Hyaluronidase inhibitors from Keiskea japonica

Article abstract of DOI:10.1248/cpb.60.121

An extract of Keiskea japonica MIQ. showed an inhibitory effect on hyaluronidase activity. From the extract, four new phenylpropanoids, two new maltol glycosides, two new monoterpene glycosides, and two new phenolic compounds were isolated together with 19 known compounds. Among these constituents, two phenylpropanoids and a flavone glucuronide were revealed as hyaluronidase inhibitors.

Full text of DOI:10.1248/cpb.60.121

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January 2012
Regular Article
Chem. Pharm. Bull. 60(1) 121 128 (2012)
121
Hyaluronidase Inhibitors from Keiskea japonica
,a
Toshihiro Murata,* Toshio Miyase,b and Fumihiko Yoshizakia
a Department of Pharmacognosy, Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai
981–8558, Japan: and b School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka
422–8526, Japan. Received September 23, 2011; accepted October 28, 2011; published online November 4, 2011
An extract of Keiskea japonica MIQ. showed an inhibitory effect on hyaluronidase activity. From the
extract, four new phenylpropanoids, two new maltol glycosides, two new monoterpene glycosides, and two
new phenolic compounds were isolated together with 19 known compounds. Among these constituents, two
phenylpropanoids and a flavone glucuronide were revealed as hyaluronidase inhibitors.
Key words Keiskea japonica; Lamiaceae; hyaluronidase inhibitor; phenylpropanoid
Keiskea japonica, a herbaceous perennial with white flow- scopic data as 3′-O-methyl-rosmarinic acid (14),5) isovitexin
ers that belongs to the family Lamiaceae, grows in the moun- (19),6) vicenin-2 (20),7) maltol 6′-O-(5-O-p-coumaroyl)-(1 6)-
→
tainous areas of western Japan.1) As the ice that attaches to old β-D-apiofranosyl-β-D-glucopyranoside (21),8) (Z)-3-hexenyl
stems of Keiskea japonica MIQ. can produce artistic forma- β-D-glucopyranoside (23),9) perilloside E (25),10) benzyl β-D-
glucopyranoside (26),11) (6S,9S)-roseoside (27),12) (−)-(1R,2R)-
“
”
tions, the plant is named Shimobashira in Japanese.
We have searched for hyaluronidase inhibitory extracts of 5′-β-D-glucopyranosyloxyjasmonic acid (28),13) and (3R)-O-
Lamiaceae plants and reported phenylpropanoids and flavone β-D-glucopyranosyloxy-5-phenylvaleric acid (29).14) Other
—
4)
glucuronides.2
An extract of K. japonica also showed in- known compounds were directly compared with those isolated
hibitory activity (IC50 608μg/mL) and 29 compounds (1 29) previously and identified as caffeic acid (11),15) clinopodic acid
were isolated (Figs. 1, 2) from an 80% acetone extract of A (12),16) rosmarinic acid (13),16) luteolin (15),15) chrysoeriol
the plant. Known compounds were identified from spectro- (16),15) apigenin 7-O-β-D-glucuronopyranoside (17),3) acacenin
—
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Fig. 1. Structures of 1 10
*To whom correspondence should be addressed. e-mail: murata-t@tohoku-pharm.ac.jp
© 2012 The Pharmaceutical Society of Japan

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Fig. 2. Structures of 11 29
7-O-β-D-glucuronopyranoside (18),2) protocatechualdehyde fect (NOE) spectrum, and showed the presence of a methoxy
(22),15) and 3-O-[β-D-xylopyranosyl-(1-6)-β-D-glucopyranosyl]- group at C-3′. The 13C-NMR spectrum showed 19 carbon sig-
(3R)-1-octen-3-ol (24).17)
nals, and high resolution (HR)-FAB-MS [m/z 373.0928 (Calcd
Compounds 1 and 3 10 were new (Fig. 1), and 2 was iso- for C19H17O8: 373.0923)] indicated the molecular formula of 1
—
—
—
lated as a natural product for the first time. 1 4 and 11 14 to be C19H16O8. The H-7 proton signal (δ 4.55, brs) was long
were phenylpropanoids and 17 and 18 were flavone glucuro- range coupled with aromatic carbons at δ 133.7 (C-1), 115.1
nides (Fig. 2).
(C-2), and 119.4 (C-6) and an olefinic carbon at δ 122.5 (C-8′)
1
In the H-NMR spectrum of shimobashiric acid A (1), two and two carbonyl carbons at δ 173.7 (C-9) and 174.5 (C-9′) in
sets of ABX proton signals at (δ 6.77, d, J=2.0Hz; 6.77, d, the heteronuclear multiple bond correlation (HMBC) spectrum
J=8.5Hz; 6.67, dd, J=8.5, 2.0Hz) and (δ 6.89, d, J=2.0Hz; (Fig. 3). The olefinic proton signal at δ 7.68 (H-7′) was long
6.75, d, J=8.5Hz; 6.99, dd, J=8.5, 2.0Hz), and an olefinic range coupled with aromatic carbons at δ 126.9 (C-1′), 114.3
proton signal at δ 7.68 (d, J=2.0Hz) were observed in the (C-2′), and 128.0 (C-6′) and an aliphatic carbon at δ 49.5 (C-7).
aromatic region. In the aliphatic region, there were two proton In the H–1H correlation spectroscopy (COSY) spectrum, H-7
1
signals at δ 4.55 (brs) and 4.78 (overlapped) and a methoxy correlated with δ 4.78 (H-8) corresponding to an oxygenated
proton signal at δ 3.56 (3H, s). The methoxy proton signal carbon at δ 83.6 (C-8). H-8 was long range coupled with two
correlated with H-2′ (δ 6.89) in the nuclear Overhauser ef- carboxyl carbons at δ 173.7 and 174.5, which suggested that 1

January 2012
123
Fig. 3. Key HMBC and NOE Correlations for 1
—
Fig. 4. Key HMBC and NOE Correlations for 2 10
has a five-member ring as shown in Fig. 1. This planar struc- flicted with 8R-configurations in rosmarinic acid derivatives.
ture represents a state in which C-7 is linked with C-8′ in 14. Examples of known absolute configuration about the PGME
In the NOE spectra (Fig. 3), H-7 was correlated with H-2′ and method for α-oxy-α-monosubstituted acetic acids have been
H-6′ showing that C-7′ and C-8′ had an E-configuration. The too small to be conclusive. Hence, absolute configurations of 1
relative configurations of C-7 and C-8 were determined as rel- were not determined.
(7R,8R) based on the NOE correlation between H-8 and H-2
1H- and 13C-NMR spectra of shimobashiric acid B (2) are
(Fig. 3). For the phenylglycine methyl ester (PGME) method to shown in the experimental section. They were similar to those
determine absolute configurations of α-oxy-α-monosubstituted of rosmarinic acid (13) except for the existence of a methoxy
acetic acid including a γ-lactone, (S)- and (R)-PGME amides signal. HR-FAB-MS suggested that the molecular formula of
of 1 (1a,b) were synthesized. The difference in 1H-NMR 2 was C19H18O8. The methoxy proton signal at δ 3.90 (3H, s)
Δ
chemical shifts between 1a and b [ δ=δ(S)−δ(R)] suggested correlated with an aromatic proton at δ 7.00 (1H, d, J=8.5Hz,
C-8 to have an S-configuration.18) However, this result con- H-5′) in the NOE spectrum (Fig. 4). These data showed that 2

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was 4′-methoxy rosmarinic acid. The absolute configurations configurations of both C-8 and C-8″ were determined as R, as
of C-8 was determined to be R from the retention time of in the case of 2.
