponent defensive response (Dixon & Harrison, 1994).
The individual components of this response include the
hypersensitive response, chemical weapons such as
antimicrobial phytoalexins and hydrolytic enzymes,
and structural defensive barriers such as lignin and
hydroxyproline-rich cell proteins (Dixon & Harrison,
1994; Kuc, 1994).
We are evaluating the role of phytoalexins as a part
of a research program aimed at understanding the
mechanisms of disease resistance of fruits, since some
fruits in unripe stage showed resistance to infection by
pathogens (Hirai, Ishida & Koshimizu, 1994; Kamo et
al., 1998a, b). We have investigated the resistance
mechanism in nectarine (Prunus persica ) fruit, and
found that unripe nectarine fruit produced phytoalex-
ins upon wounding and inoculation with Colletotricum
musae. In this report we describe the induction, iso-
2. Results and discussion
The unripe fruit of nectarine was wounded and
inoculated with a conidia suspension of C. musae
strain No. 1679 since Colletotrichum shows a broad
host range (Simmonds, 1965). A bioautography with a
TLC plate of extracts from peels of the fruit revealed
antifungal zones against the fungi mentioned pre-
viously, which were not detected in the extracts of
*
Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00316-7

6
24
H. El Lahlou et al. / Phytochemistry 52 (1999) 623±629
non-treated fruit. This ®nding showed that the unripe
fruit produced antimicrobial compounds as phytoalex-
ins.
ylene proton at d 2.50 was assigned to one of H-11,
the low ®eld chemical shift will be explained later.
Three methine protons at d 3.50 (d, J = 9.6 Hz), d
3.65 (dd, J = 9.6 and J = 3.1 Hz) and d 3.85 (d,
J = 3.1 Hz), and methylene protons at d 3.44 and 3.70
(1H each, d, J = 11.4 Hz) suggested that 1 has at least
four hydroxyl groups. Compound 1 gave a mono-
methyl ester (1a) on treatment with diazomethane, and
acetylation of 1a a€orded a tetra-acetate (1b), con®rm-
ing that 1 has one carboxyl and four hydroxyl groups.
A mass spectrum of 1a showed a molecular ion at m/z
518, and its high resolution mass spectrum gave a mol-
The antifungal compounds 1±7 were isolated, of
which 3 and 4 were isolated as methyl esters 3a and 4a
after methylation of a mixture of 3 and 4 since separ-
ation of them was dicult. Compounds 1, 2, mixtures
of 3 and 4, and 5±7 showed antifungal activity against
C. musae at 30 mg in bioautography, of which 1 and 2
gave large antifungal zones compared to that of 3±7.
Compounds 3±7 were characterized as 2a,3a,24-trihy-
droxyolean-12-en-28-oic acid (3) (Kojima & Ogura,
1989; Fang & Ying, 1986), 2a,3a,24-trihydroxyurs-12-
ecular formula C31H O for 1a. Fragment ions at m/z
50 6
en-28-oic acid (4) (Kojima & Ogura, 1989), 2a,3b,24-
trihydroxyolean-12-en-28-oic acid (5) (Yamagishi et
al., 1988), 2a,3a-dihydroxyolean-12-en-28-oic acid (6)
262, 203, 189, and 133 were also observed in the mass
spectrum. The fragment ion at m/z 262 which had a
formula C17H O would be formed by a retro-Diels-
26 2
(
Cheung & Yan, 1970), and 2a,3a-dihydroxyurs-12-en-
8-oic acid (7) (Kojima & Ogura, 1989) by comparison
Alder fragmentation between C-9 and C-11, and
between C-8 and C-14, as shown in Fig. 1, since such
cleavage is characteristic of pentacyclic triterpenoids
having a double bond at C-12 like a urs-12-ene skel-
2
of their physical constants and spectroscopic data with
those reported in the literature. These compounds were
previously reported as constitutive natural products
from other plants, but were never described as phytoa-
lexins. Compounds 1 and 2 were new triterpenoids.
eton (Budzikiewicz, Wilson
&
Djerassi, 1963;
Ngninzeko, David & Lucas, 1987). Formation of the
other fragment ions was explained by fragmentation of
this ion. Further, the fragment ion at m/z 262 revealed
that neither of the four hydroxyl groups were present
in the rings C, D and E, consequently these hydroxyl
groups must be attached on the rings A and/or B. The
position of the hydroxyl groups was established on the
basis of HMBC and HMQC spectra of 1.
