Citrusosides A-D and furanocoumarins with cholinesterase inhibitory activity from the fruit peels of citrus hystrix

Article abstract of DOI:10.1021/np100531x

Four new compounds, citrusosides A-D (1-4), and 15 known compounds were isolated from the hexanes and CH₂Cl₂ extracts of the peels of Citrus hystrix fruits. Compound 1 is a 1-O-isopropyl-6-O-β-d- glucopyranosyl ester of 5″,9″-dimethyl-2″,8″-decadienoic acid. Compounds 2-4 possess a 1-O-isopropyl-β-d-glucopyranosyl and a dihydroxyprenylfuranocoumarin moiety conjugated to the 3-hydroxy-3- methylglutaric acid as diesters. Several furanocoumarins were evaluated for their cholinesterase inhibitory activity. (R)-(+)-6′-Hydroxy-7′- methoxybergamottin, (R)-(+)-6′,7′-dihydroxybergamottin, and (+)-isoimparatorin showed IC₅₀ values of 11.2 ± 0.1, 15.4 ± 0.3, and 23 ± 0.2 μM, respectively. Bioassay results indicated that the presence of a dioxygenated geranyl chain in the test compounds is crucial for the inhibitory activity.

Full text of DOI:10.1021/np100531x

J. Nat. Prod. 2010, 73, 1879–1883
1879
Citrusosides A-D and Furanocoumarins with Cholinesterase Inhibitory Activity from the Fruit
Peels of Citrus hystrix
Juthamanee Youkwan,† Somyote Sutthivaiyakit,*,† and Pakawadee Sutthivaiyakit‡
Department of Chemistry and Center for InnoVation in Chemistry, Faculty of Science, Ramkhamhaeng UniVersity, Hua Mark,
Bangkapi, Bangkok 10240, Thailand, and Department of Chemistry and Center for InnoVation in Chemistry, Faculty of Science, Kasetsart
UniVersity, Chatuchak, Bangkok 10903, Thailand
ReceiVed July 30, 2010
Four new compounds, citrusosides A-D (1-4), and 15 known compounds were isolated from the hexanes and CH2Cl2
extracts of the peels of Citrus hystrix fruits. Compound 1 is a 1-O-isopropyl-6-O-ꢀ-D-glucopyranosyl ester of 5′′,9′′-
dimethyl-2′′,8′′-decadienoic acid. Compounds 2-4 possess a 1-O-isopropyl-ꢀ-D-glucopyranosyl and a dihydroxypre-
nylfuranocoumarin moiety conjugated to the 3-hydroxy-3-methylglutaric acid as diesters. Several furanocoumarins were
evaluated for their cholinesterase inhibitory activity. (R)-(+)-6′-Hydroxy-7′-methoxybergamottin, (R)-(+)-6′,7′-
dihydroxybergamottin, and (+)-isoimparatorin showed IC50 values of 11.2 ( 0.1, 15.4 ( 0.3, and 23 ( 0.2 µM,
respectively. Bioassay results indicated that the presence of a dioxygenated geranyl chain in the test compounds is
crucial for the inhibitory activity.
Citrus hystrix DC (syn. C. papeda Miq.),1 leech lime of the
Rutaceae family, is commonly found in Southeast Asia. It is a small
plant of about 3-8 m in height. The leaves and fruits of this plant
have long been used as an ingredient in several Thai cooking
recipes. The fruits are pear-shaped with a rough, warty skin. Its
essential oil was reported to exhibit antibacterial activity.2 Extracts
of fruit peels were found to inhibit implantation, produce abortion,
and slightly hasten labor time in pregnant rats.3 Previous phy-
tochemical investigations revealed the presence of terpenes,4
phenolic compounds,5 glyceroglycolipids,6 and coumarins.7 We
report herein the isolation and spectroscopic data of compounds
1-4 from the fruit peels of this plant and cholinesterase inhibitory
activity of some of the isolates.