1
the PGME amide derivative of 3-(3,4-dihydroxyphenyl)-2-hy-
The H- and 13C-NMR spectra of shimobashirasides A (5)
droxypropanoic acid, which was obtained by acidic hydrolysis and B (6) were almost superimposable onto those of 21,8) ex-
of 2, in (S)-2-phenylglycine methyl ester (see Experimental).2)
cept for the aromatic region. For 5, ABX aromatic proton sig-
The molecular formula of shimobashiric acid C (3) was nals at δ 7.04 (1H, d, J=1.5Hz, H-2′), 6.78 (1H, d, J=8.0Hz,
established as C36H32O16 based on HR-FAB-MS [m/z 743.1597 H-5′), and 6.95 (1H, dd, J=8.0, 1.5Hz, H-6′) suggested a caf-
(Calcd for C36H32O16Na: 743.1587)]. In the 1H-NMR spec- feoyl group, instead of the p-coumaroyl group in 21. HR-FAB-
trum, four sets of ABX aromatic protons at δ 6.54 (1H, d, MS [m/z 583.1646 (Calcd for C26H31O15: 583.1662)] supported
J=2.0Hz, H-2), 6.62 (1H, d, J=8.0Hz, H-5), 6.34 (1H, dd, this conclusion. For 6, there were ABX system aromatic
J=8.0, 2.0Hz, H-6), 6.64 (1H, d, J=2.0Hz, H-2′), 6.57 (1H, d, proton signals at δ 7.20 (1H, d, J=2.0Hz, H-2′), 6.82 (1H, d,
J=8.0Hz, H-5′), 6.31 (1H, dd, J=8.0, 2.0Hz, H-6′), 6.60 (1H, J=8.0Hz, H-5′), and 7.08 (1H, dd, J=8.0, 2.0Hz, H-6′) and a
d, J=2.0Hz, H-2″), 6.65 (1H, d, J=8.0Hz, H-5″), 6.43 (1H, methoxy proton at δ 3.90 (3H, s), which correlated with the
dd, J=8.0, 2.0Hz, H-6″), 6.72 (1H, d, J=2.0Hz, H-2‴), 6.63 H-2′ proton in the NOE spectrum (Fig. 4). These data sug-
(1H, d, J=8.0Hz, H-5‴), 6.41 (1H, dd, J=8.0, 2.0Hz, H-6‴) gested that 6 has a feruloyl group. Again HR-FAB-MS [m/z
were observed. The H-2 and H-6 protons were correlated with 597.1804 (Calcd for C27H33O15: 597.1819)], supported this con-
methylene and methine protons at δ 2.50 (overlapped, H-7), clusion. Sugar identifications suggested that both 5 and 6 have
2.58 (1H, dd, J=14.0, 6.0Hz, H-7), and 4.43 (1H, t, J=6.0Hz, D-glucose and D-apiose.8,21) The coupling constant of Glc-1 [6:
H-8) in the NOE spectra (Fig. 4). The H-2″ and H-6″ protons δ 4.77, d, J=7.5Hz (Glc-1)] and chemical shifts of 13C-NMR
were also correlated with another methylene and methine [5: δ 105.4, 75.0, 78.5, 71.4, 77.5 (Glc-1-5) and δ 110.6 (Api-1);
protons at δ 2.71 (1H, dd, J=14.0, 7.0Hz, H-7″), 2.76 (1H, dd, 6 δ 110.6 (Api-1)] showed that the anomeric carbons of these
J=14.0, 6.0Hz, H-7″), and 4.58 (1H, t, J=6.0Hz, H-8″) in the sugars have β-configurations.8,22)
NOE spectra. The 13C-NMR spectrum showed that the pres-
Shimobashiraside C (7) was revealed to have the molecu-
ence of four carbonyl carbons at δ 170.2, 170.2, 171.1, and lar formula C20H20O11 based on HR-FAB-MS [m/z 437.1090
170.0, eight aliphatic carbons, and 24 aromatic carbons. These (Calcd for C20H21O11: 437.1083)]. In the 1H-NMR spectrum,
results suggested that 3 has four phenylpropanoid units includ- ABX system proton signals at δ 6.72 (1H, d, J=9.0Hz, H-3),
ing two 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid 7.19 (1H, dd, J=9.0, 3.0Hz, H-2), and 7.41 (1H, d, J=3.0Hz,
1
moieties. Although the H- and 13C-NMR data (in D2O) of 3 H-6) and ABCD system proton signals at δ 6.95 (overlapped,
were similar to those of sagerinic acid,19) they were not iden- H-3′), 7.54 (1H, m, H-4′), 6.97 (overlapped, H-5′), and 7.77
tical. The 1H1H COSY spectrum showed that four methine (1H, dd, J=7.5, 2.0Hz, H-6′) were observed in the aromatic
protons at δ 4.05 (1H, dd, J=10.5, 7.0Hz, H-7′), 3.76 (1H, dd, region. In the HMBC spectrum (Fig. 4), H-4 was long range
J=10.5, 7.0Hz, H-8′), 4.13 (1H, dd, J=10.5, 7.0Hz, H-7‴), and coupled with an aromatic carbon at C-2 (δ 156.4), H-6 was
3.67 (1H, dd, J=10.5, 7.0Hz, H-8‴) construct a cyclobutane coupled with the carbonyl carbon at δ 171.3, and an anomeric
ring. In the HMBC spectrum (Fig. 4), H-2′ and 6′ were cor- proton at δ 4.84 (1H, d, J=7.5Hz, H-Glc-1) was coupled with
related with C-7′ (δ 39.7), and H-2‴ and H-6‴ were correlated C-5 (δ 149.1). These data showed that an aglycone moiety
with C-7‴ (δ 40.8). These results showed that 3 was a dimer of of 7 was a gentisic acid, which was similar to the gentisic
two rosmarinic acid moieties as shown in Fig. 1. In the NOE acid 5-O-β-D-glucopyranoside.23) 1H–1H COSY and 13C-NMR
spectra, the H-2′ and -6′ protons were correlated with H-8′ spectra showed a sugar moiety that was a 6-acylated glucose.
and 7‴; H-2‴ and -6‴ protons were correlated with H-7′ and The sugar analysis21) and the coupling constant of H-Glc-1
8‴, which suggested the conformation revealed in Fig. 1.19) suggested that the glucose was β-D-glucopyranose. H-Glc-6
The conformation of cyclobutane ring was same as that of protons at δ 4.33 (1H, dd, J=11.5, 7.5Hz) and 4.61 (1H, dd,
1
α-truxillic acid,20) and the two sets of H-, 13C-NMR chemical J=11.5, 2.0Hz) were shifted downfield relative to the H-6 in
shift values for
9 and 1′ 9′ and 1″ 9″ and 1‴ 9‴
5-O-β-D-glucopyranoside.23) The H-6 protons were long range
“ —
1
— ” ”
“ —
—
are interchangeable. The absolute configuration of both C-8 coupled with a carbonyl carbon at δ 168.6 (C-7′) in the HMBC
and C-8″ was determined to be R as in the case of 2.
