Medicinal flowers. XXXX.1) Structures of dihydroisocoumarin glycosides and inhibitory effects on aldose reducatase from the flowers of Hydrangea macrophylla var. thunbergii

Article abstract of DOI:10.1248/cpb.c13-00160

Six dihydroisocoumarin glycosides, florahydrosides I and II, thunberginol G 8-O-β-D-glucopyranoside, thunberginol C 8-O-β-D-glucopyranoside, 4-hydroxythunberginol G 3′-O-β-D-glucopyranoside, and thunberginol D 3′-O-β-D-glucopyranoside, have been isolated from the flowers of Hydrangea macrophylla SERINGE var. thunbergii MAKINO (Saxifragaceae) together with 20 known compounds. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, acylated quinic acid analog, neochlorogenic acid, was shown to substantially inhibit aldose reductase [IC₅₀=5.6 μM]. In addition, the inhibitory effects on aldose reductase of several caffeoylquinic acid analogs were examined for structure-activity relationship study. As the results, 4,5-O-trans-p-dicaffeoyl-D-quinic acid was found to exhibit a potent inhibitory effect [IC₅₀=0.29 μM].

Full text of DOI:10.1248/cpb.c13-00160

June 2013
Regular Article
Chem. Pharm. Bull. 61(6) 655–661 (2013)
655
Medicinal Flowers. XXXX.1) Structures of Dihydroisocoumarin
Glycosides and Inhibitory Effects on Aldose Reducatase from the
Flowers of Hydrangea macrophylla var. thunbergii
Jiang Liu, Seikou Nakamura, Yan Zhuang, Masayuki Yoshikawa, Ghazi Mohamed Eisa Hussein,
Kyohei Matsuo, and Hisashi Matsuda*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received February 21, 2013; accepted March 18, 2013
Six dihydroisocoumarin glycosides, florahydrosides I and II, thunberginol G 8-O-β-d-glucopyranoside,
thunberginol C 8-O-β-d-glucopyranoside, 4-hydroxythunberginol G 3′-O-β-d-glucopyranoside, and thun-
berginol D 3′-O-β-d-glucopyranoside, have been isolated from the flowers of Hydrangea macrophylla Seringe
var. thunbergii Makino (Saxifragaceae) together with 20 known compounds. The chemical structures of the
new compounds were elucidated on the basis of chemical and physicochemical evidence. Among the constitu-
ents, acylated quinic acid analog, neochlorogenic acid, was shown to substantially inhibit aldose reductase
[IC50=5.6µM]. In addition, the inhibitory effects on aldose reductase of several caffeoylquinic acid analogs
were examined for structure–activity relationship study. As the results, 4,5-O-trans-p-dicaffeoyl-d-quinic
acid was found to exhibit a potent inhibitory effect [IC50=0.29µM].
Key words florahydroside; Hydrangea macrophylla var. thunbergii; medicinal flower; dihydroisocoumarin
glycoside; aldose reductase; acylated quinic acid analog
The Saxifragaceae plant, Hydrangea (H.) macrophylla (1-BuOH) to give a 1-BuOH (12.5%) and a H2O (11.0%)
Seringe var. thunbergii Makino, is native to Japan. The pro- soluble fraction. The EtOAc- and 1-BuOH-soluble fractions
cessed leaves of this plant (Hydrangea Dulcis Folium) are were subjected to normal-phase and reversed-phase silica gel
currently used as a natural medicine for an oral refrigerant column chromatography and repeated HPLC to give flora-
and as a sweetener for diabetic patients. In addition, these hydrosides I (1, 0.068%) and II (2, 0.014%), thunberginol G
preparations are listed in the Japanese Pharmacopoeia. Previ- 8-O-β-d-glucopyranoside (3, 0.035%), thunberginol C 8-O-
ously, we reported the isolation and structure determination of β-d-glucopyranoside (4, 0.045%), 4-hydroxythunberginol
many chemical constituents with anti-diabetic, anti-allergic, G 3′-O-β-d-glucopyranoside (5, 0.020%), and thunberginol
and anti-bacterial activities from the processed leaves and the
D
3′-O-β-d-glucopyranoside (6, 0.027%) together with
dried leaves of this plant.2–6) However, a full chemical analysis (3R)-phyllodulcin 8-O-β-d-glucopyranoside (7, 0.034%),16)
of the flowers from this plant has not yet been performed. In (3S)-phyllodulcin 8-O-β-d-glucopyranoside (8, 0.0059%),16)
the course of our chemical and pharmacological studies on (+)-hydrangenol 4′-O-β-d-glucopyranoside (9, 0.0040%),17)
Hydrangea plants2–8) and medicinal flowers,9–14) we recently (3R)-thunberginol I 4′-O-β-d-glucopyranoside (10, 0.056%),18)
reported the isolation and structure elucidation of secoiridoid (+)-3-(4-methoxyphenyl)-8-hydroxy-3,4-dihydroisocoumarin
glycosides named hydrangeamines A and B with a pyridine (11, hydrangenol monomethyl ether, 0.011%),19) phyllodulcin
ring from the flowers of H. macrophylla var. thunbergii cul- (12, 0.19%),20,21) hydrangenol 8-O-β-d-glucopyranoside (13,
tivated in Nagano prefecture, Japan.15) As a continuing study, 2.87%),16,18) thunberginol G 3′-O-β-d-glucopyranoside (14,
six dihydroisocoumarin glycosides, florahydrosides I (1) and II 0.16%),16,22) hydrangenol (15, 4.79%),23,24) thunberginol G
(2), thunberginol G 8-O-β-d-glucopyranoside (3), thunberginol (16, 0.067%),5,25) neochlorogenic acid (17, 0.075%),26,27) 3-O-
C 8-O-β-d-glucopyranoside (4), 4-hydroxythunberginol G trans-p-coumaroyl-d-quinic acid (18, 0.27%),28,29) 3-O-cis-p-
3′-O-β-d-glucopyranoside (5), and thunberginol D 3′-O-β-d- coumaroyl-d-quinic acid (19, 0.029%),29) chlorogenic acid (20,
glucopyranoside (6), were isolated from the flowers of H. mac- 0.054%),30) chlorogenic acid methyl ester (21, 0.049%),9,31)
rophylla var. thunbergii together with 20 known compounds taxiphyllin (22, 0.076%),8,32,33) umbelliferone glucoside (23,
including five acylated quinic acid analogs. Furthermore, the 0.11%),34,35) α-morroniside (24, 0.021%),36) trans-p-coumaric
inhibitory effects of the isolated compounds and its related acid (0.013%),30,37) and shikimic acid (0.36%)38) (Fig. 1).