Our study on bioactive compounds from C. hystrix led to the
isolation of four new 1-O-isopropyl-ꢀ-D-glucopyranoside conju-
gates, citrusosides A-D (1-4), in addition to six known furano-
coumarins, (+)-oxypeucedanin (5),7,8 (R)-(+)-oxypeucedanin hy-
drate (6),9 (+)-isoimparatorin (7),10 (R)-(+)-6′-hydroxy-7′-
methoxybergamottin (8),10 (R)-(+)-6′,7′-dihydroxybergamottin (9),7,11
and (+)-bergomottin (10),7,11 a eudesmane sesquiterpene, (+)-4-
epi-crytomeridiol (eudesmane-4ꢀ,11-diol),12 five monoterpenes,
citronellal,13 citronellol,13 citronellol acetate,13 isopulegol,14 and
neo-isopulegol,14 as well as 1-O-isopropyl-ꢀ-D-glucopyranoside,15
sitosteryl-ꢀ-D-glucopyranoside,16 and ꢀ-sitosterol.16
4.38 was assigned to H2-6 of the glucopyranose ring connected to
Results and Discussion
an O-COR group. The two doublets at δH 1.21 and 1.17 (both J )
Compound 1 was isolated as a liquid. The HRESIMS spectrum
6.2 Hz), which coupled to a broad quintet at δH 3.95 (J ) 6.2 Hz),
showed an [M + Na]+ ion at m/z 423.2361 corresponding to an
indicated the presence of an O-isopropyl group. The 5′′,9′′-dimethyl-
elemental formula of C21H36O7Na. The FT-IR spectrum revealed
2′′,8′′-decadienoyl unit was detected from the 1H-1H COSY
absorption maxima for a hydroxy (3407 cm-1) and double-bond
spectrum, which showed cross-peaks between two double triplet
(1652 cm-1) functions. The 13C NMR spectrum exhibited 21 carbon
signals at δH 5.82 (J ) 15.6, 1.4 Hz, H-2′′) and 6.95 (J ) 15.3, 7.5
signals, comprising five methyl, four methylene, 10 methine, and
Hz, H-3′′) and sequential correlations from H-4′′ to H-8′′. Con-
two quaternary carbons, including one carbonyl and one olefinic
nectivity of an isopropyl group to an oxygen atom at C-1 and the
carbon. The presence of a ꢀ-D-glucopyranosyl unit was evident from
presence of an ester linkage between the carboxylic C-1′′ of the
NMR signals of a dioxygenated methine group at δH 4.31 (1H, d,
long-chain acid and an oxygen atom at C-6 of a ꢀ-D-glucopyranose
J ) 7.7 Hz, H-1) and δC 101.2 (C-1), in addition to signals of four
were detected from HMBC correlations between H-1/C-1′ and H-6/
oxymethine groups at δH 3.53-3.33 and δC 76.1, 73.9, 73.4, and
C-1′′, respectively. Compound 1 was named citrusoside A and
70.3, and of an oxymethylene group at δH 4.38 (d, J ) 3.9 Hz,
identified as 1-O-isopropyl-6-O-(5′′,9′′-dimethyl-2′′,8′′-decadienoyl)-
H-6) and δC 63.5 (C-6). The low-field two-proton doublet at δH
ꢀ-D-glucopyranoside. Reaction of 1 with Ac2O in pyridine/DMAP
1
gave a triacetate derivative of 1. Full assignment of H and 13C
* To whom correspondence should be addressed. Tel: (662) 319-0931.
NMR chemical shifts of 1 was as indicated in the Experimental
Fax: (662) 319-1900. E-mail: s_somyote@ru.ac.th.
† Ramkhamhaeng University.
‡ Kasetsart University.
Section (for the triacetate derivative, see Supporting Information,
Table S2).
10.1021/np100531x  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/21/2010

1880 Journal of Natural Products, 2010, Vol. 73, No. 11
Youkwan et al.
and C-5′′ to O-C-6′′′ were established from HMBC correlations of
H-6/C-1′′ and of H-6′′′/C-5′′, respectively. The structure of
compound 2 was assigned accordingly as shown and was given
the name citrusoside B. Upon acetylation, a triacetate derivative
with well-defined 1H NMR signals of H-2 (δH 4.90, t, J ) 8.4 Hz),
H-3 (δH 5.17, t, J ) 9.6 Hz), and H-4 (δH 5.01, t, J ) 9.8 Hz) was
1
obtained (for H and 13C NMR data of triacetate of 2, see Table
S3, Supporting Information).
On the basis of the configuration at C-6′ in 6′,7′-dihydroxyber-
gamottin as R using the Mosher ester method (see Figure 2),19,20
and on the proposal that 2 was biosynthesized from 3-hydroxy-3-
methylglutaric acid, 6′R,7′-dihydroxybergamottin, and 1-O-isopro-
pyl-ꢀ-D-glucopyranoside, the absolute configuration at C-6′′′ in 2
could thus be assigned as R.