spectrum. The ABCD spin system protons and an oxygenated
The 1H- and 13C-NMR spectra of shimobashiric acid D aromatic carbon at δ 160.2 suggested that the acyl moiety of 7
(4) were similar to those of 3 (in CD3OD). HR-FAB-MS [m/z was a salicylic acid. Hence, the structure of 7 was identified
771.1899 (Calcd for C38H36O16Na: 771.1900)] showed that as shown in Fig. 1.
the molecular formula of 4 was C38H36O16, which was C2H4
1H- and 13C-NMR spectra of shimobashiraside D (8) were
more than that of 3, indicating the presence of two methoxy similar to those of phlorein 6,8-bis-C-β-D-glucopyranosides7)
groups. Two methoxy proton signals δ 3.83 (3H, s) and 3.84 except for signals of the B-ring in the dihydrochalcone moiety.
(3H, s) were correlated with the aromatic carbons at δ 148.8 A spin system at δ 7.23 (4H, m, H-2′,3′,5′,6′) and 7.14 (1H,
and 148.9 in the HMBC spectrum, respectively. In the NOE m, H-4′) suggested that 8 has a phenyl moiety instead of the
spectra (Fig. 4), the methoxy protons were correlated with δ p-hydroxyphenyl moiety in 6,8-bis-C-β-D-glucopyranosides.
6.80 (1H, d, J=2.0Hz. H-2′) and 6.91 (1H, d, J=2.0Hz, H-2‴), HR-FAB-MS [m/z 583.2032 (Calcd for C27H35O14: 583.2027)],
respectively, suggesting 3-methoxy-4-hydroxyphenyl moieties. supported the conclusion.
Additionally the H-2′ and -6′ protons were correlated with
The molecular formula of shimobashiraside E (9) was de-
H-8′ and 7‴; H-2‴ and -6‴ protons were correlated with H-7′ termined as C16H25O8 based on HR-FAB-MS [m/z 345.1548
and 8‴, which suggested the conformation of cyclobutane ring (Calcd for C16H25O8: 345.1550)]. In the 1H- and 13C-NMR
was same as that of α-truxillic acid.20) Hence, the structure spectra, coupling patterns of aromatic protons of the furan
of 4 was determined to be that shown in Fig. 1. The absolute ring signal at δ 8.36 (1H, brs, H-2), 6.77 (1H, d, J=1.0Hz,

January 2012
125
H-4), and 7.57 (1H, dd, J=1.5, 1.0Hz, H-5), aromatic carbon Ueno, School of Pharmaceutical Sciences, University of
signals at δ 150.0 (C-2, having a corresponding proton at δ Shizuoka. A voucher specimen has been deposited in the her-
8.36), 128.8 (C-3), 109.3 (C-4), and 145.8 (C-5, having a cor- barium of Tohoku Pharmaceutical University, No.20090701.
responding proton at δ 7.57), and carbonyl carbon signal at δ
Extraction and Isolation Powdered aerial parts of K.
198.3 (C-6) suggested that 9 was a perillaketone type mono- japonica (300g) were extracted with acetone–water (8:2) at
terpene as shown in Fig. 1.24) An anomeric proton at δ 4.25 room temperature for two weeks (6L). The extract was con-
1
(1H, d, J=8.0Hz, H-Glc-1), and the H–1H COSY spectrum centrated at reduced pressure (22.0g), suspended in water
showed a glucose moiety. The sugar analysis21) and the cou- (1.5L) and subjected to extraction with diethyl ether (1.0L)
pling constant of H-Glc-1 suggested that the glucose was β-D- three times. The aqueous layer extract (11.8g) was dissolved
glucopyranose. The anomeric proton was long range coupled in water and passed through a porous polymer gel (Mitsubishi
with an oxygenated carbon at δ 75.5 (C-10). These results Diaion HP-20, 70×180mm) eluted with EtOH–water (95:5)
suggested that 9 was a glucoside of perillaketone and had the after being washed with water (10L). The 95% EtOH frac-
structure shown in Fig. 1. The absolute stereochemistry of C-9 tion (4.5g) was subjected to HPLC [TSKgel ODS-80Ts;
is unclear.
mobile phase, acetonitrile–0.1% trifluoroacetic acid (TFA)
→
The molecular formula of shimobashiraside F (10) was (15:85) (40:60)], to give 44 fractions. Each fraction was
determined as C16H25O9 based on HR-FAB-MS [m/z 361.1504 subjected to HPLC [columns; AR-II, mobile phases 15%,
(Calcd for C16H25O9: 361.1499)]. In the 1H- and 13C-NMR 17.5%, 20%, 25%, 27.5%, and 30% acetonitrile in 0.2%
spectra, signals of a furan ring, carbonyl carbon, and glucose trifluoroacetic acid (TFA); 5PE-MS, mobile phases 12.5%,
moiety in 10 were almost superimposable onto those of 9. 22.5%, 25%, and 30% acetonitrile in 0.2% TFA; AR-II,
Compound 10 has a hydroxyl moiety at C-8 revealed by an mobile phases 12.5%, 17.5%, 27.5%, and 30% acetonitrile
oxygenated proton signal at δ 4.16 (1H, dd, J=9.0, 3.0Hz, H-8) in 0.2% TFA and 30% MeOH in 0.2% TFA] to yields com-
and the 1H–1H COSY spectrum. The anomeric proton at δ pounds 1 (1.7mg), 2 (2.4mg), 3 (9.6mg), 4 (1.3mg), 5 (1.1mg),
4.54 (1H, d, J=8.0Hz, H-Glc-1) was long range coupled with 6 (3.1mg), 7 (0.8mg), 8 (4.2mg), 9 (39.8mg), 10 (3.4mg), 11
the quaternary carbon signal at δ 80.8 (C-9). From these data, (43.4mg), 12 (3.2mg), 13 (639.1mg), 14 (27.1mg), 15 (57.5mg),
the structure of 10 was determined as shown in Fig. 1. The 16 (195.2mg), 17 (287.8mg), 18 (125.7mg), 19 (2.9mg), 20
absolute stereochemistry of C-8 is still unknown. (49.7mg), 21 (6.5mg), 22 (3.6mg), 23 (4.6mg), 24 (2.1mg), 25
The hyaluronidase inhibitory activity measured for (6.0mg), 26 (2.3mg), 27 (1.3mg), 28 (6.3mg), 29 (6.9mg).
—
—
compounds 3, 9 18, and 20 29 is shown in Table 1.