compounds on aldose reductase were investigated. This paper
Structures of New Dihydroisocoumarin Glycosides (1–6)
deals with the structure elucidation of the new dihydroiso- Florahydrosides I (1) and II (2) were isolated as a white amor-
coumarin glycosides (1–6) and the inhibitory effects of the phous powder with negative optical rotation (1: [α]D20 −8.0°,
constituents and their related compounds, acylated quinic acid 2: [α]D25 −21.0°, in MeOH). Their IR spectra showed absorp-
analogs, on aldose reductase.
tion bands due to hydroxy (1 and 2: 3400cm−1), lactone (1:
A methanol (MeOH) extract (32.2% from the flowers of 1684 cm−1, 2: 1686cm−1), aromatic ring (1: 1610, 1508cm−1,
H. macrophylla var. thunbergii cultivated in Nagano prov- 2: 1618, 1510cm−1), and ether functions (1: 1071cm−1, 2:
ince) was partitioned between EtOAc–H2O (1:1) to furnish 1073cm−1). In the FAB-MS spectra of 1 and 2, the common
an EtOAc-soluble fraction (8.8%) and an aqueous layer. quasimolecular ion peak was observed at m/z 487 ([M+Na]+)
The aqueous layer was further extracted with 1-butanol and the molecular formula C22H24O11 was determined by high-
resolution (HR) MS measurement. Acid hydrolysis of 1 and 2
with 5% aqueous H2SO4 liberated d-glucose, which was iden-
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: matsuda@mb.kyoto-phu.ac.jp

656
Vol. 61, No. 6
Fig. 1. Structures of Constituents from the Flower of Hydrangea macrophylla
tified by HPLC analysis using an optical rotation detector. The dihydroisocoumarin.18) On the basis of all this evidence, the
1H-NMR (methanol-d4) and 13C-NMR (Table 1) spectra of 1, chemical structure of florahydroside I (1) was determined to
which were assigned by various NMR experiments,39) showed be (3S)-thunberginol E 8-O-β-d-glucopyranoside.
signals assignable to a dihydroisocoumarin moiety [δ 2.93
On the other hand, the 1H-NMR (methanol-d4) and
(dd, J=16.5, 2.1Hz, H-4a), 3.14 (dd, J=16.5, 12.4Hz, H-4b), 13C-NMR (Table 1) spectra of 2,39) showed signals assignable
5.29 (dd, J=12.4, 2.1Hz, H-3), 6.45 (brs, H-5), 6.78 (brs, to thunberinol E with a β-d-glucopyranosyl moiety as well
H-7)], ABX-type aromatic ring [δ 6.90 (1H, dd, J=8.2, 1.4Hz, as 1. In addition, on the basis of the comparison of the NMR
H-6′), 6.92 (1H, d, J=8.2Hz, H-5′), 6.94 (1H, d, J=1.4Hz, data for 2 with those of 1 and the DQF-COSY and HMBC
H-2′)], a methoxy group [δ 3.85 (s, OCH3)], and a β-d- experiments on 2 (Fig. 2), the position of the glucoside linkage
glucopyranosyl moiety [δ 4.81 (1H, d, J=7.6Hz, H-1′)]. The on 2 was determined to be the 6-position. Next, the absolute
proton and carbon signals of the 3-phenyldihydroisocoumarin configuration at the 3-position of 2 was found to be S by the
1
part in the H- and 13C-NMR spectra of 1 were superimpos- CD spectrum as well as 1. On the basis of all this evidence,
able on those of (3R)-thunberginol E [(3R)-3,4-dihydro-6,8- the chemical structure of florahydroside II (2) was determined
dihydroxy-3-(3-hydroxy-4-methoxyphenyl)isocoumarin],22) to be (3S)-thunberginol E 6-O-β-d-glucopyranoside.
