Monoterpene glycosides and triterpene acids from Eriobotrya deflexa

Article abstract of DOI:10.1021/np0100237

A phytochemical study on a methanolic extract of leaves of Eriobotrya deflexa led to the isolation and characterization of nine terpenoid compounds. Four of these are new chemical entities, including two monoterpene glycosides, (3S)-O-α-L-rhamnopyranosyl-(1→3)-[4-O-(E)-coumaroyl]- α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl-linalool (1) and (3S)-O-α-L-rhamnopyranosyl-(1→3)-[4-O-(Z)-coumaroyl]-α-L- rhamnopyranosyl-(1→6)-β-D-glucopyranosyl-linalool (2), and two triterpene acids, 1β,2α,19α-trihydroxy-3-oxo-12-ursen-28-oic acid (3) and 2α,3α,19α-trihydroxy-12-oleanen-28-oic acid (4). Their structures were elucidated on the basis of spectroscopic analysis. The activities of these isolates in an in vitro antiproliferation test were also determined.

Full text of DOI:10.1021/np0100237

J . Nat. Prod. 2001, 64, 865-869
865
Mon oter p en e Glycosid es a n d Tr iter p en e Acid s fr om Er iobotr ya d eflexa
†
‡
§
,†,⊥
Tzong-Huei Lee, Shoei-Sheng Lee, Yuh-Chi Kuo, and Chang-Hung Chou*
Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan 115, School of Pharmacy, College of Medicine, National
Taiwan University, Taipei, Taiwan 100, National Research Institute of Chinese Medicine, Shih-Pai, Taipei, Taiwan 112, and
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 804, Republic of China
Received J anuary 26, 2001
A phytochemical study on a methanolic extract of leaves of Eriobotrya deflexa led to the isolation and
characterization of nine terpenoid compounds. Four of these are new chemical entities, including two
monoterpene glycosides, (3S)-O-R-L-rhamnopyranosyl-(1f3)-[4-O-(E)-coumaroyl]-R-L-rhamnopyranosyl-
(
1f6)-â-D-glucopyranosyl-linalool (1) and (3S)-O-R-L-rhamnopyranosyl-(1f3)-[4-O-(Z)-coumaroyl]-R-L-
rhamnopyranosyl-(1f6)-â-D-glucopyranosyl-linalool (2), and two triterpene acids, 1â,2R,19R-trihydroxy-
3
elucidated on the basis of spectroscopic analysis. The activities of these isolates in an in vitro
-oxo-12-ursen-28-oic acid (3) and 2R,3R,19R-trihydroxy-12-oleanen-28-oic acid (4). Their structures were
antiproliferation test were also determined.
Eriobotrya deflexa N. (Rosaceae), an erect shrub indig-
enous to Taiwan, is widely distributed in hardwood forests
1
at about 1500 m in elevation throughout the island. Leaves
of E. deflexa have been used in traditional Chinese medi-
2
cine as an expectorant and antitussive. A closely related
3
species, E. japonica, is commonly known as “loquat”. A
4
-6
7
series of sesquiterpene glycosides,
triterpene acids, and
triterpene esters7 have been isolated from E. japonica, and
,8
among them, several components were found to show
8
significant bioactivities including antiviral, antiinflam-
9
10,11
matory, and hypoglycemic properties.
To determine if
E. deflexa can be used as a substitute for E. japonica, its
leaf chemical constituents were investigated. Additionally,
to determine the immunomodulatory activity of the E.
deflexa isolates obtained, these substances were evaluated
in an in vitro antiproliferation assay.
contained three sugar units, as evident from the presence
of three anomeric proton signals at δ 4.33 (d, J ) 7.8 Hz),
4
.77 (brs), and 4.82 (d, J ) 1.5 Hz). Of these three sugars,
Resu lts a n d Discu ssion
two were assigned as R-L-rhamnose units, supported by
signals typical for anomeric protons and two secondary
methyl signals (δ 1.16 and 1.24, d, J ) 6.2 Hz). Besides
the sugar proton regions, the 1H NMR spectrum of 1
A methanolic extract of the dried leaves of E. deflexa was
fractionated by liquid-liquid partitioning into fractions
soluble in n-hexane, chloroform, and n-butanol. The n-
butanol- and chloroform-soluble fractions were then sub-
jected to Sephadex LH-20 column chromatography and
HPLC to give two monoterpene glycosides (1 and 2) and
seven triterpene acids (3-9), respectively. Of these, com-
pounds 5-9 were found to be known, based on spectral
comparison with reported data. Compounds 5-8 are ursolic
acid analogues and were identified as euscaphic acid (5),
8
showed signals for a trans-coumaroyl moiety, an AA′XX′
system at δ 6.80 (H-6′′′′ and H-8′′′′) and 7.47 (H-5′′′′ (J AX
)
8.6 Hz), and an AX system at δ 6.33 (H-2′′′′) and 7.65 (H-
3′′′′) (J trans ) 15.9 Hz). In addition, this spectrum also
1
7
displayed signals for a linalool moiety, three methyl
singlets at δ 1.34, 1.56, and 1.61, two methylenes at δ 1.62
and 2.04 (2H, m), and four olefinic protons at δ 5.08 (1H,
t, J ) 7.0 Hz), 5.21 (2H, m), and 6.06 (1H, dd, J ) 10.7,
18.0 Hz). The presence of this moiety was supported by
7
12
obtained previously from E. japonica and Rosa laevigata,
â-hydroxy-2-oxopomolic acid (6) and 2-oxopomolic acid (7),
1
1
3
13
both isolated from R. woodsii, and 2R,19R-dihydroxy-3-
oxo-12-ursen-28-oic acid (8), a constituent of Geum japoni-
cum.14 Compound 9 is an oleanolic acid analogue and was
identified as arjunic acid, having been isolated from the
bark of Terminalia arjuna15 and T. macroptera.
