Phytochemical and Phar ma co log i cal Studies on Chi nese ...

<strong>Phytochemi<strong>cal</strong></strong> <strong>and</strong> <strong>Phar</strong> <strong>ma</strong> <strong>co</strong> <strong>log</strong> i <strong>cal</strong> <strong>Studies</strong> on Chi nese Paeonia Spe cies
Hsiou-Yu Ding a ( ), Hang-Ching Lin* b ( ),
Che-Ming Teng c ( ) <strong>and</strong> Yang-Chang Wu a * ( )
a Grad u ate In sti tute of <strong>Phar</strong> <strong>ma</strong> ceu ti <strong>cal</strong> Sci ences, Kaohsiung Med i <strong>cal</strong> Uni ver sity,
Kaohsiung 807, Tai wan, R.O.C.
b School of <strong>Phar</strong> <strong>ma</strong>cy, Na tional De fense Med i <strong>cal</strong> Cen ter, Tai pei 114, Tai wan, R.O.C.
c <strong>Phar</strong> <strong>ma</strong> <strong>co</strong> <strong>log</strong> i <strong>cal</strong> In sti tute, Col lege of Med i cine, Na tional Tai wan Uni ver sity,
Tai pei 100, Tai wan, R. O.C.
A new monoterpene gly<strong>co</strong>side, mudanpioside J (3), was iso lated from the root <strong>co</strong>r tex of Paeonia
suffruti<strong>co</strong>sa. Its struc ture has been es tab lished on the ba sis of spec tral ev i dence. In ad di tion, six known <strong>co</strong>m -
pounds, campesterol (4), 4-hydroxyacetopenone (5), kaempferol (6), 3-hydroxy-4-methoxybenzoic acid (7),
hederagenin (8) <strong>and</strong> galloylpaeoniflorin (9) were also iso lated. A screen ing test of monoterpene gly<strong>co</strong>sides <strong>and</strong>
ar o <strong>ma</strong>tic ac ids de rived from P. suffruti<strong>co</strong>sa <strong>and</strong> P. lactiflora on arachidonic acid, thrombin, <strong>co</strong>l la gen <strong>and</strong> PAF
in duced rab bit platelet ag gre ga tion sug gests ar o <strong>ma</strong>tic ac ids <strong>ma</strong>y play an im por tant role in in hi bi tion of platelet
ag gre ga tion, in flam <strong>ma</strong> tion <strong>and</strong> hem or rhage due to bac te rial in fec tion.
IN TRO DUC TION
The Chi nese herbs, mudanpi ( , root <strong>co</strong>r tex of
Paeonia suffruti<strong>co</strong>sa Andr, Paeoniaceae) <strong>and</strong> chishao ( ,
root of P. lactiflora Pall, Paeoniaceae) have the same phar <strong>ma</strong> -
<strong>co</strong> <strong>log</strong> i <strong>cal</strong> ef fects in tra di tional med i cine. They were clin i <strong>cal</strong>ly
used as an al ge sic, anti-inflam<strong>ma</strong>tory, bac te ri cidal drugs. 1-4 In
ad di tion, they also treated dis sem i nated intravascular <strong>co</strong> ag u -
la tion (DIC) or hem or rhage due to bac te rial in fec tion. 5,6 Both
herbs have the same <strong>ma</strong> jor po lar <strong>co</strong>n stit u ent, paeoniflorin (1),
which is a wa ter sol u ble pinane monoterpene gly<strong>co</strong>side de riv -
a tive, in ad di tion to dif fer ent gallotannin <strong>co</strong>n tent. On the other
h<strong>and</strong>, they have dif fer ent <strong>ma</strong> jor non-polar <strong>co</strong>m po nents. The
<strong>ma</strong> jor <strong>co</strong>n stit u ents of P. suffruti<strong>co</strong>sa are acetophenone de riv a -
tives whereas that of P. lactiflora is ben zoic acid (2). 1-14 In pre -
vi ous stud ies, we re ported the iso la tion of acetophenones,
monoterpene gly<strong>co</strong>sides, flavonoid <strong>and</strong> triterpenes. 15-18 In ad -
di tion, we re ported that acetophenones were resposible for in -
hi bi tion of in flam <strong>ma</strong> tion <strong>and</strong> platelet ag gre ga tion. 19 In a <strong>co</strong>n -
tin u a tion of our phytochemi<strong>cal</strong> <strong>and</strong> phar <strong>ma</strong> <strong>co</strong> <strong>log</strong> i <strong>cal</strong> stud ies,
a new monoterpene gly<strong>co</strong>side, mudanpioside J (3), to gether
with six known <strong>co</strong>m pounds, campesterol (4), 4-hydroxy -
acetophenone (5), kaempferol (6), 3-hydroxy-4- methoxy -
benzoic acid (7), hederagenin (8) <strong>and</strong> galloylpaeoniflorin (9)
were iso lated. Ad di tionally, we re port that ben zoic acid,
p-hydroxybenzoic acid or gal lic acid, de rived from mono -
terpene gly<strong>co</strong>sides or gallotannins or in free form in mudanpi
or chishao, show a sig nif i cantly in hib i tory ef fect against
platelet ag gre ga tion in duced by thrombin, arachidonic acid,
or <strong>co</strong>l la gen. Thus, the <strong>ma</strong> jor ar o <strong>ma</strong>tic ac ids (ben zoic acid <strong>and</strong>
gal lic acid) are im por tant <strong>co</strong>m pounds that <strong>ma</strong>y show an in hib -
Jour nal of the Chi nese Chem i <strong>cal</strong> So ci ety, 2000, 47, 381-388 381
i tory ef fect on DIC <strong>and</strong> hem or rhage due to bac te rial in fec tion
as well as an ef fect on in flam <strong>ma</strong> tion.