Shimobashiric acid A (1): Colorless amorphous solid,
Phenylpropanoids and flavone glucuronides showed similar [α]D22 +205.0 (c=0.16, MeOH), UV (MeOH) λmax (logε):
activities (IC50 3: 594μM, 14: 737μM, 18: 267μM) to in- 201 (5.22), 293 (4.21), 333 (4.37). CD (c=0.016, MeOH) nm
°
—
hibitors in previous reports.2
All these active compounds ([θ]): 205 (−15600), 234 (−6500), 250 (5200), 301 (8300),
4)
in Lamiaceae plants have carboxylic acid in phenylpro- 332 (9700). HR-FAB-MS (positive): m/z 373.0928 [M+H]+
1
panoid oligomers or glucuronic acid in flavonoid glycoside. (Calcd for C19H17O8: 373.0923). H-NMR: (CD3OD, 400MHz),
Carboxylic acids in limited moieties are suggested to be a key δ: 6.77 (1H, d, J=2.0Hz, H-2), 6.77 (1H, d, J=8.5Hz, H-5),
functional group for hyaluronidase inhibitory activity, and the 6.67 (1H, dd, J=8.5, 2.0Hz, H-6), 4.55 (1H, brs, H-7), 4.78
level of activity seems to depend on the number of carboxylic (overlapped, H-8), 6.89 (1H, d, J=2.0Hz, H-2′), 6.75 (1H, d,
acids in the structure and structural features around the acids. J=8.5Hz, H-5′), 6.99 (1H, dd, J=8.5, 2.0Hz, H-6′), 7.68 (1H,
d, J=2.0Hz, H-7′), 3.56 (3H, s, H-OMe). 13C-NMR: (CD3OD,
100MHz), δ: 133.7 (C-1), 115.1 (C-2), 147.4 (C-3), 146.3 (C-4),
Experimental
General Procedures Optical rotations were recorded on 116.4 (C-5), 119.4 (C-6), 49.5 (C-7), 83.6 (C-8), 173.7 (C-9),
a Jasco P-2300 polarimeter. Circular dichroism (CD) spectra 126.9 (C-1′), 114.3 (C-2′), 149.1 (C-3′), 150.7 (C-4′), 117.2 (C-
were recorded on a Jasco J-700 spectropolarimeter; and UV, 5′), 128.0 (C-6′), 141.7 (C-7′), 122.5 (C-8′), 174.5 (C-9′), 56.4
on a Shimadzu MPS-2450. 1H-NMR (400MHz), 13C-NMR (C-OMe).
(100MHz), 1H–1H COSY, heteronuclear multiple quantum
Shimobashiric acid B (2): Colorless amorphous solid, [α]D21
1
°
correlation (HMQC) (optimized for JC–H=145Hz) and HMBC +47.0 (c=0.2, MeOH), UV (MeOH) λmax (logε): 204 (4.60),
n
(optimized for JC–H=8Hz) spectra were recorded on a Jeol 288 (4.13), 324 (4.09). CD (c=0.020, MeOH) nm ([θ]): 247
JNM-AL400 FT-NMR spectrometer, and chemical shifts were (9600), 297 (6400). HR-FAB-MS (positive): m/z 375.1082
given as δ values with TMS as an internal standard. HR-FAB- [M+H]+ (Calcd for C19H19O8: 375.1080). H-NMR: (acetone-
1
and HR-electron ionization (EI)-MS data were obtained on a d6, 400MHz), δ: 6.86 (1H, d, J=2.0Hz, H-2), 6.75 (1H, d,
Jeol JMS700 mass spectrometer, using a m-nitrobenzyl alco- J=8.0Hz, H-5), 6.69 (1H, dd, J=8.0, 2.0Hz, H-6), 3.04 (1H,
hol or a glycerol matrix. A porous polymer gel (Mitsubishi dd, J=14.5, 8.5Hz, H-7), 3.13 (1H, dd, J=14.5, 4.5Hz, H-7),
Chemical, Diaion HP-20, 60×300mm) and octadecyl silica 5.23 (1H, dd, J=8.5, 4.5Hz, H-8), 7.18 (1H, d, J=2.0Hz, H-2′),
(ODS) (Cosmosil 140 C18-OPN, Nacalai Tesque, 150g) were 7.00 (1H, d, J=8.5Hz, H-5′), 7.13 (1H, dd, J=8.5, 2.0Hz,
used for column chromatography. Preparative HPLC was H-6′), 7.59 (1H, d, J=16.0Hz, H-7′), 6.37 (1H, d, J=16.0Hz,
performed on a Jasco 2089 and detected with UV at 320 H-8′), 3.90 (3H, s, H-OMe). 13C-NMR: (acetone-d6, 100MHz),
or 210nm (columns, TSKgel ODS-80Ts, 55×600mm×3; δ: 129.2 (C-1), 117.3 (C-2), 145.6 (C-3), 144.7 (C-4), 115.9 (C-5),
Cosmosil AR-II, Nacalai tesque, 20×250mm; Cosmosil 5PE- 121.7 (C-6), 37.5 (C-7), 73.7 (C-8), 171.0 (C-9), 128.5 (C-1′),
MS, Nacalai Tesque, 20×250mm; Mightisil RP-18 GP, Kanto 114.6 (C-2′), 147.7 (C-3′), 150.8 (C-4′), 112.4 (C-5′), 122.5 (C-
Chemical, 10×250mm).
Plant Material K. japonica was collected in July 2009
6′), 146.3 (C-7′), 115.8 (C-8′), 166.7 (C-9′), 56.3 (C-OMe).