Compound 3 was obtained as a liquid with the molecular formula
1
C31H40O15 by HRESIMS. The H and 13C NMR spectra showed
sets of signals of a 1-O-isopropyl-ꢀ-D-glucopyranosyl moiety, as
well as of a furanocoumarin and a 3-hydroxy-3-methylglutaryl
moiety, as found in 2. The presence of a 2′′′,3′′′-dihydroxyprenyl
group, in place of the 6′′′,7′′′-dioxygenated geranyl group, was
implied from the absence of an olefinic proton signal at ca. δH 5.51
Figure 1. Selected HMBC and COSY correlations of 1 and 2.
In order to clarify the configuration at C-5′′ in 1, compound 1
was transesterified using NaOEt/EtOH17 to give 5,9-dimethyldeca-
2E,8-dienoic acid ethyl ester (1a) and 1-O-isopropyl-ꢀ-D-glucopy-
ranoside. The specific rotation of the pure 1a in CHCl3 was found
to be [R]D -7.83 (c 0.3, CHCl3). The specific rotations of the 5S,9-
dimethyldeca-2E,8-dienoic acid ethyl ester and 5R,9-dimethyldeca-
2E,8-dienoic acid ethyl ester, prepared from Wittig reaction of the
triphenylphosphonium salt of ethyl R-chloroacetate with S-(-)- and
R-(+)-citronellal, respectively,18 were found to be +2.40 (c 0.35,
CHCl3) and -8.65 (c 0.22, CHCl3), respectively. On the basis of
these data, the configuration at C-5′′ in 1 was therefore concluded
to be R (see Supporting Information, Table S1, for NMR data, and
Scheme S1, Syntheses of 1a). In addition the 2:1 mixture of ꢀ-
1
with the presence of H NMR signals at δH 5.34 (dd, J ) 8.4, 2.5
Hz, H-2′′′), 4.67 (dd, J ) 10.0, 2.5 Hz, H-1′′′a), 4.56 (dd, J )
10.0, 8.4 Hz, H-1′′′b), 1.31 (s, H3-4′′′), and 1.30 (s, H3-5′′′) and
13C NMR signals at δC 78.2, 71.5, 71.0, 25.9, and 26.5. The long-
range HMBC correlations of the less-shielded carbinolic proton at
δH 5.34 with carbon signals of C-1′′′, C-3′′′, C-4′′′, C-5′′′, and C-5′′
at δC 71.0, 71.5, 25.9, 26.4, and 171.1, respectively, revealed
connectivity of the oxygen atom at C-2′′′ to a carboxylic carbon
(C-5′′) of the 3′′-hydroxy-3′′-methylglutaric acid moiety. The ether
linkage between C-1′′′ and C-5′′′′ was detected from HMBC
correlation between H-1′′′/C-5′′′′. The structure of compound 3
(citrusoside C) was therefore proposed as shown, and the assigned
1H and 13C NMR chemical shifts of 3 are given in Table 1 (for 1H
and 13C NMR data of the triacetate of 3, see Table S3, Supporting
Information).
1
and R-D-glucose detected by H NMR, with [R]D +34.5 (c 0.4,
H2O), was obtained after aqueous hydrolysis of 1-O-isopropyl-ꢀ-
D-glucopyranoside using ꢀ-D-glucosidase (from almonds, EC
3.2.1.21).
Compound 2 was obtained as a sticky liquid, and its HRESIMS
spectrum revealed a molecular formula of C36H48O15. The FT-IR
spectrum exhibited absorption maxima for OH (3368 cm-1) and
On the basis of the configuration at C-2′ in (+)-oxypeucedanin
hydrate, which was found to be R using the Mosher ester method
(see Figure 2),19,21 and on the proposal that 3 was biosynthesized
from 3-hydroxy-3-methylglutaric acid, 2′R-oxypeucedanin hydrate,
and 1-O-isopropyl-ꢀ-D-glucopyranoside, it could be concluded that
the absolute configuration at C-2′′′ in 3 was also R.