except for the signals around the 8-position, while the pro-
Thunberginol G 8-O-β-d-glucopyranoside (3) and thun-
ton and carbon signals due to the dihydroisocoumarin part berginol C 8-O-β-d-glucopyranoside (4), which were obtained
including glycoside moiety were very similar to those of as a white amorphous powder and a mixture of diastereo-
(3S)-scorzocreticoside I [(3S)-3,4-dihydro-6,8-dihydroxy-3-(4- isomers, showed absorption bands due to hydroxy, lactone,
methoxyphenyl)isocoumarin 8-O-β-d-glucopyranoside].40) As aromatic ring, and ether functions in their IR spectra. In the
shown in Fig. 2, the double quantum filter correlation spectros- FAB-MS spectra of 3 and 4, the common quasimolecular ion
copy (DQF-COSY) experiment of 1 indicated the presence of peak was observed at m/z 457 ([M+Na]+) and the molecular
certain structures (highlighted by bold lines). Heteronuclear formula C21H22O10 was determined by HR-MS measurement.
multiple bond connectivity spectroscopy (HMBC) experi- Acid hydrolysis of 3 and 4 liberated d-glucose, which was
ments revealed long-range correlations between the following identified by HPLC analysis using an optical rotation detec-
protons and carbons: H-3 and C-4a, 1′, 6′; H-4 and C-5, 8a; tor. The 1H-NMR (methanol-d4) and 13C-NMR (Table 1)
H-5 and C-4, 8a; H-7 and C-6, 8, 8a; H-2′ and C-4′; H-5′ and spectra of 3, which were assigned by various NMR experi-
C-1′; H-6′ and C-3, 1′, 2′, 4′; H-1′ and C-8; OCH3 and C-4′. In ments, revealed the products to be a ca. 2:1 mixture of two
1
addition, the position of the glucoside linkage was confirmed diastereoisomers. Namely, the H-NMR showed double signals
by a nuclear Overhauser effect spectroscopy (NOESY) experi- assignable to a dihydroisocoumarin moiety {[major diaste-
ment, which was showed NOE correlations between H-7 and reoisomer]: δ 3.09 (dd, J=16.5, 2.8Hz, H-4a), 3.28 (m, H-4b),
H-1′. Next, the absolute configuration at the 3-position of 1 5.36 (dd, J=11.7, 2.8Hz, H-3), 7.10 (brd, J=8.2Hz, H-5), 7.40
was identified by circular dichroism (CD) spectrum. Namely, (brd, J=8.2Hz, H-7), 7.40 (dd, J=8.2, 8.2Hz, H-6), [minor
1 showed a positive Cotton effect at 236nm (Δε +8.62) diastereoisomer]: δ 3.16 (0.33H, dd, J=16.5, 2.8Hz, H-4a),
and a negative Cotton effect at 255nm (Δε −0.79) for (3S)- 3.28 (1H, m, H-4b), 5.41 (0.33H, dd, J=11.7, 2.8Hz, H-3), 7.04

June 2013
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Table 1. 13C-NMR (125MHz) Spectroscopic Data for Compounds 1–6 in Methanol-d4
Position
1
2
3
4
5
6
1
3
166.4
80.9
171.4
82.0
166.0/165.4
81.8/81.0
166.7/166.7
81.1/80.5
171.8/171.8
85.1/84.7
171.7/171.7
81.8/81.9
4
37.2
35.9
37.1/36.7
37.3/36.9
75.7/75.3
35.7/35.7
4a
5
6
7
8
8a
1′
2′
3′
4′
5′
6′
145.5
110.3
165.4
105.3
163.2
107.2
132.7
114.6
147.7
149.4
112.5
119.1
56.4
143.4
108.3
165.2
103.6
165.2
104.1
132.6
114.5
147.7
149.5
112.5
119.0
56.4
143.7/143.2
123.2/122.7
136.6/136.3
118.2/117.0
160.8/160.3
116.0/115.8
131.1/131.3
114.8/114.6
146.6/146.5
147.1/146.8
116.3/116.3
119.4/119.1
—
145.5/145.2
110.7/110.2
166.2/166.2
105.6/104.6
163.3/162.8
106.5/106.5
130.8/130.9
129.1/128.9
116.3/116.3
159.1/158.9
116.3/116.3
129.1/128.9
—
150.2/150.3
116.8/116.8
137.0/137.1
115.6/115.7
158.0/158.0
113.5/113.6
132.1/131.9
118.3/117.4
146.5/146.4
148.5/148.1
116.6/116.4
124.0/123.9
—
143.6/143.7
108.0/108.0
166.4/166.4
102.2/102.2
165.6/165.7
101.7/101.7
131.5/131.6
117.2/117.4
146.7/146.7
148.8/148.9
117.0/117.1
123.0/123.2
—
OCH3
Glu-1″
2″
105.1
74.9
101.4
74.7
105.1/103.1
74.7/75.0
105.2/103.3
74.9/74.5
104.5/104.1
74.9/74.8
104.0/104.3
74.9/74.9
3″
77.1
77.9
77.2/77.8
77.2/77.7
77.7/77.6
77.6/77.6
4″
71.1
71.2
71.3/71.2
71.2/71.1
71.3/71.3
71.4/71.5
5″
78.7
78.4
78.8/78.5
78.7/78.4
78.2/78.4
78.3/78.4
6″
62.5
62.4
62.6/62.5
62.5/62.5
62.4/62.5
62.5/62.5
Compounds 3–6 were obtained as 3-epimeric mixtures.
