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In Vitro and In-Silico: Selectivities of Seychellene
Compound as Candidate Cycloo)rygenase
Isoenzyme Inhibitor or1. . .
Articte
Current Enzyme lnhibition
.
December 2015
DOI: 10.2U4l1573408012666151202211035
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I
RESEARCH ARTICLE
In
Wtro and In silicoz Selectivities of Seychellene Compound as Candidate
cyclooxygenase Isoenzyme Inhibitor on Pre-osteoblast cells
J. Raharjol2,.chanif
!*tot
Fatchiyah
Mahdi3, Nurdiana Nurdianaa, Takheshi Kikuchis and
Fatchiyah6'
lBiotW
Docto.ral Program, Faculty of Science, Brawijaya University, Jawa Timttr, Indonesia; 2Academic of pharmacy
and Food Analysis, Pttra Indo,nesia Malang, Indonesia: 3Chemistry bepartment, Faculty of Science, Brawii:aya lJniveisity. Jawa riyun l\d9n91ia; lMedicine Pharmacologlt. Faculty oj'Meltine, Brawijaya lJniversity, Jawa Timur. Indones,ia.: "Graduate Life Science School, Ritsumeikan (lniversity. Biwaka Kufriasu-CLmpus, Sttiga fre.lecture, Japan;
"Biolag Department. Faculty of science, Brawijaya {Iniversity, Jawa Timur, Indonesia
Abstrtct: objectiva To determine seleaivity's single urd clusGr of the
(CID519743) compound such as cycloorygenase (COX-I
vitrro and.In si/ico analysis.
t
seychellene
COX-2) inhibitor by in-
r
Merho*z Fraction of seychellene obtained fractional-vacuum distillation patchouli
oil by Pilodist-IO4. The fraction was determined through IC50 COX-I/ COX-2 value
by colorimetric test coXovine 7607ffi and ihe effect decreases lhe expression of
cox-l/ coX-2
ARTIC:LE IIISTORY
R@[cd: S€ptmbcr 26,
2015
REYEcd: NoY@bcr 26, 201 5
A@Frd: ,sEry 2t,2016
DOI:
I A2 I 71/ I 573.t080t26661 5 I 2022 I
tts5
on pre+steoblast cell to induce LpS. Molecular interaction shrdies
single/ cluster of seychellene and other compounds with C.OX-I and COX-2 used
the molecular docking tools by Hex 8.0 and the inleractions were further visualized
and the binding energr was calculated (with Generalized Born Molecular volume
(GBMD model solvent) usingDiscovery Studio Client 3.5 software.
Fatchiyah Fatchiyah
Resn& Fraction of seychellene obtained from patchouli oil in fraction-5 es.osyo). The IC56 values of
fraction-S were: COX-I (73.47 u. M) and COX-2 (73.3 I p M). The fraction of seychellene decreased the
expression of COX-I/ COX-2 isoenzym. ln silico analyses of scoring binding energy calculation (with
GBMV model solvent) of single and cluster seychellene compourds alFected the s€lectivity of COX-I
and COX-2 inhibitor.
Condaston: Collectivety, selectiviry's
of the
seychellene compound as non-seleaive inhibitor
cycloorygenase.
Keywords: ICso,in sl/ico analyses of scoring binding energy, inhibitor selective, selectivity, single/ cluster seychellene
com-
pounds.
T.INTRODUCTION
Patomechanism osteoporosis is a disorder of bone furnover involving cell function to achieve coordination and balance resorption of old bone (osteoclasts) and the formation
of new bone (osteoblasts) to the local conditions. coordinated and sequentially as the coupling process. Bone reorganization were initiated by the recruitnent and activation of
osteoclasts. Osteoblast cells as bone-forming also play a role
in producing Prost4glandin-E} (PGEZI, mainly related to
inflammation and act as a stimulator of bons resorption [t].
Furthermore, Osteoclastogenesis leads to osteoblasts to secr€te Receptor Activated Nuclear Kappa Ligand (RANKL),
which responded by osteoclasts with the formation of R*eptor
rAddrcss correspondcnce to this
aulhor at thc Biology Deportncnt, Facutty
of Science. Bra*ijaya Univcrsity. . Jawa Timur, Indonesia:
Tcl: +62 341 575841; Fax: +62341 5758/l.,Bmail: farchivarOub.ac.id
l573408{i.ll7
Ss8.tx)+.m
Activated Nuclear Kappa (RANK), due to the interaction of
RANKL and RANK, which turns into then mature osteoclasts, so that it can function in the process of bone resorption. When bone resorption is considered suffrcient, then
osboblasts secrete Osteoprotegerin (OPG) for binding
RANKL, losing RANK-RANKL and osteoclasrs, thus induc-
ing apoptosis. Pre-osteoblast cells have differentiated charac-
teristics of polygonal shape, fusiform cells become mature
osteoblasts. The mature osteoblast cells rvill undergo miner-
alization with hydroxyapatite deposition, therby producing
osteoid matrix. Pre-osteoblast cells expressing alkaline
phosphatase play a role in bone mineralization
[2].
