Aphrodisiac Activity of Ethanol Extract of Cratoxylum sumatranum ( Jack ) Blume Stems on Isolated Rat Corpus cavernosum

Cratoxylum sumatranum (Jack) Blume of the Hypericaceae family is known as “Bentaleng” by Dayak Benuaq. In ethnobotany, Cratoxylum sumatranum stems (CSS) is used as energy drink or aphrodisiac, but its effect has not been scientifically proven. The objective of this research was to evaluate the aphrodisiac activity of CSS extract by screening the aphrodisiac activity in vitro. CSS was collected from Kutai Kertanegara Regency, East Kalimantan Province. Extraction was by maceration with ethanol solvent for three days. Remaceration was done twice. In vitro screening of aphrodisiac activity used isolated rat corpus cavernosum. The organ was placed into a 10 mL chamber containing Krebs-Henselheit solution at pH 7.4, 37°C and aerated with carbogen gas. After acclimation, a contraction test was performed with phenylephrine solution and after reaching the peak of contraction at plateau the Control (solvent extract) or CSS ethanol extract was administered at cumulatively increased concentration. Vasodilation activity was known if the contraction response was decreased after the extract’s administration and expressed in percent contraction with negative value. The result of this study showed that CSS ethanol extract induce vasodilatory response on rat Corpus cavernosum blood vessels. Vasodilation activity was increasing with increasing concentration of extract given compared to Control. This study concluded that CSS ethanol extract has aphrodisiac activity and it act directly through the vasodilatation of blood vessels in the rat corpus cavernosum.


INTRODUCTION
Erectile dysfunction (ED) is a sexual dysfunction disorder on men due to the inability to achieve and maintain an erection of the penis for a satisfactory sexual relationship [1].Prevalence of ED in the world is estimated at about 2% in adult men <40 years old and reaches 86% in older men >80 years [2].There are many options to deal with ED, phosphodiesteration inhibitor under the name sildenafil is most widely used but its raw materials are still imported and the price is expensive so the national pharmaceutical industry is difficult to develop.The global drug market for erectile dysfunction through 2020 is promising because it will reach a market size of 4 billion dollars and sildenafil (Viagra®) dominates the market by 47% in 2014 [3,4].Research for invention of new drug's raw material to overcome ED is urgently needed for the sake of national pharmaceutical needs in order to compete and enlarge its market share.
A potential plant to be studied is

Animal
Young male Wistar rats (4-6 month old) weighing 200-250 g were obtained from the Animal House Unit, Faculty of Medicine, Mulawarman University.The animal had free access to food and drinking water, and were kept at room temperature of 25 + 1°C.Study protocol was reviewed and approved by the institutional ethics committee of Faculty of Medicine Mulawarman University before the start of the work.

Plant material and extraction
C. sumatranum stems was picked from Soeharto Hill, Kutai Kertanegara Reagency of East Kalimantan Province and authenticated by the taxonomist of Foresty Faculty of Mulawarman University.Specimen samples were stored at Pharmacology Laboratory, Faculty of Medicine Mulawarman University repository with specimen number CS01/XI/2016.The stems were washed, cut in small pieces and dried in an oven at 60°C, then ground into powder.Extraction was by maceration using ethanol solvent with a ratio of 1:4 for three days.Remaceration was done twice.The extract was dried in a vacuum evaporator at 50°C until a constant yield of 17.1% on repeated weighing was obtained.

Effects of CSS ethanol extract on corpus cavernosum
Six male albino rats were used in this experiment.The animals were anesthetized with Ketamine (100 mg/kg).The rat's isolated corpus cavernosum was prepared according to the method described by Keegan et al. (1999) and Paskaloglu et al. (2004) [8,9].The penises were removed and the corpus cavernosa was carrefully dissected in a dish containing chilled Krebs-Henselheit solution at pH 7.4 and aerated with carbogen gas (95% O2 + 5% CO2).Each corpus cavernosum yielded a single strip of 2x2x15 mm.Strips of rat corpus cavernosum placed in a 10 mL organ chamber.Tissue baths containing Krebs-Henselheit solution at pH 7.4 and 37 o C, aerated with carbogen gas.The rat corpus cavernosum strips were connected to isometric force-displacement transducers (model 7003, Ugo Basile), and changes in the tension were recorded continuously by using an octal bridge amplifier and Power Lab/16 SP digital recorder (AD Instrument).Data acquisition and processing were carried out with Chart v.5 software (AD Instrument).Tissues were preloaded with 1 g of tension and allowed to equilibrate for 90 min in Krebs-Henselheit solution that was changed at 15 min interval.Each strip was submaximally contracted with PE (10 -5 M).After the contractile response due to PE has stabilized, relaxation responses to the different treatment were assessed as a percentage of the PE-induced contractile responses = ((submaximal contraction with PE only -contraction in the presence of extract) / submaximal contraction with PE only) x 100.

Data analysis
The results obtained were expressed as mean + standard error of mean (SEM), data were analyzed statistically using student's t-test at significance level of p<0.05.