1
carbonyl (1731, 1626 cm-1) groups. The H NMR signals of an
anomeric proton at δH 4.33 (d, J ) 7.7 Hz, H-1), oxymethine
protons at δH 3.47-3.30, and oxymethylene protons at δH 4.40 (H-
6) indicated the presence of a ꢀ-D-glucopyranosyl moiety, and
signals at δH 1.20 and 1.16 (both d, J ) 6.4 Hz) and a quintet at
δH 3.94 (J ) 6.2 Hz) could be assigned to an O-isopropyl group,
as found in 1. The 1H NMR spectrum, which showed two pairs of
doublet signals at δH 8.15 and 6.25 (both with J ) 9.9 Hz), and
another two pairs at δH 7.58 and 6.93 (both with J ) 2.3 Hz), in
addition to 13C NMR signals (Table 1) at δC 161.4, 158.1, 152.6,
148.9, 114.3, 107.6 (all qC), 145.1, 139.8, 112.6, 105.0, and 94.4
(all CH), indicated compound 2 to possess a furanocoumarin unit.7
The 1H-1H COSY spectrum, which showed sequential connectivity
from H-1′′′ to H-6′′′, and HMBC correlations of H-1′′′/C-5′′′′, C-3′′′;
H-5′′′/C-3′′′, C-6′′′ (δC 80.0); and H3-8′′′, H3-9′′′/C-6′′′, C-7′′′ (δC
72.3) indicated the presence of a 6′′′,7′′′-dioxygenated geranyl
moiety connected to O-C-5′′′′ of the furanocoumarin nucleus, as
found in 6′,7′-dihydroxybergamottin,7,11 which was also isolated
Compound 4 was isolated as a colorless liquid with a similar
1
elemental formula, C31H40O15, to 3. The H and 13C NMR spectra
exhibited similar sets of signals for a furanocoumarin, a 1-O-
isopropyl-ꢀ-D-glucopyranosyl, a 2,3-dihydroxyprenyl, and a 3-hy-
droxy-3-methylglutaryl unit. Connectivity among units was detected
by HMBC correlations and found to be rather similar to those in
compounds 2 and 3. The 1H,1H-COSY spectrum indicated,
however, that H-3 resonated at a less-shielded position (δH 4.98, t,
J ) 9.5 Hz) and for H-6 at higher field (δH 3.87 and 3.81, both as
dd, J ) 12.0, 3.8 Hz) than those of the corresponding protons in 3,
thus indicating the bonding of an O-acyl group at C-3, instead of
at C-6 as in 3. The presence of an ester linkage between the
carboxylic C-1′′ of the 3′′-hydroxy-3′′-methylglutaric acid and an
oxygen atom at C-3 of a ꢀ-D-glucopyranose moiety was further
supported by HMBC correlations between H-3/C-4, C-1′′. The
structure of 4 (citrusoside D) was thus elucidated as shown. Full
assignments of 1H and 13C NMR resonances are given in Table 1.
1
in this study. The less-shielded H NMR resonances at δH 4.83
(dd, J ) 9.9 and 2.6 Hz) assigned to H-6′′′ required the bonding
of C-6′′′ to an O-acyl group. Key NMR signals of two methylene
groups at δH 2.68 and 2.63 (both doublets with J ) 14.7 Hz, H-2′′),
δC 46.0 (C-2′′) and at δH 2.79 and 2.54 (both doublets with J )
14.6 Hz, H-4′′), δC 45.1 (C-4′′) with methyl signals at δH 1.36 (H-
6′′), δC 27.4 (C-6′′), and three quaternary carbon signals at δC 171.6
(C-1′′), 171.9 (C-5′′), and 70.1 (C-3′′), in conjunction with HMBC
correlations between H-2′′/C-1′′, C-3′′, C-4′′, and C-6′′ and between
H-4′′/C-2′′, C-3′′, C-5′′, and C-6′′, indicated the presence of a
3-hydroxy-3-methylglutaryl unit. Connectivities from C-1′′ to O-C-6
Due to a previous report on the anticholinasterase properties of
several coumarins,22 butyrylcholinesterase inhibitory activity of the
isolates was investigated. The results are shown in Table 2.
Compound 8 was found to possess the highest potency, showing
an IC50 value of 11.2 ( 0.1 µM, whereas compounds 9 and 7
showed IC50 values of 15.4 ( 0.3 and 23 ( 0.2 µM, respectively.