On pre-osteoblast cell culture, NSAID compounds were
capable of inhibiting the synthesis of PGE2 and once served
suppress osteoclastic bone resorption RANKl{ependent
associated with inflammation [3]. In such a mechanism,
mPGES-l also plays a role in the synthesis of pGE2 in cellGl 2017
Bcntham Science publishers
2
CurcN Enume Inhibirion,zol7, VoL
13,
Na
I
mediated differentiation of osteoblasts and osteoclasts PGE
[4]. Analysis of in-vitro, in lipopolysaccharide-induced osteoblast cells showed that mPGES-l plays a role in the synthesis of PGE2 in bone degradation due to inflammation [],
51. Prostaglandim (PG) are known to play a role in the
padrogercsis of inflammation and inflammatory pain. pG
plays a c€ntral role in the peripheral and inJlammatory processes, nociception sensitization, and the generation of pain.
PG are a taryet h the inhibition of Cyclooxygenase (COX),
inhibiting the hflammatory process and aoalgesic. Two isoforms of COX have been identified; COX-I is consitutiveh,,
distributed in most tissues and is responsible for the phvsiological production of PG, whereas COX-2 is induced in a
variety of cell types, ircludhg chondrocytes cells lhen exposed to c}1okilles, mitogens, and endotoxins. Conventional
NSAID drugs have the ability to act as inhibitors of COX-I
and COX-2 as anti-inflammatory dose and this dual inhibition has gastrointestinal side effects. Effects ofNSAIDs a.re
corsidered as an abilil.r, of the drug in inhibiting the Cyclooxygenase (COX) and dre production of prcstagtandins
that arc important mediators of pain and inflammatory rcsponse. COX enz-vme facilitates the metabolism of arachidonic acid to beclme prostaglandins H2, and then metabolized by prostaglandin-E synthase thus producing prostaglandio-E2 (PGE2). NSAID compounds, such as Celecoxib
(SC-58 635) are selectiye COX-2 inhibitors in the teatment
of OA. The drug was the only dnrg that has been approved
by the FDA [6, 7]. However, the lack of sel€ctive aOX-2
irhibitors causes side effects, such as increase in blood pressure and increase in the risk ofhea( afiack [8,9]. ThJ side
eff€cts caused great concem, so there is a need for methods
and compounds of natural ingredients that can be developcd
to design selective COX-I and COX-2 inhibitors.
Seychellene compound
is a
class
of
sesquiterpenoid
compoutrds as one of the main components
of paichouli oil
comtrnunds Pogostemoh cqblin Bonlh. The other major
patlhoulr oil component uas alpha-patchouli alcohol- alplabulneseoe. and alpha-guaiene. Alfa-patchouli alcohol had
anti-inflammatory artivit-y in macrophages RAW264.7 cells
Uol. Alfa-bulnesene had the abiliq as an anri-aggregation
plalete on rabbit blood
bv inhibiting rhc Coi"eriamc
mechanisms and PAF (Pla6let Activaiing
factor)
1t
t, iZ1.
The diversity of frmtion or isolation from naurral mareriali,
such as seychellene require exploration activities as a selective inhibitor of the enz-vme COX-I/ COX-2 on the preost€oblast cells. The purpose of this research is to explore
the potenlial of seychellene as COX-I/ COX-2 inhibitor.
includiog isolation/ fractlonation and determinaoon of lCro
values, and also molecular interaction 1fi silico studies sitrgle/ cluster ofseychellene and other compoulds with COX-l
and COX-2 inhibiton.
2.
2.1. Fractionation and Identification of Seychellene
Patchouti
bra4, in the Organic Chem isrry Laboraton - erawijayi Univorsity.
2.2. Selectivity of [Ce of Frsction-s as COX-I/ COX-2
Inhibitor
The value of IC56 fraction-5 using colorimetric COX
(Ovine) Inhibitor Screening Assay Kit was determined. The
conc.€nh'ation inhibitor fraction-5 (seychelleae in fraction-s),
i.e: 20pM,60pM and l80pM was added to the solvent
DMSO. Pre-test preparatior! protocol, and analyses for the
determined IC56 are done by manual tests Colorimetric COX
(Ovine) Inhibitor Screening Assay Kit Series ?60700 [ l3].
2.3. The Affect Offraction-S on Erpressions COX-I and
COX-2 on Pre.osteoblast Cells
Pre-osteoblasts cell used preosteoblasts cells MC3T3EI
sub clone
4 from the
Americaa Type Cutture Cell.
Preosteoblasts c.ells were grown in complete medium alphaMEM. 2mM L-glutamine. I mM sodium pyruvate, l0o/o FBS
and l0% pensrep in tlre bonle flask 25 cm2 and incubaled rn
ar incubator CO2 5%, temperaturc of 37o C until 70-80%
colfluent cells. Orce conflueot, the cells is roady to be
treated [l4]. Culture of pre-osteoblast cdlls is cultured in
rrell plates to achieve optimal grorvth. Then the cell cultures
were divided into eight groups, i.e.: normat group, LpS
group ( t0 mg/ mL), th€ treatnent grcup fraction of seychellene (concetrtration: 20, 40, and 60 FM) and LpS ae;ment
group (10 mg/ mL) + frartion-s (concentratioD 20, 40. and
60 pM) to the p.otein COX-ll COX-2 expression. Immunohistochemical staining performed siagte staining to COX-I
and COX-2 proteins. The rcsults of immurohistochemical
staiuing were visualized and measured color values were
used, Corfocal I-aser Scanning Microscope (CLSM) to determine the target COX-I and COX-2 protein expressiots.