the C NMR and DEPT data, in which the C-3 signal in
linalool was significantly shifted downfield (δ 81.4 vs 72.9),
suggesting that the hydroxy group at C-3 is glycosylated.
Acid hydrolysis of 1 yielded S-(+)-linalool,18 rhamnose, and
glucose, with the sugars being analyzed as their TMS
derivatives by GC-MS.19 The HMBC spectrum of 1 showed
the anomeric proton signal H-1′ (glc) (δ 4.33, d, J ) 7.8
Hz) coupled to C-3 of linalool (δ 81.4, s), the H-1′′ (rha-1)
16
The molecular formula for 1, C37
54
H O16, was determined
by 1 C NMR, DEPT, and FABMS data. The H NMR
3
1
spectrum of 1 (Table 1) indicated that the molecule
(
(
δ 4.77) signal coupled to C-6′ (glc) (δ 68.0, t), and the H-1′′′
rha-2) (δ 4.82) signal coupled to C-3′′ (rha-1) (δ 78.5, d).
*
To whom correspondence should be addressed. Tel: 886 2 27899590,
These correlations helped to determine the main part of
the structure of 1 as (3S)-O-R-L-rhamnopyranosyl-(1f3)-
R-L-rhamnopyranosyl-(1f6)-â-D-glucopyranosyllinalool. The
coumaroyl moiety in 1 was assigned at C-4′′ of the central
ext 451. Fax: 886 2 27827954. E-mail: chou@gate.sinica.edu.tw.
†
Institute of Botany, Academia Sinica.
School of Pharmacy, National Taiwan University.
National Research Institute of Chinese Medicine.
‡
§
⊥
Department of Biological Sciences, National Sun Yat-Sen University.
1
0.1021/np0100237 CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/06/2001

8
66 J ournal of Natural Products, 2001, Vol. 64, No. 7
Lee et al.
structure was supported by the FABMS data, which
showed prominent fragments at m/z 639, 439, 293, and 147.
1
13
Complete H and C NMR data (Table 1) assignment of
, especially in the sugar region, was accomplished by
1
analysis of two-dimensional COSY-45, NOESY, TOCSY,
HMQC, and HMBC NMR spectra.
Compound 2 was assigned the same molecular formula
as 1, as deduced from its FABMS and 13C NMR data. It
possessed spectroscopic data closely comparable to those
of 1 except for those in the coumaroyl part of the molecule.
1
Its H NMR exhibited signals for two cis-conjugated olefinic
protons at δ 5.77 and 6.92 (J ) 12.9 Hz) in contrast to the
trans-conjugated signals (δ 6.33 and 7.65, J ) 15.9 Hz)
observed for 1. Hence, 2 contained a cis-coumaroyl moiety,
which was also supported by the evidence that H-5′′′′ and
1
H-9′′′′ appeared at a lower field in the H NMR spectrum
8
relative to those same signals in 1 (Table 1). Therefore,
the structure of 2 was established as (3S)-O-R-L-rham-
nopyranosyl-(1f3)-[4-O-(Z)-coumaroyl]-R-L-rhamnopyrano-
syl-(1f6)-â-D-glucopyranosyllinalool.