1
The H NMR spectrum of compound 1 was similar
to that of 4, and displayed four tertiary methyl groups
at d 0.87 (H-26), 1.02 (H-25), 1.10 (H-23 or H-24) and
1
0
.17 (H-27), and two secondary methyl groups at d
.92 (H-29) and 1.01 (H-30) on an ursane skeleton. A
doublet of one proton at d 2.23 and a triplet of one
proton at d 5.12 were assigned to H-18 and H-12, re-
spectively, suggesting an urs-12-ene skeleton. A meth-
In the HMBC spectrum (Fig. 2), the hydroxymethy-
lene protons H-24 or H-23 showed a correlation with
Fig. 1. Proposed EI mass spectral fragmentation of compound 1a.

H. El Lahlou et al. / Phytochemistry 52 (1999) 623±629
625
Ê
is close to the H-11b, the distance is about 2.13 A,
which explains the low chemical shift d 2.50 observed
for the methylene proton in the H NMR spectrum.
1
Thus, the structure of a new phytoalexin 1 was ident-
i®ed as 1b,2a,3a,24-tetrahydroxyurs-12-en-28-oic acid.
Another triterpene 2 gave a monomethyl ester 2a on
treatment with diazomethane. Compound 2a had the
same molecular formula C H O as compound 1a,
31
50
6
and its mass spectrum was identical to that of 1a. A
1
H NMR spectrum of 2 was similar to that of 1, but
six methyl singlets were observed instead of the four
methyl singlets and two secondary methyl groups as in
1
, and H-18 appeared as double doublets instead of a
doublet in 1. These showed that 2 has an olean-12-ene
skeleton. Signals corresponding to H-1, H-2, H-3 and
H-24 showed the similar chemical shifts and the same
1
multiplicities as 1 in the H NMR spectrum, indicating
that 2 has the same substitutions at the same position
on the ring A as those of 1. From these data, the
structure of 2 was elucidated as 1b,2a,3a,24-tetrahy-
droxyolean-12-en-28-oic acid.
Triterpenoids with four hydroxyl groups in the ring
A have been already reported in the literature, but
those compounds were accompanied with a 19a-hy-
droxyl group in the ring E in the case of compounds
with an ursane skeleton (Zhang & Yang, 1994), and
with a 3b-hydroxyl group in the case of compounds
with oleanane and ursane skeletons (Zhang & Yang,
Fig. 2. Three and two-bond correlations for compound 1 in the
HBMC spectrum.
the C-23 or C-24 and with a methine carbon at d 73.2.
The methine carbon was linked to a methine proton at
d 3.85 in the HMQC spectrum, which was assigned to
the H-3. The H-3 was correlated with the methine car-
bon signals at d 47.6 (C-5) and d 69.7 (C-2) through
three bonds and two bonds, respectively, in the
HMBC spectrum, so the methine carbon was assigned
to the C-2. The double doublets at d 3.65, assigned to
H-2, were two bonds away from a methine carbon at d
1
994; Gupta & Singh, 1989). The antimicrobial activity
reported here for tetrahydroxy-triterpenoids is the ®rst
example as far as we now.
79.4 (C-1). The methine proton at d 3.50 which was
assigned to H-1 was con®rmed to be attached to the
methine carbon C-1 in the HMQC spectrum. Both the
C-5 and the C-25 showed a correlation with the H-1
through three bonds in the HMBC spectrum, and this
proton had also a correlation with the C-2. All these
correlations indicated clearly that the four hydroxyl
groups were attached to C-1, C-2, C-3, and C-24 or C-
Compound 3 is known to occur as a constitutive
glucosyl ester in Polygala japonica Houtt (Fang &
Ying, 1986), so compounds 1±7 might be present as
glucosyl esters or glucosides in the fruits before treat-
ment, and released by action of b-glucosidase after
infection by fungi (Schonbeck & Schlosser, 1976).
Aqueous material from non-treated fruits was hydro-
lysed with b-glucosidase to examine the presence of the
conjugates. However, compounds 1±7 were not
detected, indicating that the conjugates do not occur
in the non-treated fruits. The activities of 1±7 meant
that 1,2,3,24-tetrahydroxytriterpenoids have higher ac-
tivities than 2,3,24-trihydroxytriterpenoids and 2,3-
dihydroxytriterpenoids. This suggested another possi-
bility that the triterpenoids with low degree of oxi-
dation may occur as precursors in the fruits before
treatment. In order to examine this possibility, ma-
terial that was soluble in an EtOAc fraction from non-
treated fruits was analysed, and ursolic and oleanolic
acids were found (Kojima & Ogura, 1986). This ®nd-
ing suggested that the fruit might induce enzymes
hydroxylating C-1, C-2 and C-24 when the fruit is
exposed to micro-organism, in order to produce more
active triterpenoids than triterpenoids with low degree
of oxidation.