Furthermore, 3-field was observed and then tlte aver4ge expression of proteins in three replications sample preosteoblast c€lls waws calculated
2.4. Sel€ctivity Binding Energy of Seychellene (Single/Cluster) as COX-1/ COX-2 Inhibitor
Hex8.0 software was us€d to calculate the rigid docking
interaction betweetr COX-I and COX-2 with single seycheli
lene (CID519743) [t5]. We also were docking clusrei sey-
chellene-seychellene and alpha-guaiene (CIDl07l52')seychellene to COX-I and COX-2. The ou$ul ofth€ docking analyzed the 2D interaction active site using Discovery
Studio Client 3.5 software to simulate complei suucture;
sirgle/ cluster (CID519743) by interacting COX-I
MATERIAL AND METHODS
oil is produced from
patchouli leaves (Pogostemon cablin
Java, Indonesia. The fractiouation results were analvzed bv
Gas Chmmatography Mass Spectrometry Shimadzu Qi2010 with RTX-Wa.r column, detector MS ard WilevS Li-
steam distillation of
Be hl in home indus-
m Trenggalek-Easr Java, Indonesia. Fraction of sevchellene
used fractional-vacuum disrillation bv pilobisrl04-
Gcrmanl in Chemical Research Center. Llirl. Scrpong. Easr
and
COX-2 widr MM-GBMV model solvent. Furtlrermore. energl analysis (complex. protein aDd hgand) as a candidate
model of COX-I and COX-2 inhibitors selective was calculated by the Discovery Strrdio 3.5 software and the binding
energl was deoermined usmg equalon AG = 6*,6- lG.-*
- Crisedl It6-181. Selecrivit] of the binding
ofSeych_elles was determined by the equatioD: {og "neig1 energy
[bhding
COX-2/ binding eneryy COX-ll.
In l7fio and In silico: Selectivities ol Seychellene Compound
Cunetd Enzyme
Fndon{: u$rarene
6
(6,71%),
Inhibitio6z0ll,
rbl
13, No.
1
3
seydr{hm {ta3!1),
0-palchoulone (23,01%), 0-pahhoulene {8.68%), 8caqpphrlcns (8.76%), aM o.br.dneee (38,616)
I
Fndbn5:
o-gu(unerc (0,86%), oguarene (2i.02%),
(25,0511, o-patchoulsn0 (28,04%), F-
!.rchrll.m
5
patclnulene (7,06%), p.ccyopnyletnn (1,60%), o.lr*csene
(
10,50%), ar$ o-gathouli
Fncton
A
q
l;
ffi
(2,22%)
unknofln-l (4,3596), urknowrr2 (1,a4%), o-
guriunene (5.49%), oguaene (32.15%),
(fioJlfl o{ddn
d[fu
3
F+atdulene !zfi,h't. F
q&fpsone ('19,98%), gemacrsn&A
caFlodryleno (2,51%),
(7.1 7%) and
I
rcyclrllrno
rlen6 (9,r,1%),
oj*tu.ili
ahdrd
(4,60%)
Fncdon-3: w*no*n.l (11,?2%). unfnovn2 (6,13%), o-
gqunae
I
(16.06%),
ogrlar
lryldho
(lt,58tt),
(11,57!t), and otdnesene (6.17Y0)
Fnctior.2: unknown.l (1,69%). oguaene
2
rydtdhm (2J7tl,
q{atdroulert
(31,45%),
atlnosene
(15.16%),
{33,98%}. gonlacr0nsA(8,07%}, V-paHuJsne(3,91%}.
ild
onddofiahotrl(3,27%)
Frlcdo+l: ur*no*nl
I
g,ai€ne ({0,47%),
(8,87%.
qptUur
ogi4tmne (6,08%), o.
('l0p0tl}, ofatchodene
(5,52%), Fcaqoprry&mo {3,,l7%), otutneeone (t9,0?4).
ll
and
o-patdurlr abohd (5,97%)
Prdrouf O[ ogriurrne (t,61%], ograCIne (12,0{1(},
!.ydxllu. (dll.Al, ofeiod€ne (1,50%), p{atdtor1qB
PO
(1,96%), Fcaryophy,lenen (2,60%),
o{fncene
gfxrscrwteA( 1.904)and o-potdnuli alcdpl
fiS. (1). Isolate
lodistl04).
of
seyclrellene and the ottrer compounds
(CI,7S%).
(49,29961
of palchouli oil from fraction prcduct by fractional-vacuum d.istillation (pi-
3. RESULT
3.1. Fractionation and Identification of Seychellene
Fractionation of seychellene from patchouli oil was used
vacuum fractional distillation by piloDist-I04, Germany.
