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

Abstract

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 μ g/ml) showed a significant inhibition for ROCK-II activity (62.8-81%) at P < 0.001 with an IC 50 (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 IC 50 (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 , 6 α - 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 μ g/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 μ g/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.

Full text

Research Article
Rho-Kinase II Inhibitory Potential of Eurycoma longifolia New
Isolate for the Management of Erectile Dysfunction
Shahira M. Ezzat ,1,2 Mona M. Okba ,1Marwa I. Ezzat,1
Nora M. Aborehab,3andShanazO.Mohamed
4
1Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Ainy Street, Cairo 11562, Egypt
2Pharmacognosy Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA),
6𝑡ℎ October 12566, Egypt
3Biochemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 6th October 12566, Egypt
4School of Pharmaceutical Sciences, Universiti Sains Malaysia, Malaysia
Correspondence should be addressed to Mona M. Okba; mona.morad@pharma.cu.edu.eg
Received 31 January 2019; Revised 18 March 2019; Accepted 21 April 2019; Published 15 May 2019
Guest Editor: Arielle Cristina Arena
Copyright ©  Shahira M. Ezzat et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Eurycoma longifolia Jack (Fam.: Simaroubaceae), known as Tongkat Ali (TA), has been known as a symbol of virility
and sexual power. e aim of the study was to screen E. longifolia aqueous extract (AE) and isolates for ROCK-II inhibition.
Results. e AE (- 𝜇g/ml) showed a signicant inhibition for ROCK-II activity (.-%) at P<. with an IC50 (.
±. ng/ml) compared to Y- ([(+)-(R)-trans--(-aminoethyl)-N-(-pyridyl)cyclohexanecarboxamide dihydrochloride])
(.-. %) at same concentrations with an IC50 ( ±. ng/ml). Chromatographic purication of the aqueous extract (AE)
allowed the isolation of eight compounds; stigmasterol T1,trans-coniferyl aldehyde T2, scopoletin T3,eurycomalactoneT4,𝛼-
hydroxyeurycomalactone T5, eurycomanone T6,eurycomanolT7, and eurycomanol--O-𝛽-D-glucopyranoside T8. is is the
rst report for the isolation of T1 and T3 from E. longifolia and for the isolation of T2 from genus Eurycoma.eisolates(at
𝜇g/ml) exhibited maximum inhibition % of ROCK-II . ±. (T), . ±. (T), . ±. (T), . ±. (T), . ±
. (T), . ±. (T), . ±. (T), and . ±. (T), 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. e standard
Y-  ( 𝜇g/ml) showed . ±. % inhibition for ROCK-II activity when compared to control at P<.. Conclusion. e
traditional use ofE. longifolia as aphrodisiac and for male sexual disorders might be in part due to the ROCK-II inhibitory potential.
1. Introduction
Libido refers to a uctuating state of sexual desire [].
e st century has seen the evolution of a lot of rms
andclinicsthatclaimtotreatreducedlibidoinmales[].
Studies have reported a prevalence of the Hypoactive Sexual
Desire Disorder (HSDD) in men between  and % [].
It is estimated that -% of people around the world
experience lack of sexual interest for at least several months
in any given year []. Nowadays, sexual desire is controlled
by some external factors including psychiatric disorders as
depression, some types of medications including antide-
pressants, 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 []. Alteration in libido also may be due
to some biochemical messengers, such as levels of serum
steroid hormone (mainly testosterone), feedback aer sexual
stimulation, and disturbances in the brain neurotransmitters
[]. Till this moment, the only available medicines indicated
to increase male libido are some herbal drugs and hormonal
therapy in cases of testosterone deciency [].
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
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2019, Article ID 4341592, 8 pages
https://doi.org/10.1155/2019/4341592

 Evidence-Based Complementary and Alternative Medicine
are twisted and long. e plant (particularly roots) has been
traditionally used for reducing fever and fatigue and for its
uniqueantimalarial,antipyretic,antiulcer,anditsaphrodisiac
properties. Body builders have been recently focusing on
regular intake of its root extracts to improve muscular mass
and strength [–].