Compound 3 was assigned a molecular formula of
1
13
C
30
H
46
O
6
, as deduced from the HREIMS and H and
C
NMR data, one more oxygen atom than that of 8. Its IR
-1
spectrum indicated the presence of a carbonyl (1716 cm ),
-
1
a carboxylic acid group (1688 cm ), and a double bond
-
1
1
(
1653 cm ). The H NMR spectra of both 3 and 8 showed
characteristic signals for the olefinic proton H-12 (δ 5.73,
brs), H-18 (δ 3.08, s), and seven methyls including six
singlets and one doublet (H -29), consistent with a 19R-
3
hydroxyursolic acid skeleton.20 In addition, the H NMR
spectrum of 3 exhibited two mutually coupled carbinoyl
protons at δ 3.70 (d) and 4.88 (d), instead of only one
carbinoyl proton (H-2) in 8, which appeared as a double
doublet (δ 4.84, J ) 6.4, 12.6 Hz). This difference was also
reflected in their 13C NMR spectra, which indicated that a
methylene signal (C-1, δ 50.6, t) in 8 was replaced by an
oxygenated methine (δ 87.1, d) in 3. To account for this
difference, C-1 of 3 must be hydroxylated, and OH-1 and
OH-2 should be â- and R-oriented, respectively, to fit the
diaxial coupling constant between H-1 and H-2 (J ) 9.6
Hz). The NOESY spectrum of 3 exhibited mutual correla-
1
tions between H-2, H
axially oriented. The HMBC spectrum revealed a three-
bond coupling of H -25 (δ 1.43) to C-1 (δ 87.1), which was
3 3
-24, and H -25, confirming H-2 to be
3
directly coupled to H-1, as observed in the HMQC spec-
trum, confirming C-1 to be hydroxylated. Furthermore,
correlations between the carbonyl carbon (δ 214.1) and two
3 3
methyl protons (δ 1.02, H -24; δ 1.24, H -23) in the HMBC
spectrum confirmed the ketone group to be located at the
C-3 position. Further analysis of 2D NMR data allowed the
1
13
complete assignments of the H and C NMR spectra of
, and the results are listed in Table 2. Thus, compound 3
3
was established as 1â,2R,19R-trihydroxy-3-oxo-12-ursen-
2
8-oic acid.
Compound 4 was assigned a molecular formula of
C H O , by HREIMS, the same as that of 9. Similar to
30 48 5
1
rhamnose residue (rha-1) on the basis of observation of the
downfield-shifted signal of the H-4′′ (δ 5.18 vs 3.35) with
respect to the corresponding signal of the nonsubstituted
rhamnose unit (rha-2). This proposal was also confirmed
by the HMBC spectrum, which revealed a three-bond
coupling between H-4′′ and C-1′′′′ (δ 168.4) of the coumaroyl
unit. In addition, this coumaroyl moiety was in the trans
form, deduced from the coupling constant between H-2′′′′
and H-3′′′′ (J ) 15.9 Hz). Hence, 1 was established to be
that of 9, its H NMR spectrum exhibited characteristic
2
0
signals for a 19R-hydroxyoleanolic acid skeleton, namely,
seven methyl singlets and signals for an olefinic H-12 (δ
5.57, brs), and the mutually coupled H-18 (δ 3.64, brs) and
H-19 (δ 3.61, brs), as determined by the COSY-45 spec-
trum. Both spectra also displayed another two carbinoyl
proton signals, however, having quite different coupling
patterns. These two protons, H-2 and H-3, in 9 are diaxially
oriented, thus having a large coupling constant (J ) 9.5
Hz), with the corresponding signals in 4 exhibiting a much
smaller coupling constant. In addition, the coupling con-
(
3S)-O-R-L-rhamnopyranosyl-(1f3)-[4-O-(E)-coumaroyl]-R-
L-rhamnopyranosyl-(1f6)-â-D-glucopyranosyllinalool. This

Monoterpene Glycosides from Eriobotrya
J ournal of Natural Products, 2001, Vol. 64, No. 7 867
Ta ble 1. 1H and 13C NMR and 2D NMR Data for Compound 1 (methanol-d4, 500 MHz)
position
linalool
δC (ppm) mult.a
δH mult. (J /Hz)b
HMBC (HfC)
NOESY (HfH)
1
2
3
4
5
6
7
8
9
1
3
1
2
3
4
5
6
6
6
1
2
3
4
5
6
3
1
2
3
4
5
6
4
1
2
3
4
5
6
7
8
9
115.3 t
144.4 d
81.4 s
41.8 t
5.21 m
6.06 dd (10.7, 18.0)
2,3
3,4,9
2
1
1.62 m
2.04 m
5.08 t (7.0)
2,3,6,9
3,4,6,7
4,5,8,10
23.7 t
125.7 d
132.1 s
17.8 q
23.6 q
25.9 q
1.56 s
1.34 s
1.61 s
6,7,10
2,3,4
6,7,8
0
-O-glc
′
′
′
′
′
′a
′b
′-O-rha
′′
′′
′′
′′
′′
′′
′′-O-rha
′′′
′′′
′′′
′′′
′′′
′′′
99.2 d
75.1 d
78.3 d
71.7 d
76.4 d
68.0 t
4.33 d (7.8)
3.18 dd (7.8, 8.9)
3.32
3.27
3.27
2′, 3′,3
1′,3′
2′,4′
3′, 5′
3′, 4′, 6′
1′′,5′
2′, 3′, 5′
1′
1′
1′, 6′a, 6′b
1′′,5′, 6′b