Identification of seychellene from fiaction-5, pcchouli oil,
and other fraction using GC-MS, as presented in Fig. (l)"
The fragmentation analysis of seychellen€ from fraction-S
was used GC-MS, is shown in Fig. (2). The analyses of GC-
MS vr.ith TIC retention time = 21.82 minutes. EI mass
spectrum analysis results with mlz: 204 (base), lg9, 175,
16l- 147, 133, 122, 107, 93, 79, 67, 55. Simularion of rhe
fr4gmentation pattem is presented in Fig. (2). The spectral
dda of the EI-MS analysis results in accordance wi&
l,Pr:n.q data seychellene [19]. The purity of seychellene
(CID519743) in fraction-S was 25.0-5%.
3.2. Selectivity ICe Value of Fraction-S to COX-U COX-2
ICsovalue of fraction-S (Seychelles 25.05o/o in the fraction-5) was calculated to the COX-Ovine test kit. The regression linier analyses to determine IC5q fraction-5 to COX-I
and COX-2, is shown in Fig (3) The IC5e value of fraction-
5 from COX-I
73.31FM.
was 73.47pM and from COX-2
3.3. The Aflect Fraction-S
to
COX-2 on Pre.Osteoblast Cell
Expression
COX-I
was
and
Expression of COX-I and COX-2 in the preosteoblast
cells in eight groups, i.e.: normal group (COX-ll C,OX-2 +
FITC), LPS group (LPS induction of l0 mg / mL + COX-I/
COX-2 + FITC), a group of fraction-S (concentration of
201601180 pM + COX-ll COX-} + FITC), and a group of
LPS_+ fraction-s (concentrdion of 20/ 601 180 pM + COXI I COX-} + FITC)) were incubate d for 24 hours for monitor-
ing using Confocal Single Laser Microscope
(CSLM).
Aaalysis of differences in all groups in fraction 5, is shorm
in Fig. (4). Fraction-5 showed a decreased expression of
COX-I and COX-2.
33. Selectivity to Binding Energr of Single and Ctuster of
Seychellene
to proteins) ligand single seychellene
- Docking (ligands
(CID519743)
to COX-I and COX-2 were useda rigid docking
4
Vol
C,.rrcnl Erzyme Inhibilion,20l1,
13, No.
I
Raharjo et
al
IB]
ot
.ffi
-o\
I
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Itf:
a
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'lt
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t
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'h
ratr
fig.
ftiiltri,
(2). Fragmentation
f\
,h
C\A
7
lie . t!
*
of s€ychellene (A) anatysis (B) pauem
!o
t0
,0
lC-50
60
:
?0
13 41 uM
ta 30
3't tdyl
ta 50
,It {0
!a
t
c t0
a0
P 50
t0
f:09561
l0
10
0
C.50'73
60
l0
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50
0
ls
150
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050rm$0
200
Omrrdoll*0
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..i.C0l.l'tydrcE
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Fig. (3). Regression linier anatyses of IC5e fracrion-S to COX-I and COX-2
r30
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t 2m
librml
a!
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rtP5s.9"lmlM
{()x/
liq {l). Eight groups of exprcssion COx-l ad COX-2 for fiaction-5 with nonnal, LpS, F-5-20, F-5{0, F-5-180, LPS-F-5-20. LPS-F-5{0,
LPS-F 5'180. and induced LPS r0 mg/ mL, Frrc coloron pre-osteoblast cells.
In Vitro od In silico: Selzaivirtcs ot'Seychellcne Conpound
Trbk
l.
C\crent Enryme Inhibitiorr,2OlT,
,bL
13, No.
I
5
Compleres etrer$/r protein ener$/, lisatrd eoergJi ard binding €ner$' shgle CID5I9743{OX-I1 COX-Z oompteres utd
cluster ClD5l9743+ther oompounds-COx-l1 COX-2 aompterg (GBMV Modet Sohent) were calculate discorrery studio
35 Clhnt softwarc.
Complcr energr
Ligend-C0X-l/ COX-2
No,
Proteln energr
Compleres
Ligend energr
Binding Energr
KkaU mol
I
cID5 19743-COX- 1-CIDs I 9743-CID5
19743
-ffiJW.6s
-60.121.94
201.51
-107.23
)
cIDst 9743-COX-2{ID5 t 9743-C1D5197 43
-6142l.El
-61514.4s
194.23
-l0l.6l
3.
cID519743-COX- l-CIDs 19743
-60.095.60
-60.137.95
133.?0
-91.36
4.
cIDs I 9743{OX -2-CID5
-61,4E9.98
-67,531.6
13122
-83.54
-60.t49.56
-60.137. I
l
60.76
-l
197 43
5
cID519743-COX-I
6.
cIDsI9743-COX-2
41-567.53
-61,560.02
60.76
{0.08
7
cID5 I 9743-COX- I -CIDI
071 52
-61.018.91
-60,052.63
127.69
-93.98
8.
cID5 19743-COX-2-CIDl07 ts2
-61520.31
-61,575.13
125.37
-70.57
9.
cIDl07l52-COX-1
-60,165.85
-6$214.N)
65.95
-78.40
10.
cIDl07l52-cox-2
-60.560.97
-61.561.17
65.95
-73.47
1.89
Note: CIDs19?43 = sydrell6n6; CIDIf/152 = alphrguiem
*
npsl3?${ox.rfi0ar152
$dEila'-
il
clDSr 9741{OX:iCDs197G
fi*o&rrl
eltllBrild
ltr;L iacryc31- !t.:{i.d,Br :'
i*
{3
s**
et
,
CEt0?152-COX"'
6."x
H{igfft!:$3ilEilrl
cto5l97.$clox.'re
5t
*dry-p.'r36s11o1
,Bft'(i
ir&
-lt
*il6!
fl ofi gr&cox. r.ct03l
9ai34r0s$il*
g{t r/=
Iis-.(s)'. linding eners/ and 2D-interaction active sit€ single/ cluser scychetlere compourds with CoX-l protein rcceptor by Discovery
Studio 3.5 software.
