Cyclic spermidine alkaloids and flavone glycosides from Meehania fargesii

Article abstract of DOI:10.1248/cpb.58.696

The dried whole plant of Meehania fargesii (H. LEV.) C. Y. WU is utilized as an antipyretic and antidote for poison in China. Water soluble fractions, obtained from an 80% acetone extract of the whole plant, showed hyaluronidase inhibitory activity. From these fractions, hyaluronidase inhibitors were isolated along with two new spermidine alkaloids (meefarnines A and B), four new flavone glycosides (fargenins A-D), and 10 known compounds. The structure of each was elucidated by spectroscopic methods.

Full text of DOI:10.1248/cpb.58.696

6
96
Regular Article
Chem. Pharm. Bull. 58(5) 696—702 (2010)
Vol. 58, No. 5
Cyclic Spermidine Alkaloids and Flavone Glycosides from
Meehania fargesii
,
a
b
a
Toshihiro MURATA,* Toshio MIYASE, and Fumihiko YOSHIZAKI
a
Department of Pharmacognosy, Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai 981–8558,
b
Japan: and School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526,
Japan. Received January 22, 2010; accepted February 9, 2010; published online February 10, 2010
The dried whole plant of Meehania fargesii (H. LÉV.) C. Y. WU is utilized as an antipyretic and antidote for
poison in China. Water soluble fractions, obtained from an 80% acetone extract of the whole plant, showed
hyaluronidase inhibitory activity. From these fractions, hyaluronidase inhibitors were isolated along with two
new spermidine alkaloids (meefarnines A and B), four new flavone glycosides (fargenins A—D), and 10 known
compounds. The structure of each was elucidated by spectroscopic methods.
Key words spermidine alkaloid; polyamine; flavone glycoside; Meehania fargesii; Lamiaceae; hyaluronidase inhibitor
Meehania fargesii (H. LÉV.) C. Y. WU is a climbing herba- showed strong inhibitory activity, and diosmetin-7-O-b-D-
ceous plant (Lamiaceae) known as Chinese herb. The dried glucuronopyranoside and apigenin-7-O-b-D-glucuronopyra-
or fresh whole plant and the dried root have been used to noside had moderate inhibitory activity as shown in Table 4.
1
)
treat colds, pain and poisoning. Two caffeic acid esters in
Meefarnines A (1) and B (2) were revealed to have the
2)
this plant were reported. However, studies on other con- molecular formula C H N O based on high resolution
2
5
31
3
4
stituents and the biological activities of this plant are not suf- (HR)-FAB-MS [m/z 438.2411 (1) and 438.2394 (2), Calcd
1
3
ficient. The water soluble fractions obtained from an 80% for C H N O , 438.2395]. However, the C-NMR spec-
2
5
32
3
4
acetone extract of M. fargesii were found to have an in- trum acquired in methanol-d at 30 °C showed more than 25
4
1
hibitory effect on hyaluronidase. Hyaluronidase degrades signals and the H-NMR spectrum showed the presence of
hyaluronic acid and its inhibitors are known to have anti- two or more sets of closely spaced resonances (Table 1).
3,4)
allergic activity. Moreover, they are potent anti-inflamma- These spectra were attributed to the restricted rotation of the
5)
18—21)
1
13
tory, anti-aging, and anti-cancer agents. In the present amide bonds.