1′′,5′, 6′a,
3.56 dd (6.0, 10.4)
3.93 dd (3.6, 10.4)
1′′,5′
101.9 d
72.0 d
78.5 d
74.2 d
67.9 d
17.9 q
4.77 brs
3.98
4.00
5.18 t (9.6)
3.95 dq (9.6, 6.2)
1.16 d (6.2)
2′′,3′′,5′′,6′
3′′, 4′′
1′′′,4′′
1′′′′,3′′, 5′′, 6′′
4′′,3′′, 6′′
4′′,5′′
2′′,5′′,6′a,6′b
1′′
1′′′, 4′′,5′′′
3′′,5′′, 6′′
1′′, 4′′,6′′
4′′,5′′
104.1 d
72.4 d
72.1 d
73.9 d
70.2 d
18.1 q
4.82 d (1.5)
3′′,3′′′,5′′′
3′′′, 4′′′
4′′′
2′′′, 6′′′
3′′′, 4′′′,6′′′
4′′′, 5′′′
2′′′,3′′
1′′′
4′′′
3′′′,5′′′, 6′′′
3′′,4′′′
4′′′, 5′′′
3.68 dd (1.5, 3.3)
3.70 dd (3.3, 9.4)
3.35
3.77 dq (9.4, 6.2)
1.24 d (6.2)
′′-coumaroyl
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
168.4 s
114.6 d
147.4 d
127.1 s
131.4 d
116.8 d
161.5 s
116.8 d
131.4 d
6.33 d (15.9)
7.65 d (15.9)
1′′′′, 3′′′′, 4′′′′
1′′′′,2′′′′, 4′′′′, 5′′′′, 9′′′′
3′′′′, 5′′′′, 9′′′′
2′′′′,5′′′′, 9′′′′
7.47 d (8.6)
6.80 d (8.6)
3′′′′,6′′′′, 7′′′′,8′′′′, 9′′′′
4′′′′,7′′′′,8′′′′
2′′′′,3′′′′, 6′′′′
5′′′′
6.80 d (8.6)
7.47 d (8.6)
4′′′′, 6′′′′,7′′′′
3′′′′,5′′′′,6′′′′,7′′′′,8′′′′
9′′′′
2′′′′,3′′′′, 8′′′′
a
Multiplicities were obtained from DEPT experiments. b Signals without multiplicity were picked up from COSY-45 or HMQC spectra.
stants between H-2 and H
2
-1 were found to be 12.1 and
from E. deflexa were tested for their antiproliferation
activity on human mononuclear cells involving T lympho-
cytes, B lymphocytes, and macrophages isolated from
peripheral blood.23 Compounds 3, 5, 6, 7, and 8, having IC50
values of 26.9, 28.8, 32.5, 38.1, and 40.0 µM, respectively,
were found to be moderately active in contrast to cy-
closporin A, with a IC50 value of 0.012 µM. However,
compounds 1 and 2 did not show any activity up to 100
µM concentration. The quantities of 4 and 9 obtained were
insufficient to perform this biological investigation.
3
.9 Hz, corresponding to a diaxial and an axial-equatorial
coupling, respectively. Thus, H-2 was axially oriented.
Consequently, H-3 was deduced to be equatorially oriented.
In the NOESY spectrum, the signal of H-2 showed cor-
3 3
relations with those of H-3, H -24, and H -25, confirming
both H-2 and H-3 to be â-oriented. Its HMBC spectrum
displayed the correlation of H-3 to C-1, C-2, C-4, C-5, C-23,
and C-24, confirming H-3 to be hydroxylated. Further
analysis of these 2D NMR data allowed the complete
1
13
assignment of its H and C NMR spectra, and the results
are listed in Table 2. Accordingly, compound 4 was assigned
as 2R,3R,19R-trihydroxy-12-oleanen-28-oic acid.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured using a J ASCO DIP-180 digital spectropola-
rimeter. UV spectra were measured in MeOH on a Hitachi
U-2000 spectrophotometer. The IR spectra were recorded on
a Nicolet 510P FT-IR spectrometer. The NMR spectra were
The inflammatory response is a nonspecific immune
response triggered by pathogenic microorganism infection
or tissue injury and provides early protection in restricting
2
1
the tissue damage to the site of infection or tissue injury.
4 5
recorded in MeOH-d or pyridine-d at room temperature on
Several immune cells including lymphocytes, neutrophils,
monocytes, eosinophils, and basophils are involved in the
inflammatory response, which in response to invasive
organisms, if sufficiently intense or inappropriately pro-
longed, could paradoxically aggravate the injury or even
cause death. The use of antiinflammatory medications
must therefore be discreet. Blockade of the lymphocyte
activation and proliferation is an antiinflammatory mech-
anism.22 In this investigation the isolated pure compounds
a Bruker DMX-500 SB spectrometer, and the solvent reso-
nances were used as internal shift references. The 2D NMR
spectra were recorded using standard pulse sequences. FABMS
were recorded on a J EOL SX-102A instrument using m-
nitrobenzyl alcohol (NBA) as the matrix. Sephadex LH-20
(Pharmacia Biotech) was used for open column chromatogra-
phy. TLC was performed using silica gel 60 F254 plates (200
µm, Merck). HPLC was performed using an ODS column
(Hyperprep ODS, 10 mm i.d. × 250 mm, Keystone Scientific

8
68 J ournal of Natural Products, 2001, Vol. 64, No. 7
Lee et al.