Shimobashiric acid C (3): Colorless amorphous solid, [α]D22
°
in Shizuoka, Japan. The plant was identified by Prof. Akira −4.1 (c=1.21, MeOH), UV (MeOH) λmax (logε): 204 (4.92),

126
Vol. 60, No. 1
284 (4.05). CD (c=0.012, MeOH) nm ([θ]): 252 (11900). H-8), 6.80 (1H, d, J=2.0Hz, H-2′), 6.72 (1H, d, J=8.0Hz,
HR-FAB-MS (positive): m/z 743.1597 [M+Na]+ (Calcd for H-5′), 6.54 (1H, dd, J=8.0, 2.0Hz, H-6′), 4.29 (1H, dd, J=11.0,
C36H32O17Na: 743.1587). 1H-NMR: (DMSO-d6, 400MHz), δ: 7.0Hz, H-7′), 3.96 (1H, dd, J=11.0, 7.0Hz, H-8′), 3.83 (3H,
6.54 (1H, d, J=2.0Hz, H-2), 6.62 (1H, d, J=8.0Hz, H-5), 6.34 s, H-3′-OMe), 6.73 (1H, d, J=2.0Hz, H-2″), 6.75 (1H, d,
(1H, dd, J=8.0, 2.0Hz, H-6), 2.50 (overlapped, H-7), 2.58 (1H, J=8.0Hz, H-5″), 6.57 (1H, dd, J=8.0, 2.0Hz, H-6″), 2.82 (1H,
dd, J=14.0, 6.0Hz, H-7), 4.43 (1H, t, J=6.0Hz, H-8), 6.64 (1H, dd, J=14.0, 8.0Hz, H-7″), 2.89 (1H, dd, J=14.0, 4.5Hz, H-7″),
d, J=2.0Hz, H-2′), 6.57 (1H, d, J=8.0Hz, H-5′), 6.31 (1H, 4.79 (1H, dd, J=8.0, 4.5Hz, H-8″), 6.91 (1H, d, J=2.0Hz,
dd, J=8.0, 2.0Hz, H-6′), 4.05 (1H, dd, J=10.5, 7.0Hz, H-7′), H-2‴), 6.74 (1H, d, J=8.0Hz, H-5‴), 6.63 (1H, dd, J=8.0,
3.76 (1H, dd, J=10.5, 7.0Hz, H-8′), 6.60 (1H, d, J=2.0Hz, 2.0Hz, H-6‴), 4.34 (1H, dd, J=11.0, 7.0Hz, H-7‴), 3.90 (1H,
H-2″), 6.65 (1H, d, J=8.0Hz, H-5″), 6.43 (1H, dd, J=8.0, dd, J=10.5, 7.0Hz, H-8‴), 3.84 (3H, s, H-3‴-OMe). 13C-NMR:
2.0Hz, H-6″), 2.71 (1H, dd, J=14.0, 7.0Hz, H-7″), 2.76 (1H, (CD3OD, 100MHz), δ: 129.1 (C-1), 117.6 (C-2), 146.1a (C-3),
dd, J=14.0, 6.0Hz, H-7″), 4.58 (1H, dd, J=7.0, 6.0Hz, H-8″), 145.4a (C-4), 116.4b (C-5), 122.1 (C-6), 37.7 (C-7), 75.2 (C-8),
6.72 (1H, d, J=2.0Hz, H-2‴), 6.63 (1H, d, J=8.0Hz, H-5‴), 172.9 (C-9), 131.7 (C-1′), 112.8 (C-2′), 148.8 (C-3′), 146.3 (C-
6.41 (1H, dd, J=8.0, 2.0Hz, H-6‴), 4.13 (1H, dd, J=10.5, 4′), 116.4b (C-5′), 120.5 (C-6′), 42.6 (C-7′), 48.1 (C-8′), 173.6
7.0Hz, H-7‴), 3.67 (1H, dd, J=10.5, 7.0Hz, H-8‴). 13C-NMR: (C-9′), 56.5 (C-3′-OMe), 128.9 (C-1″), 117.8 (C-2″), 145.5a (C-
(DMSO-d6, 100MHz), δ: 116.4 (C-1), 119.9 (C-2), 145.0b (C-3), 3″), 145.3a (C-4″), 116.5b (C-5″), 122.1 (C-6″), 38.0 (C-7″), 75.2
144.2a (C-4), 115.2 (C-5), 120.3 (C-6), 35.9 (C-7), 73.1 (C-8), (C-8″), 173.6 (C-9″), 131.8 (C-1‴), 112.4 (C-2‴), 148.9 (C-3‴),
170.2 (C-9), 129.2 (C-1′), 115.4 (C-2′), 144.7b (C-3′), 144.0a (C- 146.4 (C-4‴), 116.7b (C-5‴), 121.3 (C-6‴), 43.2 (C-7‴), 49.0
4′), 115.4c (C-5′), 117.8c (C-6′), 39.7 (C-7′), 46.2 (C-8′), 171.1 (C-8‴), 172.9 (C-9‴), 56.6 (C-3‴-OMe). a,b: Assignments are
(C-9′), 126.8 (C-1″), 119.6 (C-2″), 145.0b (C-3″), 144.1a (C-4″), interchangeable.
115.4c (C-5″), 120.3 (C-6″), 36.2 (C-7″), 73.2 (C-8″), 170.0 (C-
Shimobashiraside A (5): Colorless amorphous solid, [α]D21
a
9″), 129.3 (C-1‴), 114.7 (C-2‴), 144.8b (C-3‴), 144.0 (C-4‴), −70.0 (c=0.1, MeOH), UV (MeOH) λmax (logε): 202 (4.52),
°
115.4 (C-5‴), 118.5 (C-6‴), 40.8 (C-7‴), 46.5 (C-8‴), 170.2 (C- 251 (4.20), 331 (4.15). HR-FAB-MS (positive): m/z 583.1646
1
1
9‴). a,b,c: Assignments are interchangeable. H-NMR: (CD3OD, [M+H]+ (Calcd for C26H31O15: 583.1662). H-NMR: (CD3OD,
400MHz), δ: 6.78 (1H, d, J=2.0Hz), 6.70 (1H, d, J=8.0Hz), 400MHz), δ: 6.42 (1H, d, J=5.5Hz, H-5), 7.94 (1H, d,
6.68 (1H, d, J=8.0Hz), 6.67 (1H, d, J=8.0Hz), 6.66 (1H, d, J=5.5Hz, H-6), 2.44 (3H, s, H-7), 4.76 (overlapped, H-Glc-1),
—
J=2.0Hz), 6.66 (1H, d, J=2.0Hz), 6.64 (1H, d, J=8.0Hz), 3.3 3.45 (overlapped, H-Glc-2,3,4,5), 3.62 (1H, dd, J=11.5,
6.63 (1H, d, J=2.0Hz), 6.53 (1H, dd, J=8.0, 2.0Hz), 6.48 (1H, 6.5Hz, H-Glc-6), 3.96 (overlapped, H-Glc-6), 4.98 (1H, d,
dd, J=8.0, 2.0Hz), 6.45 (1H, dd, J=8.0, 2.0Hz), 6.35 (1H, J=1.5Hz, H-Api-1), 3.89 (1H, brs, H-Api-2), 3.82 (1H, d,
dd, J=8.0, 2.0Hz), 4.73 (1H, dd, J=7.5, 5.0Hz), 4.59 (1H, J=9.5Hz, H-Api-4), 3.96 (1H, d, J=9.5Hz, H-Api-4), 4.21
dd, J=6.0, 6.0Hz), 4.21 (2H, brdd, J=10.5, 7.0Hz), 3.91 (1H, (1H, d, J=11.5Hz, H-Api-5), 4.25 (1H, d, J=11.5Hz, H-Api-5),
dd, J=10.5, 7.0Hz), 3.75 (1H, dd, J=10.5, 7.0Hz). 2.86 (1H, 7.04 (1H, d, J=1.5Hz, H-2′), 6.78 (1H, d, J=8.0Hz, H-5′), 6.95
dd, J=14.0, 5.0Hz), 2.81 (1H, dd, J=14.0, 7.5Hz), 2.71 (1H, (1H, dd, J=8.0, 1.5Hz, H-6′), 7.58 (1H, d, J=16.0Hz, H-7′),
dd, J=14.0, 6.0Hz), 2.61 (1H, dd, J=14.0, 6.0Hz), 13C-NMR: 6.28 (1H, d, J=16.0Hz, H-8′). 13C-NMR: (CD3OD, 100MHz),
(CD3OD, 100MHz), δ: 173.4, 173.2, 172.8, 172.7, 146.2, 146.1, δ: 164.7 (C-2), 143.6 (C-3), 177.2 (C-4), 117.3 (C-5), 157.2 (C-
146.0, 145.9, 145.5, 145.3, 145.2, 145.2, 131.7, 131.6, 128.9, 6), 15.9 (C-7), 105.4 (C-Glc-1), 75.0 (C-Glc-2), 78.5 (C-Glc-3),
128.6, 122.2, 122.2, 120.5, 119.5, 117.8, 117.6, 116.5, 116.4, 71.4 (C-Glc-4), 77.5 (C-Glc-5), 68.6 (C-Glc-6), 110.6 (C-Api-1),
116.4, 116.3, 116.0, 115.6, 74.9, 74.9, 48.7, 48.0, 42.8, 42.1, 37.9, 78.0 (C-Api-2), 79.0 (C-Api-3), 75.4 (C-Api-4), 67.4 (C-Api-5),
37.6. 1H-NMR: (D2O, 400MHz), δ: 6.68 (1H, d, J=2.0Hz), 127.8 (C-1′), 115.3 (C-2′), 146.9 (C-3′), 149.8 (C-4′), 116.6 (C-
6.66 (1H, d, J=8.0Hz), 6.64 (1H, d, J=8.0Hz), 6.63 (1H, d, 5′), 123.1 (C-6′), 147.5 (C-7′), 114.8 (C-8′), 168.9 (C-9′).
