Inhibitory effects of glycosides from the leaves of Melaleuca quinquenervia on vascular contraction of rats

Article abstract of DOI:10.1055/s-2002-32563

Two new glycosides, 3-hydroxy-5-methoxy-4-methylphenyl β-D-glucopyranoside (1) and 4-benzoyl-2-C-β-glucopyranosyl-3,5-dihydroxy-6-methylphenyl β-D-glucopyranoside (2), together with four known glycosides, 2-endo-β-o-glucopyranosyloxy-1,8-cineole (3a), 2-exo-β-D-glucopyranosyloxy-1,8-cineole (3b), roseoside (4), and citroside A (5), were isolated from the methanolic extract of leaves of Melaleuca quinquenervia. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1, 2a and 3 inhibited contractile response induced by phenylephrine in aortic rings from Sprague-Dawley rats. This inhibition was independent of the endothelium. Compounds 2 and 4 significantly relaxed precontracted aortic rings, in an endothelium-dependent manner. Pretreatment of N^ω-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, partially attenuated the vasorelaxation induced by both compounds, suggesting that nitric oxide was likely the responsible mediator. The rank-order potency (EC₅₀ value) of vasorelaxing activities of these compounds is 4 > 2 > 2a > 3 > 1.

Full text of DOI:10.1055/s-2002-32563

Tzong-Huei Lee1
Guei-Jane Wang2
Ching-Kuo Lee3
Yueh-Hsiung Kuo4
Chang-Hung Chou1,5
Inhibitory Effects of Glycosides from the Leaves
of Melaleuca quinquenervia on Vascular Contraction
of Rats
Abstract
hibition was independent of the endothelium. Compounds 2 and
4 significantly relaxed precontracted aortic rings, in an endothe-
Two new glycosides, 3-hydroxy-5-methoxy-4-methylphenyl b-D- lium-dependent manner. Pretreatment of Nw-nitro-L-arginine (L-
glucopyranoside (1) and 4-benzoyl-2-C-b-glucopyranosyl-3,5- NNA), a nitric oxide synthase inhibitor, partially attenuated the
dihydroxy-6-methylphenyl b-D-glucopyranoside (2), together vasorelaxation induced by both compounds, suggesting that ni-
with four known glycosides, 2-endo-b-D-glucopyranosyloxy-1,8- tric oxide was likely the responsible mediator. The rank-order
cineole (3a), 2-exo-b-D-glucopyranosyloxy-1,8-cineole (3b), ro- potency (EC50 value) of vasorelaxing activities of these com-
seoside (4), and citroside A (5), were isolated from the methano- pounds is 4 > 2 > 2a > 3 > 1.
lic extract of leaves of Melaleuca quinquenervia. Their structures
were elucidated on the basis of spectroscopic analysis. Com- Key words
pounds 1, 2a and 3 inhibited contractile response induced by Melaleuca quinquenervia ´ Myrtaceae ´ glycosides ´ leaves ´ vaso-
phenylephrine in aortic rings from Sprague-Dawley rats. This in- relaxing activities
Introduction
the new compounds as well as their biological activity toward
blood vessels.