Fig. 2. Important 2D-NMR Correlations of Compounds 1–6
(0.33H, brd, J=7.6Hz, H-5), 7.29 (0.33H, brd, J=7.6Hz, H-7), part including glycoside moiety were very similar to those of
7.53 (0.33H, dd, J=7.6, 7.6Hz, H-6)}, an ABX-type aromatic 7,16) 8,16) and 13.16,18) As shown in Fig. 2, the HMBC experi-
ring {[major diastereoisomer]: δ 6.79 (d, J=8.5Hz, H-5′), 6.82 ments revealed long-range correlations between the following
(dd, J=8.5, 2.0Hz, H-6′), 6.93 (d, J=2.0Hz, H-2′), [minor dia- protons and carbons: H-4 and C-5, 8a; H-5 and C-4, 8a; H-6
stereoisomer]: δ 6.79 (0.33H, m, H-5′), 6.82 (0.33H, m, H-6′), and C-8; H-7 and C-5, 8a; H-2′ and C-3, 4′; H-5′ and C-1′;
6.88 (0.33H, brs, H-2′)} and a β-d-glucopyranosyl moiety H-6′ and C-3, 2′, 4′; H-1″ and C-8. Furthermore, the 3-posi-
{[major diastereoisomer]: δ 4.90 (d, J=7.6Hz, H-1′), [minor tion of dihydroisocoumarins with the 4′-hydroxyphenyl group
diastereoisomer]: δ 4.94 (d, J=8.3Hz, H-1′)}. The proton and such as hydrangenol (15), thunberginols B, C, and G (16),
1
carbon signals of the aglycon part in the H- and 13C-NMR were reported to display tautomer-like behavior.18) Therefore,
spectra of 3 were superimposable on those of thunberginol 3 was displayed to be a 3-epimeric mixture of thunberiginol
G (16),5,25) except for the signals around the 8-position, while G glycoside. On the basis of all this evidence, the chemical
the proton and carbon signals due to the dihydroisocoumarin structure of 3 was determined to be as shown.

658
Vol. 61, No. 6
On the other hand, the 1H-NMR (methanol-d4) and
13C-NMR (Table 1) spectra of 4 revealed the products to be
a ca. 7:3 mixture of two diastereoisomers. The proton and
carbon signals of the dihydroisocoumarin part in the 1H-
and 13C-NMR spectra of 4 were superimposable on those of
1, while the proton and carbon signals due to the 3-phenyl
part were very similar to those of hydrangenol 8-O-β-d-
glucopyranoside (13).16,18) On the basis of the DQF-COSY
and HMBC experiments (Fig. 1), the structure of 4 was de-
Fig. 3. Structures of Caffeoylquinic Acid Analogs 25–28
termined to be thunberginol C17,20,22) with glucoside. Next, the effects on aldose reductase of the isolated constituents from
position of the glucoside linkage in 4 was confirmed based on the flowers of H. macrophylla var. thunbergii were examined.
HMBC and NOESY experiments, which was showed long- Among the constituents, neochlorogenic acid (17) inhibited
range correlations between H-1″ and C-8 and NOE correla- aldose reductase [IC50=5.6 µM]. In addition, chlorogenic acid
tions between H-7 and H-1″, respectively. Consequently, the methyl ester (21) showed the inhibitory effect [IC50=2.9µM]
chemical structure of 4 was determined to be as shown.
4-Hydroxythunberginol 3′-O-β-d-glucopyranoside (5) dihydroisocoumarin glycosides (1–10, 13, 14) and dihydroiso-
in agreement with the previous study.44) On the other hand,
G
and thunberginol D 3′-O-β-d-glucopyranoside (6), which were coumarins (11, 12) lacked the inhibitory effects [IC50>100 µM].
