Rho-Kinase II Inhibitory Potential of Eurycoma longifolia New Isolate for the Management of Erectile Dysfunction

Background. Eurycoma longifolia Jack (Fam.: Simaroubaceae), known as Tongkat Ali (TA), has been known as a symbol of virility and sexual power. The aim of the study was to screen E. longifolia aqueous extract (AE) and isolates for ROCK-II inhibition. Results. The AE (1-10 μg/ml) showed a significant inhibition for ROCK-II activity (62.8-81%) at P < 0.001 with an IC50 (651.1 ± 32.9 ng/ml) compared to Y-27632 ([(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) (68.15-89.9 %) at same concentrations with an IC50 (192 ± 8.37 ng/ml). Chromatographic purification of the aqueous extract (AE) allowed the isolation of eight compounds; stigmasterol T1, trans-coniferyl aldehyde T2, scopoletin T3, eurycomalactone T4, 6α- hydroxyeurycomalactone T5, eurycomanone T6, eurycomanol T7, and eurycomanol-2-O-β-D-glucopyranoside T8. This is the first report for the isolation of T1 and T3 from E. longifolia and for the isolation of T2 from genus Eurycoma. The isolates (at 10 μg/ml) exhibited maximum inhibition % of ROCK-II 82.1 ± 0.63 (T2), 78.3 ± 0.38 (T6), 77.1 ± 0.11 (T3), 76.2 ± 3.53 (T4), 74.5 ± 1.27 (T5), 74.1 ± 2.97 (T7), 71.4 ± 2.54 (T8), and 60.3 ± 0.14 (T1), where the newly isolated compound trans-coniferyl aldehyde T2 showed the highest inhibitory activity among the tested isolated compounds and even higher than the total extract AE. The standard Y-27632 (10 μg/ml) showed 89.9 ± 0.42 % inhibition for ROCK-II activity when compared to control at P < 0.0001. Conclusion. The traditional use of E. longifolia as aphrodisiac and for male sexual disorders might be in part due to the ROCK-II inhibitory potential.


Introduction
Libido refers to a fluctuating state of sexual desire [1]. The 21st century has seen the evolution of a lot of firms and clinics that claim to treat reduced libido in males [2]. Studies have reported a prevalence of the Hypoactive Sexual Desire Disorder (HSDD) in men between 1 and 20% [3]. It is estimated that 30-40% of people around the world experience lack of sexual interest for at least several months in any given year [2]. Nowadays, sexual desire is controlled by some external factors including psychiatric disorders as depression, some types of medications including antidepressants, some diseases as diabetes and hypothyroidism, social and interpersonal problems, and other conditions causing inhibited or decreased dopamine release, leading to sexual dysfunction, general lack of sexual desire, and decreased libido [1]. Alteration in libido also may be due to some biochemical messengers, such as levels of serum steroid hormone (mainly testosterone), feedback after sexual stimulation, and disturbances in the brain neurotransmitters [4]. Till this moment, the only available medicines indicated to increase male libido are some herbal drugs and hormonal therapy in cases of testosterone deficiency [5].
E. longifolia (Tongkat Ali, Genus: Eurycoma; Family: Simaroubaceae) is one of the most well-known tropical plants, indigenous to Southeast Asian countries like Vietnam, Malaysia, and Indonesia. It is known as 'Tongkat Ali' where in Malaysia 'Ali' refers to "walking stick" because this plant roots 2 Evidence-Based Complementary and Alternative Medicine are twisted and long. The plant (particularly roots) has been traditionally used for reducing fever and fatigue and for its unique antimalarial, antipyretic, antiulcer, and its aphrodisiac properties. Body builders have been recently focusing on regular intake of its root extracts to improve muscular mass and strength [6][7][8].
Around the world, there has been a gigantic increment in the utilization of this plant. There are about two hundred Tongkat Ali products, mostly focusing on the sexual enhancing properties. It is available either as crude root powder, in capsules blended with different aphrodisiac drugs, as an added substance blended with ginseng or coffee, or in other healthcare products as a substitute for ginseng [8].
Corpus cavernosum smooth muscle (CCSM) and penile arteries relaxation results in blood trapping in the penis leading to raised intracavernous pressure (ICP) which plays a pivotal role as penile erection [16].
RhoA and ROCK are found in different tissues in the body and responsible for regulating many functions. In spite of their presence in the neural and endothelial tissues of the human corpora, but their prominent effects are obvious in penile erection through modulation of cavernous sinusoidal and arteriolar smooth-muscle cells contractile state [17].
Although Tongkat Ali traditional use as an aphrodisiac herb is well-recognized, there is no sufficient information on the possible underlying mechanisms. Therefore, this study was designed to evaluate E. longifolia AE and isolated biophytochemicals potential in management of erectile dysfunction (ED).