J=8.0Hz), 6.62 (1H, d, J=2.0Hz), 6.59 (1H, d, J=8.0Hz), 6.56
Shimobashiraside B (6): Colorless amorphous solid, [α]D22
°
(1H, d, J=2.0Hz), 6.55 (1H, d, J=2.0Hz), 6.47 (1H, dd, J=8.0, −60.0 (c=0.28, MeOH), UV (MeOH) λmax (logε): 202
2.0Hz), 6.37 (1H, dd, J=8.0, 2.0Hz), 6.35 (1H, dd, J=8.0, (4.34), 244 (4.07), 327 (4.11). HR-FAB-MS (positive): m/z
2.0Hz), 6.09 (1H, dd, J=8.0, 2.0Hz), 4.59 (1H, dd, J=8.5, 597.1804 [M+H]+ (Calcd for C27H33O15: 597.1819). H-NMR:
1
4.5Hz), 4.52 (1H, dd, J=7.0, 5.0Hz), 4.05 (2H, m), 3.74 (1H, (CD3OD, 400MHz), δ: 6.42 (1H, d, J=5.5Hz, H-5), 7.94 (1H,
dd, J=10.5, 7.5Hz), 3.65 (1H, dd, J=10.5, 6.0Hz). 2.80 (1H, d, J=5.5Hz, H-6), 2.44 (3H, s, H-7), 4.77 (1H, d, J=7.5Hz,
—
dd, J=14.5, 4.5Hz), 2.68 (1H, dd, J=14.5, 8.5Hz), 2.62 (1H, H-Glc-1), 3.3 3.4 (overlapped, H-Glc-2,3,4,5), 3.62 (1H, dd,
dd, J=14.5, 7.5Hz), 2.57 (1H, dd, J=14.5, 5.5Hz), 13C-NMR: J=11.5, 6.5Hz, H-Glc-6), 3.96 (1H, J=11.5, 2.0Hz, H-Glc-6),
(D2O, 100MHz), δ: 174.0, 173.9, 173.7, 173.5, 144.6, 144.5, 4.99 (1H, d, J=2.0Hz, H-Api-1), 3.89 (1H, d, J=2.0Hz,
144.4, 144.4, 143.8, 143.7, 143.6, 143.6, 131.4, 131.2, 129.1, H-Api-2), 3.82 (1H, d, J=9.5Hz, H-Api-4), 3.97 (1H, d,
128.8, 122.7, 122.5, 120.6, 119.4, 117.7, 117.5, 116.8, 116.7, J=9.5Hz, H-Api-4), 4.22 (1H, d, J=11.5Hz, H-Api-5), 4.25
116.7, 116.7, 116.2, 115.7, 74.8, 74.7, 47.8, 46.8, 41.7, 41.2, 36.7, (1H, d, J=11.5Hz, H-Api-5), 7.20 (1H, d, J=2.0Hz, H-2′), 6.82
36.4.
(1H, d, J=8.0Hz, H-5′), 7.08 (1H, dd, J=8.0, 2.0Hz, H-6′),
Shimobashiric acid D (4): Colorless amorphous solid, [α]D21 7.64 (1H, d, J=16.0Hz, H-7′), 6.38 (1H, d, J=16.0Hz, H-8′),
−7.7 (c=0.13, MeOH), UV (MeOH) λmax (logε): 204 (4.95), 3.90 (3H, s, H-OMe). 13C-NMR: (CD3OD, 100MHz), δ: 164.7
°
284 (4.19). CD (c=0.013, MeOH) nm ([θ]): 250 (11600). (C-2), 143.5 (C-3), 177.2 (C-4), 117.3 (C-5), 157.2 (C-6), 15.9
HR-FAB-MS (positive): m/z 771.1899 [M+Na]+ (Calcd for (C-7), 105.4 (C-Glc-1), 75.0 (C-Glc-2), 78.5 (C-Glc-3), 71.4
1
C38H36O16Na: 771.1900). H-NMR: (CD3OD, 400MHz), δ: 6.70 (C-Glc-4), 77.5 (C-Glc-5), 68.6 (C-Glc-6), 110.6 (C-Api-1),
(1H, d, J=2.0Hz, H-2), 6.73 (1H, d, J=8.0Hz, H-5), 6.50 (1H, 78.0 (C-Api-2), 79.0 (C-Api-3), 75.4 (C-Api-4), 67.5 (C-Api-5),
dd, J=8.0, 2.0Hz, H-6), 2.65 (1H, dd, J=14.0, 5.0Hz, H-7), 127.7 (C-1′), 115.1 (C-2′), 149.5 (C-3′), 150.8 (C-4′), 116.6 (C-
2.74 (1H, dd, J=14.0, 7.0Hz, H-7), 4.66 (1H, dd, J=7.0, 5.0Hz, 5′), 124.3 (C-6′), 147.3 (C-7′), 111.8 (C-8′), 168.9 (C-9′), 56.5

January 2012
127
(C-OMe).
H-Glc-5), 3.62 (1H, dd, J=12.0, 5.0Hz, H-Glc-6), 3.82 (1H,
Shimobashiraside C (7): Colorless amorphous solid, [α]D23 dd, J=12.0, 2.0Hz, H-Glc-6). 13C-NMR: (CD3OD, 100MHz),
°
−65.0 (c=0.08, MeOH), UV (MeOH) λmax (logε): 205 (5.08), δ: 150.6 (C-2), 129.7 (C-3), 109.3 (C-4), 145.9 (C-5), 197.1 (C-
310 (4.02). HR-FAB-MS (positive): m/z 437.1090 [M+H]+ 6), 43.6 (C-7), 74.7 (C-8), 80.8 (C-9), 24.0 (C-10), 22.1 (C-11),
(Calcd for C20H21O11: 437.1083). 1H-NMR: (DMSO-d6, 98.6 (C-Glc-1), 75.4 (C-Glc-2), 78.2 (C-Glc-3), 71.6 (C-Glc-4),
400MHz), δ: 6.72 (1H, d, J=9.0Hz, H-3), 7.19 (1H, dd, J=9.0, 77.9 (C-Glc-5), 62.9 (C-Glc-6).