13
23 of the ring A. In the C NMR spectrum of 1, the
signal corresponding to the hydroxymethylene carbon
C-24 or C-23 appeared at d 63.7. It is known that in
1
3
the C NMR spectra of cyclic triterpenoids the signal
due to an axial hydroxymethylene carbon at C-4
appears up®eld shifted at d 63±66 than an equatorial
hydroxymethylene carbon at C-4, 68±71 (Kojima &
Ogura, 1989; Zhang & Yang, 1994; Ahmed, Bano &
Bano, 1986). Therefore, the hydroxymethylene carbon
was assigned to the axial C-24, meaning that the ter-
1
tiary methyl group at d 1.10 in the H NMR is the H-
23. The chemical shifts for the protons H-2 and H-3
were within the range for a b axial-b equatorial pos-
ition (Kojima & Ogura, 1989), and the values of the
coupling constant for the H-1 and H-2, and H-2 and
H-3 showed a axial-b axial and b axial-b equatorial
couplings, respectively.
A
calculation using the
MNDO program showed that the 1b-hydroxyl group

6
26
H. El Lahlou et al. / Phytochemistry 52 (1999) 623±629
A pentacyclic triterpenoid has been reported as a
phytoalexin from the sapwood of apple infected with
ness. Antifungal zones lacking aerial mycelia were
detected by the absence of hyphae turning brown on
exposure of the plate to iodine vapour.
Chondrostereum purpureum Pouzar (Kemp, Holloway
&
Burden, 1985). However, triterpenoids are rarely
recognised as phytoalexins. The phytoalexins from nec-
tarine fruit are the second example as far as we know.
3.5. Isolation
The MeOH extract from the treated fruits was con-
centrated to give 100 ml of an aqueous solution, and
the solution was extracted with EtOAc three times.
The EtOAc extract was concentrated to dryness to
give 1.9 g of crude material which was subjected to
silica gel (100 g) column. Elution was carried out using
gradients of n-hexane±EtOAc, EtOAc, and MeOH.
The material eluted with n-hexane±EtOAc (4:6) was
chromatographed on silica gel (8 g) column using gra-
dient of n-hexane±EtOAc (6:4), and the fraction con-
taining compounds 6 and 7 was applied to prep. TLC
developed with n-hexane±EtOAc (1:1). The material at
3
. Experimental
3.1. Instruments
1
13
H and C NMR, and HMQC and HMBC: TMS
as international standard using Bruker ARX 500
500 MHz) and AC 300 (300 MHz); MS: JEOL JMS-
00H; IR: Shimadzu FTIR-8100 AI spectrometer;
Optical Rotation: JASCO DIP-1000 polarimeter.
(
6
3.2. Materials
an R 0.7 was recovered and puri®ed by HPLC in an
f
Unripe fruits of P. persica cv Fantasia were collected
A-311 column (ODS, 6 Â 100 mm; YMC, Kyoto,
in Okayama prefecture, Japan on 16 July 1997. C.
musae Berk and Curt Arx. Strain No. 1679 was
obtained from Department of Scienti®c and Research,
Mount Albert Research Centre, Auckland, New
Zealand, and cultured on potato±sucrose±agar med-
ium at 238C in darkness.
Japan), eluting with CH CN±H O (6:4) at a ¯ow rate
3
2
�
1
of 1.0 ml min with detection at 205 nm. The ma-
terials eluted at R 14.6 min and at R 16.0 min were
t
t
collected, and concentrated to a€ord 6 (4.8 mg) and 7
(2.8 mg), respectively, as white powder.
The material eluted with 100% EtOAc was subjected
to a silica gel (50 g) column eluted with gradients of
CHCl ±acetone to give three fractions. The ®rst one
3
.3. Treatments and extraction
3
which was eluted with 40% acetone was chromato-
graphed on silica gel (5 g) column using gradients of
CHCl ±MeOH. The material eluted with 5% MeOH
Unripe fruits, 120, were washed with water, wiped
with 70% ethanol, and rubbed with sand-paper (G 60
or 80). Conidia of the fungus were suspended in steri-
3
was applied to a Sephadex LH-20 (8 g) column, pre-
viously equilibrated with n-hexane±CHCl ±MeOH
6
� 1
lised water at density 6 Â 10 ml . The injured fruits
were soaked in the suspension of conodia in a plastic
bag for 10 s, and incubated in the plastic box at 258C
in darkness for 4 days. Ten unripe fruits were incu-
bated under the same conditions without wounding
and inoculation. After incubation, the peels of non-
treated and treated fruits, 80 and 768 g, respectively,
were soaked in MeOH for 3 days at room temp. The
MeOH extracts were ®ltered, and used for the bioauto-
graphy and isolation of compounds.