RE SULTS AND DIS CUS SION
The 1 H NMR <strong>and</strong> 13 C NMR spec tral data for new <strong>co</strong>m -
pound 3 re sem bled known monoterpene gly<strong>co</strong>sides with ben -
zo ate moi ety which were pre vi ously iso lated from P.
Suffruti<strong>co</strong>sa (Ta ble 1). 16,18 The NMR data clearly in di cates
that <strong>co</strong>m pound 3 was <strong>co</strong>m posed of dif fer ent moi eties, one glu -
<strong>co</strong>se <strong>and</strong> two ar o <strong>ma</strong>tic rings. Com pound 3 gave rise to a
quasimolecular ion peak at m/z 629.1870 ([M-H] ) on neg a -
tive FAB <strong>ma</strong>ss spec tros <strong>co</strong>py <strong>and</strong> es tab lished the molecular
for mula as C31H34O14. The 1 H NMR spec trum re vealed sig nals
as sign able to a monoterpene, i.e. sig nals for: two methylenes
at 2.31 <strong>and</strong> 2.49 (d, J =12.8 Hz) (H-3) <strong>and</strong> 2.31 (d, J = 10.6
Hz) <strong>and</strong> 2.90 (dd, J = 10.6, 6.6 Hz) (H-6), a meth y lene bear ing
an acyloxy moi ety at 5.03 <strong>and</strong> 5.18 (d, J = 12.6 Hz) (H-8),
one methine at 3.05 (d, J = 6.6 Hz) (H-5), a methine ad ja cent
to two heteroatoms at 5.92 (s) (H-9) <strong>and</strong> a methyl at 1.70
(s) (H-10). In ad di tion, the glu <strong>co</strong>se moi ety ac <strong>co</strong>unted for sig -
nals at 4.04 (t, J = 8.4 Hz) for H-2 , 4.09 (t, J = 8.4 Hz) for
H-4 , 4.13 (t, J = 8.4 Hz) for H-5 , 4.23 (t, J = 8.4 Hz) for H-3 ,
5.15 (d, J = 8.4 Hz) for H-1 , <strong>and</strong> 4.96 (dd, J = 10.8, 8.4 Hz)
<strong>and</strong> 5.26 (d, J = 10.8 Hz) for two H-6 . The re <strong>ma</strong>in ing sig nals
dis closed the pres ence of two aryloxy groups; one set for a
sim ple benzoyloxy unit at 7.29 (t, J = 8.0 Hz, H-3 <strong>and</strong>
H-5 ), 7.45 (t, J = 8.0 Hz, H-4 ) <strong>and</strong> 8.11(d, J = 8.0 Hz, H-2
<strong>and</strong> H-6 ), <strong>and</strong> the other sig nals for 4-hydroxy-3- methoxy -
benzoyloxy unit at 7.24 (d, J = 8.2 Hz, H-5 ), 7.93 (d, J = 2.0

382 J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 Ding et al.
Table 1. 13 C NMR <strong>and</strong> 1 H NMR Spectral Data <strong>and</strong> HMBC, NOESY Correlations of Mudanpioside J
(Pyridine-d5)
No c H (J in Hz ) HMBC NOESY
1 88.8 C1/H3a,5,6a,6b,10,1
2 86.1 C2/H3a,6a,6b,9,10
3 44.7 2.31(1H, d, 12.8 Hz)
C3/H10 H3a/H3b,10
2.49(1H, d, 12.8 Hz)
H3b/H5,10
4 105.9 C4/H3a,3b,6a,6b,9
5 43.8 3.05(1H, d, 6.6 Hz) C5/H6a,6b,8a H5/H6a,6b,8b
6 23.0 2.31(1H, d, 10.6 Hz)
H6a/H6b,8b
2.90(1H, dd, 10.6, 6.6 Hz)
H6b/H8b
7 71.4 C7/H8a,8b
8 61.3 5.03(1H,d, 12.6 Hz)
5.18(1H, d, 12.6 Hz)
9 101.6 5.92(1H, s) C9/H8a,8b H9/H8a,8b
10 19.8 1.70(3H, s)
1 100.3 5.15(1H, d, 8.4 Hz) C1 /H2 H1 /H3 ,5
2 74.8 4.04(1H, t, 8.4 Hz)
3 78.2 4.23(1H, t, 8.4 Hz) C3 /H4 H3 /H5
4 71.9 4.09(1H, t, 8.4 Hz)
5 75.3 4.13(1H, t, 8.4 Hz) C5 /H4
6 64.9 4.96(1H, dd, 10.8, 8.4 Hz)
5.26(1H, d, 10.8 Hz)
1 130.6 C1 /H2 ,6
2 129.9 8.11(1H, d, 8.0 Hz) C2 /H3 ,4 ,6 H2 /H3
3 128.7 7.29(1H, t, 8.0 Hz) C3 /H5
4 133.3 7.45(1H, t, 8.0 Hz) C4 /H2 ,6 H4 /H3 ,5
5 128.7 7.29(1H, t, 8.0 Hz) C5 /H3 H5 /H6
6 129.9 8.11(1H, d, 8.0 Hz) C6 /H4 ,5
7 166.6 C7 /H8a,2 ,6
1 121.7 C1 /H5
2 113.4 7.93(1H, d, 2.0 Hz) C2 /H6 H2 /OCH3
3 148.4 C3 /H5 ,OCH3
4 153.4 C4 /H2 ,6
5 116.2 7.24(1H, d, 8.2 Hz) H5 /H6
6 124.8 8.00(1H, dd, 8.2, 2.0 Hz) C6 /H2
7 166.6 C7 /H6 b,2 ,6
3 -OCH3 55.9 3.79(3H, s)
Hz, H-2 ), 8.00 (dd, J = 8.2, 2.0 Hz, H-6 ), <strong>and</strong> 3.79 (s,
3 -OCH3). The pres ence of these groups was also sup ported
by the frag ment ion peaks at m/z 105, 122, 151, <strong>and</strong> 168, the
first two ions due to benzoyl <strong>and</strong> ben zoic acid frag ments, the