3.0Hz, H-4), 7.41 (1H, d, J=3.0Hz), 4.84 (1H, d, J=7.5Hz,
(S)-PGME and (R)-PGME Amides of 1 for Determining
H-Glc-1), 3.1-3.5 (3H, overlapped, H-Glc-2-4), 3.74 (1H, m, the Stereochemistry of C-8 To 1 (each 0.8mg) in N,N-
H-Glc-5), 4.33 (1H, dd, J=11.5, 7.5Hz, H-Glc-6), 4.61 (1H, dd, dimethylformamide (DMF) (0.5mL) was added (S)- or
J=11.5, 2.0Hz, H-Glc-6), 6.95 (overlapped, H-3′), 7.54 (1H, (R)-PGME (5mg), and then benzotriazol-1-yl-oxy-tris-pyrro-
m, H-4′), 6.97 (overlapped, H-5′), 7.77 (1H, dd, J=7.5, 2.0Hz, lidinophonium hexafluorophosphate (PyBOP) (10mg), 1-hy-
H-6′). 13C-NMR: (DMSO-d6, 100MHz), δ: 113.5 (C-1), 156.4 droxybenzotriazole (HOBT) (5mg), and N-methylmorpholine
(C-2), 117.3 (C-3), 124.7 (C-4), 149.1 (C-5), 117.2 (C-6), 171.3 (20µL) were added and the mixture was stirred for 10h at
(C-7), 101.3 (C-Glc-1), 73.1 (C-Glc-2), 76.1 (C-Glc-3), 70.1 room temperature. The reactions gave (S)-amide: (1a) and (R)-
(C-Glc-4), 73.4 (C-Glc-5), 64.6 (C-Glc-6), 112.8 (C-1′), 160.2 amide (1b).16)
(C-2′), 119.3 (C-3′), 135.8 (C-4′), 117.3 (C-5′), 130.0 (C-6′),
168.6 (C-7′).
(S)-PGME Amide of 1 (1a): Colorless amorphous solid,
FAB-MS (positive): m/z 520 [M+H]+, 542 [M+Na]+,
Shimobashiraside D (8): Colorless amorphous solid, [α]D22 1H-NMR: (CD3OD, 400MHz), δ: 6.70 (1H, d, J=2.0Hz, H-2),
°
+58.5 (c=0.39, MeOH), UV (MeOH) λmax (logε): 202 (4.40), 6.72 (1H, d, J=8.5Hz, H-5), 6.59 (1H, dd, J=8.5, 2.0Hz,
231 (4.12), 287 (3.97), 326 (3.71). HR-FAB-MS (positive): m/z H-6), 4.54 (1H, brs, H-7), 4.86 (overlapped, H-8), 6.86 (1H,
1
583.2032 [M+H]+ (Calcd for C27H35O14: 583.2027). H-NMR: d, J=2.0Hz, H-2′), 6.74 (1H, d, J=8.5Hz, H-5′), 6.98 (1H, dd,
(CD3OD, 400MHz), δ: 2.96 (1H, m, H-2), 3.39 (1H, dd, J=7.5, J=8.5, 2.0Hz, H-6′), 7.69 (1H, d, J=2.0Hz, H-7′), 3.54 (3H, s,
3.0Hz, H-3), 3.42 (overlapped, H-3), 7.23 (4H, m, H-2′,3′,5′,6′), H-OMe).
7.14 (1H, m, H-4′), 4.94 (1H, d, J =10.0Hz, H-1″), 3.61 (1H,
(R)-PGME Amide of 1 (1b): Colorless amorphous sol-
dd, J=10.0, 9.0Hz, H-2″), 3.51 (1H, dd, J=9.0, 9.0Hz, H-3″), id, FAB-MS (positive): m/z 520 [M+H]+, 542 [M+Na]+,
3.51 (1H, dd, J=9.0, 9.0Hz, H-4″), 3.42 (overlapped, H-5″), 1H-NMR: (CD3OD, 400MHz), δ: 6.79 (1H, d, J=2.0Hz, H-2),
3.80 (1H, dd, J=12.0, 4.0Hz, H-6″), 3.85 (1H, dd, J=12.0, 6.77 (1H, d, J=8.5Hz, H-5), 6.71 (1H, dd, J=8.5, 2.0Hz,
2.0Hz, H-6″), 4.94 (1H, d, J=10.0Hz, H-1‴), 3.61 (1H, dd, H-6), 4.59 (1H, brs, H-7), 4.86 (overlapped, H-8), 6.90 (1H,
J=10.0, 9.0Hz, H-2‴), 3.51 (1H, dd, J=9.0, 9.0Hz, H-3‴), 3.51 d, J=2.0Hz, H-2′), 6.75 (1H, d, J=8.5Hz, H-5′), 6.99 (1H, dd,
(1H, dd, J=9.0, 9.0Hz, H-4‴), 3.42 (overlapped, H-5‴), 3.80 J=8.5, 2.0Hz, H-6′), 7.68 (1H, d, J=2.0Hz, H-7′), 3.58 (3H, s,
(1H, dd, J=12.0, 4.0Hz, H-6‴), 3.85 (1H, dd, J=12.0, 2.0Hz, H-OMe).
H-6‴). 13C-NMR: (CD3OD, 100MHz), δ: 31.7 (C-2), 47.4 (C-3),
(S)-PGME and (R)-PGME Amides of 3-(3,4-Di-
206.8 (C-4), 162.2 (C-5), 104.3 (C-6), 163.1 (C-7), 104.3 (C-8), hydroxyphenyl)-2-hydroxypropanoic Acid To 3-(3,4-di-
162.2 (C-9), 106.1 (C-10), 143.1 (C-1′), 129.5 (C-2′,6′), 129.3 hydroxyphenyl)-2-hydroxypropanoic acid (5mg, each) obtained
(C-3′,5′), 126.8 (C-4′), 76.6 (C-1″), 74.1 (C-2″), 79.0 (C-3″), 71.0 from rosmarinic acid16) in DMF (1.0mL) was added (S)- or
(C-4″), 82.7 (C-5″), 61.8 (C-6″), 76.7 (C-1‴), 74.1 (C-2‴), 79.0 (R)-PGME (10mg), and then benzotriazol-1-yl-oxy-tris-pyrro-
(C-3‴), 71.0 (C-4‴), 82.7 (C-5‴), 61.8 (C-6‴).
lidinophonium hexafluorophosphate (PyBOP) (15mg), 1-hy-
Shimobashiraside E (9): Colorless amorphous solid, [α]D23 droxybenzotriazole (HOBT) (5mg), and N-methylmorpholine
°
−18.7 (c=3.12, MeOH). HR-FAB-MS (positive): m/z 345.1548 (20µL) were added and the mixture was stirred for 10h at
1
[M+H]+ (Calcd for C16H25O8: 345.1550). H-NMR: (CD3OD, room temperature. The reactions gave (S)-amide and (R)-
400MHz), δ: 8.36 (1H, brs, H-2), 6.77 (1H, d, J=1.0Hz, H-4), amide.16) The retention time of (S)-amide was 19.4min and
7.57 (1H, dd, J=1.5, 1.0Hz, H-5), 2.87 (2H, m, H-7), 1.57 (1H, that of (R)-amide was 20.1min. The analytical HPLC was per-
m, H-8), 1.84 (overlapped, H-8), 1.84 (overlapped, H-9), 3.39 formed on a Shiseido Capcell Pak C18 column (4.6×250mm)
(1H, m, H-10), 3.80 (1H, dd, J=9.5, 6.5Hz, H-10), 0.97 (3H, d, using acetonitrile–0.2% TFA in water (22.5:77.5) as the mo-
J=7.0Hz, H-11), 4.25 (1H, d, J=8.0Hz, H-Glc-1), 3.18 (1H, dd, bile phase (flow rate, 1mL/min; detector, UV 210nm).