3
(2:1:1), and was eluted with the same mixture.
Fractions containing 5 were concentrated, and puri®ed
by prep. TLC developed with a mixture of CHCl3±
MeOH (9:1) to give 5 (7 mg). The second fraction
which was eluted with 100 ml of 60% acetone was sub-
mitted to a chromatographic separation on a silica gel
(8 g) column eluted with gradients of CHCl ±MeOH.
3
The material eluted with 5% MeOH was subjected to
a Sephadex LH-20 (8 g) column eluted with n-hexane±
CHCl ±MeOH (2:1:1). Fractions containing 1 and 2
3
3.4. Bioautography
were combined, and injected to an A-311 column
(
(35:65) at a ¯ow rate of 1.0 ml min with detection at
ODS, 6 Â 100 mm; YMC) eluted with CH CN±H O
3
2
�
1
The ®ltered MeOH extracts corresponding to 0.2 g
fresh weight of the peels, 0.4 mg for the non-treated
fruits, 2.2 mg for the wounded fruits and 2.1 mg for
the wounded and inoculated fruits, were applied to
thin layer silica gel plate, and developed with CHCl3±
acetone (4:6) until 11 cm. After drying the solvent,
conidia of C. musae strain No. 1679 suspended in a
205 nm. The materials eluted at R 20.8 min and at Rt
t
23.3 min were collected, and concentrated to give 1
(1.2 mg) and 2 (1.0 mg), respectively, as white powder.
The third fraction which was eluted with further 50 ml
of 60% acetone was concentrated, methylated with
CH N , and chromatographed in an A-311 column
2
2
6
� 1
Czapek-Dox medium at a density of 10 ml
sprayed on the thin layer plate, which was then incu-
were
(ODS, 6 Â 100 mm; YMC) eluted with CH CN±H O
3
2
�
1
(55:45) at a ¯ow rate of 1.0 ml min with detection at
bated in a moist chamber at 238C for 2 days in dark-
205 nm. The materials eluted at R 25.8 min and at Rt
t

H. El Lahlou et al. / Phytochemistry 52 (1999) 623±629
627
2
6.8 min were collected to give 3a (2.8 mg) and 4a
0.26 (3H, s,H-26), 0.85 (3H, d, J = 6.4 Hz, H-29), 0.96
(3H, d, J = 6.7 Hz, H-30), 1.11 (3H, s, H-23), 1.18
(
0.7 mg), respectively.
(
3H, s, H-27), 1.25±1.78 (18H, m, H-5, 6, 7, 9, 11, 15,
3
.6. 1b,2a,3a,24-Tetrahydroxyurs-12-en-28-oic acid (1)
16, 19, 20, 21, 22), 1.93, 1.98, 2.09 and 2.16 (3H each,
s, 4 Â OAc), 3.60 (3H, s, 17-COOMe), 4.07 (1H, d,
J = 11.6 Hz, H-24a), 4.25 (1H, d, J = 11.6 Hz, H-
24b), 5.15 (2H, bs, H-2 and H-1), 5.36 (2H, br s, H-12
and H-3).
1
H NMR (500 MHz, CD OD): d 0.87 (3H, s, H-26),
3
0.92 (3H, d, J = 6.4 Hz, H-29), 1.01 (3H, d,
J = 7.5 Hz, H-30), 1.02 (3H, s, H-25), 1.10 (3H, s, H-
2
9
3), 1.17 (3H, s, H-27), 1.19±2.00 (17H, m, H-5, 6, 7,
, 11a, 15, 16, 19, 20, 21, 22), 2.23 (1H, d, J = 9.6 Hz,
3.9. 1b,2a,3a,24-Tetrahydroxyolean-12-en-28-oic acid
(2)
H-18), 2.50 (1H, m, H-11b), 3.44 (1H, d, J = 11.4 Hz,
H-24a), 3.50 (1H, d, J = 9.6 Hz, H-1), 3.65 (1H, dd,
J = 9.6 and 3.1 Hz, H-2), 3.70 (1H, d, J = 11.4 Hz,
H24b), 3.85 (1H, d, J = 3.1 Hz, H-3), 5.12 (1H, t,
1