last two ions to 4-hydroxy-3-methoxybenzoyl <strong>and</strong> 4- hydroxy-
3 - methoxybenzoic acid frag ments. COSY, HETCOR,
HMBC, <strong>and</strong> NOESY ex per i ments (HMBC <strong>and</strong> NOESY, Ta -
ble 1) also sup ported the <strong>co</strong>m pleted <strong>co</strong>nnectivities <strong>and</strong> as sign -
ments of 1 H <strong>and</strong> 13 C NMR spec tral data <strong>and</strong> es tab lished the lo -
ca tion of sub sti tu tion on the monoterpene <strong>and</strong> ar o <strong>ma</strong>tic ring
sys tems. Fur ther, the HMBC ex per i ment showed a no ta ble
cross peak be tween C-7 <strong>and</strong> H-8 as well as a peak be tween
C-7 <strong>and</strong> H-6 . These phe nom ena clearly proved the at tach -
ment of the benzoyloxy group to C-8 of the monoterpene <strong>and</strong>
the other 4-hydroxy-3-methoxybenzoyloxy group to H-6 of
glu <strong>co</strong>se. Based on the above spec tral anal y ses, the struc ture of
mudanpioside J was de duced as 3 with six chiral cen ters. On
the ba sis of the spe cific ro ta tional value of <strong>co</strong>m pound 3, [ ]D
-11.5 , <strong>co</strong>m pared to those of known monoterpene gly<strong>co</strong>sides
(1, 10-17) from this herb, -5 to -25 , this new <strong>co</strong>m pound had
to have the same ab so lute <strong>co</strong>n fig u ra tion as the known <strong>co</strong>m -
pounds 1, 10-17. 16,18 As shown in the ex per i men tal sec tion, in
ad di tion to mudanpioside J (3), six known <strong>co</strong>m pounds,
campesterol (4), 4-hydroxyacetophenoe (5), kaempferol (6),
3-hydroxy-4-methoxybenzoic acid (7), hederagenin (8) <strong>and</strong>
galloylpaeoniflorin (9) were iso lated <strong>and</strong> iden ti fied. Of these
<strong>co</strong>m pounds, 5, 6, 7, <strong>and</strong> 8 are iso lated for the first time from
this herb.
As to phar <strong>ma</strong> <strong>co</strong> <strong>log</strong> i <strong>cal</strong> ac tiv i ties of mudanpi <strong>and</strong>
chishao re lated to in hi bi tion of platelet ag gre ga tion or hem or -

Phytochemistry of Paeonia Spe cies J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 383
Table 2. Effect of Monoterpene Gly<strong>co</strong>sides <strong>and</strong> Other Minor Constituents from Mudanpi <strong>and</strong>
Chishao on Washed Rabbit Platelet Aggregation Induced by Thrombin (Thr),
Arachidonic Acid (AA), Collagen (Col), <strong>and</strong> Platelet-Activating Factor (PAF) a
Compd Conc. ( ug/mL ) Aggregation ( % )
Thr AA Col PAF
Control 89.9 2.2 83.4 1.4 84.0 1.1 88.9 0.7
1 100 92.0 1.5 79.3 2.5 83.0 1.0 87.2 1.1
7 100 88.0 2.1 81.3 0.3 80.5 0.9 b
88.5 0.7
9 150 89.9 1.4 83.4 1.1 83.5 1.0 89.0 1.0
10 100 86.4 2.4 81.2 1.9 82.0 0.5 87.4 1.5
11 100 81.9 4.4 72.4 2.7 c
78.1 1.0 c
81.3 0.2 d
12 100 80.9 0.9 b
72.0 2.5 d
81.4 0.7 85.7 0.2 c
13 100 85.9 4.6 78.1 1.7 79.2 0.8 c
81.5 1.7 d
14 100 83.6 4.5 79.1 1.4 76.7 7.3
15 100 83.5 4.2 79.7 1.6 78.0 1.9 b
86.3 1.0
86.3 0.7 b
16 100 84.3 4.6 76.7 3.2 80.5 0.4 b
84.7 1.0 c
17 100 82.0 3.2 79.7 0.8 79.4 1.2 b
86.6 0.5
18 100 80.4 6.6 81.2 3.3 82.3 0.6 87.5 0.4
19 100 86.2 3.2 76.7 3.4 83.0 1.6 86.1 1.8
20 100 83.5 4.2 77.7 1.9 80.9 1.7 88.1 0.4
21 100 88.0 2.8 83.3 0.5 81.7 0.9 88.1 0.5
22 100 85.0 1.8 81.9 1.6 78.9 0.9 c
84.9 0.7 d
23 50 74.5 6.3 b
0.0 0.0 d
17.7 2.1 d
32.9 8.7 d
10 13.2 3.2 d
5 76.9 3.3
aspirin 50 91.9 2.5 0.0 0.0 d
85.4 3.9 90.5 1.2
a Platelets were preincubated with each <strong>co</strong>mpound, the solvent (0.5 % DMSO, <strong>co</strong>ntrol),
aspirin (positive <strong>co</strong>ntrol) at 37.0 C for 3 min, then Thr (0.1 U/mL), AA (100 uM), Col (10
ug/mL) or PAF (2 ng/mL) was added. Percentages of aggregation are presented as
means S.E. (n=3-7).