H- and C-NMR spectra of 1 were
study, we investigated constituents of fractions which showed acquired in dimethyl sulfoxide (DMSO)-d at temperatures
6
1
13
strong hyaluronidase inhibitory activity from whole plants of between 30 °C and 100 °C (Fig. 2). The H- and C-NMR
M. fargesi. Rosmarinic acid, lithospermic acid B, diosmetin- spectra at 100 °C of 1 and 2 showed one set of resonances
7
-O-b-D-glucuronide, and apigenin-7-O-b-D-glucuronide were (Table 1), which were similar to those of a cyclic spermidine
22,23)
identified as hyaluronidase inhibitors. Two new cyclic sper- alkaloid: (S)-dihydroperiphylline
midine alkaloids, meefarnines A and B (1, 2), four new and olefinic signals. For 1, analyses of the H-NMR and
flavone glycosides, fargenins A—D (3—6), and the methyl
except for the aromatic
1
1
1
H– H correlation spectroscopy (COSY) spectra in methanol-
ester (4a) of 4 have also been isolated together with 10 d showed the presence of a N–C(ꢀO)–CH –CH–N spin sys-
4
2
known compounds and we describe the elucidation of their tem [d 3.72 (overlapped, H-4), 3.79 (dd, Jꢀ7.5, 7.0 Hz, H-4),
structure. and 2.36 (br d, Jꢀ7.0 Hz, H-3)], a N–(CH ) –N spin system
2
3
Dried whole plants of M. fargesii were extracted with ace- [d 2.13 (m, H-6), 2.43 (ddd, Jꢀ12.5, 6.5, 2.0 Hz, H-6), 2.53
tone–water (8 : 2). The concentrated extract was partitioned (ddd, Jꢀ12.5, 5.5, 2.0 Hz, H-6), 1.37 (m, H-7), 1.60 (over-
between water and diethyl ether. The water layer was frac- lapped, H-7), 1.90 (overlapped, H-7), 3.40—3.52 (over-
tionated using the inhibitory activity against hyaluronidase as lapped, H-8), and 3.65 (m, H-8)], and a N–(CH ) –N spin
2
4
a marker during column chromatography (a porous polymer system [d 3.36—3.55 (overlapped, H-10), 3.69—3.77 (over-
gel column and octadecyl silica (ODS) columns). From the lapped, H-10), 1.80 (overlapped, H-11), 1.40—1.53 (over-
active fractions (Fr. 1A—D, Table 3), 21 compounds were iso- lapped, H-12), 1.61—1.70 (overlapped, H-12), 3.21 (m, H-
lated. Known compounds were identified from spectroscopic 13), and 3.36—3.46 (overlapped, H-13)]. The proton signals
6)
data as apigenin-7-O-b-D-glucuronopyranoside, apigenin- of H-6 and H-7 were divided due to the rotamer of the amide
6
,7)
7
-O-b-D-glucuronopyranoside methyl ester,
acacetin-7-O-
luteolin-7-O-b-D-glucuronopy-
diosmetin-7-O-b-D-glucuronopyra-
6,8)
b-D-glucuronopyranoside,
ranoside,
9)
10)
hyperin,
7
)
11)
12)
12)
noside methyl ester, acacetin, pectolarigenin, ladanein,
-{[6-O-(4-hydroxy-3,5-dimethoxybenzoyl)-b-D-glucopyra-
4
13)
14)
nosyl]oxy}-3-methoxybenzoic acid, rosmarinic acid, 7-
15)
16)
epiblechnic acid, and lithospermic acid B. Four principal
components of Fr. 1A and Fr. 1B, rosmarinic acid, lithosper-
mic acid B, diosmetin-7-O-b-D-glucuronopyranoside, and
apigenin-7-O-b-D-glucuronopyranoside, were assayed for
hyaluronidase inhibitory activity. Rosmarinic acid is already
17)
known as a hyaluronidase inhibitor. Lithospermic acid B Fig. 1. Structures of 1—6
∗
To whom correspondence should be addressed. e-mail: murata-t@tohoku-pharm.ac.jp.
© 2010 Pharmaceutical Society of Japan

May 2010
697
d
d
d
d
d
d
d
d

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Vol. 58, No. 5
d
d
d
d
d
d
d
d
d
d
d
d

May 2010
699
Table 3. Hyaluronidase Inhibitory Activity of Fractions of M. fargesii
Obtained with a Porous Polymer Gel and an ODS Column
Fraction
Hyaluronidase inhibition (%)
Water eluate
ꢄ6.38ꢅ3.94
26.8ꢅ4.62
82.8ꢅ1.52
86.9ꢅ3.97
92.8ꢅ2.16
65.5ꢅ4.07
65.1ꢅ6.92
31.9ꢅ2.55
30% MeOH eluate
MeOH eluate
Fr. 1A
Fr. 1B
Fr. 1C
Fr. 1D
Fr. 1E
All values represent the meanꢅS.E. (nꢀ3).