H NMR (300 MHz, CD OD): d 0.84 (3H, s, H-26),
3
0.93 (3H, s, H-29), 0.98 (3H, s, H-30), 1.01 (3H, s, H-
23), 1.21 (3H, s, H-27), 1.32-2.13 (17H, m, H-5, 6, 7, 9,
11a, 15, 16, 19, 21, 22), 2.88 (1H, dd, J = 9.0 and
3.5 Hz, H-18), 2.46 (1H, m, H-11b), 3.44 (1H, d,
J = 11.4 Hz, H-24a), 3.48 (1H, d, J = 9.7 Hz, H-1),
3.64 (1H, dd, J = 9.7 and 3.1 Hz, H-2), 3.70 (1H, d,
J = 11.4 Hz, H-24b), 3.85 (1H, d, J = 3.1 Hz, H-3),
13
J = 3.3 Hz, H-12); C NMR (125 MHz, CD OD): d
3
79.4 (C-1), 69.7 (C-2) 73.2 (C-3), 42.9 (C-4), 47.6 (C-
5), 16.2 (C-6), 32.8 (C-7), 39.5 (C-8), 42.3 (C-9), 38.6
(
(
C-10), 26.1 (C-11), 125.6 (C-12), 137.0 (C-13), 47.9
C-14), 27.4 (C-15), 36.3 (C-16), 41.2 (C-17), 52.5 (C-
1
2
8), 30.0 (C-19), 23.5 (C-20), 27.4 (C-21), 38.5 (C-22),
1.1 (C-23), 63.7 (C-24), 11.6 (C-25), 15.7 (C-26), 22.1
13
5.26 (1H, t, J = 3.2 Hz, H-12); C NMR (75 MHz,
CD OD): d 79.2 (C-1), 69.7 (C-2), 73.2 (C-3), 42.9 (C-
(
C-27), 178.0 (C-28), 19.8 (C-29), 17.4 (C-30). The dis-
3
tance between 1b-OH and 11b-H of 1 was measured
after minimisation of its energy by an MNDO method
of CS Chem3D Pro version 3.5.1 (Cambridge Soft
Corporation, Massachusetts, USA).
4), 47.8 (C-5), 22.3 (C-6), 33.2 (C-7), 39.2 (C-8), 42.3
(C-9), 31.7 (C-10), 26.2 (C-11), 122.2 (C-12), 142.7 (C-
13), 47.6 (C-14), 27.0 (C-15), 32.0 (C-16), 40.8 (C-17),
52.5 (C-18), 24.5 (C-19), 32.4 (C-20), 28.8 (C-21), 29.7
(C-22), 21.1 (C-23), 63.7 (C-24), 11.4 (C-25), 16.1 (C-
26), 12.1 (C-27), 178.0 (C-28), 17.5 (C-29), 16.1 (C-30).
3.7. Methylation of compound 1
Compound 1 (1.2 mg) was dissolved in 0.8 ml of
MeOH, and treated with an ether soln of CH N at
3.10. Methylation of compound 2
2
2
room temp. for 2 h. After evaporation of the solvent,
�
Compound 2 (1.0 mg) was methylated by the same
1
1
.2 mg of methyl ester 1a was obtained. IR nmax cm
:
400, 2700, 1750, 1600, 1450; EIMS (probe) 70 eV, m/
method as that for 1 to give a methyl ester 2a. IR n
max
cm : 3300, 1730, 1600, 1100; EIMS (probe) 70 eV, m/
�
1
3
z (rel. int.): 518 [M] (7), 500 (50), 262 (92), 203 (100),
+
+
z (rel. int.): 518 [M] (10), 500 (20), 262 (62), 203
(100), 189 (36), 133 (21); HR-EIMS (probe) 70 eV:
+
89 (35), 133 (47); HR±EIMS (probe) 70 eV: [M] at
1
m/z 518.3605 (C H O requires 518.3607); H NMR
1
+
[M] at m/z 518.3548 (C H O requires 518.3607);
3
1
50
6
31 50
6
1
(
J = 7.2 Hz, H-29), 0.88 (3H, s, H-23 and H-26), 0.97
300 MHz, CDCl ): d 0.73 (3H, s, H-25), 0.87 (3H, d,
H NMR (300 MHz, CDCl ); d 0.70 (3H, s, H-26),
3
3
0.85 (3H, s, H-29), 0.89 (3H, s, H-30), 0.93 (3H, s, H-
25), 0.97 (3H, s, H-23), 1.14 (3H, s, H-27), 1.32±2.13
(17H, m, H-5, 6, 7, 9, 11a, 15, 16, 19, 21, 22), 2.85
(1H, br d, J = 11.0 Hz, H-18), 3.47 (1H, d, J = 9.5 Hz,
H-1), 3.51 (1H, d, J = 12.0 Hz, H-24a), 3.66 (3H, s,
17-COOMe), 3.70 (2H, m, H-2 and H-24b), 3.97 (1H,
br s, H-3), 5.28 (1H, br s, H-12).