Galanthamine, a positive control, showed an IC50 value of 3.2 (
0.2 µM. This bioassay indicated the presence of a dioxygenated

Citrusosides from the Fruit Peels of Citrus hystrix
Journal of Natural Products, 2010, Vol. 73, No. 11 1881
a
Table 1. 1H and 13C NMR Data of 2-4 in CDCl3
2
3
4
position
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
4.33 d (7.7)
3.47
3.30 t (8.1)
3.47
101.1
76.3
73.4
70.2
73.5
63.7
4.30 d (7.8)e
3.48
101.1
76.2
73.5
70.1
73.3
63.7
4.42 d (7.7)
3.41g
4.98 t (9.5)
3.67 t (9.5)
3.40g
101.2
75.5
78.0
69.0
71.9
62.2
3.30 t (8.2)
3.46f
3.47
3.45f
4.42 d (12.0)
4.38 d (12.0)
3.94 quint (6.4)
1.20 d (6.4)
4.39 d (10.9)
4.30 d (10.9)e
3.90 quint (6.2)
1.12 d (6.2)
1.15 d (6.2)
3.87 dd (12.0, 3.4), 3.81 dd (12.0, 4.3)
1′
72.2
22.0d
23.4d
171.6b
46.0e
72.3
21.9
3.99 quint (6.2)
1.19 d (6.2)
1.23 d (6.2)
72.5
21.9
2′
3′
1.16 d (6.4)
23.4
23.4
1′′
2′′
171.6c
45.7
171.8d
46.1
2.68 d (14.7)
2.63 d (14.7)
2.62 d (14.7)
2.60 d (14.7)
2.73 d (14.2)
2.69 d (14.2)
3′′
4′′
70.1
70.1
45.4
70.4
44.6
2.79 d (14.6) 2.54 d (14.6)
45.1e
2.79 d (14.8)
2.72 d (14.8)
2.90 d (15.1) 2.57 d (15.1)
5″
6′′
1′′′
171.9b
27.4
69.6
171.1c
27.2
71.0
171.5d
26.8
71.5
1.36 s
4.91 brd (6.8)
1.34 s
1.39 s
4.67 dd (10.0, 2.5) 4.56 dd (10.0, 8.5)
4.64 dd (10.3, 2.9)
4.58 dd (10.3, 8.0)
5.34 dd (8.0, 2.9)
2′′′
5.51 t (6.8)
119.7
141.8
35.9
27.9
80.0
5.34 dd (8.5, 2.5)
78.2
71.5
25.9
26.5
78.1
71.2
25.8
27.7
3′′′
4′′′
2.04
1.31 s
1.30 s
1.32 s
1.32 s
5′′′
1.65h
6′′′
4.83 dd (9.9, 2.6)
7′′′
72.3
8′′′
1.169
1.173
1.65h
26.5f
24.1f
16.6
9′′′
10′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
7′′′′
8′′′′
9′′′′
10′′′′
11′′′′
12′′′′
161.4
112.6
139.8
148.9
114.3
158.1
94.4
152.6
107.6
105.0
145.1
161.4
112.8
139.4
148.1
113.1
158.1
94.2
152.4
106.7
104.9
145.3
161.5
113.2
139.1
148.4
113.3)
158.2
94.6
152.5
106.8
104.7
145.0
6.25 d (9.9)
8.15 d (9.9)
6.24 d (9.8)
8.03 d (9.8)
6.29 d (9.8)
8.07 d (9.8)
7.12 s
7.05 s
7.14 s
6.93 d (2.3)
7.58 d (2.3)
6.95 d (2.4)
7.57 d (2.4)
6.93 d (2.3)
7.59 d (2.3)
a Coupling constants are listed in parentheses in Hz. b-d Interchangeable signals. e-h Overlapping signals.
separated by CC using hexanes-CH2Cl2 (90:10 to 80:20) to give three
subfractions (1.1-1.3). Subfraction 1.2 after CC (silica gel,
hexanes-CH2Cl2, 90:10 to 40:60) gave five subfractions (1.2.1-1.2.5).
Isopulegol (48.9 mg) was obtained from subfraction 1.2.5. Purification
of subfraction 1.2.2 using silica gel CC (hexanes-CH2Cl2, 85:15) gave
citronellal (21.7 mg). Subfraction 1.2.3 after CC (silica gel,
hexanes-CH2Cl2, 85:15 to 80:20) gave citronellyl acetate (27.5 mg)
and neo-isopulegol (8.3 mg). Fraction 3 (6.88 g) was purified by CC
(silica gel, hexanes-EtOAc, 96:4 to 93:7) to give isoimperatorin (7,
55.1 mg), [R]29D +0.41 (c 0.53, CHCl3), and bergomottin (10, 7.0 mg),
geranyl chain in the test compounds to be crucial for the inhibitory
activity. Our study is among the few reports on the occurrence of
1-O-isopropyl-ꢀ-D-glucopyranoside15 in Nature.