Table 4. Hyaluronidase Inhibitory Activity of Compounds from M.
fargesii
a)
Compound
Rosmarinic acid
Lithospermic acid B
Apigenin-7-b-D-glucuronide
Diosmetin-7-b-D-glucuronide
DSCG
Content (%)
IC50 (mM)
1.36
1.63
0.96
1.94
309
164
548
644
297
1
Fig. 2. H-NMR Spectra of 1 in DMSO-d6 at Temperatures from 30 to
00 °C
1
a) g/g of the methanol eluate fraction.
bond. These spin system protons and corresponding carbons
displayed the presence of a cyclic spermidine 13-membered
22)
ring moiety, which was either a periphylline-type skeleton
24)
(
1,4,9-triazacyclotridecan-4-one) or a celacinnine-type skele-
ton (1,4,9-triazacyclotridecan-2-one). Both skeletons were
possible via some biosynthetic pathway of naturally occur-
25)
ring macrocyclic spermidine alkaloids. In the heteronu-
clear multiple bond correlation (HMBC) spectrum, the H-6
signals were long-range coupled with the carbon of C-4 (d
61.2). Other long-range couplings included the H-8 signals
with the carbon of C-10 (d 51.0), and H-13 signals with the
carbon of C-2 (d 175.0). These data demonstrared that the
1
1
1
3-membered ring spermidine moiety was the periphylline- Fig. 3. H– H COSY, Key HMBC and Key NOE Correlations for Com-
type skeleton as shown in Fig. 3. The absolute configuration pounds 1 and 2
of C-4 was assumed to be S since all other known macro-
cyclic spermidine alkaloids have the (S)-configuration. The tified as (S)-4-(4-hydroxyphenyl)-9-[(2Z)-1-oxo-3-(4-hy-
circular dichroism (CD) spectrum of 1 revealed a weak cot- droxyphenyl)-2-propen-1-yl]-1,5,9-triazacyclotridecan-2-one.
1
13
ton effect around 210—240 nm, which was comparable to
The H- and C-NMR spectra (in methanol-d ) of 2 were
4
1
that reported for the (S)-configuration of cyclic sper- similar to those of 1 except for the olefinic signals. The H-
2
5—28)
midines.
NMR spectrum of the olefinic signals at d 6.76 (overlapped,
1
The H-NMR spectrum of the AB-type benzene ring sig- H-27) and 7.50 (d, Jꢀ15.5 Hz, H-26) suggested the presence
nals at d 6.70 (br d, Jꢀ8.5 Hz, H-22, 24), 6.71 (br d, Jꢀ of a (E)-p-coumaroyl unit. Hence, 2 was identified as (S)-4-
8
.5 Hz, H-22, 24), 7.21 (br d, Jꢀ8.5 Hz, H-21, 25), and 7.22 (4-hydroxyphenyl)-9-[(2E)-1-oxo-3-(4-hydroxyphenyl)-2-
(
(
(
br d, Jꢀ8.5 Hz, H-21, 25) and the olefinic signals at d 6.51 propen-1-yl]-1,5,9-triazacyclotridecan-2-one.
d, Jꢀ13.0 Hz, H-26), 6.56 (d, Jꢀ13.0 Hz, H-26), and 5.90 Diosmetin-7-O-glucuronide was obtained as a flavone gly-
d, Jꢀ13 Hz, H-27) suggested the presence of a (Z)-p- coside. The full assignments of the H- and C-NMR spectra
1
13
coumaroyl unit. In the HMBC spectrum, the H-8 and -10 sig- of this compound in DMSO-d are shown in Table 2. A nu-
6
nals were long-range coupled with the carbon of C-28 (d clear Overhauser effect (NOE) correlation between H-OMe
172.0, 172.1). The other AB type benzene ring signals at d (d 3.88, 3H, s) and H-5ꢁ (d 7.10, d, Jꢀ8.5 Hz) supported that
6.74 (br d, Jꢀ8.5 Hz, H-16, 18), 6.76 (br d, Jꢀ8.5 Hz, H-16, aglycone had a diosmetin skeleton. This compound seemed
18), and 7.09 (br d, Jꢀ8.5 Hz, H-15, 19) showed the presence to be a main flavone glycoside in M. fargesii.
of a p-hydroxybenzene moiety. The proton signals of H-16,
Five novel flavone glucuronides were obtained as amor-
1
13
18, 21, 22, 24, 25 and 26 were divided due to the rotamer of phous powders. The H- and C-NMR data are shown in
29)
the amide bond. In the HMBC spectrum, the H-4 methine Table 2. These spectra and the UV spectra of compounds
signals were long-range coupled with the carbons of C-14 (d 3—6 suggested that they were diosmetin-7-glycoside deriva-
1
35.5) and C-15 and 19 (d 128.4, 128.5). Hence, 1 was iden- tives. For fargenin A (3), the molecular formula C H O
2
4
22 13

7
00
Vol. 58, No. 5
the basis of HR-FAB-MS, the molecular formula C H O
28 18
2
8
was deduced (m/z 653.1373, Calcd for C H O , 653.1353).