b p

384 J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 Ding et al.
rhage due to bac te rial in fec tion, we re ported that aceto -
penones from mudanpi showed po tent ac tiv ity against rab bit
platelet ag gre ga tion in duced by AA. 19 Thus, a suf fi cient quan -
tity of plate lets in cir cu la tion pre vent hem or rhage due to bac -
te rial in fec tion. But, the ac tive <strong>co</strong>m pounds from chishao with
the above-mentioned phar <strong>ma</strong> <strong>co</strong> <strong>log</strong> i <strong>cal</strong> ac tiv i ties are still not
clear.
As shown in Ta ble 2, in the in vi tro screen ing test against
rab bit platelet ag gre ga tion in duced by thrombin, arachidonic
acid, <strong>co</strong>l la gen, <strong>and</strong> platelet ac ti vat ing fac tor (PAF), all tested
<strong>co</strong>m pounds in clud ing <strong>ma</strong> jor monoterpene gly<strong>co</strong>sides (1,
9-19) <strong>and</strong> mi nor <strong>co</strong>n stit u ents (7, 20-22) from mudanpi <strong>and</strong>
chishao show no or a lit tle ac tiv ity at the <strong>co</strong>n cen tra tion of 100
or 150 ug/mL. The weak in hib i tory ac tiv i ties of <strong>ma</strong> jor
monoterpene gly<strong>co</strong>sides (1, 9-19) in platelet ag gre ga tion are
in agree ment with those of monoterpene gly<strong>co</strong>sides (1, 10-12)
in hu <strong>ma</strong>n platelet ag gre ga tion in duced by ADP or <strong>co</strong>l la gen at
the dose of 500-1000 ug/mL. 20 Ad di tionally, the prop erty of
good wa ter sol u bil ity of monoterpene gly<strong>co</strong>sides limit rapid
ab sorp tion by the in tes tine <strong>and</strong> the poor ab sorp tion causes a
low <strong>co</strong>n cen tra tion in blood cir cu la tion. On the <strong>co</strong>n trary, the
me tab o lites (ar o <strong>ma</strong>tic acid <strong>and</strong> paeonimetabolins
(monoterpenes)) due to in tes ti nal microflora deg ra da tion
should have a high <strong>co</strong>n cen tra tion in cir cu la tion due to their
good lipophilicity. 21-23 Thus, the above-mentioned screen ing
test data <strong>and</strong> in for <strong>ma</strong> tion clearly sug gest that <strong>ma</strong> jor mono -
terpene gly<strong>co</strong>sides in chishao or mudanpi are not po tent <strong>co</strong>m -
pounds <strong>and</strong> biotransformed me tab o lites of <strong>ma</strong> jor <strong>co</strong>n stit u ents
of mudanpi or chishao <strong>ma</strong>y play an im por tant role in phar <strong>ma</strong> -
<strong>co</strong> <strong>log</strong> i <strong>cal</strong> ef fects. Ben zoic acid <strong>and</strong> p-hydroxybenzoic acid are
in free form or biotransformed me tab o lites from <strong>ma</strong> jor
paeoniflorin (1), oxypaeoniflorin (10), benzoyl paeo ni florin
(11), <strong>and</strong> benzoyloxypaeoniflorin (12) by hu <strong>ma</strong>n gut
microflora deg ra da tion. 24,25 Gal lic acid (25) is in free form or
a biotransformed me tab o lite from <strong>ma</strong> jor gallotannins by hu -
<strong>ma</strong>n gut en zy <strong>ma</strong>tic hy dro ly sis, microflora deg ra da tion or ba -
sic hy dro ly sis. 26 As shown in Ta ble 3, three ar o <strong>ma</strong>tic ac ids
show lit tle in hib i tory ef fect at 100 ug/mL but show sig nif i cant
in hi bi tion by 50 per cent at about 150 ug/mL in the screen ing
test against rab bit platelet ag gre ga tion in duced by AA, <strong>co</strong>l la -
gen, thrombin or PAF. Ben zoic acid is ac tive against
thrombin- induced platelet ag gre ga tion; p-hydroxybenzoic
acid against thrombin <strong>and</strong> <strong>co</strong>l la gen in duced platelet ag gre ga -
tion <strong>and</strong> gal lic acid against AA <strong>and</strong> thrombin in duced platelet
ag gre ga tion. Chishao <strong>and</strong> mudanpi have dif fer ent <strong>co</strong>n tents of
monoterpene gly<strong>co</strong>sides or gallotannins: paeoniflorin (1,
3.5-8.0% <strong>co</strong>n tent for chishao <strong>and</strong> mudanpi), benzoy lpaeoni -
florin (11, 0.04-0.22% <strong>co</strong>n tent for chishao, 0.08-0.52% <strong>co</strong>n -
tent for mudanpi), oxypaeoniflorin (10, 0.05-0.65% <strong>co</strong>n tent