Experimental Section
General Experimental Procedures. Melting points were measured
using an Electrothermal melting point apparatus and are uncorrected.
Optical rotations were recorded on a JASCO DIP 1020 polarimeter.
The IR spectra were obtained on a Perkin-Elmer 1760x FT-IR
1
spectrophotometer. The H and 13C NMR spectra were recorded with
[R]28 +2.89 (c 0.36, CHCl3), after CC (silica gel, CH2Cl2, then
D
a Bruker AVANCE 400 MHz spectrometer. Chemical shifts are
referenced to the residual solvent signals (CDCl3: δH 7.24 and δC 77.0
ppm). HRESIMS was recorded on a Bruker Daltonics microTOF mass
spectrometer.
reversed-phased C18, MeOH-H2O, 85:15). Fraction 5 was purified
(silica gel CC, hexanes-EtOAc, 90:10 to 50:50) and yielded five
subfractions (5.1-5.5). Subfraction 5.2 gave ꢀ-sitosterol (21.9 mg) after
CC (CH2Cl2-MeOH, 100:0 to 97:3). Subfraction 5.4 was fractionated
by CC (silica gel, CH2Cl2-MeOH, 99:1) to give three subfractions
(5.4.1-5.4.3). Subfraction 5.4.2 after further CC (reversed-phase C18,
MeOH-H2O, 70:30, then silica gel, CH2Cl2-MeOH, 99:1 to 97:3) gave
Plant Material. The fruits of Citrus hystrix DC. (Rutaceae), known
in Thailand as “Makruut”, was collected from Bangkunkong subdistrict,
Bangkruai district, Nonthaburi Province, Thailand, in June 2003. The
plant was identified by Assoc. Prof. Dr. Nijsiri Ruangrangsi, Department
of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn
University, Thailand. A voucher specimen (SSCH/2003) is maintained
at the Chemistry Department, Ramkhamhaeng University.
Extraction and Isolation. The dried, ground fruit peels (5.7 kg)
were extracted successively with hexanes, CH2Cl2, and MeOH at room
temperature to obtain, after solvent evaporation under reduced pressure,
hexanes (240.2 g), CH2Cl2 (155.0 g), and MeOH (384.8 g) extracts,
respectively.
(R)-(+)-6′-hydroxy-7′-methoxybergamottin (8, 16.6 mg), [R]28 +8.7
D
(c 0.68, CHCl3), and (+)-6′,7′-dihydroxybergamottin (9, 9.2 mg), [R]28
D
+6.1 (c 0.42, CHCl3). Fraction 7 (1.18 g) was purified using CC (silica
gel, CH2Cl2-MeOH, 99:1 to 85:15) to give five subfractions (7.1-7.5).
Subfraction 7.2 gave citronellol (39.8 mg). Subfraction 7.4 (780.5 g)
was purified using silica gel CC (hexanes-EtOAc, 60:40 to 50:50) to
give three subfractions (7.4.1-7.4.3). Subfraction 7.4.2 (190.5 g) after
CC (reversed-phase C18, MeOH-H2O, 60:40 to 100:0) yielded oxy-
peucedanin hydrate (6, 54.9 mg), [R]28 +20.4 (c 0.41, CHCl3), and
The hexanes extract (201.3 g) was fractionated using silica gel
column chromatography (CC) with a gradient of hexanes-CH2Cl2 (100:
0) to CH2Cl2-MeOH (70:30) to obtain eight fractions. Fraction 1 was
D
an additional quantity of dihydroxybergamottin (9, 17.4 mg). Subfrac-
tion 7.5 (466 mg) after CC (silica gel, hexanes-EtOAc, 70:30 to 0:100,

1882 Journal of Natural Products, 2010, Vol. 73, No. 11
Youkwan et al.
Subfraction 7.4 after purification (reversed-phase C18, MeOH-H2O,
60:40 to 100:0) gave three subfractions (7.4.1-7.4.3). Subfraction 7.4.2
(113.5 mg) was further purified using silica gel CC (CH2Cl2-MeOH,
97:3 to 95:5) to yield 3 (18.7 mg), and purification of subfraction 7.4.3
(mg) using silica gel CC (CH2Cl2-MeOH, 97:3 to 95:5) yielded 2 (11.8
mg). Fraction 9 (3.98 g) after CC (silica gel, CH2Cl2-MeOH, 95:5 to
80:20) gave three subfractions (9.1-9.3), and further purification of
subfraction 9.2 using CC (reversed-phase C18, MeOH-H2O, 60:40 to
100:0, then silica gel, EtOAc-MeOH, 97:3 to 94:4) gave 1-O-isopropyl-
ꢀ-D-glucopyranoside (10.1 mg).