6
Caneat Enqme
Inhibbion,20l7,l,ol
13, No. 1
Rahafio
*a
E&
BP
flrUcr*g''lt-cr*i*;i
3
6
"-,
cl61s?43.cox.2{Dtoii52 n
d aI
a
mlgl4t&X.t€polgr.3
srtoterrq.rUhr'qF
t*
rF
,1
._lr,:r.i,'-.,.
: trj-.r,
t1
ffi 1$il*Cor-2486rsrtclElcT.3 *
t
Ilhri";iLrfitr:erc)'
:&*"Hl'"-*rg
&
and 2D-interaction active site single,/ cluser scyctreltene
compounds with
cox-2
prcrein rcceptor by Discovery
0.rt
o,:6
O.ltl
0.rl
0.1
o.8
0.(b
o.{x
0"(}2
o
{.0,
tal|b rrd (lr*.r rrycr!.[...
Fig (7)' Selectivil-r"'s to binding
(OO9f 97at)
energy single and cluster seychellene/ alpha-guaiene with
pare with selectivity to IC5e seychellene (fiaction-5).
coX
complexes by Discovery studio 3.5, com-
ln
)fitm
aitl In silico: Selectivtias
o./
Hex 8.0 software and 2D-visualization analys€s by Discovery Studio 3.5 software. We were also docking cluster seychellene-seychellene and alpha-guaiene (CIDl07l52)seychellene to COX-I and COX-2, as shown io Figs. (5 and
6). Discovery Studio 3.5 software was also used to simulatr
complex of single/ cluster seychellene (CIDs19743) interacting through COX-I and COX-2 wi& MM-GBMV model
solvent aod calculations ofbinding energy using tie equation
AG = G*r6*- [Gr,6"a + Gfig-d], are presented in Tabte I and
Figs. (5 and 6). Setectivity
Fig. (7).
of binding energy is sho$,n in
4. DISCUSSION
Patchouli plants collected from differeqt cultivation rcgions and harvested at different times showed differenc€s in
their votatile oil compositions [20]. Generally, steam or fractional distillation is used ro obtain patchouli oil, although
these processes cause the tremal degradation of a few of the
chemical constituents present in the oil. Patchouli oit consists of rich sesquibrpenoid, mainlv the patchouli alcohol
(pa[choulol) and a tricyclic sesquiteryenoid, such as alphapatchouli alcohot, alpha-bulnesene, alpha-guaiene, seychellene and also the otfier sesquiterpenoid (alpha-patchoulene,
beta-palchoulene, beta-caryophyllene, gemacrcne-A, alphagu{unene) [l9,23]. The standard ISO 3757:2002 pa,lchouli
oil, the major constituent of patchouli oil were alphapdchouli alcohol (30-35%), alpha-bulnesene (13-2to/o) ard,
alpha-guaiene (11-16%) [211. In the res€arch, the GC-MS
analysis of patchouli oil compounds consist alpha-patchouli
alcohol (49.29W, alpha-bulnesene (20.79%), alpha-guaiene
(12.64%), seychelle[e (4.78oh\, and the other components.
According to many researchers, alpha-patchouli alcohol and
alpha-patchoulene are the rnajor constituerts which regulate
and control patchouli oit quality. Some other minor sesquiterpenoid, caryophvtlene, pogostol, alpha-, beta-, gammaand delta-patchoulene, seychellene, cyclo-seychellene, alpha- and beta-bulnesene, alpha- and beta-guaiene and norpatahoulercl are also reporied in patchouli oil [20]. The
characteristics patchouli oil compounds from TrenggalekEast Java had a major component, including alpha-patchouli
alcohol, alpha-bulnesene, alpha-guaiene and ssychellene,
respoctively
Currcn Enqrne Inhihition 2Ol7,l'ol
Sefcheuene Con?ound
.
Seychellene structure has a different sttucture with alphaguaiene and alpha-guaiene or tlte ca.lled stuctrre isomers.
The properties in Seychelles, Cr:Hz.r; rveight molecule =
204.351 [g/Mol]; XlogP = 5.1 u9,231. Seychellene was one
of the secondary metabolites found in plants of Waldheimia
glabru (Dearc.) regal (Nepat) potential as an antioxidant
[24] and also present in the essential oil (9.9%) of lndiaq
Ya]e'ja,] (Valeriana jdtamansi Jones) [25'1. Ideftificatioo of
seychellene was detemined by GC-MS analyscs with RTXWa,.( column, d€tector MS and Wiley8 Libmr),. Data EI-MS
of saychellene (Faction-5) as a member of sesquiterpenoid
were showed decre asing peak similar with Wilev8 Librarv.