for chishao, 0.15-0.90% <strong>co</strong>n tent for mudanpi), <strong>and</strong>
gallotannins (3.4-12.6% <strong>co</strong>n tent for chishao, 0.25% <strong>co</strong>n tent
for Jap a nese mudanpi). 9,10,13,14,27,28 Thus, the high lev els of
ben zoic acid <strong>and</strong> gal lic acid in the blood cir cu la tion sys tem
are likely to have phys i o <strong>log</strong> i <strong>cal</strong> ef fects <strong>and</strong> <strong>co</strong>uld re duce the
in ci dence of platelet ag gre ga tion in duced by AA or thrombin
that are in duced by bac te rial in fec tion. 29,30 More over, the in hi -
bi tion of platelet ag gre ga tion by ben zoic acid <strong>and</strong> gal lic acid is
help ful to keep ad e quate platelet <strong>co</strong>n cen tra tion in the blood
cir cu la tion which pre vents hem or rhage due to bac te rial in fec -
tion. In ad di tion, the high <strong>co</strong>n cen tra tion of ben zoic acid <strong>and</strong>
gal lic acid elicit an anti-inflam<strong>ma</strong>tory ef fect through their in -
hi bi tion of <strong>co</strong>n ver sion from arachidonic acid to pro stag -
l<strong>and</strong>ins be cause Takagi et al. re ported that paeoniflorin <strong>and</strong>
ben zoic acid showed sim i lar po tency in vivo anti- inflam -
<strong>ma</strong>tory ex per i ments. 19,31 Gal lic acid also elic its an anti-

Phytochemistry of Paeonia Spe cies J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 385
Table 3. Inhibitory Effects of Aro<strong>ma</strong>tic Acids from Paeonia Spp. on Washed Rabbit Platelet Aggregation
Induced by Arachidonic Acid (AA), Collagen (Col), Thrombin (Thr), <strong>and</strong> Platelet-Activating
Factor (PAF) a
Compd Conc.(ug/mL) Inhibition (%)
AA Col Thr PAF
Benzoic acid 100 0.0 0.0 0.0 0.0 4.4 4.0 1.5 0.9
150 5.2 1.2 d
4.7 3.3 71.1 5.3 b
30.8 5.7 d
p-OH-Benzoic acid 100 0.0 0.0 28.3 2.1 30.6 4.2 7.4 2.0
150 4.3 3.1 63.7 12.0 e
47.8 0.6 d
31.9 5.3 c
Gallic acid 100 32.3 1.7 0.0 0.0 17.3 2.4 14.1 1.3
150 58.9 3.0 b
3.8 4.3 54.4 2.6 b
18.9 5.7
Galloylpaeoniflorin
(<strong>co</strong>ntrol, 9)
150 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
a Platelets were preincubated with test <strong>co</strong>mpounds, galloylpaeoniflorin (9, positive <strong>co</strong>ntrol) or solvent
(0.5% DMSO, <strong>co</strong>ntrol) at 37.0 C for 3 min, then Thr (0.1 U/mL), AA (100 uM), Col (10 ug/mL) or
PAF (2 ng/mL) was added. Percentages of inhibition are presented as means S.D. (n=4).
b p

386 J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 Ding et al.
<strong>and</strong> Fr. 2-3 (20 g). Acetophenones were iso lated from Fr. 2-2
as re ported in a pre vi ous study. 19 The Fr. 2-3 was re peat edly
rechro<strong>ma</strong>tographed on a sil ica gel <strong>co</strong>l umn, eluted with
CHCl3-Me2CO (97:3), to give campesterol (4, 16.1 mg), <strong>and</strong>
4-hydroxyacetophenone (5, 17.4 mg) <strong>and</strong> other <strong>co</strong>n stit u ents. 17
The Fr. 3 (60 g) was <strong>co</strong>m bined with the n-BuOH ex tract (890
g) <strong>and</strong> sub jected to <strong>co</strong>l umn chro <strong>ma</strong> tog ra phy on sil ica gel with
CHCl3-MeOH (19:1, 9:1, 17:3, 7:3) as mov ing sol vents.
Rechro<strong>ma</strong>tography of the first frac tion on sil ica gel, eluted
with CHCl3-MeOH (94:6), yielded se quen tially mudan -
pioside J (3, 477.3 mg), kaempferol (6, 25.3 mg), 3-hydroxy-
4-methoxybenzoic acid (7, 131.3 mg), hederagenin (8, 32.4
mg). Kaempferol was pu ri fied by sephadex LH-20 <strong>co</strong>l umn
chro <strong>ma</strong> tog ra phy eluted with MeOH. Mudanpioside J was pu -
ri fied by pre para tive HPLC eluted with 43 % MeOH (in H2O)
on phenyl <strong>co</strong>l umn <strong>and</strong> 38 % MeOH (in H2O) on C-8 <strong>co</strong>l umn.
Rechro<strong>ma</strong>tography of the sec ond frac tion on a pre pared lo bar
RP-8 <strong>co</strong>l umn eluted with 45% MeOH (in H2O) gave galloyl -
paeoniflorin (9). The galloylpaeoniflorin was fur ther pu ri fied
by pre para tive HPLC eluted with 40 % MeOH (in H2O) on a
phenyl <strong>co</strong>l umn to yield 816.5 mg of 9.
