Effect of TD0014 on intracavernous pressure elicited with electrical stimulation of the cavernous nerve in male rats

This study aimed to determine the effects of an herbal formula named TD0014 on the electrostimulationinduced erection in a rat model. Eighteen sixteen-week-old male Wistar rats were used. The intracavernous pressure and the arterial blood pressure were simultaneously monitored during cavernous nerve electrical stimulation before and after the administration of TD0014 (1.8 g/kg).

EFFECT OF TD0014 ON INTRACAVERNOUS PRESSURE ELICITED WITH ELECTRICAL STIMULATION OF THE

CAVERNOUS NERVE IN MALE RATS Mai Phuong Thanh 1 , Pham Thi Van Anh 1 , Nguyen Trong Thong 1 ,

Nguyen Thi Huong Lien 2

1 Department of Farmacology, Hanoi Medical University, 2 Sunstar Joint Stock Company

This study aimed to determine the effects of an herbal formula named TD0014 on the electrostimulation-induced erection in a rat model Eighteen sixteen-week-old male Wistar rats were used The intracavernous pressure and the arterial blood pressure were simultaneously monitored during cavernous nerve electrical stimulation before and after the administration of TD0014 (1.8 g/kg) Statistical analysis was performed on measurements of maximal intracavernous pressure (MIP), mean arterial blood pressure (MAP) and the MIP/MAP The administration of TD0014 resulted in a significant increase in the baseline level of intracavernosal pressure (ICP) before stimulation in comparison to the control group (P < 0.05) The findings also showed that the maximal ICP and the area under the curve (AUC time × ICP curves)

in TD0014 group were greater than distilled water group, however, this difference was not significant TD0014 had no significant effect on the mean arterial pressure These results indicated that the herbal formulation TD0014 may have positive and selective effects on improving erectile functions in male rats.

Keywords: TD0014, cavernous nerve stimulation, intracavernous pressure, rat

I INTRODUCTION

Erectile dysfunction (ED) is a term

recommended by a panel of experts in

1992 to replace the term “impotence” [1]

ED is when a man is unable to get and/or

keep an erection It is not a disease, but

a symptom of some other problem, either

physiological, psychological or a mixture

of both [2] Although physiological ED

itself is not life threatening, it is a strong

predictor of high-mortality diseases such as

cardiovascular disease [3] ED does directly

and negatively impact the quality of life of afflicted men and their spouse [4] Recently developed phosphodiesterase type-5 (PDE-5) inhibitors have been widely used as first-line therapeutics to treat ED [5] PDE5 is

an enzyme found primarily in the smooth muscle of the corpus cavernosum that selectively cleaves and degrades cGMP

to 5′-GMP PDE5 inhibitors are similar in structure to cGMP; they competitively bind

to PDE5 and inhibit cGMP hydrolysis The increased amounts of cGMP enhance the effects of NO a potent vasodicator This increase in cGMP in the smooth muscle cells

is responsible for prolonging an erection [6] Although large, multicenter clinical trials have shown the efficacy and tolerability of

Corresponding author: Mai Phuong Thanh, Department

of Pharmacology, Hanoi Medical University

Email: maiphuongthanh@hmu.edu.vn

Received: 05 June 2017

Accepted: 16 November 2017

these drugs in patients with ED of various

etiologies and a broad range of severity;

however, 30% to 35% of patients fail to

respond The use of PDE - 5 inhibitors may

result in several side effects, including visual

disturbances, headache, facial flushing,

rhinitis, and indigestion Other treatments

for ED include penile injection therapy or

penile implants However, such methods

are invasive and irreversible, and are

therefore not widely used [7] Thus, there

is a continued need for the development of

new noninvasive and effective therapies to

treat patients with ED

Despite the remarkable developments

of modern medicine, many people are

still favorably disposed towards herbal

medicines owing to the aggressive

treatment protocols, toxicity, and drug

tolerance associated with modern therapies

The widespread use of herbal medicines,

however, requires scientific verification of

their effects

TD0014 is a preparation which

comprises of thirty-three medicinal plants

The composition of TD0014 has several

medicinal herbs that have been studied

and used since ancient times in traditional

folk medicine as an aphrodisiac [8 - 15]