—
Acidic Hydrolysis of Compounds 2 4 and Their (S)-
J=9.0, 8.0Hz, H-Glc-2), 3.35 (1H, m, H-Glc-3), 3.29 (1H, m,
H-Glc-4), 3.29 (1H, m, H-Glc-5), 3.67 (1H, dd, J=12.0, 5.0Hz, PGME Amides Each compound (2, 3, 4: each 1.0mg)
H-Glc-6), 3.87 (1H, dd, J=12.0, 2.0Hz, H-Glc-6). 13C-NMR: was dissolved in 7% HCl (1mL) and stirred for 2h at 90 C.
°
(CD3OD, 100MHz), δ: 150.0 (C-2), 128.8 (C-3), 109.3 (C-4), After concentration, the residues were dissolved in DMF
145.8 (C-5), 198.3 (C-6), 38.7 (C-7), 29.6 (C-8), 34.2 (C-9), and (S)-PGME (5mg), PyBOP (7mg), HOBT (3mg), and N-
75.5 (C-10), 17.3 (C-11), 104.5 (C-Glc-1), 75.1 (C-Glc-2), 78.1 methylmorpholine (15µL) were added. The mixtures were
(C-Glc-3), 71.7 (C-Glc-4), 77.9 (C-Glc-5), 62.8 (C-Glc-6).
then stirred for 10h at room temperature to give (S)-amide;
Shimobashiraside F (10): Colorless amorphous solid, [α]D22 tR=19.4min in the HPLC analysis [column, Shiseido Capcell
°
−7.9 (c=0.28, MeOH). HR-FAB-MS (positive): m/z 361.1504 Pak C18 (4.6×250mm); mobile phase, acetonitrile–0.2%
1
[M+H]+ (Calcd for C16H25O9: 361.1499). H-NMR: (CD3OD, TFA in water (22.5:77.5); flow rate, 1mL/min; detector, UV
400MHz), δ: 8.37 (1H, brs, H-2), 6.79 (1H, d, J=1.0Hz, H-4), 210nm].
7.58 (1H, dd, J=1.5, 1.0Hz, H-5), 2.99 (1H, dd, J=15.5, 9.0Hz,
H-7), 3.05 (1H, dd, J=15.5, 3.0Hz, H-7), 4.16 (1H, dd, J=9.0,
3.0Hz, H-8), 1.29 (3H, s, H-10), 1.31 (3H, s, H-11), 4.54 (1H, 7% HCl (1mL) at 60 C for 2h. The reaction mixture was
d, J=8.0Hz, H-Glc-1), 3.16 (1H, dd, J=9.0, 8.0Hz, H-Glc-2), neutralized with an Amberlite IRA400 column, and the eluate
3.34 (1H, m, H-Glc-3), 3.26 (1H, m, H-Glc-4), 3.26 (1H, m, was concentrated. The residues were stirred with L-cysteine
Acid Hydrosis and Sugar Identification Compounds
—
—
5
7, 9, 10 and 21 (each 0.5 1.0mg) were hydrolyzed with
°

128
Table 1. Hyaluronidase Inhibitory Activity of Compounds 3, 9 18,
Vol. 60, No. 1
—
with the MS measurements.
—
20 29, and DSCG
References
Compound
Hyaluronidase inhibition (%)a)
IC50 (μM)
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3
9
88.7
5.93
−2.50
0.57
65.8
86.5
78.7
2.01
3.46
93.8
86.5
−2.97
5.54
1.43
−1.78
28.6
8.10
7.41
594
N.D.b)
N.D.
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814
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267
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N.D.
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23
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DSCG
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—
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a) Final concentration: 1.0mM. b) N.D., not determined. c) Previously re-
ported value.3)
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—
60, 732 735 (1996).
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methyl ester (5mg) and o-tolyl isothiocyanate (10µL) in pyri-
dine (0.5mL), by using the procedure reported by Tanaka et
al.21) The reaction mixtures were analyzed by HPLC (column,
Cosmosil 5C18–AR II column, 4.6×250mm; mobile phase,
CH3CN–0.2% TFA in H2O (25:75), 1.0mL/min; detector, UV
—
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°
at 210nm) at 20 C. D-Glucose (tR 15.7min) was identified as
—
Matsubara K., J. Nat. Prod., 72, 1379 1384 (2009).
—
—
17) Murata T., Miyase T., Yoshizaki F., J. Nat. Med., 65, 385 390
the sugar moieties of 5 7, 9, and 10 based on comparisons
(2011).
with authentic samples of D-glucose (tR 15.7min) and L-glu-
cose (tR 14.3min). Compound 21 has an D-apiofuranose,8) and
sugar analyses by HPLC (column, Cosmosil 5C18–AR II col-
umn, 4.6×250mm; mobile phase, CH3CN–0.2% TFA in H2O
(25:75), 0.8mL/min; detector, UV at 250nm) were conducted.
Chromatograms of 5, 6, and 21 showed peaks at 17.7min (D-
glucose) and 30.6min (suggesting D-apiose).8)
—
18) Yabuuchi T., Kusumi T., J. Org. Chem., 65, 397 404 (2000).
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19) Lu Y., Foo L. Y., Phytochemistry, 51, 91 94 (1999).
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Pharm. Bull., 55, 899 901 (2007).
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—
Tetrahedron, 35, 1427 1432 (1979).
Assay of Hyaluronidase Inhibition The assay was car-
ried out according to the Morgan–Elson method, which was
23) Yahara S., Satoshiro M., Nishioka I., Nagasawa T., Oura H., Chem.
—
Pharm. Bull., 33, 527 531 (1985).
—
27)
modified by Davidson and Aronson.25
Each compound (fi- 24) Bassoli A., Borgonovo G., Caimi S., Scaglioni L., Morini G.,
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nal concentration: 1, 0.3, 0.1, 0.03mM) was dissolved in 0.1M
acetate buffer as the sample solution. Hyaluronidase activity
was measured as described previously.2,3) Disodium cromogly-
cate (DSCG) was used as a positive control. The final concen-
tration of hyaluronidase was 400unit/mL.
—
17, 1636 1639 (2009).
25) Ippoushi K., Yamaguchi Y., Itou H., Azuma K., Higashio H., Food
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Sci. Technol. Res., 6, 74 77 (2000).
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Acknowledgments We thank Mr. S. Sato and Mr. T.
Matsuki of Tohoku Pharmaceutical University for assisting
(1967).