obtained as a mixture of diastereoisomers, showed absorp- Hydrangenol (15), thunberginol G (16), taxiphyllin (22), and
tion bands due to hydroxy, lactone, aromatic ring, and ether umbelliferone glucoside (23) exhibited moderate inhibitory
functions in their IR spectra. The common molecular formula effects [IC50=48–69µM]. Next, the inhibitory effects on aldose
(C21H22O11) of 5 and 6 was determined from the quasimolecu- reductase of caffeoylquinic acid analogs 25–28, which were
lar ion peak (m/z 473 [M+Na]+) in the positive-ion FAB-MS previously obtained from Ilex paraguariensis,48,49) were exam-
and by HR-MS measurement. Acid hydrolysis of 5 and 6 lib- ined for the structure–activity relationship study (Fig. 3, Table
1
erated d-glucose. The H-NMR (methanol-d4) and 13C-NMR 2). The inhibitory effect of d-quinic acid with trans-p-caffeoyl
(Table 1) spectra of 5,39) showed signals assignable to a dihy- group at the 5-position [chlorogenic acid (20, reference com-
droisocoumarin moiety {ca. 11:9 mixture of two diastereoiso- pound,50) IC50=0.41 µM)] was stronger than those of d-quinic
mers (3-epimeric mixture), [major diastereoisomer]: δ 4.81 (d, acids with trans-p-caffeoyl group at the 3 or 4-positions [17
J=5.5Hz, H-4), 5.60 (d, J=5.5Hz, H-3), 6.53 (brd, J=7.9Hz, or 4-O-trans-p-caffeoyl-d-quinic acid (25, IC50=11.8 µM)]. In
H-7), 6.80 (brd, J=7.9Hz, H-5), 7.40 (dd, J=7.9, 7.9Hz, H-6), addition, quinic acids with two caffeoyl groups [3,4-di-O-
[minor diastereoisomer]: δ 4.91 (d, J=5.5Hz, H-4), 5.61 (d, trans-caffeoyl-d-quinic acid (26, IC50=0.34µM), 3,5-di-O-
J=5.5Hz, H-3), 6.82 (brd, J=7.9Hz, H-5), 7.45 (dd, J=7.9, trans-caffeoyl-d-quinic acid (27, IC50=0.31 µM), and 4,5-di-O-
7.9Hz, H-6), 6.74 (overlap, H-7); ABX-type aromatic ring, and trans-caffeoyl-d-quinic acid (28, IC50=0.29µM)] exhibited
a β-d-glucopyranosyl moiety. The proton and carbon signals potent inhibitory effects. Those biological effects were equal
1
in the H- and 13C-NMR spectra of 5 were superimposable on to or stronger than that of a reference compound, chlorogenic
those of thunberginol G 3′-O-β-d-glucopyranoside (14),16,22) acid (20). Further study for the development of acylated quinic
except for the signals around the 4-position. The structure of acid analogs as potent anti-cataract agents are expected.
dihydroisocoumarin moiety including the two hydroxy groups
on 5 was confirmed based on DQF and HMBC experiments,
Experimental
which was showed long-range correlations between the fol-
General Experimental Procedures The following instru-
lowing protons and carbons: H-3 and C-4; H-4 and C-3, 5; H-5 ments were used to obtain physical data: specific rotations,
and C-8a; H-6 and C-8; H-7 and C-8a. On the basis of all this a Horiba SEPA-300 digital polarimeter (l=5cm); IR spectra,
evidence, the chemical structure of 5 was determined to be as a Shimadzu FTIR-8100 spectrometer; CD spectra, a JASCO
shown.41)
J-720WI spectrometer; EI-MS and HR-EI-MS, JEOL JMS-
On the other hand, the proton and carbon signals of the GCMATE mass spectrometer; FAB-MS and HR-FAB-MS, a
1
aglycon part in the H- and 13C-NMR spectra of 6 were su- JEOL JMS-SX 102A mass spectrometer; 1H-NMR spectra,
perimposable on those of thunberginol D,20,22) except for the JEOL JNM-ECA600 (600MHz) spectrometers; 13C-NMR
signals around the 3′-position, while the proton and carbon spectra, JEOL JNM-ECA600 (150MHz) spectrometers with
signals due to the 3-benzene ring including a glucoside moiety tetramethylsilane as an internal standard; and HPLC, a Shi-
were very similar to those of 5. In addition, on the basis of the madzu RID-6A refractive index and SPD-10Avp UV-VIS
DQF-COSY and HMBC experiments (Fig. 1), the chemical detectors. YMC-Pack ODS-A (YMC), COSMOSIL-5C18-
structure of 6 were determined to be as shown.