Ta ble 2. 1H and 13C NMR Data for Compounds 3 and 4 (pyridine-d5, 500 MHz)
3
4
position
1
δC (ppm) mult.a
δH mult. (J /Hz)b
δC (ppm) mult.a
δH mult. (J /Hz)b
1.80 t (12.1)
.95 dd (3.9, 12.1)
87.1 d
3.70 d (9.6)
43.1 t
1
2
3
4
5
6
76.4 d
214.1 s
48.5 s
53.9 d
19.5 t
4.88 d (9.6)
66.6 d
79.9 d
39.4 s
49.4 d
19.1 t
4.34 dt (3.9, 12.1)
3.79 brs
1.16
1.52 m
1.69
1.42 m
1.58 m
1.39 m
1.61 m
1
.59 m
1.41 m
.57 m
7
33.9 t
33.8 t
1
8
9
0
1
41.5 s
48.9 d
44.0 s
28.4 t
40.7 s
48.7 d
39.3 s
24.7 t
2.23 dd (7.4, 10.8)
2.65 m
2.13
1
1
2.12 m
5.57 brs
3
.28 m
1
1
1
1
2
3
4
5
129.8 d
139.4 s
42.6 s
5.73 brs
124.0 d
145.4 s
42.7 s
29.9 t
1.31 m
29.6 t
2.10-2.20
2
.35 m
2.10 m
.15 m
1
6
26.9 t
28.9 t
2.16 m
2.83 m
3
1
1
1
2
2
7
8
9
0
1
48.8 s
55.0 d
73.1 s
42.8 d
27.4 t
46.5 s
45.3 d
81.7 d
36.2 s
29.7 t
3.08 s
3.64 brs
3.61 brs
1.50 m
1.36 m
2.10-2.20
2
.12 m
2.08 m
.18 m
2
2
38.9 t
34.2 t
2.05 m
2.21m
1.28 s
0.93 s
1.02 s
1.09 s
1.39 s
2
2
2
2
2
2
2
2
3
3
4
5
6
7
8
9
0
25.9 q
22.1 q
13.3 q
18.2 q
25.2 q
181.2 s
17.2 q
27.5 q
1.24 s
1.02 s
1.43 s
1.23 s
1.70 s
29.9 q
22.7 q
17.0 q
18.1 q
25.3 q
181.5 s
29.3 q
25.3 q
1.13 d (6.5)
1.45 s
1.20 s
1.13 s
a
Multiplicities were obtained from DEPT experiments. b Signals without multiplicity were picked up from COSY-45 or HMQC spectra.
Inc., Bellefonte, PA; detector, UV 254 nm) and a silica column
the monoterpene glycoside fractions from the above separation
were combined and rechromatographed on a reversed-phase
(Hyperprep HS silica, 10 mm i.d. × 250 mm, ThermoQuest
Hypersil, Runcorn, UK; detector, RI). GLC: HP gas chromato-
graph 6890; MS, HP 5973 mass selective detector; column,
HP-5MS fused silica capillary column, 0.25 mm i.d. × 30 m,
carrier gas, He; flow rate, 1 mL/min.
HPLC column with MeCN-H
(360 mg) and 2 (195 mg).
2
O (40:60) as eluent to yield 1
Fractions collected from the chloroform layer were further
purified by repetitive HPLC separations on a Hyperprep ODS
P la n t Ma ter ia l. Leaves of E. deflexa were collected from
Central Cross Island Road in central Taiwan in April 2000
and were identified by Chii-Cheng Liao, a Ph.D. candidate in
the Department of Botany of National Taiwan University.
Voucher specimens (No. 20000405) have been deposited at the
Institute of Botany, Academia Sinica, Nankang, Taipei, Tai-
wan.
Extr a ction a n d Isola tion . Dried leaves (500 g) were
ground into a powder and extracted three times with 500 mL
of MeOH for 2 days. The methanolic extract was adjusted to
column with MeCN-H
and 2. Subsequently, HPLC of subfraction 1 on a silica
column with CH Cl -EtOAc (3:7) as eluent afforded 3 (6 mg),
(4 mg), 5 (10.8 mg), 6 (12 mg), and 9 (3.5 mg). Subfraction 2
2
O (60:40) as eluent to give subfractions
1
2
2
4
was purified by using the same column with n-hexane-EtOAc
(2:1) as eluent to yield 7 (12 mg) and 8 (14 mg).
