of icariin on cGMP-specific PDE5 and cAMP-specific PDE4 activities
ZC Xin1, EK Kim2,
CS Lin3, WJ Liu1, L Tian1, YM Yuan1,
1Department of Urology,
the 1st Hospital, Peking University, Beijing 100034, China
2Department of Life Science, Pohang University of Science and
Technology, Pohang, Korea
3Knuppe Molecular Urology Laboratory, Department of Urology,
Asian J Androl
2003 Mar; 5:
icariin; PDE5; cGMP; penile erection
Aim: To clarify the
mechanism of the therapeutic action of icariin on erectlile dysfunction
(ED). Methods: PDE5 was isolated from the human platelet and PDE4
from the rat liver tissue using the FPLC system (Pharmacia, Milton Keynes,
UK) and the Mono Q column. The inhibitory effects of icariin on PDE5 and
PDE4 activities were investigated by the two-step radioisotope procedure
with [3H]-cGMP/[3H]-cAMP. Papaverine served as the
control drug. Results: Icariin and papaverine showed dose-dependent
inhibitory effects on PDE5 and PDE4 activities. The IC50 of
Icariin and papaverine on PDE5 were 0.432 µmol/L and 0.680 µmol/L,
respectively and those on PDE4, 73.50 µmol/L and 3.07 µmol/L,
respectively. The potencies of selectivity of icariin and papaverine on
PDE5 (PDE4/PDE5 of IC50) were 167.67 times and 4.54 times,
respectively. Conclusion: Icariin is a cGMP-specific PDE5 inhibitor
that may be developed into an oral effective agent for the treatment of
Penile erection is a hemodynamic
process involving relaxation of smooth muscle of the corpus cavernosum
(CC) and its associated arterioles. This relaxation process brings about
an increased flow of blood into the trabecular spaces of CC with resultant
penile erection. The process is generally accepted to be under neural
control and involves the cholinergic, adrenergic and non-adrenergic non-cholinergic
(NANC) neuroeffector system [1-3]. Nitric oxide (NO) is a gaseous messenger
molecule, which, during sexual stimulation is synthesized by NANC neurons
in the penis. It is also synthesized by the endothelial cells lining the
blood vessels and the lacunar spaces of CC and plays an important role
in mediating smooth muscle relaxation. NO activates guanylate cyclase
(GC), resulting in an increased conversion of guanosine triphosphate (GTP)
to cyclic guanosine monophosphate (cGMP). cGMP provides the signal leading
to relaxation of smooth muscle of CC and penile arterioles [4-5]. The
level of cGMP in CC is regulated through the balance between the rate
of synthesis by GC and the rate of hydrolytic breakdown to 5'-monophosphate
by cyclic nucleotide phosphodiesterase (PDE5) which is specifically distributed
in CC. Therefore, agents that inhibit cGMP hydrolysis may increase the
cGMP signal and are expected to enhance relaxation of smooth muscle in
the CC and thereby facilitate penile erectile responses [5,6]. Intracavernosal
injection of prostaglandin E1 and papaverine induces penile erection by
increase the cAMP levels in CC [7-9]. cAMP is hydrolytically broken down
to AMP by PDE4, which is nonspecifically distributed in the smooth muscles
of different organs. Thus, a PDE5 or PDE4 inhibitor may increase the cGMP
or cAMP levels and induce smooth muscle relaxation. cGMP-specific PDE5
inhibitors, such as sildenafil, may enhance penile erection after oral
administration. Although these agents can be useful drugs to treat erectile
dysfunction (ED), each has its limitation, because of the potential side
effects such as pain, priapism, cavernosal fibrosis, as well as systemic
side effects [8-9].
molecular weight: 676.67) is a flavonoid isolated from the plant drug
Epimedii herba [10-12], which is traditionally believed to have
a tonic effect and improve the sexual function. A few studies on the effects
of icariin in regard to its anti-fatigue, immuno-regulatory and liver
function improving activity have been reported [13-18]. We previously
indicated that Icariin relaxed CC, an effect that was inhibited by the
NO scavanger methyline blue and soluble GC scavanger ODQ . Icariin
significantly increased the cGMP level of CC of the penis and clitoris,
but not the cAMP level of CC. These results implied that Icariin could
mediate NO cGMP signaling pathway to enhance penile erection .
In order to further clarify the action mechanism
of icariin on penile erection, its effect on cGMP-specific PDE5 and cAMP-specific
PDE4 activities were studied by means of the two-step radioisotope procedure
2 Materials and methods
Pharmaceutical Co., China) was used as the control drug. All other chemicals
were obtained from Sigma, Poole, UK.