A large number of phytochemicals have been detected
and identied from E. longifolia roots including eury-
comanone, eurycomaoside, eurycolactone, eurycomalac-
tone, canthin--one alkaloids, quassinoid diterpenoids, 𝛽-
carboline alkaloids, tirucallane-type triterpenes, biphenylne-
olignans, laurycolactone, and squalene derivatives [, ]. E.
longifolia has gained wide appreciation for its uniqueness
in enhancing sexual power which was supported by some
literature in experimental animals [–]. It has been utilized
by Malaysian men for hundreds of years to enhance the
quality and performance of sexual exercise [, ].
Around the world, there has been a gigantic increment
in the utilization of this plant. ere are about two hundred
Tongkat Ali products, mostly focusing on the sexual enhanc-
ing properties. It is available either as crude root powder,
in capsules blended with dierent aphrodisiac drugs, as an
added substance blended with ginseng or coee, or in other
healthcare products as a substitute for ginseng [].
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 [].
RhoA and ROCK are found in dierent 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 eects are obvious in
penile erection through modulation of cavernous sinusoidal
and arteriolar smooth-muscle cells contractile state [].
Although Tongkat Ali traditional use as an aphrodisiac
herb is well-recognized, there is no sucient information on
the possible underlying mechanisms. erefore, this study
was designed to evaluate E. longifolia AE and isolated biophy-
tochemicals potential in management of erectile dysfunction
(ED).
2. Materials and Methods
2.1. Plant Material. e roots of Eurycoma longifolia Jack
were obtained from HCA products Sdn Bhd. Spring . e
plant was kindly identied in the Forest Research Institute,
Malaysia. A voucher specimen (--) was kept in
the herbarium of Pharmacognosy Department, Faculty of
Pharmacy, Cairo University, Cairo, Egypt.
2.2. Preparation of the Aqueous Extract (AE). e collected
roots were washed with running water and then dried on an
open surface and dried by exposure to sunlight for  or  days
to ensure freedom of humidity. e dried roots were then
chippedtommparticles.edriedchippedroots(kg)
were boiled with  liters of RO water (water puried with
reverse osmosis) for  hours; the extract was concentrated in
a rotary evaporator for  hours at ∘C to  liters. e extract
was then dried in a spray dryer by heating for h and  min
at a temperature of ∘C and yielded . kg powdered extract
where the extract yield is %.
2.3. Rock-II Inhibition Assay. e assay was done as
mentioned in ADP-GloKinase Assay (SER-THR KINASE
SERIES: ROCK Kinase assay) (Promega, USA) and Y-
 [(+)-(R)-trans--(-aminoethyl)-N-(-pyridyl)cyclo-
hexanecarboxamide dihydrochloride] was used as standard
drug; luminescence was recorded using Topotecan, USA,
SparkM,multimodemicroplatereader.Avehiclecontrol
for%DMSOwasusedintheassaytochecktheinterference.
Standard curve for ROCK-II enzyme was done (Figure ).
Serial dilution and IC50 of the AE was performed in triplicate.
2.4. Fractionation of the AE and Isolation of
Its Major Phytochemicals
2.4.1. General. Silica gel  ( -  mesh ASTM; Fluka,
Steinheim, Germany), Diaion HP- AG, Sephadex LH-
 (Pharmacia Fine Chemicals AB, Uppsala, Sweden), and
reversed phase silica gel (RP-) (- mesh) for column
chromatography (- 𝜇m, Mitsubishi Chemical Indus-
tries Co. Ltd). in-layer chromatography (TLC) (silica
gel GF254 precoated plates- Fluka) was done using this
solvent systems: Sa:n-Hexane: ethyl acetate (: v/v); Sb;
ethyl acetate-methanol-water-formic acid (:.:.: v/v).
Chromatograms detections were performed under UV light
(at  and  nm) and sprayed by p-anisaldehyde sulphuric
acid spray reagent. Bruker NMR was used for 13C-NMR (
MHz) and 1H-NMR ( MHz). e NMR spectra were
observed in DMSO and CD3OD.Chemicalshisaregiven
in 𝛿(ppm) relative to internal standard TMS.
2.4.2. Isolation of the Major Phytochemicals. For isolation of
the major compounds,  grams of AE were suspended in
 ml distilled water then defatted with methylene chloride
( mLx ). e organic and aqueous layers were separated.
e organic layer was evaporated using rotary evaporator
under reduced pressure at ∘Ctoyieldgmofmethylene
chloride residue (MeCl). e aqueous layer was kept for
further fractionation.