However, no studies have shown reliable

evidence of their effect on reproductive

function, or toxicity when taken in TD0014

Therefore, the purpose of this study was to

explore the impact of TD0014 on erectile

capacity in adult male rats

II MATERIALS AND METHODS

1 Materials

Herbal formula TD0014 preparation

TD0014 was manufactured as hard pills according to the quality standard of Sunstar Joint Stock Company, Vietnam The major ingredients of the herbal formula are obtained from thirty-three plants (per 7.5g of serving): Tribulus terrestris (4 g), Chrysanthemum sinense (1.83 g), Prunus persica (1.14 g), Vigna cylindrica (1.14 g), Eurycoma longifolia (0.69 g), Sophora japonica (0.57g), Dioscorea persimilis (0.43 g), Dioscorea tokoro (0.4 g), Polygonum multiflorum (0.4 g), Citrus deliciosa (0.34 g), Polyscias fruticosa (0.34 g), Tinospora sinensis (0.29 g), Chaenomeles lagenaria (0.29 g), Passiflora foetida (0.29 g), Zizyphus sativa (0.29 g), Rehmannia glutinosa (0.23 g), Angelica sinensis (0.23 g), Alisma plantago-aquatica L var orientalis Samuelsson (0.23 g), Achyranthes bidentata (0.23 g), Schizandra chinensis (0.23 g), Morinda offcinalis (0.23 g), Rosa laevigata (0.23 g), Allium sativum (0.2 g), Lycium sinense (0.17 g), Glycyrrhiza uralensis (0.14 g), Panax ginseng (0.11 g), Ligusticum wallichii (0.11 g), Cistanche tubulosa (0.11 g), Atractylodes macrocephala (0.11 g), Radix Codonopsis (0.11 g), Cuscuta sinensis (0.11 g), Psoralea corylifolia (0.06 g), Cornu Cervi parvum (7.2 mg) The experimental animals drank the test drug mixed with pure water

Animal groups

Wistar male rats (250 - 300 g), 16 weeks

of age, were used in our study They were housed in groups of six rats per cage in a 24

- hour air - conditioned room with access to standard certified rodent diet and water ad libitum They were acclimated to housing for

at least 1 week prior to investigation

The rats were randomly separated into 3 groups of 6 animals each: 1) control, 2) sildenafil treatment, and 3) TD0014 treatment In each group, animals were administered per os one-time with either distilled water (10 ml/kg b.w.), or sildenafil (6 mg/kg b.w.), or TD0014 (1.8 g/kg b.w.) 2 hours after treatment, erectile function was evaluated by measuring intracavernosal pressure (ICP) and maximal ICP/mean arterial pressure (MIP/MAP)

2 Methods

Measurement of intracavernosal pressure

Rats were anaesthetized with an intraperitoneal injection of ketamine at a dose of 25 mg/

kg (Rotexmedica, Germany) The penis was dissected and the corpus cavernosum and crus were exposed in a supine position A low-midline incision was made to access the pelvis, and the pelvic ganglion lateral to the right prostate was exposed The penile skin was degloved and the corpus cavernosum was identified To measure ICP, a heparinized 23-gauge butterfly needle was inserted into the proximal portion of the corpus cavernosum A bipolar electrical stimulator was placed on the ganglion to stimulate the cavernosal nerve for 60 seconds at 5 V and 20 Hz for 2 millisecond periods The cavernosal nerve stimulation was conducted 3 times with a 10-minute interval between stimulations Before and after each electrical stimulation, ICP was recorded on a computer by Powerlab system record software ICP data was normalized

by mean systemic arterial pressure (MAP) and the MAP was monitored simultaneously with ICP monitoring The right carotid artery was dissected via a midline cervical incision under

a microscope, and then PE-50 tubing was inserted into the carotid artery The catheter was connected to both a pressure transducer and an amplifier unit which was connected to a data acquisition module MAP was recorded on a computer by Powerlab system record software

Statistical analyses

Data was analyzed employing with the Labchart pro software and Microsoft Excel 2010 All data is presented as mean ± standard deviation Statistical significance was determined

by Student's t-test and p < 0.05 were considered to be significant

III RESULTS

* p < 0.05; ** p < 0.01; *** p < 0.001; compared with control (Student’t-test)

Figure 1 Intracavernous pressure (ICP) before and after electrical stimulation of the

cavernous nerve in rats from each experimental group

**

In sildenafil group, before and after the electrical stimulation of the cavernous nerve, the ICP significantly increased compared to the control group In the flaccid state, TD0014 elevated the ICP level which was statistically different compared to the control animals (p < 0.05) In addition, the ICP after stimulation also increased in TD0014 group, however, it was not statistically significant

**p < 0.01 vs control rats (Student’t-test); ∆∆∆p < 0.001 vs sildenafil rats ((Student’t-test)

Figure 2 Effect of TD0014 on time to the maximal ICP and response time

to the electrical stimulation of the cavernous nerve

Observing in Figure 2, the parameter of time to the maximal ICP was not different amongst treatment groups The treatment with sildenafil increased the response time to electrical stimulation (p < 0.01) The animals treated with TD0014 were not able to extend the response time

Table 1 Total ICP (ICP vs stimulation time, area under curve) in treatment rats and

control rats.

*p < 0.05, response significantly different from control rats

In Table 1, statistical analysis indicated that sildenafil group showed significantly greater total ICP compared with the control group TD0014 group exhibited an increase in total ICP, but it was not statistically significant

The effect of sildenafil and TD0014 on blood pressure is shown in Table 1 After sildenafil administration, MAP decreased by approximately 10 mmHg, but this was not a significant difference to the control (p > 0.05) MIP/MAP increased significantly compared with distilled water group and TD0014 group (p < 0.05 an p < 0.01 respectively) There was no statistical

**

ΔΔΔ

differences in MAP and MIP/MAP values between the TD0014 group and the control group.