492
Melaleuca quinquenervia (Myrtaceae), a large erect paper-bark
tree, is widely cultivated in Taiwan. Its leaves containing bioac-
tive agents have long been used in traditional folk medicine as Materials and Methods
sedative and pain-relieving agents [1]. Findings of chemical stud-
ies on leaves of the plant were reported, from which series of tri- General experimental procedures
terpenoids [2], [3], flavanones [4] and polyphenols [5] were iden- Optical rotations were measured using a JASCO DIP-180 digital
tified. However, the glycosidic constituent of M. quinquenervia was spectropolarimeter. IR spectra were recorded on a Nicolet 510P
not reported, a chemical investigation on the n-butanol soluble FT-IR spectrometer. UV spectra were measured in MeOH or
part of the methanolic extract of its leaves was thus undertaken CH2Cl2 on a Hitachi U-2000 spectrophotometer. The NMR spectra
that has led to the isolation and identification of two new phenolic were recorded in CD3OD or CDCl3 at room temperature on a Bru-
glycosides as well as four known glycosides. In an attempt to as- ker DMX-500 SB spectrometer, and the solvent resonances were
sess their potential as antihypertensive agents, in vitro experi- used as internal shift references. The 2D NMR spectra were re-
ments were first conducted to verify the vasorelaxing properties. corded using standard pulse sequences. Positive ion FAB-MS
This study describes the isolation and structural elucidation of and HR-FAB-MS data were obtained on a JOEL SX-102A mass
Affiliation
1 Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan
2 National Research Institute of Chinese Medicine, Shih-Pai, Taipei, Taiwan
3 China Junior College of Medical Technology, Tainan, Taiwan
4 Department of Chemistry, National Taiwan University, Taipei, Taiwan
5 Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
Correspondence
Prof. Chang-Hung Chou ´ Department of Biological Sciences ´ National Sun Yat-sen University ´ Kaohsiung ´
Taiwan 804 R.O.C. ´ Phone: +886 2 26510363 ´ Fax: +886 2 26510363 ´ E-Mail: choumasa@mail.nsysu.edu.tw
Received August 16, 2001 ´Accepted January 20, 2002
Bibliography
Planta Med 2002; 68: 492±496 ´ ꢀ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943

spectrometer using m-nitrobenzyl alcohol (NBA) as the matrix. MeOH); UV (MeOH): lmax (log e) = 225 (4.1), 268 (3.4) nm; IR
Sephadex LH-20 (Pharmacia Biotech) was used for open column (KBr): nmax = 3372, 1603, 1516, 1464, 1423, 1165, 1117, 1074
chromatography. HPLC was performed using an ODS column cm±1; 1H-NMR (CD3OD, 500 MHz): d = 6.27 (d, J = 2.0 Hz, H-6),
(Hyperprep ODS, 10 mm i.d.”250 mm, Keystone Scientific Inc., 6.24 (d, J = 2.0 Hz, H-2), 4.79 (d, J = 7.3 Hz, H-1¢), 3.90 (dd,
Bellefonte, PA; detector, RI) and a silica column (Hyperprep HS J = 11.9, 1.2 Hz, H-6¢a), 3.74 (s, H3 ±7), 3.69 (dd, J = 11.9, 5.4 Hz,
silica, 10 mm i.d.”250 mm, ThermoQuest Hypersil, Runcorn, H-6¢b), 3.2±3.45 (m, H-2¢, 3¢, 4¢, 5¢), 1.94 (s, H3 ±8); 13C-NMR
UK; detector, RI). TLC was performed using silica gel 60 F254 (CD3OD, 300 MHz): d = 160.4 (C-5), 158.1 (C-1), 157.2 (C-3),
plates (200 mm, Merck, Germany).
107.5 (C-4), 102.7 (C-1¢), 97.6 (C-2), 93.4 (C-6), 78.2 (C-3¢ or C-
5¢), 78.1(C-5 ¢ or C-3¢), 74.9 (C-2¢), 71.5 (C-4¢), 62.6 (C-6¢), 56.0
Plant material
(C-7), 7.9 (C-8); FAB-MS (NBA): m/z = 339 [M+Na]+ (34), 154
+
Leaves of M. quinquenervia (CAV.) S. T. Blake were collected at the (100); HR-FAB-MS (NBA): m/z = 317.1250 [M+H] calcd. for
Academia Sinica, Taipei on March 16, 2001 and were identified
by Mr. Chii-Cheng Liao, Department of Botany, National Taiwan
C14H20O8 + H: 317.1236.
University. Voucher specimens (No. 20 010316) have been de- 4-Benzoyl-2-C-b-glucopyranosyl-3,5-dihydroxy-6-methylphenyl b-
posited at the Institute of Botany, Academia Sinica, Nakang, Tai- D-glucopyranoside peracetate (2a): amorphous white powder;
pei, Taiwan.