MeCl ( g) was fractionated over a silica gel column
( g). Gradient elution was done using n-hexane-methylene
chloride then methylene chloride-methanol mixtures. e
polarity was increased by  % incriminations of methylene
chloride in n-hexane every   ml till % methylene chloride
then further % incriminations of methanol in methylene
chloride till % methanol. Fractions ( ml) were collected
to obtain  fractions which were then monitored by TLC
using solvent system (S1). Subfraction (% methylene
chloride in n-hexane) was washed with methanol to yield
pure compound T1 (white crystals,  mg). Subfraction
(%methylenechlorideinn-hexane) was chromatographed
over a silica gel column. e elution carried out using
n-hexane-ethyl acetate (: v/v). Similar fractions were
pooled together to yield compound T2 (white crystals, 
mg). Fraction (% methanol in methylene chloride) was
chromatographed over a sephadex LH using methanol-
water (: v/v) as eluent to yield one compound T3 (yellowish

Evidence-Based Complementary and Alternative Medicine 
white crystals,  mg). Fraction (% methanol in methylene
chloride) was chromatographed over a sephadex LH using
n-butanol-isopropanol-water (:: v/v) as eluent to yield a
fraction containing two major spots with minor impurities.
is fraction was further puried by rechromatography over
silica gel column. It was gradient eluted using n-hexane-ethyl
acetate (-%) mixtures to yield two pure compounds T4
(white crystals,  mg) and T5 (white crystals,  mg).
e defatted aqueous solution was chromatographed on
diaion HP- AG ( g) column. Elution was carried out
with water, followed by methanol-water (%), methanol-
water (%), and methanol (%) to give four fractions (D-
D), respectively. e solvent in each case was evaporated
using rotary evaporator to yield solid residues weighing
, , and  g, respectively. Methanol-water (%) (D)
fraction (g) was further fractionated over a silica gel
( g) column where elution was carried out by n-hexane:
ethyl acetate. Gradient elution was carried out by n-hexane-
ethyl acetate and ethyl acetate-methanol-water mixtures. e
polarity was increased by  % incriminations of ethyl acetate
every  ml till % ethyl acetate then further incrimination
of methanol (till .%) and water (till .%). Fractions (
ml, each) were combined to give  fractions which were
monitored by TLC using solvent systems (Sb). Subfraction
(% ethyl acetate in n-hexane) was fractionated over a silica
(RP) column. e elution carried out using water-methanol
as eluent. e fractions eluted with % and % methanol
give compounds T6 (white powder,  mg) and T7 (white
powder,  mg), respectively. Subfraction (.% methanol,
.% water in ethyl acetate) was chromatographed over a
sephadex column eluted with % methanol then silica gel
column eluted with ethyl acetate-methanol (: v/v) to give
compound T8 (white crystals,  mg).
2.5. Rock-II Inhibition Assay. e assay was repeated as
mentioned in Section . on the AE fractions and the isolates
T-T.
e assay performance measure was used to validate
the screening assay quality through calculation of Z-factor
according to methodology of Zhang et al.,  [].
2.6. Statistical Analysis. Enzyme inhibition by tested samples
is expressed as mean ±SD and analyzed using Prism program
version  (GraphPad Soware, Inc., San Diego CA); com-
parisons among tested samples were carried out using one-
way analysis of variance (ANOVA) followed by Bonferroni’s
multiple comparisons test. P*. was considered signicant.
3. Results
3.1. Evaluation of AE Rock-II Inhibition Potential. Concen-
trations at (- 𝜇g/ml)oftheAEandY-asastandard
showed a signicant inhibition for ROCK-II activity (.-
%). e inhibition of ROCK-II activity at P<. was
recorded in Table . IC50 in ROCK-II inhibition assay of AE
(. ±. ng/ml) and Y- were recorded in Table .