Citrusoside A (1): colorless, sticky liquid; [R]30 -20.6 (c 0.66,
D
MeOH); FT-IR (KBr) νmax 3407, 2969, 2918, 1723, 1652, 1454, 1380,
1316, 1270, 1183, 1087, 1038, 927, 832, 643 cm-1; 1H NMR (CDCl3,
400 MHz) δH 6.95 (1H, dt, J ) 15.3, 7.5 Hz, H-3′′), 5.82 (1H, dt, J )
15.3, 1.4 Hz, H-3′′), 5.05 (1H, q, J ) 7.1, 1.2 Hz, H-8′′), 4.38 (1H, br
d, J ) 3.6 Hz, H-6), 4.31 (1H, d, J ) 7.7 Hz, H-1), 3.95 (1H, br quint,
J ) 6.2 Hz, H-1′), 3.53 (1H, br t, J ) 9.0 Hz, H-3), 3.45 (1H, dd, J )
9.7, 3.9 Hz, H-5), 3.37 (1H, br t, J ) 9.7 Hz, H-4), 3.33 (1H, m, H-2),
2.20 (1H, m, H-4′′a), 2.00 (1H, m, H-4′′b), 1.93 (1H, m, H-7′′a), 1.65
(3H, s, H-10′′), 1.61 (1H, m, H-5′′), 1.59 (1H, m, H-7′′b), 1.57 (3H, s,
H-11′′), 1.31 (1H, m, H-6′′a), 1.22 (1H, d, J ) 6.2 Hz, H-2′), 1.17
(1H, d, J ) 6.2 Hz, H-3′), 1.14 (1H, m, H-6′′b), 0.87 (3H, d, J ) 6.7
H, H-12′′); 13C NMR (CDCl3, 100 MHz) δC 167.1 (C, C-1′′), 149.5
(CH, C-3′′), 131.5 (C, C-9′′), 124.4 (CH, C-8′′), 121.7 (CH, C-2′′),
101.2 (CH, C-1), 76.1 (CH, C-3), 73.9 (CH, C-5), 73.4 (CH, C-2),
72.5 (CH, C-1′), 70.3 (CH, C-4), 63.5 (CH2, C-6), 39.7 (CH2, C-4′′),
36.7 (CH2, C-6′′), 32.0 (CH, C-5′′), 25.7 (CH3, C-10′′), 25.5 (CH2,
C-7′′), 23.4 (CH3, C-2′), 22.1 (CH3, C-3′), 19.5 (CH3, C-12′′), 17.6
(CH3, C-11′′); HRESIMS [M + Na]+ ion m/z 423.2361 (calcd for
C21H36O7Na, 423.2349).
Citrusoside B (2): colorless, sticky liquid; [R]30 -16.2 (c 0.40,
D
MeOH); FT-IR (KBr) νmax 3368, 2921, 1731, 1626, 1579, 1455, 1382,
1
1205, 1131, 1074, 825, 749 cm-1; H NMR (CDCl3, 400 MHz) and
13C NMR (CDCl3, 100 MHz) see Table 1; HRESIMS m/z 743.2885
[M + Na]+ (calcd for C36H48O15Na, 743.2877).
Citrusoside C (3): colorless, sticky liquid; [R]30 -2.8 (c 0.45,
Figure 2. Distribution of ∆δS-R values of the S- and R-MTPA esters
of (+)-6′-hydroxy-7′-methoxybergamottin (8), (+)-6′,7′-dihydroxy-
bergamottin (9),20 and (+)-oxypeucedanin hydrate (6).21
D
MeOH); FT-IR (KBr) νmax 3401, 2975, 2925, 1731, 1623, 1579, 1455,
1
1355, 1285, 1204, 1134, 1081,87, 751 cm-1; H NMR (CDCl3, 400
MHz) and 13C NMR (CDCl3, 100 MHz) see Table 1; HRESIMS m/z
675.2266 [M + Na]+ (calcd for C31H40O15Na, 675.2253).