PAQ (Nacalai Tesque) and COSMOSIL-Cholester (Nacalai
Inhibitory Effect on Rat Lens Aldose Reductase Al- Tesque) {[250×4.6mm i.d. (5µm) for analytical purposes] and
dose reductase, a key enzyme in the polyol pathway, has [250×20mm i.d. (5µm) for preparative purposes]} columns
been reported to catalyze the reduction of glucose to sorbitol. were used. The following experimental conditions were used
Sorbitol does not readily diffuse across cell membranes, and for chromatography: ordinary-phase silica gel column chroma-
the intracellular accumulation of sorbitol has been implicated tography (CC), Silica gel BW-200 (Fuji Silysia Chemical, Ltd.,
in the chronic complications of diabetes such as cataract. 150–350 mesh); reverse-phase silica gel CC, Chromatorex
Previously, we have reported that various constituents such as ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh);
acylated quinic acids, flavonoids, and terpenoids from natural TLC, precoated TLC plates with Silica gel 60F254 (Merck,
medicines and medicinal foods inhibited aldose reductase 0.25mm) (ordinary phase) and Silica gel RP-18 F254S (Merck,
inhibitory effect.1,9,42–47) In the present study, the inhibitory 0.25mm) (reverse phase); reversed-phase HPTLC, precoated

June 2013
659
Table 2. Inhibitory Effects on Aldose Reductase of Compounds from the
Flowers of H. macrophylla var. thunbergii and Caffeoylquinic Acid Ana-
logs
droxythunberginol G 3′-O-β-d-glucopyranoside (5, 5.8mg),
hydrangenol 8-O-β-d-glucopyranoside (13, 42mg), and
trans-p-coumaric acid (3.7mg). A part of Fr. B1-6 was puri-
fied by HPLC {MeOH–H2O ([1] 40:60, YMC-Pack ODS-A),
[2] 40:60, COSMOSIL-Cholester} to give thunberginol G
8-O-β-d-glucopyranoside (3, 4.1mg), thunberginol C 8-O-β-
d-glucopyranoside (4, 5.3mg), and (3R)-phyllodulcin 8-O-β-
d-glucopyranoside (7, 4.0mg). A part of Fr. B1-8 was purified
by HPLC {MeOH–H2O ([1] 40:60, YMC-Pack ODS-A),
[2] 35:65, COSMOSIL-Cholester} to give florahydroside
I (1, 36mg), florahydroside II (2, 6.3mg), thunberginol D
3′-O-β-d-glucopyranoside (6, 14mg), (3S)-phyllodulcin 8-O-
β-d-glucopyranoside (8, 3.2mg), (+)-hydrangenol 4′-O-β-d-
glucopyranoside (9, 2.1mg), and thunberginol G 3′-O-β-d-
glucopyranoside (14, 102mg). A part of Fr. B1-9 was purified
by HPLC [MeOH–H2O (45:55, YMC-Pack ODS-A)] to give
(3R)-thunberginol I 4′-O-β-d-glucopyranoside (10, 34mg). A
part of the EtOAc-soluble fraction (20.0g) was subjected to
normal-phase silica gel CC [300g, n-hexane–EtOAc (10:1→
5:1→2:1, v/v)→CHCl3–MeOH (20:1→10:1)→MeOH] to
Sample
Hydrangenol (15)
Thunberginol G (16)
Neochlorogenic acid (17)
IC50 (µM)
47.8
58.3
5.6
3-O-trans-Coumaroyl-d-quinic acid (18)
3-O-cis-Coumaroyl-d-quinic acid (19)
Taxiphyllin (22)
11.5
55.2
54.1
68.6
11.8
0.34
0.31
0.29
2.4
Umbelliferone glucoside (23)
4-O-trans-Caffeoyl-d-quinic acid (25)
3,4-Di-O-trans-caffeoyl-d-quinic acid (26)
3,5-Di-O-trans-caffeoyl-d-quinic acid (27)
4,5-Di-O-trans-caffeoyl-d-quinic acid (28)
Chlorogenic acid methyl ester (21)44)
Chlorogenic acid (20) (positive control)50)
0.41
Each value represents the mean of 2–4 experiments. The IC50 values of compounds
1–14, and 24 were less than 100µM.
TLC plates with Silica gel RP-18 WF254S (Merck, 0.25mm). give 10 fractions [Fr. E1-1, Fr. E1-2, Fr. E1-3, Fr. E1-4 (2.67g),
Detection was achieved by spraying with 1% Ce(SO4)2–10% Fr. E1-5, Fr. E1-6 (0.75g), Fr. E1-7, Fr. E1-8, Fr. E1-9, Fr.
aqueous H2SO4 followed by heating.
E1-10]. A part of Fr. E1-4 was purified by HPLC [MeOH–H2O
Plant Material Flowers of Hydrangea macrophylla (60:40, YMC-Pack ODS-A)] to give hydrangenol (15, 921mg).
Seringe var. thunbergii Makino (Saxifragaceae), which were A part of Fr. E1-6 was purified by HPLC [MeOH–H2O
cultivated in Nagano prefecture, Japan, in 2010, were pur- (60:40, YMC-Pack ODS-A)] to give phyllodulcin (12, 39mg),
chased from Kurohime Medical Herb Tea Co., Ltd. (Nagano, hydrangenol (15, 37mg), and thunberginol G (16, 13mg).
Japan). A voucher of the plant is on file in our laboratory
Florahydroside I (1): A white amorphous powder; [α]D20
−8.0° (c=0.50, MeOH); IR (KBr): νmax 3400, 1684, 1610,
(KPU Medicinal Flower-2010-HM).
Extraction and Isolation Dry flowers of H. macrophylla 1508, 1071cm−1; CD (MeOH) λmax (Δε) 236 (+8.62), 255
1
var. thunbergii (647g) were extracted three times with MeOH (−0.79); H-NMR (methanol-d4, 600MHz) δ 2.93 (dd, J=16.5,
for 3h under reflux. Evaporation of the solvent under reduced 2.1Hz, H-4a), 3.14 (dd, J=16.5, 12.4Hz, H-4b), 3.85 (s, OCH3),
pressure provided a MeOH extract (209g, 32.2%). A part of 4.81 (d, J=7.6Hz, H-1′), 5.29 (dd, J=12.4, 2.1Hz, H-3), 6.45
the MeOH extract (198g) was partitioned into an EtOAc– (brs, H-5), 6.78 (brs, H-7), 6.90 (dd, J=8.2, 1.4Hz, H-6′),
H2O (1:1, v/v) mixture to furnish an EtOAc-soluble fraction 6.92 (d, J=8.2Hz, H-5′), 6.94 (d, J=1.4Hz, H-2′); 13C-NMR:
(53.9g, 8.8%) and aqueous phase, which was extracted with given in Table 1; positive-ion FAB-MS: m/z 487 [M+Na]+;
1-BuOH to give 1-BuOH- (76.9g, 12.5%) and H2O- (67.5g, HR-FAB-MS: m/z 487.1214 (Calcd for C22H24O11Na [M+Na]+:
11.0%) soluble fractions as reported previously.15) A part of m/z 487.1216).