2
8
28 18
The anomeric carbon at d 104.3 suggested the presence of
another monosaccharide moiety. The corresponding anomeric
1
1
proton at d 4.55 (1H, d, Jꢀ7.5 Hz, H-1ꢃ), and the H– H
1
3
COSY and C-NMR signals at d 74.1 (C-2ꢃ), 75.5 (C-3ꢃ),
1.6 (C-4ꢃ), 75.8 (C-5ꢃ), and 169.7 (C-6ꢃ) showed the pres-
7
ence of a b-glucuronopyranosyl moiety. Sugar analysis
30)
showed D-glucuronic acid. These data suggested that 5 had
a b-D-glucuronic acid moiety instead of the acetyl group of
3
. In the HMBC spectrum, the H-1ꢃ signal was long-range
coupled with the carbon of C-2ꢂ (d 82.3). Hence, the struc-
ture of 5 was identified as shown in Fig. 1.
1
13
For fargenin D (6), the H- and C-NMR data suggested
the presence of a malonyl moiety on an acylated glucuronic
acid similar to 4. The H-3ꢂ resonance at the d 4.99 (1H, t, Jꢀ
9.5 Hz) proton signal of 6 was shifted downfield relative to
the H-3ꢂ signal of diosmetin-7-O-glucuronide. In the HMBC
spectrum, the H-3ꢂ signal was long-range coupled with the
1
carbon of C-1ꢃ (d 166.3). The H-NMR signal at d 3.42 (2H,
1
3
s) and the C-NMR signals at d 41.4, 166.3, and 167.7 sug-
gested that 6 had a malonyl group on C-3ꢂ. The molecular
formula C H O was confirmed on the basis of HR-FAB-
2
5
22 15
MS (m/z 563.1031, Calcd for C H O , 563.1036). Thus,
2
5
23 15
the structure of 6 was established as shown in Fig. 1.
From the whole plant of Meehania fargesii, hyaluronidase
inhibitors, two new spermidine alkaloids and four new
flavone glycosides were isolated. Some phenylpropanoid
Fig. 4. Structure of Known Compounds
1
7,31)
oligomers are potent hyaluronidase inhibitors.
Lithosper-
was deduced by HR-FAB-MS (m/z 517.0997, Calcd for mic acid B is a phenylpropanoid tetramer with inhibitory
1
13
C H O , 517.0981). The H- and C-NMR spectra of 3, activity comparable to that of the oligomers. Some flavo-
2
4
21 13
32,33)
with an anomeric proton signal at d 5.57 (1H, d, Jꢀ8.5 Hz, noids have been reported to inhibit of hyaluronidase,
H-1ꢂ), suggested the presence of a monosaccharide. The cor- and diosmetin-7-O-b-D-glucuronide and apigenin-7-O-b-D-
responding anomeric carbon (d 96.7, C-1ꢂ), four oxymethine glucuronide were also revealed as active compounds. Previ-
carbons [d 73.0 (C-2ꢂ), 72.9 (C-3ꢂ), 71.2 (C-4ꢂ), 75.3 (C-5ꢂ)] ously, in phytochemical studies of the Meehania genus, we
and a carbonyl carbon (d 169.5, C-6ꢂ) indicate the 2-acylated found 23 spermidine alkaloidal glycosides in M. urticifo-
2
7,28)
glucuronic acid moiety. The H-2ꢂ resonance at the d 4.82 lia.
Polyamine alkaloids have a rich diversity of skele-
2
1,34,35)
(
1H, dd, Jꢀ8.5, 9.0 Hz) proton signal of 3 was shifted down- tons and biological activities.
The 13-membered ring
field relative to the H-2ꢂ signal of diosmetin-7-O-gluc- moieties of 1 and 2 were a periphylline-type skeleton, and
uronide. Singlet methyl protons at d 2.03 (3H) suggested the different from that of cyclic spermidine alkaloidal glycosides
presence of an acetyl group. In the HMBC spectrum, the H- from M. urticifolia, which have a celacinnine-type skeleton.