According to Silverste; i2005) decreasing peak mlz = i4
was showrl drat the compound as saturated hydrocarbon
group. itrcluding sesquiterpenoid group. The fiagmetrlation
patem of this compound was simulated as fotlow: start€d
wilh termiDation of these compounds tirough the release of
CH3 radicals in the peak molecutar ion (M'), so that the peak
ll,
No.
1
1
rn/z = (M+-15) = 189. Furthermore, the peak (m/z =189) occurs reduction of mlz = 14 to obtain peak mlz = 175, thert
release the C2H{ thus obtained peak m/z = 133. The peak m/z
= 189 split again by releasrng C5HT to obtain peak I22. The
peak m/z = 107 was obtained through C2H2 release of peak
ntlz = 133. Furthermore, the peak m/z 133 was broken with
the release of C2H4 to produce a peak mlz = 79. The peak
m/z = 79 underyo breakdon'n by releasilg C2H2 to obtain
peak mlz = 53. Data of EI-MS spaat"a was fairly accorded
*ith experimental results reportod in literdure (seychellene)
[9]. The purity of seychellene (CID519743) from fraction-5
was 25.05o/o. The other components of a fraction-S were alpha-patchoulene (28.04%), alpha-guaiene (21.02%), and
alpha-bulnesene (10.50%). These compounds Fedicted the
effect IC5q value and the expression ofCOX-l/ COX-2.
In this research, IC5e of &action-5 (seychellene 25.057o)
from COX-I was 73.41pM and from COX-2 was 73.3IUM.
If &e calculated of value logaritlmic (raro tcto COX-2/
COX-I) obtajnable to the value -59 x l0{, it's the nrean
suggested ron seleative COX inhibitor. It's similar, such as
aspiin also had the ability as COX non-selective inhibitor
[26]. It is aiso supported by the observation ofthe influeace
&action-5 to the expression of COX-I/ COX-2 on preosteoblast cell with eight different groups wcre used in this
research: nomal, LPS induction, &roe differellt concentrations of fracrion-s (20, 50, and 180 pM) and LPS togcthor
with three different cpncentrations of fraction-S. The measurement results of tle inteoslty of the expression of COX-I
and COX-2 were statisticall] (SPSS 15.0) showed significant
differences b€tween trealment and bet$,een conc€ntrations
groups (p> 0.05). The analysis of frmtions-5 \ras candidate
reduce the er
ffl tsstltrru munilil.oil
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In Vitro and In-Silico: Selectivities of Seychellene
Compound as Candidate Cycloo)rygenase
Isoenzyme Inhibitor or1. . .
Articte
Current Enzyme lnhibition
.
December 2015
DOI: 10.2U4l1573408012666151202211035
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I
RESEARCH ARTICLE
In
Wtro and In silicoz Selectivities of Seychellene Compound as Candidate
cyclooxygenase Isoenzyme Inhibitor on Pre-osteoblast cells
J. Raharjol2,.chanif
!*tot
Fatchiyah
Mahdi3, Nurdiana Nurdianaa, Takheshi Kikuchis and
Fatchiyah6'
lBiotW
Docto.ral Program, Faculty of Science, Brawijaya University, Jawa Timttr, Indonesia; 2Academic of pharmacy
and Food Analysis, Pttra Indo,nesia Malang, Indonesia: 3Chemistry bepartment, Faculty of Science, Brawii:aya lJniveisity. Jawa riyun l\d9n91ia; lMedicine Pharmacologlt. Faculty oj'Meltine, Brawijaya lJniversity, Jawa Timur. Indones,ia.: "Graduate Life Science School, Ritsumeikan (lniversity. Biwaka Kufriasu-CLmpus, Sttiga fre.lecture, Japan;
"Biolag Department. Faculty of science, Brawijaya {Iniversity, Jawa Timur, Indonesia
Abstrtct: objectiva To determine seleaivity's single urd clusGr of the
(CID519743) compound such as cycloorygenase (COX-I
vitrro and.In si/ico analysis.
t
seychellene
COX-2) inhibitor by in-
r
Merho*z Fraction of seychellene obtained fractional-vacuum distillation patchouli
oil by Pilodist-IO4. The fraction was determined through IC50 COX-I/ COX-2 value
by colorimetric test coXovine 7607ffi and ihe effect decreases lhe expression of
cox-l/ coX-2
ARTIC:LE IIISTORY
R@[cd: S€ptmbcr 26,
2015
REYEcd: NoY@bcr 26, 201 5
A@Frd: ,sEry 2t,2016
DOI:
I A2 I 71/ I 573.t080t26661 5 I 2022 I
tts5
on pre+steoblast cell to induce LpS. Molecular interaction shrdies
single/ cluster of seychellene and other compounds with C.OX-I and COX-2 used
the molecular docking tools by Hex 8.0 and the inleractions were further visualized
and the binding energr was calculated (with Generalized Born Molecular volume
(GBMD model solvent) usingDiscovery Studio Client 3.5 software.