the 1-BuOH-soluble fraction (72.5g) was subjected to normal-
Florahydroside II (2): A white amorphous powder; [α]D25
phase silica gel CC [500g, CHCl3–MeOH–H2O (20:3:1→ −21.0° (c=0.50, MeOH); IR (KBr): νmax 3400, 1686, 1618,
10:3:1→7:3:1→6:4:1)→MeOH] to give 2 fractions [Fr. B1 1510, 1073cm−1; CD (MeOH) λmax (Δε) 231 (+3.88), 249
1
(44.2g) and Fr. B2 (16.4g)]. Fraction B1 (42.6g) was subjected (−3.14); H-NMR (methanol-d4, 600MHz) δ 3.01 (dd, J=16.5,
to reversed-phase silica gel CC [740g, MeOH–H2O (10:90→ 2.0Hz, H-4a), 3.15 (dd, J=16.5, 12.4Hz, H-4b), 3.77 (s, OCH3),
20:80→30:70→40:60→50:50→MeOH)] to give 10 fractions 4.92 (d, J=7.6Hz, H-1′), 5.40 (dd, J=12.4, 2.0Hz, H-3), 6.46
[Fr. B1-1 (2.23g), Fr. B1-2, Fr. B1-3, Fr. B1-4 (4.69g), Fr. B1-5 (brs, H-7), 6.47 (brs, H-5), 6.83 (d, J=8.2Hz, H-5′), 6.85 (brd,
(3.54g), Fr. B1-6 (6.23g), Fr. B1-7, Fr. B1-8 (6.11g), Fr. B1-9 J=8.2Hz, H-6′), 6.85 (brs, H-2′); 13C-NMR: given in Table 1;
(0.92g), Fr. B1-10]. A part of Fraction B1-1 was separated positive-ion FAB-MS: m/z 487 [M+Na]+; HR-FAB-MS: m/z
by HPLC [MeOH–H2O (20:80, COSMOSIL-5C18-PAQ)] to 487.1220 (Calcd for C22H24O11Na [M+Na]+: m/z 487.1216).
give shikimic acid (413mg). A part of Fr. B1-3 was purified
Thunberginol G 8-O-β-d-Glucopyranoside (3): A white
by HPLC {MeOH–H2O ([1] 30:70, YMC-Pack ODS-A), [2] amorphous powder; IR (KBr): νmax 3400, 1684, 1618, 1509,
1
20:80, COSMOSIL-Cholester} to give neochlorogenic acid 1070cm−1; H-NMR (methanol-d4, 600MHz, ca. 2:1 mixture
(17, 23mg), 3-O-trans-p-coumaroyl-d-quinic acid (18, 70mg), of two diastereoisomers): [major diastereoisomer] δ 3.09 (dd,
3-O-cis-p-coumaroyl-d-quinic acid (19, 8.8mg), chlorogenic J=16.5, 2.8Hz, H-4a), 3.28 (m, H-4b), 4.90 (d, J=7.6Hz, H-1′),
acid (20, 17mg), taxiphyllin (22, 24mg), umbelliferone glu- 5.36 (dd, J=12.4, 2.8Hz, H-3), 6.79 (d, J=8.5Hz, H-5′), 6.82
coside (23, 14mg), and α-morroniside (24, 6.4mg). A part (dd, J=8.5, 2.0Hz, H-6′), 6.93 (d, J=2.0Hz, H-2′), 7.10 (brd,
of Fr. B1-4 was purified by HPLC {MeOH–H2O ([1] 40:60, J=8.2Hz, H-5), 7.40 (brd, J=8.2Hz, H-7), 7.40 (dd, J=8.2,
YMC-Pack ODS-A), [2] 30:70, COSMOSIL-Cholester} to 8.2Hz, H-6), [minor diastereoisomer] δ 3.16 (dd, J=16.5,
give hydrangenol 8-O-β-d-glucopyranoside (13, 197mg) and 2.8Hz, H-4a), 3.28 (m, H-4b), 4.94 (d, J=8.3Hz, H-1″), 5.41
chlorogenic acid methyl ester (21, 5.1mg). A part of Fr. B1-5 (dd, J=10.4, 2.8Hz, H-3), 6.79 (m, H-5′), 6.82 (m, H-6′), 6.88
was purified by HPLC {MeOH–H2O ([1] 45:55, YMC-Pack (brs, H-2′), 7.04 (brd, J=7.6Hz, H-5), 7.29 (brd, J=7.6Hz,
ODS-A), [2] 40:60, COSMOSIL-Cholester} to give 4-hy- H-7), 7.53 (dd, J=7.6, 7.6Hz, H-6); 13C-NMR: given in Table

660
Vol. 61, No. 6
1; positive-ion FAB-MS: m/z 457 [M+Na]+; HR-FAB-MS: m/z
457.1105 (Calcd for C21H22O10Na [M+Na]+: m/z 457.1111).