Mudanpioside J (3)
Col or less pow der; mp 132-136 C; [ ]D 11.5 (c 0.1,
MeOH); UV (MeOH) <strong>ma</strong>x nm (<strong>log</strong> ): 297.4 (3.78), 222.6
(4.46); IR (KBr) <strong>ma</strong>x cm -1 : 3418 (OH), 1710 (C=O); the 1 H
<strong>and</strong> 13 C NMR data shown in Ta ble 1; Neg a tive HRFAB-MS
m/z: 629.1870 (<strong>cal</strong>c. for C31H33O14, 629.1854 [M-H] ); EI-MS
m/z (rel. int.): 168 (16), 151 (42), 122 (60), 105 (100).
4-Hydroxyacetophenone (5)
Col or less Pow der; mp 107-108 C; UV (MeOH) <strong>ma</strong>x
nm (<strong>log</strong> ): 278.6 (3.56), 222.8 (3.38); 1 H NMR (CD3OD)
7.87 (each 1H, d, J = 8.8 Hz, H-2, H-6), 6.81 (each 1H, d, J =
8.8 Hz, H-3, H-5), 2.51 (3H, s, COCH3); 13 C NMR (CD3OD)
26.2 (C-8), 116.6 (C-3, C-5), 129.5 (C-1), 132.2 (C-2, C-6),
165.4 (C-4), 199.5 (C=O); EI-MS m/z (rel. int.): 136([M] + ,
30), 121 (100), 93 (48). Spec tral data ( 1 H NMR) <strong>and</strong> mp are
<strong>co</strong>n sis tent with val ues in the lit er a ture. 33
Kaempferol (6)
Yel low nee dles; mp 275-277 C; UV (MeOH) <strong>ma</strong>x nm
(<strong>log</strong> ): 365.0 (4.64), 267.5 (4.57), 222.0 (4.58); 1 H NMR
(C5D5N) 8.54 (each 1H, dd, J = 8.8 Hz, H-2 , H-6 ), 7.32
(each 1H, dd, J = 8.8 Hz, H-3 , H-5 ), 6.85 (1H, d, J = 2.0 Hz,
H-8), 6.76 (1H, d, J = 2.0 Hz, H-6); 13 C NMR (C5D5N) 94.3
(C-8), 99.3 (C-6), 104.4 (C-10), 116.3 (C-3 , C-5 ), 123.5
(C-1 ), 130.5 (C-2 , C-6 ), 137.8 (C-3), 147.5 (C-2), 157.5
(C-9), 160.7 (C-4 ), 162.4 (C-5), 165.6 (C-7), 177.3 (C-4);
EI-MS m/z (rel. int.): 286 ([M] + , 92). Spec tral data (UV, 13 C
NMR) <strong>and</strong> mp are <strong>co</strong>n sis tent with val ues in the lit er a ture. 34,35
3-Hydroxy-4-methoxybenzoic acid (7)
Pale pow der; mp 250-252 C; UV (MeOH) <strong>ma</strong>x nm (<strong>log</strong>
): 291.2 (3.66), 259.0 (3.99), 217.0 (4.18); 1 H NMR
(CD3OD) 7.56 (1H, dd, J = 8.8, 2.0 Hz, H-6), 7.55 (1H, d, J
= 2.0 Hz, H-2), 6.84 (1H, d, J = 8.8 Hz, H-5), 3.89 (3H, s,
OCH3); 13 C NMR (CD3OD) 56.4 (OCH3), 113.8 (C-5), 115.8
(C-2), 123.1 (C-1), 125.3 (C-6), 148.6 (C-3), 152.6 (C-4),
170.0 (C=O); EI-MS m/z (rel. int.): 168 ([M] + , 100), 153 (73),
125 (38). Spec tral data ( 1 H NMR) <strong>and</strong> mp are <strong>co</strong>n sis tent with
val ues in the lit er a ture. 36
Hederagenin (8)
White pow der; mp 291-293 C; [ ]D 73.5 (c 0.5,
C5H5N); 1 H NMR (C5D5N) 5.50 (1H, t, J = 3.4 Hz, H-12),
4.21 (1H, m, H-3), 4.18 <strong>and</strong> 3.72 (each 1H, d, J = 10.4 Hz,
H-23), 1.24, 1.06, 1.06, 1.01, 0.98 <strong>and</strong> 0.93 (each 3H, s, 6
CH3); 13 C NMR (C5D5N) 13.2 (C-24), 16.0 (C-25), 17.6
(C-26), 18.7 (C-6), 23.8 (C-11, 16), 23.9 (C-30), 26.2 (C-27),
27.7 (C-2), 28.4 (C-15), 31.0 (C-20), 33.0 (C-7), 33.3 (C-22,
C-29), 34.3 (C-21), 37.3 (C-10), 38.9 (C-1), 39.8 (C-8), 42.1
(C-18), 42.3 (C-14), 43.0 (C-4), 46.5 (C-17), 46.8 (C-19),
48.2 (C-9), 48.6 (C-5), 67.9 (C-23), 73.4 (C-3), 122.7 (C-12),
144.9 (C-13), 180.3 (C-28); EI-MS m/z (rel. int.): 472 ([M] + ,
0.1), 248 (68), 203 (100). Spec tral data ( 1 H NMR, 13 C NMR,
<strong>ma</strong>ss) <strong>and</strong> mp are <strong>co</strong>n sis tent with val ues in the lit er a ture. 37
Monoterpene gly<strong>co</strong>sides (1, 10-18) from P. suffruti<strong>co</strong>sa
Paeoniflorin (1), oxypaeoniflorin (10), benzoyl pae o -
niflorin (11), benzoyloxypaeoniflorin (12), mudanpioside A
(13), mudanpioside B (14), mudanpioside C (15), mudan -
pioside D (16), mudanpioside E (17), <strong>and</strong> mudanpioside F
(18) were ob tained from our pre vi ous stud ies on P.