m.p. 125±1268C; [a]D25: ±1 7.89 (c 0.48, CH2Cl2); UV (CH2Cl2):
lmax (log e) = 256 (4.6), 230 (4.3) nm; IR (KBr): nmax = 2942,
1755, 1674, 1599, 1435, 1369, 1223, 1039 cm ; H-NMR (CDCl3,
±1
1
Extraction and isolation
Fresh leaves of M. quinquenervia (650 g) were successively ex- 500 MHz): d = 7.77 (d, J = 7.5 Hz, H-3¢, -7¢), 7.56 (t, J = 7.5 Hz,
tracted three times with 3 L of MeOH at room temperature. The H-5¢), 7.44 (t, J = 7.5 Hz, H-4¢, -6¢), 5.47 (t, J = 9.5 Hz, H-2¢¢¢),
methanolic extract was adjusted to 85% in aqueous solution for 5.43 (t, J = 8.2 Hz, H-2¢¢), 5.35 (t, J = 9.5 Hz, H-3¢¢¢), 5.26 (t,
an n-hexane partition, which generated two fractions soluble in J = 8.2 Hz, H-3¢¢), 5.25 (d, J = 9.5 Hz, H-1¢¢¢), 5.24 (t, J = 8.2 Hz,
aqueous methanol and n-hexane. Subsequently, the aqueous me- H-4¢¢), 5.00 (t, J = 9.5 Hz, H-4¢¢¢), 4.84 (d, J = 8.2 Hz, H-1¢¢), 4.48
thanol-soluble fraction was then evaporated in vacuum to dry- (dd, J = 12.5, 3.2 Hz, H-6¢¢a), 4.28 (dd, J = 12.6, 4.8 Hz, H-6¢¢¢a),
ness (52 g) and further partitioned between chloroform (500 3.88 (br d, J = 12.5, H-6¢¢b), 3.76 (br d, J = 12.6, H-6¢¢¢b), 3.72 (m,
mL”2) and water (500 mL), and the remaining water solution H-5¢¢¢), 3.65 (m, H-5¢¢), 2.08, 2.07, 2.04, 2.01, 2.00, 1.98, 1.95, 1.94,
was extracted three times with n-butanol (500 mL). The n-buta- 1.85, 1.78 (s, OAc”10), 2.06 (s, H ±7); 13C-NMR (CDCl3, 300
3
nol layer was evaporated to a brown residue and redissolved in MHz): 948; = 191.1 (C-1¢), 170.3, 170.2”3, 169.5, 169.4, 169.2,
MeOH for chromatographic separation. The first separation step 168.7, 167.8, 167.7 (OCOCH3 ”10), 154.1 (C-1), 148.7 (C-3), 146.3
was carried out using gel filtration chromatography on a Sepha- (C-5), 136.8 (C-2¢), 133.5 (C-5¢), 129.8 (C-3¢, -7¢), 128.4 (C-4¢, -6¢),
dex LH-20 column (3”55 cm) and eluted by MeOH with a flow 126.0 (C-4), 122.9 (C-6), 122.5 (C-2), 101.7 (C-1¢¢), 75.8 (C-5¢¢¢),
rate of 13 mL/min. Each fraction (15 mL) collected from then-bu- 74.4 (C-3¢¢¢), 73.9 (C-1¢¢¢), 72.8 (C-3¢¢), 72.2 (C-5¢¢), 71.7 (C-2¢¢),
tanol layer was checked for their compositions by TLC using 70.4 (C-2¢¢¢), 68.6 (C-4¢¢¢), 67.2 (C-4¢¢), 62.2 (C-6¢¢¢), 60.9 (C-6¢¢),
EtOAc/HCO2H/H2O (85:10:15) for development. Observation 20.7, 20.6”4, 20.5”2, 20.4, 20.2, 20.1(OCO CH3 ”10), 10.1 (C-7);
under UV 254 nm and dipping in vanillin-sulfuric acid (brown FAB-MS (NBA): m/z = 989 [M+H]+ (12), 947 (7), 617 (8), 331
or orange spots) were used in the detection of glycosides. Subse- (70), 169 (100), 109 (37); HR-FAB-MS (NBA): m/z = 989.2935
quently, the glycoside fractions (#fr. 11 ±19) from above se- [M+H]+ calcd for C46H52O24 + H: 989.2927.
493
paration were combined to give subfraction 1and 2. HPLC of sub-
fraction 1on a reversed-phase column with MeCN/H O (20:80) Compound 3, [a]D25: ±40.28 (c 0.64, MeOH), was characterized as a
2
as eluent, 2 mL/min, afforded 3 (56 mg), 4 (45 mg), and 5 (6 mg), mixture (3a: 3b = 4:1 ) of 2e-ndo-b-D-glucopyranosyloxy-1,8-ci-
retention time: 16.2, 11.5 and 12.4 min, respectively. Subfraction neole (3a) and 2-exo-b-D-glucopyranosyloxy-1,8-cineole (3b), ob-
2 was purified by using the same column with MeCN/H2O tained previously from Eucalyptus perriniana [6] and Salvia
(15:85) as eluent to yield 1 (11 mg) and crude mixture of2 (25 bucharica [7], respectively. Compound 4, [a]D25: + 83.58 (c 0.50,
mg), retention time: 10.1 and 10.8 min, respectively. Crude mix- MeOH), was identified as roseoside, having been isolated from aer-
ture of 2 (10 mg) was dissolved in pyridine (3 mL) with Ac2O (3 ial parts of Epimedium grandiflorum [8]. Compound 5, [a]D25: ±858
mL), and the mixture left overnight at room temperature. Then, (c 0.25, MeOH), was determined to be citroside A, and its spectral
ice water (30 mL) was added to the reaction mixture, and the re- data were in good agreement with the published data [9].