3.2. Fractionation of AE and Isolation of the Major Phy-
tochemicals. Chromatographic fractionation of E. longifolia
roots AE allowed the isolation of one sterol: stigmasterol,
T1;aphenoliccompound:trans-coniferyl aldehyde T2;
one coumarin: scopoletin T3; and  known quassinoids
namely eurycomalactone T4,𝛼-hydroxyeurycomalactone
T5, eurycomanone T6,eurycomanolT7, and eurycomanol-
-O-𝛽-D-glycopyranoside T8.eisolatedcompoundswere
identied via their co-TLC comparison to authentic reference
samples, physicochemical characters, and spectroscopic anal-
ysis and through comparing their D and D NMR data with
the previously published data. 1HNMRand13CNMR data
of the isolated phytochemicals are presented in Tables S and
S in the supplementary le. e structures of the isolated
phytochemicals are shown in Figure .
3.3. Evaluation of Rock-II Inhibition Potential of AE Fractions
and Isolates. All tested samples and Y- standard at
concentration range (.- 𝜇g/ml) showed a signicant
inhibition for ROCK-II activity.
At dose  𝜇g/ml,MeCl,D,D,D,Dshoweda
maximum inhibition % of (. ±.), (. ±.), (.
±.), (. ±.), (. ±.), respectively.
e isolates (at  𝜇g/ml) exhibited maximum inhibition
%of.±. (T), . ±. (T), . ±. (T),
. ±. (T), . ±. (T), . ±. (T), .
±. (T), and . ±. (T). e standard Y-
( 𝜇g/ml) showed (. ±.) inhibition % for ROCK-
II activity when compared to vehicle control at P<..
Nonsignicant dierence was found between MeCl, D, D,
D, D at concentration  𝜇g/ml compared to Y- at
the same concentration against the inhibition of ROCK-II
activity at P<. as presented in Table .
IC50 of ROCK-II inhibition assay of all tested AE frac-
tions, isolates, and Y- were recorded in Table .
Nonsignicant dierence was found between MeCl, D,
D, D and D with IC (. ±.,  ±.,  ±
., . ±., and . ±., respectively) compared to Y-
 IC50 ( ±.); these fractions showed a prominent
eect as the same eect as Y- in ROCK-II inhibition.
e assay performance measure was evaluated by calcu-
lation of Z-factor which was equal to . which indicated
that it is an excellent assay [].
4. 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 [, ]. Although
traditional use of E. longifolia asanaphrodisiacherbiswell-
recognized, there is a paucity of information on the possible
underlying mechanisms. erefore, the present study aimed
at substantiating the aphrodisiac activity of E. longifolia.
ROCK-II inhibition assay was performed using ADP-
GloKinase Assay and Y- 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.

 Evidence-Based Complementary and Alternative Medicine
T : Eect of aqueous extract (AE), fractions and isolates on percentage inhibition of ROCK-II activity.
Treatment (s) Concentrations (𝜇g/ml) % of inhibition of ROCK-II ±SD
Control -0
Vehi c l e c o n t r o l % . ±.
AE   ±.∗
AE .±.∗
AE . . ±.∗
AE . . ±.
MeCl  86.3±.∗
MeCl  . ±.∗
MeCl . . ±.∗
MeCl . . ±.∗
D1  90.1±.∗
D1  . ±.∗
D1 . . ±.∗
D1 . . ±.∗
D2  86.1±.∗
D2  . ±.∗
D2 . . ±.∗
D2 . . ±.∗
D3  90.25±.∗
D3  . ±.∗
D3 . . ±.∗
D3 . . ±.∗
D4  87.05±.∗
D4  . ±.∗
D4 . . ±.∗
D4 . . ±.∗
T1  . ±.∗
T1 .±.∗
T1 . . ±.∗
T1 . . ±.
T2  . ±.∗
T2  . ±.∗
T2 . . ±.∗
T2 . . ±.∗
T3  . ±.∗
T3  . ±.∗
T3 .  ±.∗
T3 . . ±.∗
T4  . ±.∗
T4 ±.∗
T4 . . ±.∗
T4 . . ±.∗
T5  . ±.∗
T5 .±.∗
T5 . . ±.∗
T5 . . ±.∗
T6  . ±.∗
T6 .±.∗
T6 . . ±.∗
T6 . . ±.
T7  . ±.∗
T7 .±.   ∗

Evidence-Based Complementary and Alternative Medicine 
T  : C ontinu e d .
Treatment (s) Concentrations (𝜇g/ml) % of inhibition of ROCK-II ±SD
T7 . . ±.∗
T7 . . ±.