(
(
3H, d, J = 11.6 Hz, H-30), 1.09 (3H, s, H-23), 1.13
3H, s, H-27), 1.20±2.02 (17H, m, H-5, 6, 7, 9, 11a, 15,
16, 19, 20, 21, 22), 3.47 (1H, d, J = 9.5 Hz, H-1), 3.51
(
1H, d, J = 12.0 Hz, H-24a), 3.66 (3H, s, 17-COOMe),
3
5
.70 (2H, m, H-24b and H-2), 3.97 (1H, br s, H-3),
.25 (1H, br s, H-12).
3.8. Acetylation of compound 1a
3.11. Acetylation of compound 2a
Compound 1a (1.0 mg) was treated with 1 ml of
Ac O±pyridine (1:1). The reaction mixture was kept
Compound 2a (0.8 mg) was acetylated by the same
method as that for 1a to give a tetra-acetate 2b.
2
31
for 36 h at room temp. The solution was treated as
usually and the organic fraction was concentrated to
[a] +25.08 (MeOH; c 0.07); EIMS (probe) 70 eV, m/z
D
+
(rel. int.): 686 [M] (7), 627 (77), 626 (100), 566 (71),
3
1
1
506 (68), 262 (45), 203 (53); H NMR (300 MHz,
give a tetra-acetate (1b). [a] +27.08 (MeOH; c 0.06);
D
+
EIMS (probe) 70 eV, m/z (rel. int.): 686 [M] (3), 627
CDCl ): d 0.72 (3H, s, H-26), 0.88 (3H, s, H-29), 0.90
3
(
60), 626 (100), 566 (58), 506 (62), 262 (39), 203 (51);
H NMR (300 MHz, CDCl ): d 0.71 (3H, s, H-25),
(6H, s, H-30 and H-25), 0.93 (3H, s, H-23), 0.95 (3H,
s, H-27), 1.25±1.78 (18H, m, H-5, 6, 7, 9, 11, 15, 16,
1
3

6
28
H. El Lahlou et al. / Phytochemistry 52 (1999) 623±629
1
4
9, 20, 21, 22), 1.93, 1.97, 2.01, 2.08 (3H each, s,
 OAc), 2.85 (1H, br d, J = 8.4 Hz, H-18), 3.63 (3H,
3.15. 2a,3a-Dihydroxyolean-12-en-28-oic acid (6) and
its methyl ester (6a)
s, 17-COOMe), 4.07 (1H, d, J = 11.6 Hz, H-24a), 4.21
1H, d, J = 11.6 Hz, H-24b), 5.14 (2H, bs, H-2 and H-
23
1
(
Compound 6: [a] +1468 (MeOH; c 0.070);
H
D
1), 5.36 (2H, br s, H-12 and H-3).
NMR (300 MHz, CD OD): d 0.86 (3H, s, H-26), 0.89
3
(
3H, s, H-29), 0.93 (3H, s, H-23), 0.98 (3H, s, H-30),
3
.12. 2a,3a,24-Trihydroxyolean-12-en-28-acid methyl
1.02 (6H, s, H-24 and H-25), 1.20 (3H, s, H-27), 2.91
(1H, br d, J = 10.4 Hz, H-18), 3.34 (1H, overlapping
with methanol), 3.95 (1H, m, H-2), 5.27 (1H, br s, H-
12). Compound 6 (0.7 mg) was treated with CH N to
ester (3a)
2
3
[
a] +48.18 (MeOH; c 0.28); EIMS (probe) 70 eV,
D
2
2
+
m/z (rel. int.): 502 [M] (2), 425 (5), 368 (5), 262 (93),
give a monomethyl ester 6a. EIMS (probe) 70 eV, m/z
+
(rel. int.): 486 [M] (6), 262 (90), 203 (100), 189 (25),
1
03 (100), 189 (32), 133 (26); H NMR (300 MHz,
2
1
CDCl ): d 0.69 (3H, s, H-26), 0.92 (3H, s, H-29), 0.92
129 (25); H NMR (300 MHz, CDCl ): d 0.71 (3H,s,
3
3
(
6H, s, H-30 and H-25), 1.13 (3H, s, H-23), 1.15 (3H,
s, H-27), 2.86 (1H, dd, J = 13.8 and 4.2 Hz, H-18),
.51 (1H, d, J = 11.2 Hz, H-24a), 3.62 (3H, s, 17-
H-26), 0.88 (3H, s, H-29), 0.92 (3H, s, H-23), 0.95
(3H, s, H-30), 1.01 (3H, s, H-24), 1.13 (3H, s, H-25),
1.17 (3H, s, H-27), 2.86 (1H, dd, J = 13.6 and 4.1 Hz,
H-18), 3.43 (1H, br s, H-3), 3.62 (3H, s, 17-COOMe),
4.00 (1H, m, H-2), 5.28 (1H, t, J = 3.6 Hz, H-12).