Fatchiyah Fatchiyah
Resn& Fraction of seychellene obtained from patchouli oil in fraction-5 es.osyo). The IC56 values of
fraction-S were: COX-I (73.47 u. M) and COX-2 (73.3 I p M). The fraction of seychellene decreased the
expression of COX-I/ COX-2 isoenzym. ln silico analyses of scoring binding energy calculation (with
GBMV model solvent) of single and cluster seychellene compourds alFected the s€lectivity of COX-I
and COX-2 inhibitor.
Condaston: Collectivety, selectiviry's
of the
seychellene compound as non-seleaive inhibitor
cycloorygenase.
Keywords: ICso,in sl/ico analyses of scoring binding energy, inhibitor selective, selectivity, single/ cluster seychellene
com-
pounds.
T.INTRODUCTION
Patomechanism osteoporosis is a disorder of bone furnover involving cell function to achieve coordination and balance resorption of old bone (osteoclasts) and the formation
of new bone (osteoblasts) to the local conditions. coordinated and sequentially as the coupling process. Bone reorganization were initiated by the recruitnent and activation of
osteoclasts. Osteoblast cells as bone-forming also play a role
in producing Prost4glandin-E} (PGEZI, mainly related to
inflammation and act as a stimulator of bons resorption [t].
Furthermore, Osteoclastogenesis leads to osteoblasts to secr€te Receptor Activated Nuclear Kappa Ligand (RANKL),
which responded by osteoclasts with the formation of R*eptor
rAddrcss correspondcnce to this
aulhor at thc Biology Deportncnt, Facutty
of Science. Bra*ijaya Univcrsity. . Jawa Timur, Indonesia:
Tcl: +62 341 575841; Fax: +62341 5758/l.,Bmail: farchivarOub.ac.id
l573408{i.ll7
Ss8.tx)+.m
Activated Nuclear Kappa (RANK), due to the interaction of
RANKL and RANK, which turns into then mature osteoclasts, so that it can function in the process of bone resorption. When bone resorption is considered suffrcient, then
osboblasts secrete Osteoprotegerin (OPG) for binding
RANKL, losing RANK-RANKL and osteoclasrs, thus induc-
ing apoptosis. Pre-osteoblast cells have differentiated charac-
teristics of polygonal shape, fusiform cells become mature
osteoblasts. The mature osteoblast cells rvill undergo miner-
alization with hydroxyapatite deposition, therby producing
osteoid matrix. Pre-osteoblast cells expressing alkaline
phosphatase play a role in bone mineralization
[2].
On pre-osteoblast cell culture, NSAID compounds were
capable of inhibiting the synthesis of PGE2 and once served
suppress osteoclastic bone resorption RANKl{ependent
associated with inflammation [3]. In such a mechanism,
mPGES-l also plays a role in the synthesis of pGE2 in cellGl 2017
Bcntham Science publishers
2
CurcN Enume Inhibirion,zol7, VoL
13,
Na
I
mediated differentiation of osteoblasts and osteoclasts PGE
[4]. Analysis of in-vitro, in lipopolysaccharide-induced osteoblast cells showed that mPGES-l plays a role in the synthesis of PGE2 in bone degradation due to inflammation [],
51. Prostaglandim (PG) are known to play a role in the
padrogercsis of inflammation and inflammatory pain. pG
plays a c€ntral role in the peripheral and inJlammatory processes, nociception sensitization, and the generation of pain.
PG are a taryet h the inhibition of Cyclooxygenase (COX),
inhibiting the hflammatory process and aoalgesic. Two isoforms of COX have been identified; COX-I is consitutiveh,,
distributed in most tissues and is responsible for the phvsiological production of PG, whereas COX-2 is induced in a
variety of cell types, ircludhg chondrocytes cells lhen exposed to c}1okilles, mitogens, and endotoxins. Conventional
NSAID drugs have the ability to act as inhibitors of COX-I
and COX-2 as anti-inflammatory dose and this dual inhibition has gastrointestinal side effects. Effects ofNSAIDs a.re
corsidered as an abilil.r, of the drug in inhibiting the Cyclooxygenase (COX) and dre production of prcstagtandins
that arc important mediators of pain and inflammatory rcsponse. COX enz-vme facilitates the metabolism of arachidonic acid to beclme prostaglandins H2, and then metabolized by prostaglandin-E synthase thus producing prostaglandio-E2 (PGE2). NSAID compounds, such as Celecoxib
(SC-58 635) are selectiye COX-2 inhibitors in the teatment
of OA. The drug was the only dnrg that has been approved
by the FDA [6, 7]. However, the lack of sel€ctive aOX-2
irhibitors causes side effects, such as increase in blood pressure and increase in the risk ofhea( afiack [8,9]. ThJ side
eff€cts caused great concem, so there is a need for methods
and compounds of natural ingredients that can be developcd
to design selective COX-I and COX-2 inhibitors.
Seychellene compound
is a
class
of
sesquiterpenoid
compoutrds as one of the main components
of paichouli oil
comtrnunds Pogostemoh cqblin Bonlh. The other major
patlhoulr oil component uas alpha-patchouli alcohol- alplabulneseoe. and alpha-guaiene. Alfa-patchouli alcohol had
anti-inflammatory artivit-y in macrophages RAW264.7 cells
Uol. Alfa-bulnesene had the abiliq as an anri-aggregation
plalete on rabbit blood
bv inhibiting rhc Coi"eriamc
mechanisms and PAF (Pla6let Activaiing
factor)
1t
t, iZ1.