(3S)-O-r-L-r h am n opyr an osyl-(1f3)-[4-O-(E)-cou m ar oyl]-
r-L -r h a m n op yr a n osyl-(1f6)-â-D -glu cop yr a n osyllin a -
2
5
lool (1): amorphous white powder; mp 144-146 °C; [R]
-31.8° (c 0.67, MeOH); IR (KBr) νmax 3413, 1605, 1169, 1046
D
8
5% in aqueous solution for an n-hexane partition, which
-
1
generated two fractions soluble in methanol and n-hexane.
Subsequently, the methanol-soluble fraction was then vacuum-
evaporated to dryness (75 g) and further partitioned between
chloroform and water, and the remaining water solution was
extracted three times with n-butanol. Both the n-butanol and
chloroform layers were evaporated to dryness and redisssolved
in MeOH for individual chromatographic separation. The first
separation step was carried out using gel filtration chroma-
tography on a Sephadex LH-20 column (3 × 55 cm) and eluted
by MeOH with a flow rate of 13 mL/min.
cm ; UV λmax (MeOH) (log ꢀ) 203 (4.0), 227 (3.9), 313 (4.2)
1 13
nm; H and C NMR data, see Table 1; FABMS m/z [M +
+
Na] 777 (20), 639 (4), 585 (3), 439 (17), 293 (47), 154 (48),
+
1
7
47 (100), 136 (37), 82 (35), 69 (56); HRFABMS m/z [M + H]
55.3472 (calcd for C37 16 755.3490); R
H-H O (85:10:15)].
3S)-O-r-L-r h am n opyr an osyl-(1f3)-[4-O-(Z)-cou m ar oyl]-
r-L -r h a m n op yr a n osyl-(1f6)-â-D -glu cop yr a n osyllin a -
H
55
O
f
0.32 [EtOAc-
HCO
2
2
(
2
5
lool (2): amorphous white powder; mp 136-137 °C; [R]
87.4° (c 0.39, MeOH); IR (KBr) νmax 3401, 1605, 1163, 1046
D
-
Fractions collected from the n-butanol layer were checked
-
1
1
for their compositions by TLC using CH
2
Cl
2
-EtOAc-HCO
2
H-
cm ; UV λmax (MeOH) (log ꢀ) 203 (4.1), 312 (4.1) nm; H NMR
(CD OD) δ 7.69 (2H, d, J ) 8.7 Hz, H-5′′′′, -9′′′′), 6.92 (1H, d,
H
2
O (15:70:5:1) for development. Vanillin-sulfuric acid (yel-
3
low-green spots) and observation under long-wave UV were
used in the detection of monoterpene glycosides. Subsequently,
J ) 12.9 Hz, H-3′′′′), 6.76 (2H, d, J ) 8.7 Hz, H-6′′′′, -8′′′′),
6.04 (1H, dd, J ) 10.7, 18.0 Hz, H-2), 5.77 (1H, d, J ) 12.9

Monoterpene Glycosides from Eriobotrya
J ournal of Natural Products, 2001, Vol. 64, No. 7 869
Hz, H-2′′′′), 5.20 (2H, m, H
.09 (1H, t, J ) 7.3 Hz, H-6), 4.79 (1H, brs, H-1′′′), 4.75 (1H,
brs, H-1′′), 4.32 (1H, d, J ) 7.8 Hz, H-1′), 3.95 (1H, H-2′′), 3.92
1H, dd, J ) 3.3, 10.5 Hz, H-6′b), 3.90 (1H, H-3′′), 3.86 (1H,
dq, J ) 9.8, 6.2 Hz, H-5′′), 3.77 (1H, dq, J ) 9.4, 6.3 Hz, H-5′′′),
.71 (1H, H-2′′′), 3.70 (1H, H-3′′′), 3.54 (1H, dd, J ) 6.2, 10.5
Hz, H-6′a), 3.32 (1H, H-4′′′), 3.31 (1H, H-2′), 3.26 (1H, H-5′),
.23 (1H, H-4′), 3.17 (1H, dd, J ) 7.9, 9.0 Hz, H-2′), 2.04 (2H,
m, H -5), 1.65 (3H, s, H -10), 1.61 (2H, m, H -4), 1.58 (3H, s,
-8), 1.33 (3H, s, H -9), 1.24 (3H, d, J ) 6.2 Hz, H -6′′′), 1.16
3H, d, J ) 6.2 Hz, H OD) δ 167.2 (s, C-1′′′′),
2
-1), 5.12 (1H, t, J ) 9.8 Hz, H-4′′),
performed on a fused silica capillary column using a temper-
ature gradient of 60 to 140 °C at 25 °C/min, increased to 250
°C at 5 °C/min, and then increased to 300 °C at 10 °C/min.
When compared with the authentic standards, the retention
time of persilylated glucose and rhamnose was found to be
15.54 (15.89) and 10.08 (10.30) min, respectively.