Effects on Rat Lens Aldose Reductase The experi-
ments were performed as described in our previous reports.42)
Thunberginol C 8-O-β-d-Glucopyranoside (4): A white The supernatant fluid of rat lens homogenate was used as a
amorphous powder; IR (KBr): νmax 3400, 1684, 1617, 1509, crude enzyme. The enzyme suspension was diluted to pro-
1
1071cm−1; H-NMR (methanol-d4, 600MHz, ca. 7:3 mixture duce ca. 10 nmoL/tube of nicotinamide adenine dinucleotide
of two diastereoisomers): [major diastereoisomer] δ 2.95 (dd, phosphate (NADP) in the following reaction. The incubation
J=16.5, 2.0Hz, H-4a), 3.22 (dd, J=16.5, 13.1Hz, H-4b), 4.82 mixture contained phosphate buffer 135mM (pH 7.0), Li2SO4
(d, J=6.8Hz, H-1″), 5.34 (dd, J=13.1, 2.0Hz, H-3), 6.80 (d, 100 mM, reduced nicotinamide adenine dinucleotide phosphate
J=8.9Hz, H-3′,5′), 7.31 (d, J=8.9Hz, H-2′,6′), 6.43 (brs, H-5), (NADPH) 0.03mM, dl-glyceraldehyde 1mM as a substrate,
6.75 (brs, H-7), [minor diastereoisomer] δ 3.03 (dd, J=16.5, and 100µL of enzyme fraction, with 25µL of sample solution,
2.0Hz, H-4a), 3.24 (m, H-4b), 4.82 (d, J=6.8Hz, H-1″), 5.41 in a total volume of 0.5mL. The reaction was initiated by the
(dd, J=11.0, 2.8Hz, H-3), 6.78 (d, J=8.9Hz, H-3′,5′), 7.27 (d, addition of NADPH at 30°C. After 30min, the reaction was
J=8.9Hz, H-2′,6′), 6.40 (brs, H-5), 6.66 (brs, H-7); 13C-NMR: stopped by the addition of 150µL of HCl 0.5M. Then, 0.5mL
given in Table 1; positive-ion FAB-MS: m/z 457 [M+Na]+; of NaOH 6M containing imidazole 10mM was added, and the
HR-FAB-MS: m/z 457.1107 (Calcd for C21H22O10Na [M+Na]+: solution was heated at 60°C for 20min to convert NADP into
m/z 457.1111).
a fluorescent product. Fluorescence was measured using a
4-Hydroxythunberginol G 3′-O-β-d-Glucopyranoside (5): fluorescence microplate reader (FLUOstar OPTIMA, BMG
A white amorphous powder; IR (KBr): νmax 3400, 1682, 1619, Labtechnologies) at an excitation wavelength of 360nm and
1
1508, 1073cm−1; H-NMR [methanol-d4, 600MHz, ca. 11:9 an emission wavelength of 460nm. Each test sample was dis-
mixture of two diastereoisomers (3-epimeric mixture)]: [major solved in DMSO. Measurements were performed in duplicate,
diastereoisomer] δ 4.69 (d, J=7.6Hz, H-1″), 4.81 (d, J=5.5Hz, and IC50 values were determined graphically.
H-4), 5.60 (d, J=5.5Hz, H-3), 6.53 (brd, J=7.9Hz, H-7),
6.76 (d, J=8.2Hz, H-5′), 6.80 (brd, J=7.9Hz, H-5), 6.88 (dd,
Acknowledgments This research was supported in part
J=8.2, 2.0Hz, H-6′), 7.22 (d, J=2.0Hz, H-2′), 7.40 (dd, J=7.9, by a Grant-in-Aid for Scientific Research from the Ministry of
7.9Hz, H-6), [minor diastereoisomer] δ 4.58 (d, J=7.6Hz, Education, Culture, Sports, Science and Technology (MEXT)
H-1″), 4.91 (d, J=5.5Hz, H-4), 5.61 (d, J=5.5Hz, H-3), 6.74 of Japan-Supported Program for the Strategic Research Foun-
(overlap, H-7), 6.74 (d, J=8.2Hz, H-5′), 6.82 (brd, J=7.9Hz, dation at Private Universities, by a Grant-in-Aid for Young
H-5), 6.86 (dd, J=8.2, 2.0Hz, H-6′), 7.10 (d, J=2.0Hz, H-2′), Scientists (B) from the Japan Society for the Promotion of
7.45 (dd, J=7.9, 7.9Hz, H-6); 13C-NMR: given in Table 1; Science (JSPS).
positive-ion FAB-MS: m/z 473 [M+Na]+; HR-FAB-MS: m/z
473.1056 (Calcd for C21H22O11Na [M+Na]+: m/z 473.1060).
References and Note
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