T8  . ±.∗
T8  . ±.∗
T8 .  ±.∗
T8 . . ±.
Y-2 76 32  89.9±.∗
Y-2 76 32  . ±.∗
Y-2 76 32 . . ±.∗
Y-2 76 32 . . ±.∗
AE: aqueous extract; D1:water(%); D2: methanol-water (%); D3:methanol-water(%); D4:methanol(%)diaionfractions;MeCl: methylene
chloride fraction; T1: stigmasterol; T2: trans-coniferyl aldehyde; T3: scopoletin; T4: eurycomalactone; T6:𝛼- hydroxyeurycomalactone; T6: eurycomanone;
T7:eurycomanol;T8: and eurycomanol--O-𝛽-D-glucopyranoside.
∗Signicant from Vehicle control at P<.
T  : I C 50 of aqueous extract (AE) of E. longifolia root, its
fractions, and its isolates expressed as mean ±SD. Assay was
performed in triplicates
Sample IC50 (ng/ml)
AE . ±.∗
MeCl . ±.∗
D  ±.∗
D  ±.
D . ±.∗
D . ±.
T  ±∗
T  ±.∗
T  ±.∗
T  ±.∗
T  ±∗
T  ±.∗
T  ±.∗
T  ±∗
Y-    ±.
IC values are mean ±SD. Statistical analysis was carried out by one-way
ANOVA followed by Bonferroni post-hoc test. n=
∗Signicantly dierent from Y- at P<.
 Signicant dierent from Y- at P<.
AE: aqueous extract; D1:water(%); D2: methanol-water (%); D3:
methanol-water (%); D4:methanol(%)diaionfractions;MeCl:
methylene chloride fraction; T1: stigmasterol; T2: trans-coniferyl aldehyde;
T3: scopoletin; T4: eurycomalactone; T6:𝛼- hydroxyeurycomalactone;
T6: eurycomanone; T7:eurycomanol;T8: and eurycomanol--O-𝛽-D-
glucopyranoside.
Smooth-muscle contraction is regulated by the cytoso-
lic Ca2+ concentration and by the calcium sensitivity of
myolaments. e major mechanism of Ca2+ sensitization
of smooth-muscle contraction is achieved by the inhibition
of the myosin light chain phosphatase (MLCP) that dephos-
phorylatestheMyosinlightchaininsmoothmusclethrough
RhoA/Rho-kinasepathway.eactive,GTPboundformof
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 Ca2+ sensitivity.
MLCP converts the active phosphorylated myosin light chain
(MLC) to inactive one so relaxation of the muscle occurs [].
AE purication led to the isolation of eight compounds.
Compound T2 was isolated as needle crystals. Its 1HNMR
spectrum showed three aromatic protons arranged in ABX
system which was characterized by three doublets at 𝛿H.
(H, d, J=. Hz), . (H,d, J=. Hz), and . (H, dd,
J=.,. Hz) assigned to H-, H-, and H-. In addition two
trans-olenic protons appeared at 𝛿H.   (  H , d , J=. Hz,
H-) and . (H, dd, J=.,. Hz, H-) and an aldehydic
group which appeared as a doublet at 𝛿H. (H, d, J=.
Hz,H-)andnallyamethoxygroupat𝛿H. as a singlet.
e coupling constants J7,8 and J8,9 indicated that Δ7,8 is trans
and that CHO is linked to H-; this was conrmed from
HMBC correlations between . (H, d, J=. Hz, H-) and
C- at 𝛿C. and CHO at 𝛿C.andalsothecorrelations
of 𝛿H. (H, d, J=. Hz, H-) with C- at 𝛿C. and
C- at 𝛿C.. e position of OCH3at C- was deduced
from long-range coupling between 𝛿H. and C- at 𝛿C
.. e assignments of carbons were deduced from 1H-
13C correlations in HSQC. is compound was identied as
trans-coniferyl aldehyde [], which is isolated here for the
rst time from genus Eurycoma.
CompoundsT,T-Tspectraldatawereinagree-
ment with the reported data of stigmasterol [], scopoletin
[], eurycomalactone, 𝛼-hydroxyeurycomalactone[],
eurycomanone [], eurycomanol [], and eurycomanol--
O-𝛽-D-glycopyranoside []. is is the rst report for the
isolation of T and T from E. longifolia and for the isolation
of T from genus Eurycoma.