5
(
3
3
Ack n ow led gm en t. This research was supported by grants
to C.-H.C. from the National Science Council (NSC-89-2311-
B001-026) and a postdoctoral fellowship to T.-H.L. from
Academia Sinica, Taiwan. We are grateful to Ms. Shoou-Ling
Huang for the NMR data acquisition in the Instrumentation
Center of the College of Science, National Taiwan University.
We also want to thank Dr. Kay-Hooi Khoo (Institute of
Biochemistry, Academia Sinica, Taipei, Taiwan) for the
monosaccharide composition data.
2
3
2
H
(
1
1
3
3
3
3
1
3
-6′′); C NMR (CD
3
60.3 (s, C-7′′′′), 146.8 (d, C-3′′′′), 144.3 (d, 134.0 (d, C-5′′′′, -9′′′′),
32.1 (s, C-7), 127.5 (s, C-4′′′′), 125.8 (d, C-6, 115.9 (d, C-6′′′′,
-
8′′′′), 115.8 (d, C-2′′′′), 115.3 (t, C-1), 104.1 (d, C-1′′), 101.9 (d,
C-1′′), 99.2 (d, C-1′), 81.4 (s, C-3), 78.6 (d, C-3′′), 78.3 (d, C-3′),
6.5 (d, C-5′), 75.1 (d, C-2′), 73.9 (d, C-4′′′), 73.8 (d, C-4′′), 72.5
d, C-2′′′), 72.1 (d, C-3′′′), 72.0 (d, C-2′′), 71.9 (d, C-4′), 70.2 (d,
C-5′′′) 68.2 (t, C-6′), 68.0 (d, C-5′′), 41.8 (t, C-4), 25.9 (q, C-10),
3.7 (t, C-5), 23.6 (q, C-9), 18.1 (q, C-6′′′), 18.0 (q, C-6′′), 17.8
7
(
Refer en ces a n d Notes
2
+
(1) Liu, T. S. In Flora of Taiwan; Li, H. L., Liu, T. S., Huang, T. C.,
Koyama, T.;, DeVol, C. E., Eds.; Epoch Publishing Co., Ltd.: Taipei,
Taiwan, 1977; Vol. 3; p 65.
(2) Chiu, N. Y., Chang, K. H., Eds. The Illustrated Medicinal Plants of
Taiwan; SMC Publishing Inc.: Taipei, Taiwan, 1995; Vol. 4.
(
2
q, C-8); FABMS m/z [M + Na] 777 (23), 639 (3), 439 (10),
93 (25), 176 (14), 154 (52), 147 (80), 136 (40), 82 (48), 69 (100);
+
HRFABMS m/z [M + H] 755.3478 (calcd for C37
55.3490); R 0.38 [EtOAc-HCO H-H O (85:10:15)].
â,2r,19r-Tr ih yd r oxy-3-oxo-12-u r sen -28-oic a cid (3):
H
55
O
16
7
f
2
2
(
3) Chiu, N. Y., Chang, K. H., Eds. The Illustrated Medicinal Plants of
1
Taiwan; SMC Publishing Inc.: Taipei, Taiwan, 1995; Vol. 5.
2
5
amorphous white powder; mp 218-220 °C; [R]
0
1
D
+29.7° (c
(4) de Tommasi, N.; Simone, F. D.; Aquino, R.; Pizza, C.; Liang, Z. Z. J .
Nat. Prod. 1990, 53, 810-815.
(5) de Tommasi, N.; Aquino, R.; Simone, F. D.; Pizza, C. J . Nat. Prod.
.59, MeOH); IR (KBr) νmax 2934, 1716, 1688, 1653, 1558, 1541,
456 cm ; H and C NMR data, see Table 2; FABMS m/z
-
1
1
13
1
992, 55, 1025-1032.
+
[
(
(
M + Na] 525 (100), 439 (18), 307 (12), 233 (8), 187 (18), 154
(
6) Yanagisawa, H.; Ohshima, Y.; Okada, Y.; Takahashi, K.; Shibata, S.
+
42), 119 (40), 91 (50), 56 (52); HREIMS m/z [M] 502.3256
Chem. Pharm. Bull. 1988, 36, 1270-1274.
(7) Shimizu, M.; Uemitsu, N.; Shirota, M.; Matsumoto, K.; Tezuka, Y.
calcd for C30
H
46
O
6
502.3294); R
f
0.5 [EtOAc-CH
2
Cl
2
(7:3)].
Chem. Pharm. Bull. 1996, 44, 2181-2182.
2
r,3r,19r-Tr ih yd r oxy-12-olea n en -28-oic a cid (4): amor-
(
8) de Tommasi, N.; Simone, F. D.; Pizza, C.; Mahmood, N.; Moore, P.
S.; Conti, C.; Orsi, N.; Stein, M. L. J . Nat. Prod. 1992, 55, 1067-
1073.
2
5
phous white powder; mp 215-217 °C; [R]
D
+19.6°(c 0.1,
1 1
-
MeOH); IR (KBr) νmax 2932, 1693, 1682, 1454 cm ; H and
1
3
+
(9) Shimizu, M.; Fukumura, H.; Tsuji, H.; Tanaami, S.; Hayashi, T.;
Morita, N. Chem. Pharm. Bull. 1986, 34, 2614-2617.