3
COOMe), 3.69 (1H, d, J = 11.2 Hz, H-24b), 3.85 (1H,
d, J = 2.4 Hz, H-3), 3.97 (1H, m, H-2), 5.28 (1H, t,
J = 3.5 Hz, H-12).
3.16. 1a, 2a-Dihydroxyurs-12-en-28-oic acid (7) and its
methyl ester (7a)
3
(
.13. 2a,3a,24-Trihydroxyurs-12-en-28-acid methyl ester
4a)
2
1
1
Compound 7: [a] +25.68 (MeOH; c 0.050);
H
D
2
3
[
a] +56.58 (MeOH; c 0.04); EIMS (probe) 70 eV,
NMR (300 MHz, CD OD): d 0.89 (3H, s, H-26), 0.90
D
3
+
m/z (rel. int.): 502 [M] (14), 262 (100), 203 (90), 189
(3H, s, H-23), 0.92 (3H, d, J = 6.6 Hz, H-29), 1.01
(3H, d, J = 7.2 Hz, H-30), 1.02 (3H, s, H-24), 1.04
(3H, s, H-25), 1.16 (3H, s, H-27), 2.26 (1H, d,
J = 11.1 Hz, H-18), 3.34 (1H, overlapping with
1
(
(
(
(
19), 133 (45); H NMR (300 MHz, CDCl ): d 0.71
3
3H, s, H-26), 0.85 (3H, d, J = 6.4 Hz, H-29), 0.94
3H, s, H-25), 0.95 (3H, d, J = 2.7 Hz, H-30), 1.09
3H, s, H-23), 1.15 (3H, s, H-27), 2.23 (1H, d,
CD OD), 3.96 (1H, m, H-2), 5.26 (1H, t, J = 3.4 Hz,
3
J = 11.7 Hz, H-18), 3.51 (1H, d, J = 11.0 Hz, H-24a),
H-12). Compound 7 (1.0 mg) was methylated with
CH N to give a monomethyl ester 7a. EIMS (probe)
3
2
.62 (3H, s, 17-COOMe), 3.69 (1H, d, J = 11.0 Hz, H-
4b), 3.86 (1H, s, H-3), 3.97 (1H, m, H-2), 5.26 (1H, t,
2
2
+
70 eV, m/z (rel. int.): 486 [M] (21), 262 (100), 263
1
J = 3.4 Hz, H-12).
(100), 203 (100), 189 (95), 133 (100), 119 (65);
H
NMR (300 MHz, CDCl ): d 0.73 (3H, s, H-26), 0.85
3
3
.14. 2a,3b,24-Trihydroxyolean-12-en-28-oic acid (5)
(6H, d, J = 5.6 Hz, H-29 and H-23), 0.96 (3H, d,
J = 5.4 Hz, H-30), 1.02 (3H, s, H-24), 1.09 (3H, s, H-
and its methyl ester (5a)
2
5); 1.23 (3H, s, H-27), 2.33 (1H, d, J = 9.6 Hz, H-
2
4
1
Compound 5: [a] +38.38 (MeOH; c 0.30);
H
18), 3.43 (1H, br s, H-3), 3.60 (3H, s, 17-COOMe),
4.00 (1H, m, H-2), 5.25 (1H, t, J = 3.5 Hz, H-12).