2
ꢂ signal was long-range coupled with the acetyl group car- Periphylline and celacinnine had been isolated from Celas-
2
2,24)
bon of C- 1ꢃ (d 169.1). From these data, the structure of 3 traceae plants.
was identified as shown in Fig. 1.
Meehania genus plants also have two
types of skeletons. Compounds 3, 4, and 6 were esters of
The H- and C-NMR spectra of fargenin B (4) were simi- diosmetin-7-O-glucuronide which have hyaluronidase in-
1
13
1
lar to those of 3. The H-NMR signal at d 3.40 (overlapped) hibitory activity, and 5 was diosmetin-diglucuronide.
1
3
and the C-NMR signals at d 41.5, 165.8, and 167.5 sug-
gested that 4 had a malonyl group instead of the acetyl group
of 3. In the HMBC spectrum, the H-2ꢂ signal (d 4.86, dd, Jꢀ
Experimental
General Procedures Optical rotations were recorded on a Jasco P-2300
polarimeter. CD spectra were recorded on a Jasco J-700 spectropolarimeter;
UV, on a Shimadzu MPS-2450; and IR on a Perkin Elmer Spectrum One
8.5, 8.0 Hz) was long-range coupled with the carbonyl car-
ꢆ
1
13
1
1
bon of C-1ꢃ (d 165.8). The compound 4 showed an [MꢆH]
FT-IR spectrometer. H-NMR (400 MHz), C-NMR (100 MHz), H– H
ion peak in HR-FAB-MS at 563.1043 corresponding to a COSY, heteronuclear multiple quantum correlation (HMQC) (optimized
1
n
for JC–Hꢀ145 Hz) and HMBC (optimized for JC–Hꢀ8 Hz) spectra were
recorded on a Jeol JNM-AL400 FT-NMR spectrometer, and chemical shifts
were given as d values with tetramethylsilane (TMS) as an internal standard.
HR-FAB-MS data were obtained on a Jeol JMS700 mass spectrometer,
molecular formula of C H O (Calcd for C H O ,
m/z 563.1036). Hence, the structure of 4 was determined
as shown in Fig. 1. The compound 4a was obtained as
2
5
22 15
25 23 15
the 6ꢂ,3ꢃ-dimethyl ester of 4. In the HMBC spectra, two using a m-nitrobenzyl alcohol or a glycerol matrix. A porous polymer gel
Mitsubishi Chemical, Diaion HP-20, 60ꢇ300 mm) and an ODS (Cosmosil
40 C -OPN, Nacalai Tesque, 150 g) were used for column chromatogra-
(
1
methoxy singlet proton signals at d 3.52 (3H, s) and 3.70
3H, s) were correlated with the carbonyl carbon signals at d
67.4 (C-3ꢃ) and 169.7 (C-6ꢂ), respectively.
1
8
(
1
phy. Preparative Yamazen Cartridge Column Chromatography (YCCC) was
performed on a Jasco 2089 (column, Ultra Pack ODS-SM-50C-M, Yamazen,
1
13
H- and C-NMR spectra showed that fargenin C (5) was 37ꢇ100 mm; detector, UV at 210 nm). Preparative HPLC was performed on
a 2ꢂ-substituted diosmetin glucuronic acid similar to 3. On a Jasco 2089 and detected with UV at 210 nm (columns, ODS-100V, Tosoh,

May 2010
0ꢇ250 mm; 5C -AR-II, Cosmosil, 20ꢇ250 mm; Capcell-Pak Ph, Shi- NMR, see Table 2.
701
2
18
2
3
seido, 20ꢇ250 mm; YMC-Pack ODS-AM, YMC, 10ꢇ300 mm; Mightysil
RP-18 GP, Kanto Chemical, 10ꢇ250 mm).
Plant Material Meehania fargesii (H. LÉV.) C. Y. WU was collected in
Fargenin B (4): Colorless amorphous powder. [a]D ꢄ27.1 (cꢀ0.48,
DMSO). UV lmax (MeOH) nm (log e): 342 (4.52), 267 (4.47), 253 (4.49),
207 (4.77). IR (KBr) cm : 3402, 1718, 1660, 1611, 1500, 1444, 1261, 1177,
ꢄ1
ꢆ
May 2008 in JiuJiang City Lushan of Jiangxi Province, China. The plant was 1064. FAB-MS m/z: 563.1043 [MꢆH] (Calcd for C H O : 563.1036).