suffruti<strong>co</strong>sa. 16
Mi nor <strong>co</strong>n stit u ents (20, 21, 22, 23) from P. suffruti<strong>co</strong>sa
Mi nor <strong>co</strong>n stit u ents, apiopaeonoside (20), mudanoside
B (21), trans-caffeic acid stearyl es ter (22) <strong>and</strong> quercetin
(23) were ob tained from our pre vi ous stud ies on P.
suffruti<strong>co</strong>sa. 16,17
Free ar o <strong>ma</strong>tic acid from P. suffruti<strong>co</strong>sa
Free ar o <strong>ma</strong>tic acid, ben zoic acid (2), p-hydroxybenzoic
acid (24), <strong>and</strong> gal lic acid (25) were ob tained from our pre vi -
ous stud ies on P. suffruti<strong>co</strong>sa. 17,18
Ben zoic acid (2) <strong>and</strong> monoterpene gly<strong>co</strong>sides (11, 19)
from P. lactiflora
The root (10 Kg) of P. lactiflora was ex tracted with al <strong>co</strong> -

Phytochemistry of Paeonia Spe cies J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 387
hol, par ti tioned with n-hexane <strong>and</strong> EtOAc in the same pro ce -
dure as used for P. suffruti<strong>co</strong>sa. The EtOAc ex tract (50 g) was
sub jected to sil ica gel <strong>co</strong>l umn chro <strong>ma</strong> tog ra phy <strong>and</strong> eluted
with CHCl3, CHCl3-MeOH (97:3) <strong>and</strong> CHCl3-MeOH (95:5).
The 8.6 g of ben zoic acid (2) was iso lated from CHCl3-MeOH
(97:3) elu tion. The frac tion of CHCl3-MeOH (95:5) elu tion
was sep a rated on a re verse C-8 lo bar <strong>co</strong>l umn eluted with
MeOH-H2O (60:40) sol vent, to get lactiflorin (19) <strong>and</strong>
benzoylpaeoniflorin (11). Both <strong>co</strong>m pounds were fur ther pu ri -
fied by pre para tive HPLC on re verse C-18 <strong>co</strong>l umn us ing
MeOH-H2O (58:42) as mo bile phase to get 0.997 g of
lactiflorin (19) <strong>and</strong> 1.03 g of benzoylpaeoniflorin (11).
Platelet Ag gre ga tion Tests
Washed rab bit plate lets were pre pared from EDTAanti<strong>co</strong>agulated
platelet-rich plas<strong>ma</strong> ac <strong>co</strong>rd ing to pro ce dures
de scribed pre vi ously. 38,39 Plate lets were <strong>co</strong>unted by a Coul ter
Coun ter (Model ZM), ad justed to 4.5 10 8 plate lets/mL, <strong>and</strong>
sus pended in Tyrode so lu tion <strong>co</strong>n tain ing (mM) : NaCl
(136.8), KCl (2.8), NaHCO3 (11.9), MgCl2 (2.1), NaH2PO4
(0.33), CaCl2 (1.0), <strong>and</strong> glu <strong>co</strong>se (11.2) with bo vine se rum al -
bu min (0.35 %). Ag gre ga tion was mea sured by the
turbidimetric method, de signed with the absorbance of plate -
lets in sus pen sion at 0 % ag gre ga tion <strong>and</strong> the absorbance of
platelet-free Tyrode so lu tion as 100 % ag gre ga tion. 40 The ag -
gre ga tion was mea sured by a Lumi-aggregometer (Chrono-
Log Co.) <strong>co</strong>n nected to dual chan nel re <strong>co</strong>rd ers. The platelet
sus pen sion was stirred at 1200 rpm. Plate lets were pre -
incubated with the test <strong>co</strong>m pounds or the sol vent (DMSO) in
the <strong>co</strong>n trol ex per i ment for 3 min be fore the ad di tion of ag gre -
ga tion in duc ers. To elim i nate the ef fect of the sol vent on the
ag gre ga tion, the fi nal <strong>co</strong>n cen tra tion of DMSO was fixed at 0.5
%. Per cent in hi bi tion test <strong>co</strong>m pounds were <strong>cal</strong> cu lated from
platelet ag gre ga tion as sum ing the <strong>co</strong>n trol val ues were 100 %.
The re sults (Ta ble 2) were ex pressed as means S.D.. Oneway
anal y sis of vari ance (ANOVA) was car ried out for mul ti -
ple <strong>co</strong>m par i sons, <strong>and</strong> if there was a dif fer ence (p

388 J. Chin. Chem. Soc., Vol. 47, No. 2, 2000 Ding et al.
14. Lang, H. Y.; Li, S. Z.; MaCabe, T.; Clardy, J. C. Planta
Med. 1984, 59, 501-504.
15. Lin, H. C.; Chern, H. M. Chim. <strong>Phar</strong>m. J. 1991, 43,
175-177.