sultant suspension was extracted with ethyl acetate (30 mL”2).
The ethyl acetate layer was purified using a normal-phase col- Bioassays
umn with n-Hex/CHCl3/EtOAc (1:1:1) as eluent, 2 mL/min, to In vitro vascular tension study: The details of the experimental
yield the pure peracetylated derivative 2a (9.5 mg).
procedures have been described previously [10]. Briefly, Spra-
gue-Dawley rats were sacrificed by decapitation, and sections of
Acid hydrolysis: Compounds 1 and 2 (5 mg) were hydrolyzed by the thoracic aorta between the aortic arch and the diaphragm
2N HCl (2 mL) at room temperature overnight. The reaction mix- were excised carefully. Isolated aortic rings 3 to 4 mm in length
ture was then partitioned with EtOAc (2 mL”2). The lower layer were fixed isometrically in organ chambers under passive ten-
was neutralized using Amberlite IRA-400 resin (Fluka, Switzer- sion of 1.8 g for 60 min. The changes of vascular tension were re-
land), filtered with glass wool, and the filtrate was evaporated corded with a polygraph (Gould, model 2400, Vally View, OH,
to give D-glucose: [a]D25: + 47.18 (c 0.075, H2O).
USA) via a force displacement transducer (Grass FTO3, Quincy,
MASS, USA) and simultaneously displayed on the monitor of an
3-Hydroxy-5-methoxy-4-methylphenyl b-D-glucopyranoside (1): IBM-compatible computer after being digitized (PowerLab,
amorphous white powder; m.p. 112±1148C; [a]D25: ±658 (c 0.30, ADInstruments Pty Ltd., Sydney, Australia). After equilibration,

near maximal contraction was induced by phenylephrine (0.3
mM). When the rings achieved a stable contractile tension, acetyl-
choline (1 mM) was added to the bath to assess endothelial in-
tegrity. In some preparations, the intima was gently frayed with
a cotton swab to disrupt the endothelium. The absence of acetyl-
choline-induced relaxation indicated successful endothelial de-
nudation.
Relaxation of phenylephrine-induced contraction: For the evalual-
tion of relaxation, acetylcholine (1 mM, endothelium-depen-
dent) and sodium nitroprusside (0.1 mM, endothelium-inde-
pendent) were used as positive controls in this experiment.
Compounds 1, 2, 2a, 3, 4 (0.01±100 mM) or vehicle (0.1%,
DMSO) was added in a cumulative manner during the tonic
phase of contraction induced by phenylephrine (0.3 mM) in
both endothelium-intact and -denuded aortic rings. A 10-min
time interval was required to obtain the maximal effect with
each concentration of various compounds. The construction of
concentration-response curves for compounds was based on (1H”4, m), a glucopyranosyl anomeric proton at d = 4.79 (1H, d,
the percentage of relaxation of the agonist-induced maximal J = 7.3 Hz), and two meta-aromatic protons at d = 6.27 (1H, d,
contraction. The concentration evoking 50% relaxation (EC50 J = 2.0 Hz) and 6.24 (1H, d, J = 2.0 Hz), suggesting the presence
value) for each experiment was calcuated. To investigate the of a benzene ring and a b-D-glucopyranose. The b-D-glucopyrano-
possible involvement of nitric oxide in the vasorelaxing effects syl, methoxy, methyl, and hydroxy groups were determined to be
of compounds, endothelium-intact aortic rings were preincu- linked at C-1, C-5, C-4 and C-3, respectively, since correlations
bated with the nitric oxide synthase inhibitor Nw-nitro-L-argi- between glucopyranosyl H-1¢ and C-1, H3 ±7 and C-3, and H3 ±
nine (L-NNA, 100 mM) for 10 min. Cumulative concentrations of 8 and C-3, -4, -5 were observed in the HMBC spectrum. Three
each of these compounds (0.01±100 mM) were then applied singlets at d = 160.4, 158.1 and 157.2 in the13C-NMR spectrum
during the sustained phase of phenylephrine (0.3 mM)-induced of 1 further supported the oxygenation of the benzene ring at C-
contraction. The effects of the inhibitor were studied by com- 1, C-5 and C-3, respectively. Thus, the structure of 1 was deter-
paring the degrees of vasorelaxation induced by compounds in mined as 3-hydroxy-5-methoxy-4-methylphenyl b-D-glucopyra-
the absence or presence of this inhibitor. The concentrations of noside.