Result and Discussion
The addition of PE (10 -5 M) to the strip of rat corpus cavernosum produced contraction responses.The extracts solvent containing DMSO-ethanol 10% was used as Controls.On the other hand, the C. sumatranum stems extract elicited a concentration-dependent vasodilation responses on the PE-precontracted rat corpus cavernosum (Fig 1).As the concentration of the extract put into the chamber increased, the vasodilation activity of the corpus cavernosum was also increased.
The test results of the effects of Control and C. sumatranum ethanol extract on strip of rat corpus cavernosum can be seen in the Figure 1.The extract solvent could induce percent contraction with a small negative value.Increasing concentration of the solvent that were put into the chamber containing strip of rat corpus cavernosum could cause percent contraction with increasing negative value and at Control concentration which was equivalent to extract concentration of 3 mg/ml, a (-1,82 ± 0.53)% response was obtained.This indicated that the extract solvent could cause mild vasodilation on blood vessels in the corpus cavernosum.
C. sumatranum stems ethanol extracts could cause percent contraction with a mild negative value.Increasing concentration of extracts administered into the chamber containing the strip of rat corpus cavernosum could cause percent contraction with an increasingly negative value and at extract's concentration of 3 mg/ml a response of (-7.85 ± 0.66)% was obtained.This indicated that the extract of C. sumatranum stems could cause vasodilation on blood vessels in the corpus cavernosum.
The result of statistical test by ttest with p>0.05 at concentrations of 0.03, 0.1, and 0.3 mg/ml showed no significant difference between the control group and the C. sumatranum stems extract group at the same concentration.The result of statistical test by t-test with p<0.05 at concentration of 1 and 3 mg/ml showed significant different result between the control group and the C. sumatranum stems extract group at the same concentration.
Log (mg/ml) - Erection on penis occurs due to the helicin arterioles vasodilatation in the corpus cavernosum such that the arterial blood flows faster to fill the lacunar spaces on the corpus cavernosum causing the penis to become tense.When the helicin arterioles in the corpus cavernosum contracts, it causes the arterial blood flow into the lacunar space to be inhibited and so the penis becomes flaccid.The balance between factors affecting vasodilation or vasoconstriction in the lacunar cavities and arteriole smooth muscles in the corpus cavernosum will determine if the penis will be in an erect or flaccid state [10].Nitric oxide (NO) plays an important role in the erection response, it was released from the vascular endotehelium or from the nonadrenergic non-cholinergic neurons (NANC) from the central or peripheral nervous system [11,12].Nitric oxide synthase (NOS) is an enzyme that converts L-arginine to NO and Lcitrulline.NOS is present in the endothelium (eNOS), neuronal tissue (nNOS), and epithelial tissue within the pelvic and urogenital structures of males * * [13,14].eNOS activation by an agonist in the vascular endothelium will produce NO which can diffuse easily into the blood vessels' smooth muscle in the corpus cavernosum.NO activates soluble guanylate cyclases (sGS) such as those present in the blood vessels which induce guanosine triphosphate (GTP) conversion to cyclic guanosine monophosphate (cGMP).cGMP stimulates calcium ions out from the blood vessels smooth muscle in the corpus cavernosum, resulting in blood vessels vasodilation that causes penis erection.
NO can also be released if there is sexual stimulation or the influence of drugs.Decreased calcium ions in the blood vessels' smooth muscle in the corpus cavernosum can also be caused by inhibition of the phosphodiesterase (PDE) enzyme.This enzyme converts cyclic adenosine monophosphate (cAMP) and cGMP to AMP or GMP.Inhibition of PDE enzyme will cause cGMP levels to remain high such that the blood vessels in the corpus cavernosum remain in a state of relaxation that results in more blood coming into the corpus cavernosum and the penis will remain erect or tense [15].
This study was still in its early stages to scientifically prove the local ethnic knowledge of local aphrodisiacs.In this study CSS extracts was proved to be aphrodisiacs with direct action mechanisms of causing blood vessels vasodilation in the corpus cavernosum.It is necessary to further investigate the action mechanism of blood vessels' vasodilation in the Corpus cavernosum on CSS extract's intervention whether it is affected by NO or directly activate sGC and inhibit PDE by administration of NO antagonists or methylene blue as sGC antagonists or PDE antagonists.

CONCLUSION
The ethanol extract of C. sumatranum stems was proven in vitro to have an aphrodisiac effect as it could induce vasodilation in the isolated rats corpus cavernosum.

ECSS Figure 1 :
Figure 1: Concentration dose curve of CSS extract effect compared to control on an isolated corpus cavernosum Description: n=5 rats.Data is expressed in mean ± SEM.Negative results indicates vasodilation response.C = Control (DMSO-ethanol 10%).ECSS = extract of Cratoxylum sumatranum stems.*t-test was significantly different with p<0.05 if percent contraction in corpus cavernosum was compared with control at same concentration.