Materials and Methods
. . Plant Material. The roots of Eurycoma longifolia Jack were obtained from HCA products Sdn Bhd. Spring 2015. The plant was kindly identified in the Forest Research Institute, Malaysia. A voucher specimen (5-09-2015) was kept in the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
. . Preparation of the Aqueous Extract (AE). The collected roots were washed with running water and then dried on an open surface and dried by exposure to sunlight for 1 or 2 days to ensure freedom of humidity. The dried roots were then chipped to 5 mm particles. The dried chipped roots (40 kg) were boiled with 200 liters of RO water (water purified with reverse osmosis) for 3 hours; the extract was concentrated in a rotary evaporator for 3 hours at 60 ∘ C to 20 liters. The extract was then dried in a spray dryer by heating for 6h and 30 min at a temperature of 120 ∘ C and yielded 1.6 kg powdered extract where the extract yield is 4%.
. . Rock-II Inhibition Assay. The assay was done as mentioned in ADP-Glo6 Kinase Assay (SER-THR KINASE SERIES: ROCK2 Kinase assay) (Promega, USA) and Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] was used as standard drug; luminescence was recorded using Topotecan, USA, Spark 10 M, multimode microplate reader. A vehicle control for 5% DMSO was used in the assay to check the interference. Standard curve for ROCK-II enzyme was done ( Figure 2). Serial dilution and IC 50 of the AE was performed in triplicate. . . . Isolation of the Major Phytochemicals. For isolation of the major compounds, 500 grams of AE were suspended in 800 ml distilled water then defatted with methylene chloride (300 mLx 3). The organic and aqueous layers were separated. The organic layer was evaporated using rotary evaporator under reduced pressure at 40 ∘ C to yield 8 gm of methylene chloride residue (MeCl). The aqueous layer was kept for further fractionation.
MeCl (8 g) was fractionated over a silica gel column (80 g). Gradient elution was done using n-hexane-methylene chloride then methylene chloride-methanol mixtures. The polarity was increased by 10 % incriminations of methylene chloride in n-hexane every 50 ml till 100% methylene chloride then further 1% incriminations of methanol in methylene chloride till 7% methanol. Fractions (20 ml) were collected to obtain 60 fractions which were then monitored by TLC using solvent system (S 1 ). Subfraction (60% methylene chloride in n-hexane) was washed with methanol to yield pure compound T1 (white crystals, 25 mg). Subfraction (80% methylene chloride in n-hexane) was chromatographed over a silica gel column. The elution carried out using n-hexane-ethyl acetate (85:15 v/v). Similar fractions were pooled together to yield compound T2 (white crystals, 20 mg). Fraction (1% methanol in methylene chloride) was chromatographed over a sephadex LH20 using methanolwater (7:3 v/v) as eluent to yield one compound T3 (yellowish Evidence-Based Complementary and Alternative Medicine 3 white crystals, 34 mg). Fraction (6% methanol in methylene chloride) was chromatographed over a sephadex LH20 using n-butanol-isopropanol-water (4:1:5 v/v) as eluent to yield a fraction containing two major spots with minor impurities. This fraction was further purified by rechromatography over silica gel column. It was gradient eluted using n-hexane-ethyl acetate (10-30%) mixtures to yield two pure compounds T4 (white crystals, 68 mg) and T5 (white crystals, 52 mg).
. . Rock-II Inhibition Assay. The assay was repeated as mentioned in Section 2.3 on the AE fractions and the isolates T1-T8.
The assay performance measure was used to validate the screening assay quality through calculation of Z-factor according to methodology of Zhang et al., 1999 [18].
. . Statistical Analysis. Enzyme inhibition by tested samples is expressed as mean ± SD and analyzed using Prism program version 6 (GraphPad Software, Inc., San Diego CA); comparisons among tested samples were carried out using oneway analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test. P * 0.05 was considered significant.

Results
. . Evaluation of AE Rock-II Inhibition Potential. Concentrations at (1-10 g/ml) of the AE and Y-27632 as a standard showed a significant inhibition for ROCK-II activity (62.8-81%). The inhibition of ROCK-II activity at P < 0.001 was recorded in Table 1. IC 50 in ROCK-II inhibition assay of AE (651.1 ± 32.9 ng/ml) and Y-27632 were recorded in Table 2.
IC 50 of ROCK-II inhibition assay of all tested AE fractions, isolates, and Y-27632 were recorded in Table 2.
The assay performance measure was evaluated by calculation of Z-factor which was equal to 0.802 which indicated that it is an excellent assay [18].