2
5
23 15
1
13
identified by Ceming Tan of the Forestry Administration of Jiujiang county.
A voucher specimen was deposited at the herbarium of Tohoku Pharmaceu-
tical University, No. 20080601.
H- and C-NMR, see Table 2.
2
3
Compound 4a: Colorless amorphous powder; [a]D ꢄ30.0 (cꢀ0.08,
DMSO). UV l (MeOH) nm (log e): 342 (4.55), 266 (sh), 252 (4.55), 207
Extraction and Isolation Powdered whole plants (792 g) of M. fargesii (4.81). FAB-MS m/z: 613.1188 [MꢆNa] (Calcd for C H O Na:
were extracted with acetone–water (8 : 2), (7 l) twice at room temperature for 613.1168). H- and C-NMR, see Table 2.
a month. The extract was concentrated at reduced pressure, suspended in
water (1.5 l), and extracted with ether (1.0 l) three times. The water layer DMSO). UV lmax (MeOH) nm (log e): 342 (4.64), 268 (4.61), 254 (4.62),
extract (80.13 g) was dissolved in water and passed through a porous poly- 207 (4.90). IR (KBr) cm : 3394, 1655, 1611, 1500, 1442, 1401, 1262,
max
ꢆ
27
26 15
1
13
2
4
Fargenin C (5): Colorless amorphous powder. [a]D ꢄ37.0 (cꢀ0.40,
ꢄ1
ꢆ
mer gel column and eluted with water, 30% MeOH, and MeOH. The MeOH 1181, 1073. FAB-MS m/z: 653.1373 [MꢆH] (Calcd for C H O :
28
29 18
1
13
eluate extract (4.96 g) was chromatographed on a reversed-phase column 653.1353). H- and C-NMR, see Table 2.
using ODS and eluted with 30%, 40%, 50%, 60%, 80% MeOH, and MeOH
Fargenin D (6): Colorless amorphous powder. [a]D ꢄ17.8 (cꢀ0.36,
fractions 1A—F). Fraction 1A (2.10 g) was subjected to Yamazen Cartridge DMSO). UV lmax (MeOH) nm (log e): 343 (4.50), 267 (4.44), 253 (4.47),
2
3
(
ꢄ1
Column Chromatography (YCCC) [mobile phase, methanol–0.2% TFA 209 (4.68). IR (KBr) cm : 3421, 1657, 1611, 1500, 1443, 1261, 1177,
ꢆ
(
2
25 : 75), (35 : 65) and (45 : 55)] to give 15 fractions (Frs. 2A—O). Fractions
D and 2E (394.1 mg) were subjected to HPLC [ODS-100V, mobile phase,
1063. FAB-MS m/z: 563.1031 [MꢆH] (Calcd for C H O : 563.1036).
25 23 15
1
13
H- and C-NMR, see Table 2.