Table 2 Effect of TD0014 on maximal ICP/MAP ratio

Group 1 (control group) 106.34 ± 18.08 0.40 ± 0.11

*p < 0.05 as compared with control; ∆∆p < 0.01 as compared with sildenafil

Penile erection is a hemodynamic

process which penile arteries, penis and

penile venous system induced by

neuro-endocrine regulation [16] Change in

ICP induced by penile cavernous nerve

stimulation is often used as an objective

assessment index for the impact of drugs

on erectile function in vivo [17]

Stimulation of penile cavernous nerve

could activate nitric oxide synthase activity

of non-adrenergic and non-cholinergic

nerve endings, promote production and

release of NO No leads increased to cGMP

concentration, which induces the relaxation

of smooth muscle and reults in erection [18]

Sildenafil is a potent and selective inhibitor

of cyclic guanosine monophosphate (cGMP)

specific phosphodiesterase type 5 (PDE5)

which is responsible for the degradation

of cGMP in the corpus cavernosum of the

penis Therefore, increased cGMP within

the corpus cavernosum can lead to smooth

muscle relaxation [19] The results of Fig 1,

Fig 2, and Tab 1 clearly demonstrated the

beneficial effects of sildenafil on erectile

dysfunction, as shown by the ability to

significantly increase ICP before and after

electrostimulation (53% and 25% increase, respectively), prolong response time with stimulation, and increase total ICP (61% increase) compared to the control group

In this study, the administration of TD0014 at 1.8 g/kg/day showed an improvement of ICP level before the start of electrostimulation, which means the herbal preparation has a pro-erectile activity Subsequently TD0014 can improve the ICP value during erection After electrostimulating the cavernosal nerve, compared to the control group, TD0014 tended to have an elevation in the maximal ICP (12% increase), total ICP (25% increase), and a prolonged response time

to the stimulus, i.e prolong the duration of penile tumescence, however, these results showed no signitificant differences (p > 0.05) Thus, in the current study, TD0014 given once before nerve stimulation markedly increased baseline ICP, slightly increased both the MIP and the response time to the stimulus These results suggested that TD0014 may be beneficial for patients with erectile dysfunction, and can improving the patient's sexual life

IV DISCUSSION

TD0014 preparation contains several

medicinal plants that have been studied for

the effects on sexual functions, including

erectile capacity, which can partly explain

the impacts of this herbal formula on the

enhancement of penile erection Results

published by Do J et al (2013) indicated

that Tribulus terrestris extract caused

relaxation of the cavernous smooth muscle

in a concentration-dependent manner,

and the mechanism included a reaction

involving the NO/NOS pathway in the

corpus cavernosum endothelium [8]

Echinacoside, a phenylethanoid glycoside

isolated from the medicinal herb Cistanche

tubulosa, had an effect on nitric oxide

(NO)-cGMP signaling pathway and resulted in

an increased cGMP in corpus cavernosum

smooth muscle [9] The neurotransmitter

inducing penile erection, NO release was

shown to be enhanced by ginsenosides,

saponins from Panax ginseng in rabbit’s

corpus cavernosum in vitro Ginsenosides

enhanced both acetylcholine-induced and

transmural nerve stimulation-activated

relaxation associated with increased tissue

cGMP [10] Some other medicinal plants in

TD0014 were proved to cause an increase

in serum testosterone level [11 - 15],

therefore ameliorated ED by reducing ROS

production and increasing the activity of the

eNOS/cGMP [20]

The MIP/MAP indicates the extent of

relaxation of corpus cavernosum and deep

arteries in the penis, while MAP represents

the capacity of pumping blood into the

penis Generally, drugs with an effect of

increasing MAP will decrease the MIP/MAP

because of an associated contraction of corpus cavernosum and deep arteries in the penis On the contrary, drugs with an effect of decreasing MAP will increase the MIP/MAP The optimal charge in MIP should

be determined by a balanced change in the MIP/MAP and MAP The present study in rats showed that sildenafil administration slightly decreased MAP by approximately

10 mmHg, and subsequently increased MIP/MAP (Table 2) Therefore, in clinical practice, sildenafil should be prescribed with caution in patients with cardiovascular risk factors [19] This current study results show that TD0014 could increase ICP without significantly impacting on MAP The data suggests that TD0014 could selectively enhance erectile function without effecting systemic blood pressure Therefore it could limit the side effects on blood pressure and cardiovascular diseases, which are common undesirable effects of drugs currently used

to treat ED

V CONCLUSION

On the basis of our results, we conclude that the herbal formulation TD0014 may have a positive and selective effect on improving the erectile functions in male rats without impacting systemic blood pressure Treatment with TD0014 may significantly increase the baseline ICP, improve maximal ICP and the duration of penile tumescence

Acknowledgements

This work was supported by the Department of Physiology, Military Medical University

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