the inhibitor used had been reported to be adequate to produce
the necessary nitric oxide inhibition [11].
Compound 2 was purified as its peracetylated derivative 2a.
Compound 2a was obtained as an amorphous powder, whose
molecular formula was confirmed to be C46H52O24 by the HR-
494
Statistical analyses
The data are presented as mean  S.E. and n represents the num- FAB-MS and 13C-NMR spectrum. The 1H-NMR spectrum of 2a
ber of experiments. In line graphs, S.E. values are indicated by er- showed signals for fourteen sugar protons, and five aromatic
ror bars (in some cases the error bars were so small they were protons in A2X2Y system, both being corroborated by a COSY
obliterated by the line symbols). Statistical analyses were carried spectrum, in addition to a signal for an aryl methyl group
out by Student's unpaired t test when applicable. P values of less (d = 2.06). Analysis of the signals of fourteen protons suggested
than 0.05 were considered to be significant.
two b-glucopyranosyl units with the anomeric protons at
d = 5.25 (d, J = 9.5 Hz, H-1¢¢¢) and 4.84 (d, J = 8.2 Hz, H-1¢¢). The
1H-NMR spectrum of 2a also revealed ten acetyl methyl singlets,
suggesting that there existed two aromatic hydroxy groups in
addition to eight hydroxys on two glucosyl moieties. In the
Results and Discussion
A methanolic extract of the fresh leaves of M. quinquenervia was HMBC spectrum, the anomeric protons at d = 5.25 and 4.84
fractionated by liquid-liquid partitioning into fractions soluble in showed interactions with aryl C-2 and C-1at d = 122.5 and
n-hexane, chloroform, and n-butanol, successively. The n-buta- 154.1, respectively, the glucose H-1¢¢¢ signal at d = 5.25 correlated
nol-soluble fraction was then subjected to Sephadex LH-20 col- with the aryl C-1and C-3 resonances at d = 154.1 and 146.3,
umn chromatography and HPLC to give six glycosides. Of these, respectively, and the aryl methyl signal H3 ±7 at d = 2.06 cor-
compounds 1 and 2 were identified to be two novel glycosides related with the aryl C-1and C-5 resonances at d = 154.1 and
based on their spectral analysis.
148.7, respectively, through a three-bond coupling. These evi-
dences established the two glucosyl moieties to be attached at
The molecular formula for 1, C14H20O8, was determined by 13C- C-1and C-2, the two hydroxys at C-3 and C-5, and the methyl
NMR and HR-FAB-MS data. The IR spectrum of 1 indicated the group at C-6. The consecutive five phenyl protons with A2X2Y
presence of hydroxy (3372 cm±1) and aromatic (1603 and 1516 system were considered as a benzoyl moiety, which was discern-
cm-1) groups. Its UV spectrum exhibited maxima at 225 and 268 ible from their chemical shifts and coupling constants. Based on
nm. The 1H-NMR spectrum of 1 showed the signals for methylene these findings, the structure of 2a was concluded to be 4-ben-
protons at d = 3.90 (1H, dd, J = 11.9, 1.2 Hz) and 3.69 (1H, dd, zoyl-2-C-b-glucopyranosyl-3,5-dihydroxy-6-methylphenyl b-D-
J = 11.9, 5.4 Hz), methyl protons atd = 1.94 (3H, s), methoxyl pro- glucopyranoside peracetate.
tons at d = 3.74 (3H, s), four methine protons at d = 3.2±3.45

Fig. 1 Vasorelaxing effects of 1,
2a, and 3 on endothelium-intact
(+E) and endothelium-denuded (±E)
Sprague-Dawley rat thoracic aortic
rings contracted with phenylephr-
ine (0.3 mM). The tensions devel-
oped in the absence of compounds
in intact and denuded rings were
1.50  0.17 and 1.75  0.15 g,
respectively. Values are mean 
S.E.; n = 5 to 6 for each group.