Among the dierent doses used for the AE, MeCl,
fractions and isolates, all of them exhibited more than %
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 (T)

 Evidence-Based Complementary and Alternative Medicine
T1: Stigmasterol
H3CO
O
H
T2: Coniferyl aldehyde
H3CO
T3: Scopoletin
O
O
H
R
O
O
T4: Eurycomalactone R=H
T5: 6- hydroxyeurycomalactone R=OH
T6: Eurycomanone T7: Eurycomanol
HO
T8: Eurycomanol-2-O--D-glucopyranoside
HO
HO
OHO O
OH
HO
O
OH
O
OH
OH
OH
O
O
H
HO
H
O
OH
HO
O
OH
OH
OH
H
H
H
O
O
OH
H
HO
OH
O
OH
OH
H
O
O
H
OH
H
HO
H
H
OH
H
O
F : Structure of the isolated compounds (T-).
.% which is isolated from Eurycoma for the rst time. Pre-
vious studies reported the potential antimutagenic, antiox-
idant [], and anti-inammatory properties of coniferyl
aldehyde [], but its eect on erectile dysfunction was not
studied before.
Although the ROCK-II inhibitory potential of E. longi-
folia crude extract was studied once before [], this is
the rst report to evaluate the inhibition activity of E.
longifolia isolates (T-T) on ROCK-II that manage erectile
dysfunction.

Evidence-Based Complementary and Alternative Medicine 
ROCK II y = 20198Ln(x) + 23361
RLU
Log. (RLU)
0
20000
40000
60000
80000
100000
120000
RLU
10 20 30 40 500
ng
F : ROCK-II enzyme standard curve. x-axis represents the concentration from ( ng/ml,  ng/ml,  ng/ml,  ng/ml, . ng/ml, .
ng/ml and . 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 [].
All E. longifolia previously isolated compounds activities on
ROCK-IIthatmanageerectiledysfunctionwerenotreported.
Compoundsisolatedinourstudyexhibitedotheractivi-
ties rather than improvement of sexual behavior; euryco-
malactone and eurycomanol--O-𝛽-D-glycopyranoside anti-
malarial activity [], 𝛼-hydroxyeurycomalactonecytotoxic
activity [], and eurycomanol are the regulators of signaling
pathways involved in proliferation, cell death, and inam-
mation [], except for eurycomanone which was reported
to improve sexual behavior by other mechanisms more than
aecting erectile dysfunction.
Beside the herein reported potent eect in managing the
erectiledysfunction,thepositiveeectofE. 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 identied
in the present work. Eurycomanone was reported to induce
testosterone production [] and was also reported to enhance
testosterone steroidogenesis at the Leydig cells through its
inhibitory eect on the nal step of transformation of testos-
terone to estrogen through aromatase enzyme inhibition
[]. Moreover, high concentration of eurycomanone has
inhibitory eect on phosphodiesterase [].
It is worth mentioning that the IC50 of the MeCl and
the diaion fractions (D–D) is less than that of the isolated
pure compounds (Table ). Hence, these fractions have better
ROCK inhibitory potential than the isolated compounds
(T-). Further studies are highly recommended to verify
if this is due to the synergistic eects of the compounds
in the mentioned fractions or there are much more potent
compoundstobeisolatedfromthesefractions.
5. 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 conrm
our hypothesis, our future work is to study the in vivo
aphrodisiac eect of the plant in animal model.
Data Availability
edatausedtosupportthendingsofthisstudyare
included within the article.
Disclosure
e study was not funded by a third party.
Conflicts of Interest
e authors declare no conicts of interest.
Acknowledgments
is research was made possible as part of the Ministry of
AgricultureMalaysiainitiativeundertheNewKeyEconomic
Areas Entry Point Project on High Value Herbals awarded to
Natural Wellness Biotech (M) Sdn Bhd. e authors express
their gratitude and appreciation for the trust and opportunity
given. Authors extend their gratitude to Cairo University for
their collaboration and excellent teamwork.
Supplementary Materials
1H NMR data of the isolated phytochemicals are presented
in Tables S. 13CNMR data of the isolated phytochemicals are
presented in Tables S. (Supplementary Materials)
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