2.2 Isolation of icariin
Dried aerial parts of
Epimedii herba was extracted 3 times with ethanol, yielding an
ethanol extract upon removal of the solvent in vacuo. The ethanol
extract was then suspended in water and partitioned successively with
n-hexane, CHCl3 and n-BuOH to obtain different fractions. The n-BuOH fraction
was spread out using silica gel column chromatography to isolate icariin,
which was purified through repeated re-crystallization from MeOH. It was
an amorphous yellow powder [Rf=0.23 (a solvent system of CHCl3:
MeOH was used as the developer), M.P: 231 ~233
] and consisted
of pure icariin (98.8 %)  as verified by HPLC.
2.3 Isolation of PDE5 and
PDE5 was isolated from
the human platelet and PDE4 from the rat liver tissue through the following
procedure. One ml ice-cold homogenization buffer [20 mmol/L HEPES containing
0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L phenyl-methylsulfonyl fluoride
(PMSF), pH 7.2] was added to 10 mL of platelets or liver samples and were
disrupted by sonication (Kontes Microultrasonic Cell Disrupter, Brukard
Scientific, UK). The homogenate was centrifuged for 60 minutes at 4
and the supernatants were recovered
and filtered through a 0.2 mm filter. The soluble fractions of the tissues
were prepared using a Pharmacia FPLC system (Pharmacia Ltd, UK). Mono
Q column was pre-equilibrated with 20 mmol/L HEPES buffer (pH 7.2) containing
1 mmol/L EDTA and 0.5 mmol/L PMSF and then loaded with the tissue soluble
fraction, followed by washing with 5 mL of buffer. The PDE isozyme was
eluted using a continuous gradient of 0 to 500 mmol/L NaCl in the same
buffer (total volume 55 mL) at a flow rate of 1 mL per minute and 1mL
of each fraction was collected. The column fractions showing the highest
level of each characterized PDE activity were pooled and stored at -80
2.4 Effects of icariin and
papaverine on PDE5 and PDE4 activities
The cyclic nucleotide
PDE activity in FPLC fractions was determined using a modification of
the two-step radioisotope procedure of Thomson and Applemam. The reaction
mixture (total volume 100 µL) contained the column fraction elute
(10 µL to 25 µL), [3H]-cGMP]/ [3H]-cGMP]
(0.5 µmol/L, 2 µCi/mL). Reaction was initiated by addition
of the radio labeled substrate or the enzyme and incubated for 20 minutes
in a 30 water
bath. Immersed the sample tubes in boiling water for 2 minutes to stop
the reaction. The metabolized product was separated from the unmetabolized
substrate by ion exchange column chromatography using AG1-X2 resin (Bio-Rad)
and elution using 0.1 mol/L NaOH solution. The catalytic activity of PDE
was determined by measuring the radioactivity of the elute with a scintillation
To study the PDE isozyme
inhibition, icariin or papaverine was added to incubation mixtures in
dimethyl sulfoxide (DMSO, the highest final concentration < 2 %, V:
V) and the reaction was initiated by addition of the PDE enzyme fraction.
All inhibition experiments were conducted under conditions where the level
of cGMP/cAMP hydrolysis did not exceed 15 % and the product formation
increased linearly with the time and the amount of enzyme. [3H]-cGMP
was used as the substrate for PDE5 and [3H]-cAMP, for PDE4.
In studies on the kinetics of PDE5/PDE4 inhibition by icariin and papaverine,
[3H]-cGMP/[3H]-cGMP substrate concentrations ranged
from 0.3 µmol/L to 10 µmol/L were used and the initial rates
of hydrolysis were determined in the absence or the presence of samples
(10-10 mol/L to 10-4 mol/L).
2.5 Statistical analysis
For data analysis, fitted to plots of
enzyme activity versus log compound concentration using a curve-fitting
program and Lineweaver-Buck plots analysis were used and the IC50
values for inhibition of PDE5 were determined by sigmoid curves. P<0.05
was considered significant.
3.1 Effect on cGMP-specific
Icariin and papaverine
showed inhibitory effects on PDE5 in a dose-dependent manner. The inhibitory
effect of Icariin on PDE5 was 10-8 mol/L, 2.0 % to 10-4
mol/L, 99.70 % and the inhibitory effect of papaverine, 10-8
mol/L, 4.10 % to 10-4 mol/L, 96.40 % (Figure
1). Icariin and papaverine inhibited PDE5 activity in a concentration-dependent
manner (P<0.01). However, the inhibitory activities of icariin
and papaverine did not show significant difference (P>0.05).
