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Effects of icariin on cGMP-specific PDE5 and cAMP-specific PDE4 activities

ZC Xin1, EK Kim2, CS Lin3, WJ Liu1, L Tian1, YM Yuan1, J Fu1

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, UCSF, USA

Asian J Androl  2003 Mar; 5: 15-18             

Keywords: 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 ED.

1 Introduction

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].

Icariin (C33H40O15, 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 [19]. 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 [20].

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 with [3H]-cGMP/[3H]-cAMP.

2 Materials and methods

2.1 Reagents

Papaverine (Qinghai 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 %) [19] as verified by HPLC.

2.3 Isolation of PDE5 and PDE4

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 until use.

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 counter.

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 Results

3.1 Effect on cGMP-specific PDE5 activity

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).

Figure 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 PDE4 activity

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).

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 inhibition

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).

Figure 3. Inhibitory effects of icariin (167.67 times) and papaverine (4.54 times) on PDE5 (PDE4/PDE5 of IC50).

4 Discussion

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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Correspondence to: Prof. Zhong-Cheng XIN, Department of Urology, the 1st Hospital, Peking University, 8 Xishiku Street, Xicheng District, Beijing 100034, China.
Tel: +86-10-6612 9625, Fax: +86-10-6612 9625
E-mail: xinzc@bjmu.edu.cn
Received 2002-11-30      Accepted 2002-12-05