Table 2. Cholinesterase Inhibitory Activity of Some Isolates
Citrusoside D (4): colorless, sticky liquid; [R]30 -4.7 (c 0.40,
D
compound
IC50 (µM) ( SD
MeOH); FT-IR (KBr) νmax 3401, 2924, 2852, 1731, 1623, 1579, 1456,
1382, 1354, 1285, 1257, 1206, 1157, 1135, 1080, 1039, 826, 751
cm-1;1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz)
see Table 1; HRESIMS m/z 675.2259 [M + Na]+ (calcd for
C31H40O15Na, 675.2253).
citrusoside A (1)
citrusoside B (2)
citrusoside D (4)
oxypeucedanin (5)
376 ( 2
339 ( 3
inactiveb
64.0 ( 0.2
inactivec
23.1 ( 0.2
3.2 ( 0.2
11.2 ( 0.1
15.4 ( 0.3
inactived
oxypeucedanin hydrate (6)
isoimperatorin (7)
Bioassays. Butyrylcholinesterase inhibitory activity of the isolates
was evaluated according to Ellman and co-workers23 with some
modification using acetylthiocholine iodide (as substrate), cholinesterase
(from horse serum, EC 3.1.1.8), and galantamine hydrobromide from
Lycoris sp. Absorbance was read at 405 nm every 5 s for 2 min. The
IC50 is the concentration of inhibitor required to decrease enzyme
activity by 50% and was determined in triplicate. The data are shown
as mean ( standard deviation values (Table 2).
galanthaminea
6′-hydroxy-7′-methoxybergamottin (8)
6′,7′-dihydroxybergamottin (9)
bergomottin (10)
a Positive control compound. b Inactive at 7.2 mM. c At 65.1 µM, %
inhibition was found to be 24.6. d Inactive at 14.8 mM.
then reversed-phase C18, MeOH-H2O, 60:40 to 100:0) yielded (+)-
4-epi-crytomeridiol (eudesmane-4ꢀ,11-diol, mp 120-122 °C, 35.1 mg),
Acknowledgment. We are grateful to the Thailand Research Fund
and Ramkhamhaeng University for financial support. J.Y. acknowledges
Center for Innovation in Chemistry (PERCH-CIC), the Commission
on Higher Education, the Ministry of Education, for a scholarship. P.S.
thanks the Alexander von Humboldt Foundation for the donation of a
plate multilabel counter. We acknowledge the Chemistry Departments
of Mahidol and Chiangmai Universities for HRMS measurements.
[R]28 +13.9 (c 0.24, CHCl3).
D
The CH2Cl2 extract (155 g) was fractionated using silica gel CC
with a gradient of CH2Cl2-MeOH (100:0 to 70:30) and gave 11
fractions. Fraction 3 (2.52 g) after CC (silica gel, CH2Cl2-MeOH, 97:3
to 85:15) yielded six subfractions (3.1-3.6). Subfraction 3.4 gave
oxypeucedanin (5, 562.5 mg), [R]28D +13.2 (c 0.05, CHCl3), after silica
gel CC (CH2Cl2-MeOH, 97:3 to 90:10). Subfraction 3.2 was purified
(silica gel, hexanes-EtOAc, 60:40 to 100:0, then reversed-phase C18,
MeOH-H2O, 70:30 to 100:0) to yield dihydroxybergamottin (9, 32.4
mg). Fraction 5 (3.35 g) after CC (silica gel, hexanes-EtOAc, 40:60,
to EtOAc-MeOH, 80:20) gave four subfractions (5.1-5.4). Subfraction
5.2 was further purified (reversed-phase C18, MeOH-H2O, 60:40 to
100:0, then silica gel, CH2Cl2-MeOH, 97:3 to 90:10) and yielded
compound 1 (21.7 mg). Fraction 7 (8.45 g) was purified (silica gel,
CH2Cl2-MeOH, 95:5 to 88:12) and gave six subfractions (7.1-7.6).
Subfracion 7.3.3 was further purified (silica gel, hexanes-EtOAc, 97:3
to 70:30, then reversed-phase C18, MeOH-H2O, 70:30 to 100:0) and
yielded an additional quantity of 2 (35.1 mg), 3 (18.4 mg), and 4 (13.1
mg). Subfraction 7.5 yielded ꢀ-sitosteryl glucopyranoside (65.9 mg).
1
Supporting Information Available: H and 13C NMR spectra of
1-4, Scheme S1 Syntheses of 1a, HMBC correlations of 2-4, tabulated
1H and 13C NMR data of R-(-)-1a, S-(+)-1a, and acetate derivatives
of 1, 2, and 3. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Citrusosides from the Fruit Peels of Citrus hystrix
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NP100531X