16. Lin, H. C.; Ding, H. Y.; Wu, T. S.; Wu, P. L.
Phytochemistry 1996, 41, 237-242.
17. Lin, H. C.; Ding, H. Y.; Wu, Y. C. J. Nat. Prod. 1998, 61,
343-346.
18. Ding, H. Y.; Wu, Y. C.; Lin, H. C.; Chan, Y. Y.; Wu, P. L.;
Wu, T. S. Chem. <strong>Phar</strong>m. Bull. 1999, 47, 652-655.
19. Lin, H. C.; Ding, H. Y.; Ko, F. N.; Teng, C. M.; Wu, Y. C.
Planta Med. 1999, in press.
20. Kubo, M.; Matsuda, H.; Izumi, S.; Tani, T.; Arichi, S.;
Yoshikawa, M.; Kitagawa, I. Shoyakugaku Zasshi 1982,
36, 70-77.
21. Takeda, S.; Wakui, Y.; Mizuhara, Y.; Ishi hara, K.;
A<strong>ma</strong>gaya, S.; Maruno, M.; Hattori, M. J. Trad. Med. 1996,
13, 248-251.
22. Heikal, O. A.; Miyashiro, H.; Akao, T.; Hattori, M. J.
Trad. Med. 1997, 14, 15-19.
23. Reynolds, J. E. F.; Prasad, A. B. The Ex tra <strong>Phar</strong> <strong>ma</strong> <strong>co</strong> -
poeia (28 Edi tion); The <strong>Phar</strong> <strong>ma</strong> ceu ti <strong>cal</strong> Press: Lon don;
1982, pp 1283-1284.
24. Hattori, M.; Shu, Y. Z.; Shimizu, M.; Hayashi, T.; Morita,
N.; Kobashi, K.; Xu, G. J.; Namba, T. Chem. <strong>Phar</strong>m. Bull.
1985, 33, 3838-3846.
25. Shu, Y. Z.; Hattori, M.; Akao, T.; Kobashi, K.; Kakei, K.;
Fukuya<strong>ma</strong>, K.; Tsukihara, T.; Namba, T. Chem. <strong>Phar</strong>m.
Bull. 1987, 35, 3726-3733.
26. Reynolds, J. E. F.; Prasad, A. B. The Ex tra <strong>Phar</strong> <strong>ma</strong> <strong>co</strong> -
poeia (28 Edi tion); The <strong>Phar</strong> <strong>ma</strong> ceu ti <strong>cal</strong> Press: Lon don;
1982, p 287.
27. Yu, J.; Xiao, P. G. Acta <strong>Phar</strong><strong>ma</strong>ceutica Sinica 1985, 20,
782-784.
28. Jin, C. D.; Xu, G. J.; Jin, R. L.; Xu, L. S.; Cong, X. D. J.
China. <strong>Phar</strong>m. Univ. 1989, 20, 139-142.
29. Cotran, R. S.; Ku<strong>ma</strong>r, V. K.; Rob bins, S. L.; Schoen, F. J.
Rob bins Patho <strong>log</strong> i <strong>cal</strong> Ba sis of Dis ease (5th Edi tion); W.
B. Saunders Com pany: Phil a del phia, U. S. A.; 1994, pp
51-92.
30. Cotran, R. S.; Ku<strong>ma</strong>r, V. K.; Rob bins, S. L.; Schoen, F. J.
Rob bins Patho <strong>log</strong> i <strong>cal</strong> Ba sis of Dis ease (5th Edi tion); W.
B. Saunders Com pany: Phil a del phia, U. S. A.; 1994, pp
623-627.
31. Takagi, T.; Harada, M. Yakugaku Zasshi 1969, 89,
887-892.
32. Reynolds, J. E. F.; Prasad, A. B. The Ex tra <strong>Phar</strong> <strong>ma</strong> <strong>co</strong> -
poeia (28 Edi tion); The <strong>Phar</strong> <strong>ma</strong> ceu ti <strong>cal</strong> Press: Lon don;
1982, pp 1281-1282.
33. Pouchert, C. J. The Aldrich Li brary of NMR Spec tra (Edi -
tion II), Aldrich Chem i <strong>cal</strong> Com pany, U.S.A.; 1983; vol. 2,
p 34.
34. Kurihara, T.; Kikuchi, M. Yakugaku Zasshi 1975, 95,
1098-1102.
35. Agrawal, P. K. Car bon-13 NMR of Flavonoids, Elsevier,
Am ster dam, 1989, pp 152-153.
36. Pouchert, C. J. The Aldrich Li brary of NMR Spec tra (Edi -
tion II), Aldrich Chem i <strong>cal</strong> Com pany, U.S.A.; 1983; vol. 2,
p 208.
37. Ah<strong>ma</strong>d, V. U.; Rah<strong>ma</strong>n, A. U. H<strong>and</strong> book of Nat u ral Prod -
ucts Data-Pentacyclic Triterpenoids (vol. 2), Elsevier,
Am ster dam, 1994; pp 223-225.
38. Teng, C. M.; Chen, C. C.; Ko, F. N.; Lee, L. G. Thromb.
Res. 1988, 50, 757-765.
39. Lin, C. N.; Shieh, W. C.; Ko, F. N.; Teng, C. M. Biochem.
<strong>Phar</strong><strong>ma</strong><strong>co</strong>l. 1993, 45, 509-512.
40. O Brien, J. R. J. Clin. Pathol. 1962, 15, 452-455.