10) de Tommasi, N.; Simone, F. D.; Cirino, G.; Cicala, C.; Pizza, C. Planta
Med. 1991, 57, 414-416.
C NMR data, see Table 2; FABMS m/z [M + Na] 511 (100),
07 (10), 246 (12), 201 (37), 187 (35), 145 (37), 119 (88), 91
4
(
+
(88), 56 (98); HREIMS m/z [M] 488.3470 (calcd for C30
H
48
O
5
4
88.3502); R
f
0.46 [EtOAc-CH
2
Cl
2
(7:3)].
(11) Ivarra, M. D.; Paya, N.; Villar, A. Planta Med. 1988, 54, 282-286.
(12) Fang, J .-M.; Wang, K.-C.; Cheng, Y.-S. Phytochemistry 1991, 30,
Acid Hyd r olysis of 1. Compound 1 (80 mg) was hydrolyzed
3
383-3387.
by 2 N HCl (10 mL) at room temperature overnight. The
(
13) Kashiwada, Y.; Wang, H.-K.; Nagao, T.; Kitanaka, S.; Yasuda, I.;
Fujioka, T.; Yamagishi, T.; Cosentino, L. M.; Kozuka, M.; Okabe, H.;
Ikeshiro, Y.; Hu, C.-Q.; Yeh, E.; Lee, K.-H. J . Nat. Prod. 1998, 61,
reaction mixture was then extracted with n-hexane (10 mL ×
3
). The n-hexane layer was evaporated to give a residue, which
1
090-1095.
was chromatographed by semipreparative HPLC (Hyperprep
(
14) Xu, H.-X.; Zeng, F.-Q.; Wan, M.; Sim, K.-Y. J . Nat. Prod. 1996, 59,
HS Silica, 250 × 10 mm) using n-hexane-EtOAc (2:1) as the
6
43-645.
2
5
eluent to give S-(+)-linalool (4.6 mg): [R]
D
+2.7° (c 0.23,
(15) Anjaneyulu, A. S. R.; Rama Prasad, A. V. Phytochemistry 1982, 21,
2057-2060.
1
8
20
1
CHCl
1H, dd, J ) 1.2, 10.8 Hz, H-1a), 5.19 (1H, dd, J ) 1.2, 17.3
Hz, H-1b), 5.89 (1H, dd, J ) 10.8, 17.3 Hz, H-2), 1.55 (2H, m,
-4), 2.00 (2H, m, H -5), 5.10 (1H, t, J ) 6.5 Hz, H-6), 1.58
3H, s, H -8), 1.26 (3H, s, H -9), 1.66 (3H, s, H -10); GC-MS
m/z 154 [M] ; R
3
) (lit. [R]
D
+19.2°); H NMR (500 MHz, CDCl
3
) δ 5.04
(
(
(
(
16) Conrad, J .; Vogler, B.; Klaiber, I.; Roos, G.; Walter, U.; Kraus, W.
(
Phytochemistry 1998, 48, 647-650.
17) Crews, P.; Rodr ´ı guez, J .; J aspars, M. Organic Structure Analysis;
Oxford University Press: New York, 1998.
18) Weast, R. C., Grasselli, J . G., Eds. Handbook of Data on Organic
Compounds; CRC Press: Boca Raton, FL, 1989; Vol. 5.
19) Sweeley, C. C.; Bentley, R.; Makita, M.; Wells, W. W. J . Am. Chem.
Soc. 1963, 85, 2497-2507.
H
2
2
(
3
3
3
+
f
0.47 [n-hexane-EtOAc (2:1)].
Mon osa cch a r id e Com p osition An a lysis of 1. Compound
1
(1 mg) was methanolyzed with 0.5 M methanolic HCl at 80
(20) Doddrell, D. M.; Khong, P. W.; Lewis, K. G. Tetrahedron Lett. 1974,
2
7, 2381-2384.
°C for 16 h. Evaporation of the reaction mixture gave a residue,
(
21) J aneway, C. A.; Travers, P.; Hunt, S.; Walport, M. Immunobiology:
The Immune System in Health and Disease; Garland Publishing
Inc.: New York, 1997.
which was treated with the Sylon HTP trimethylsilylation
reagent (Supelco, Bellefonte, PA) for 20 min at room temper-
ature. After removal of the excess reagent and organic solvent
by condensation, the persilylated products were dissolved in
n-hexane, and this solution was used for GC-MS analysis.
GC-MS analysis of the trimethylsilylated derivatives was
(22) Adams, D. O.; Hamilton, T. A. Annu. Rev. Immunol. 1984, 2, 283-
18.
23) Lin, L. C.; Kuo, Y. C.; Chou, C. J . J . Nat. Prod. 1999, 62, 405-408.
3
(
NP0100237