the relaxation was 2a > 3 > 1 (Table 1). The vasorelaxing effect of
sodium nitroprusside was used as a positive control in each test
aortic rings without endothelium. The maximal relaxation was
93  5%. As shown in Fig. 2, the vasorelaxation induced by 2 or 4
was somewhat attenuated by removal of the endothelium, indi-
cating that both 2 and 4 relaxed vascular smooth muscle via
mechanisms which were partly endothelium-dependent. The
magnitude of relaxation induced in endothelium-intact aortic
rings had an order of 4 > 2 (Table 1). To investigate whether the
endothelial mediator nitric oxide is involved in the vasorelaxing
effect of 2 or 4, L-NNA, a nitric oxide synthase inhibitor, was pre-
incubated in endothelium-intact aortic rings. The concentration-
response curve of cumulative 2 or 4 before and after treatment
with L-NNA are illustrated in Figs. 2B and 2C. The results showed
that treatment with this inhibitor did not significantly affect the
basal vascular tone (data no shown). In the presence of L-NNA,
the vasorelaxing effect induced by both 2 and 4 were significant-
Table 1 Vasorelaxing effects of compounds in endothelium-intact
aortic rings isolated fromSprague-Dawley rats. Aortic rings
were precontracted with phenylephrine (0.3 mM), relaxation
was subsequently induced with cumulative concentrations
of each of various compounds
Compound
n*
EC50 (mM)
Max. relaxation (%)
1
5
7
6
5
5
80.1  7.2
21.2  6.5
31.0  6.8
71.4  4.8
16.3  5.1
52.2  9.8
68.1  4.6
57.9  7.4
51.9  6.2
73.2  7.9
2
2a
3
4
*n denotes the number of experiments.
In a vasorelaxing bioassay each test compound given alone did ly reduced in endothelium-intact aortic rings, showing endothe-
not alter the baseline tension of the aortic rings (data not lium-derived nitric oxide was involved in the vasorelaxing effect
shown). In the rat thoracic aorta, phenylephrine caused an initial of 2 and 4. The quantity of 5 obtained was insufficient to perform
phasic and then a tonic contraction, which lasted for at least 30 this biological investigation.
495
min. During the tonic phase of contraction induced by phenyl-
ephrine, all the compounds produced concentration-dependent
vasorelaxation compared with the vehicle-treated group Acknowledgements
(Fig. 2A). The EC50 and the maximal relaxation of various com-
pounds obtained by 100 mM are shown in Table 1. The vasorelax- This research was supported by grants to C.-H. C. and G.-J. W.
ing effects of 1, 2a or 3 on precontracted aortic rings showed no from the National Science Council (NSC-89-2311-B001-026 and
significant difference in the presence or absence of endothelium, NSC-89-2320-B077-009, respectively) and a postdoctoral fellow-
while the positive control acetylcholine produced a significant ship to T.-H. L. from Academia Sinica, Taiwan. We are grateful to
relaxation (95  4%) in endothelium-intact rings. The results im- Ms. Shou-Ling Huang for the NMR data acquisition in the Instru-
plied that these compounds acted directly on the arterial smooth mental Center of the College of Science, National Taiwan Univer-
muscle (Figs.1A, 1B, 1C). The rank-order potency (EC50 value) of sity.
Fig. 2 Vasorelaxing effects of vehi-
cle (0.1%, DMSO), 2, and 4 on endo-
thelium-intact (+E) and endothe-
lium-denuded (-E) Sprague-Dawley
rat thoracic aortic rings contracted
with phenylephrine (0.3 mM). The
tensions developed in the absence of
compounds in intact and denuded
rings were 1.50  0.17 and 1.75 
0.15 g, respectively. Values are mean
 S.E.; n = 5 to 7 for each group. *,
Statistically significant difference be-
tween test systemwith endothelium
(+E) and test systemwithout endo-
thelium(±E) or with endothelium
plus L-NNA (+E, L-LNNA).

8 Miyase T, Ueno A, Takizawa N, Kobayashi H, Oguchi H. Studies on the
glycosides of Epimedium grandiflorum Morr. var. thunbergianum
(Miq.) Nakai. II. Chem Pharm Bull 1987; 35: 3713±9
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