1. Inhibitory effects of icariin, papaverine and sildenafil on PDE5
according to sigmoid curves fitted plots of enzyme activity versus log
concentration using curve-fitting program.
3.2 Effect on cAMP-specific
Icariin and papaverine
inhibited PDE4 activity in a dose-dependent manner. The inhibitory effects
of Icariin on PDE4 were 10-7 mol/L, 1.00 % to 10-3
mol/L, 65.00 % and the inhibitory effects of papaverine, 10-8
mol/L, 4.00 % to 10-3 mol/L, 95.00 %. Icariin and papaverine
inhibited PDE4 activity in a concentration-dependent manner (P<0.01)
and the inhibitory activity of icariin was significantly higher than that
of papaverine (P<0.01) (Figure
2. Inhibitory effects of icariin and papaverine on PDE4 activity according
to sigmoid curves fitted plots of enzyme activity versus log concentration
using curve-fitting program.
3.3 IC50 values for PDE5/PDE4
They were determined from sigmoid curves
and the IC50 of icariin and papaverine on PDE5 were 0.43 µmol/L
and 0.68 µmol/L, respectively and the IC50 on PDE4, 73.50
µmol/L and 3.07 µmol/L, respectively. The potencies of selectivity
of icariin and papaverine on PDE5 (PDE4/PDE5 of IC50) were
167.67 times and 4.54 times, respectively and the selective inhibition
on PDE5 (PDE4/PDE5 of IC50) of icariin was 36.93 times higher
than that of papaverine (Figure 3).
3. Inhibitory effects of icariin (167.67 times) and papaverine (4.54
times) on PDE5 (PDE4/PDE5 of IC50).
The catalytic activity
of PDE5 is specific for cGMP, whereas PDE4 is more specific for cAMP and
PDE3 is rather nonselective for both cAMP and cGMP in their hydrolytic
breakdown process. With the advances of studies on the molecular bases
of ED, specific PDE5 inhibitor sildenafil (ViagraTM) and the PDE superfamily
have attracted widespread interests. PDEs are intracellular enzymes that
catalyze the hydrolysis of cAMP and cGMP. By counterbalancing adenyl and
GC, which catalyze the formation of cAMP and cGMP, respectively, PDEs
serve to adjust the cellular concentrations of cAMP and cGMP, thereby
influencing the cellular functions [22-24].
In addition to sildenafil,
there are many kinds of PDE inhibitors that generally or specifically
target various PDE isoforms. Some PDEs are specific for the hydrolysis
of either cAMP or cGMP, while others have mixed specificity. By now, 11
subtypes of PDE families have been identified and each of them has a different
tissue distribution profile in human; among them, the PDE3, 4 and 5 are
more selectively distributed in CC. Those that are specific for cAMP are
PDE4, PDE7 and PDE8, those specific for cGMP are PDE5, PDE6 and PDE9 and
those with mixed specificity are PDE1, PDE2, PDE3, PDE10 and PDE11. Most
PDE families are expressed in a wide variety of tissues, while PDE6 is
expressed only in the photoreceptors. Within each PDE family, the alternatively
spliced isoforms appear to exhibit tissue specificity and the cGMP-specific
PDE5 inhibitor may be used for the oral treatment of ED, papaverine, however,
is believed to be a nonspecific PDEs inhibitor effective only with intracavernosal
injection [25, 26].
In the present study, we
investigated the inhibitory effect of icariin on PDE5 and PDE4 with papaverine
as the control drug. Icariin and papaverine had a dose-dependent inhibitory
effect on PDE4 and PDE5. Our previous studies showed that icariin significantly
increased the cGMP levels of penile and clitoral CC in rabbits [20, 21].
In conclusion, icariin has a selective inhibitory effect on cGMP-specific
PDE5 compared to cAMP-specific PDE4 and may be developed into an oral
effective agent useful for the treatment of ED.
This study was sponsored
by China National Natural Science Foundation and Beijing City Natural
Science Foundation (2001).
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to: Prof. Zhong-Cheng XIN, Department of Urology, the 1st
Hospital, Peking University, 8 Xishiku Street, Xicheng District, Beijing
Tel: +86-10-6612 9625, Fax: +86-10-6612 9625