Acid Hydrolysis and Sugar Identification Compounds 3—6 (1 mg)
were hydrolyzed with 7% HCl (1 ml) at 60 °C for 2 h. The reaction mixture
acetonitrile–water (20 : 80), Capcell-Pak Ph, mobile phase, acetonitrile–
water (15 : 85), and YMC-Pack ODS-AM, mobile phase, acetonitrile–0.2%
TFA (20 : 80)] to yield 7-epiblechnic acid (1.1 mg). Fraction 2G (210.6 mg) was neutralized with an Amberlite IRA400 column, and the eluate was con-
was subjected to HPLC [ODS-100V, mobile phase, acetonitrile–water
25 : 75), and 5C -AR-II, mobile phase, acetonitrile–0.2% TFA (20 : 80)] to
yield 5 (3.2 mg), luteolin-7-O-b-D-glucuronopyranoside (6.9 mg), and api-
genin-7-O-b-D-glucuronopyranoside methyl ester (3.9 mg). Fraction 2H
(
centrated. The residues were stirred with L-cysteine methyl ester (5 mg) and
(
o-tolyl isothiocyanate (10 ml) in pyridine (0.5 ml), by using the procedure re-
18
30)
ported by Tanaka et al. The reaction mixtures were analyzed by HPLC
(column, Cosmosil 5C –AR II column, 4.6ꢇ250 mm; mobile phase,
1
8
132.9 mg) was subjected to HPLC [ODS-100V, mobile phase, acetoni- CH CN–0.2% TFA in H O (25 : 75), 1.0 ml/min; detector, UV at 210 nm) at
3
2
trile–water (25 : 75), and 5C -AR-II, mobile phase, acetonitrile–0.2% TFA
20 °C. D-Glucuronic acid (tR 16.8 min) was identified as the sugar moieties
of 3—6 based on comparisons with authentic samples of D-glucuronic acid
(tR 16.8 min) and L-glucuronic acid (using D-cysteine methyl ester and D-glu-
1
8
(20 : 80)] to yield hyperin (2.2 mg) and 4-{[6-O-(4-hydroxy-3,5-dimethoxy-
benzoyl)-b-D-glucopyranosyl]oxy}-3-methoxybenzoic acid (1.0 mg). Frac-
tions 2I, 2J, and 2K (328.4 mg) were subjected to HPLC [ODS-100V, mobile curonic acid, t 16.1 min).
R
phase, acetonitrile–water (25 : 75)] to yield rosmarinic acid (56.6 mg), dios-
metin-7-O-b-D-glucuronopyranoside (2.7 mg) and apigenin-7-O-b-D-gluc-
uronopyranoside (29.9 mg). Fractions 2L and 2M (206.8 mg) were subjected Aronson.
Assay of Hyaluronidase Inhibition The assay was carried out accord-
ing to the Morgan–Elson method, which was modified by Davidson and
1
7,36,37)
Each fraction (final concentration: 2.0 mg/ml) and each
to HPLC [Capcell-Pak Ph, mobile phase, acetonitrile–0.2% TFA (25 : 75)] to
yield lithospermic acid B (47.8 mg), apigenin-7-O-b-D-glucuronopyranoside
methyl ester (6.3 mg), and diosmetin-7-O-b-D-glucuronopyranoside methyl
compound (final concentration: 1, 0.3, 0.1, 0.03, 0.01 mM) was dissolved in
0.1 M acetate buffer as the sample solution. Hyaluronidase activity was
measured as described previously. Disodium cromoglycate (DSCG) (Wako
31)
ester (7.1 mg). Fraction 1B (1.26 g) was subjected to YCCC [mobile phase, Pure Chemical Industries Ltd., Osaka, Japan) was regarded to be a positive
methanol–0.2% TFA (35 : 65)→(50 : 50)], to give 15 fractions (Frs. 3A—O). control.
Fractions 3B and 3C (96.5 mg) were subjected to HPLC [ODS-100V, mobile
phase, acetonitrile–water (30 : 70), and Capcell-Pak Ph, mobile phase, ace-
Acknowledgments We thank Dr. Zhang Dong-Ming of Institute of
tonitrile–water (17.5 : 85.5)] to yield compounds 1 (5.7 mg) and 2 (2.5 mg). Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Fractions 3D and 3E (78.9 mg) were subjected to HPLC [ODS-100V, mobile Medical College, for supplying the plant material and Mr. S. Sato and Mr. T.
phase, acetonitrile–0.2% TFA (35 : 65), and Capcell-Pak Ph, mobile phase, Matsuki of Tohoku Pharmaceutical University for assisting with the MS
acetonitrile–0.2% TFA (35 : 65)] to yield apigenin-7-O-b-D-glucuronopyra-
noside (17.9 mg), rosmarinic acid (10.9 mg), and acacetin-7-O-b-D-glu-
measurements.
curonopyranoside (3.0 mg). Fractions 3G, 3H, 3I, and 3J (336.1 mg) were References
subjected to HPLC [ODS-100V, mobile phase, acetonitrile–0.2% TFA
35 : 65)] to yield diosmetin-7-O-b-D-glucuronopyranoside (93.5 mg) and
lithospermic acid B (33.1 mg). Fraction 3M (167.8 mg) was subjected to
HPLC [ODS-100V, mobile phase, acetonitrile–0.2% TFA (35 : 65), and Cap-
cell-Pak Ph, mobile phase, acetonitrile–0.2% TFA (27.5 : 72.5)] to yield 4
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