Discussion
E. longifolia roots AE has gained wide recognition for enhancing the virility and sexual prowess. It has been utilized by Malaysian men for hundreds of years to enhance the quality and performance of sexual exercises [6,7]. Although traditional use of E. longifolia as an aphrodisiac herb is wellrecognized, there is a paucity of information on the possible underlying mechanisms. Therefore, the present study aimed at substantiating the aphrodisiac activity of E. longifolia.
ROCK-II inhibition assay was performed using ADP-Glo6 Kinase Assay and Y-27632 was used as standard; this method was preferred more than ELISA technique due to the absence of several washing steps and false results that may happen due to the interference with horseradish peroxidase as the extracts have ant-oxidant activity.    Smooth-muscle contraction is regulated by the cytosolic Ca 2+ concentration and by the calcium sensitivity of myofilaments. The major mechanism of Ca 2+ sensitization of smooth-muscle contraction is achieved by the inhibition of the myosin light chain phosphatase (MLCP) that dephosphorylates the Myosin light chain in smooth muscle through RhoA/Rho-kinase pathway. The active, GTP bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho-kinase (ROCK-II), which phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity leading to contraction of smooth muscle through Ca 2+ sensitivity. MLCP converts the active phosphorylated myosin light chain (MLC) to inactive one so relaxation of the muscle occurs [19].
AE purification led to the isolation of eight compounds. Compound T2 was isolated as needle crystals. Its 1 HNMR spectrum showed three aromatic protons arranged in ABX system which was characterized by three doublets at Hz, H-9) and finally a methoxy group at H 3.82 as a singlet. The coupling constants J , and J , indicated that Δ 7,8 is trans and that CHO is linked to H-8; this was confirmed from HMBC correlations between 7.31 (1H, d, J=15.8 Hz, H-7) and C-8 at C 126.4 and CHO at C 193.6 and also the correlations of H 9.56 (1H, d, J=7.70 Hz, H-9) with C-7 at C 153.1 and C-8 at C 126.4. The position of OCH 3 at C-3 was deduced from long-range coupling between H 3.82 and C-3 at C 146.9. The assignments of carbons were deduced from 1 H-13 C correlations in HSQC. This compound was identified as trans-coniferyl aldehyde [20], which is isolated here for the first time from genus Eurycoma.
Among the different doses used for the AE, MeCl, fractions and isolates, all of them exhibited more than 50% of ROCK-II inhibition at higher dose which indicate the use of this potent herbal drug in the management of erectile dysfunction. It is worth noting that maximum inhibition of ROCK-II was recorded for trans-coniferyl aldehyde (T2)  82.1% which is isolated from Eurycoma for the first time. Previous studies reported the potential antimutagenic, antioxidant [25], and anti-inflammatory properties of coniferyl aldehyde [26], but its effect on erectile dysfunction was not studied before.
Although the ROCK-II inhibitory potential of E. longifolia crude extract was studied once before [16], this is the first report to evaluate the inhibition activity of E. longifolia isolates (T1-T8) on ROCK-II that manage erectile dysfunction. x-axis represents the concentration from (40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml and 0.625 ng/ml) and Y-axis represents ΔRLU.
In a recent review about E. longifolia chemistry and evidence-based pharmacology, many E. longifolia isolated compounds pharmacological activities were reported [6]. All E. longifolia previously isolated compounds activities on ROCK-II that manage erectile dysfunction were not reported. Compounds isolated in our study exhibited other activities rather than improvement of sexual behavior; eurycomalactone and eurycomanol-2-O--D-glycopyranoside antimalarial activity [27], 6 -hydroxyeurycomalactone cytotoxic activity [28], and eurycomanol are the regulators of signaling pathways involved in proliferation, cell death, and inflammation [29], except for eurycomanone which was reported to improve sexual behavior by other mechanisms more than affecting erectile dysfunction.
Beside the herein reported potent effect in managing the erectile dysfunction, the positive effect of E. longifolia in the improvement of sexual behavior may be attributed to its active constituents such as quassinoids and in particular the major one, eurycomanone, which was isolated and identified in the present work. Eurycomanone was reported to induce testosterone production [6] and was also reported to enhance testosterone steroidogenesis at the Leydig cells through its inhibitory effect on the final step of transformation of testosterone to estrogen through aromatase enzyme inhibition [30]. Moreover, high concentration of eurycomanone has inhibitory effect on phosphodiesterase [30].
It is worth mentioning that the IC 50 of the MeCl and the diaion fractions (D1-D4) is less than that of the isolated pure compounds (Table 2). Hence, these fractions have better ROCK inhibitory potential than the isolated compounds (T1-8). Further studies are highly recommended to verify if this is due to the synergistic effects of the compounds in the mentioned fractions or there are much more potent compounds to be isolated from these fractions.

Conclusion
Our research revealed that the traditional use of E. longifolia as aphrodisiac and for male sexual disorders might be partially due to the ROCK-II inhibitory activity. To confirm our hypothesis, our future work is to study the in vivo aphrodisiac effect of the plant in animal model.

Data Availability
The data used to support the findings of this study are included within the article.

Disclosure
The study was not funded by a third party.

Conflicts of Interest
The authors declare no conflicts of interest.