D
NMR (300 MHz, CD OD): d 0.87 (3H, s,H-26), 0.91
3
(
3H, s, H-29), 0.96 (3H, s, H-30), 0.98 (3H, s, H-25),
1.17 (3H, s, H-23), 1.26 (3H, s, H-27), 2.94 (1H, dd,
3.17. Enzymatic hydrolysis
J = 9.3 and 3.8 Hz, H-18), 3.07 (1H, d, J = 9.3 Hz,
H-3), 3.41 (1H, d, J = 11.2 Hz, H-24a), 3.67 (1H, m,
H-2), 4.06 (1H, d, J = 11.2 Hz, H-24b), 5.25 (1H, br s,
H-12). Compound 5 (0.5 mg) was methylated with
CH N to give a methyl ester 5a. EIMS (probe) 70 eV,
The MeOH extract from non-treated fruits was con-
centrated, and partitioned between H O and EtOAc
2
by the same method as for the treated fruits. The aqu-
eous layer was concentrated to give a gummy syrup
(1.4 g), and this was divided into two equal parts. One
part was dissolved in 40 ml of 100 mM AcOH±
AcONa bu€er soln (pH 5.0), and 10 ml of the bu€er
soln containing 1000 units of b-glucosidase (Sigma,
from almonds) was added into the soln. This soln was
incubated for 3 h at 378C, and then its pH was
adjusted to 3 with 2 N HCl. The solution was satu-
rated with NaCl, and partitioned with 70 ml of EtOAc
three times. The EtOAc layer was washed with a small
2
2
+
m/z (rel. int.): 502 [M] (12), 484 (6), 466 (5), 262
100), 203 (100), 189 (80), 133 (75), 119 (43); HR-
(
+
EIMS (probe) 70 eV: [M] at m/z 502.3669 (C H O
5
requires 502.3658); H NMR (300 MHz, CDCl ): d
31
50
1
3
0.71 (3H, s, H-25), 0.90 (3H, s,H-26), 0.91(3H, s, H-
29), 0.93 (3H, s,H-30), 1.07 (3H, s, H-23), 1.21 (3H, s,
H-27), 2.87 (1H, br d, J = 13.9 Hz, H-18), 3.16 (1H, d,
J = 8.6 Hz, H-3), 3.38 (1H, d, J = 10.5 Hz, H-24a),
3.60 (3H, s, 17-COOMe), 3.88 (1H, m, H-2), 4.12 (1H,
d, J = 10.5 Hz, H-24b), 5.28 (1H, br s, H-12).
amount of H O, and concentrated to give an oil
2

H. El Lahlou et al. / Phytochemistry 52 (1999) 623±629
629
(
2.3 mg). Another part was dissolved in 50 ml of the
Experimental Station of Okayama Prefecture,
Okayama, Japan) for supplying nectarine fruits.
same bu€er, and then partitioned with EtOAc after
acidi®cation by the same method as for the ®rst part
to give an oil (2.1 mg). These materials from the
EtOAc layers were analysed with a silica gel TLC
developed with n-hexane±EtOAc (3:2).
References
Ahmad, V. U., Bano, S., & Bano, N. (1986). Phytochemistry, 25,
1
487.
3.18. Isolation of oleanolic and ursolic acids from non-
treated fruits
Budzikiewicz, H., Wilson, J. M., & Djerassi, C. (1963). Journal of
American Chemical Society, 85, 3688.
Cheung, H. T., & Yan, T. C. (1970). Journal of Chemical Society
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The organic layer (160 mg) from the MeOH extract
of non-treated fruits was subjected to a silica gel (10 g)
column eluted with gradients of n-hexane±EtOAc, and
MeOH. The material eluted with n-hexane±EtOAc
Dixon, R. A., & Harrison, M. J. (1994). Annual Review of
Phytopathology, 32, 479.
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(
previously equilibrated with n-hexane±CHCl ±MeOH
9:1) was applied to a Sephadex LH-20 (4 g) column,
3
(
2:1:1), using as eluent the same mixture of solvent,
Kamo, T., Kato, N., Hirai, N., Tsuda, M., Fujioka, D., & Ohigashi,
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and the mixture of ursolic and oleanolic acids was pur-
i®ed by HPLC in an A-311 column (ODS, 6 Â 100 mm;
YMC), eluting with CH CN±H O (85:15) at a ¯ow
rate of 1.0 ml min with detection at 205 nm. The
materials eluted at R 7.8 min and at R 8.4 min were
collected to give oleanolic acid (3.8 mg) and ursolic
acid (4.8 mg), respectively. Ursolic and oleanolic acids
1
were identi®ed on the basis of H NMR data and by
3
2
Kemp, M. S., Holloway, P. J., & Burden, R. S. (1985). Journal of
Chemical Research (S), 154.
�
1
Kojima, H., & Ogura, H. (1986). Phytochemistry, 25, 729.
Kojima, H., & Ogura, H. (1989). Phytochemistry, 28, 1703.
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t
t
Ngninzeko, F. N., David, L., & Lucas, S. (1987). Phytochemistry,
27, 301.
EI mass spectra of their monomethyl esters (Kojima &
Ogura, 1986).
Schonbeck, F., & Schlosser, E. (1976). Heitefuss, R., & Williams, P.
H. (Ed.), Encyclopedia of plant physiology (4, p. 662). Springer
Berlin.
Simmonds, J. H. (1965). Queensland Journal of Agriculture and
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Acknowledgements
Yamagishi, T., Zhang, D., Chang, J., Mcphail, D. R., Mcphail, A.
T., & Lee, K. (1988). Phytochemistry, 27, 3213.
Zhang, Y., & Yang, C. (1994). Phytochemistry, 36, 997.
We thank Dr Hideo Nasu (the Agricultural