The diversity of frmtion or isolation from naurral mareriali,
such as seychellene require exploration activities as a selective inhibitor of the enz-vme COX-I/ COX-2 on the preost€oblast cells. The purpose of this research is to explore
the potenlial of seychellene as COX-I/ COX-2 inhibitor.
includiog isolation/ fractlonation and determinaoon of lCro
values, and also molecular interaction 1fi silico studies sitrgle/ cluster ofseychellene and other compoulds with COX-l
and COX-2 inhibiton.
2.
2.1. Fractionation and Identification of Seychellene
Patchouti
bra4, in the Organic Chem isrry Laboraton - erawijayi Univorsity.
2.2. Selectivity of [Ce of Frsction-s as COX-I/ COX-2
Inhibitor
The value of IC56 fraction-5 using colorimetric COX
(Ovine) Inhibitor Screening Assay Kit was determined. The
conc.€nh'ation inhibitor fraction-5 (seychelleae in fraction-s),
i.e: 20pM,60pM and l80pM was added to the solvent
DMSO. Pre-test preparatior! protocol, and analyses for the
determined IC56 are done by manual tests Colorimetric COX
(Ovine) Inhibitor Screening Assay Kit Series ?60700 [ l3].
2.3. The Affect Offraction-S on Erpressions COX-I and
COX-2 on Pre.osteoblast Cells
Pre-osteoblasts cell used preosteoblasts cells MC3T3EI
sub clone
4 from the
Americaa Type Cutture Cell.
Preosteoblasts c.ells were grown in complete medium alphaMEM. 2mM L-glutamine. I mM sodium pyruvate, l0o/o FBS
and l0% pensrep in tlre bonle flask 25 cm2 and incubaled rn
ar incubator CO2 5%, temperaturc of 37o C until 70-80%
colfluent cells. Orce conflueot, the cells is roady to be
treated [l4]. Culture of pre-osteoblast cdlls is cultured in
rrell plates to achieve optimal grorvth. Then the cell cultures
were divided into eight groups, i.e.: normat group, LpS
group ( t0 mg/ mL), th€ treatnent grcup fraction of seychellene (concetrtration: 20, 40, and 60 FM) and LpS ae;ment
group (10 mg/ mL) + frartion-s (concentratioD 20, 40. and
60 pM) to the p.otein COX-ll COX-2 expression. Immunohistochemical staining performed siagte staining to COX-I
and COX-2 proteins. The rcsults of immurohistochemical
staiuing were visualized and measured color values were
used, Corfocal I-aser Scanning Microscope (CLSM) to determine the target COX-I and COX-2 protein expressiots.
Furthermore, 3-field was observed and then tlte aver4ge expression of proteins in three replications sample preosteoblast c€lls waws calculated
2.4. Sel€ctivity Binding Energy of Seychellene (Single/Cluster) as COX-1/ COX-2 Inhibitor
Hex8.0 software was us€d to calculate the rigid docking
interaction betweetr COX-I and COX-2 with single seycheli
lene (CID519743) [t5]. We also were docking clusrei sey-
chellene-seychellene and alpha-guaiene (CIDl07l52')seychellene to COX-I and COX-2. The ou$ul ofth€ docking analyzed the 2D interaction active site using Discovery
Studio Client 3.5 software to simulate complei suucture;
sirgle/ cluster (CID519743) by interacting COX-I
MATERIAL AND METHODS
oil is produced from
patchouli leaves (Pogostemon cablin
Java, Indonesia. The fractiouation results were analvzed bv
Gas Chmmatography Mass Spectrometry Shimadzu Qi2010 with RTX-Wa.r column, detector MS ard WilevS Li-
steam distillation of
Be hl in home indus-
m Trenggalek-Easr Java, Indonesia. Fraction of sevchellene
used fractional-vacuum disrillation bv pilobisrl04-
Gcrmanl in Chemical Research Center. Llirl. Scrpong. Easr
and
COX-2 widr MM-GBMV model solvent. Furtlrermore. energl analysis (complex. protein aDd hgand) as a candidate
model of COX-I and COX-2 inhibitors selective was calculated by the Discovery Strrdio 3.5 software and the binding
energl was deoermined usmg equalon AG = 6*,6- lG.-*
- Crisedl It6-181. Selecrivit] of the binding
ofSeych_elles was determined by the equatioD: {og "neig1 energy
[bhding
COX-2/ binding eneryy COX-ll.
In l7fio and In silico: Selectivities ol Seychellene Compound
Cunetd Enzyme
Fndon{: u$rarene
6
(6,71%),
Inhibitio6z0ll,
rbl
13, No.
1
3
seydr{hm {ta3!1),
0-palchoulone (23,01%), 0-pahhoulene {8.68%), 8caqpphrlcns (8.76%), aM o.br.dneee (38,616)
I
Fndbn5:
o-gu(unerc (0,86%), oguarene (2i.02%),
(25,0511, o-patchoulsn0 (28,04%), F-
!.rchrll.m
5
patclnulene (7,06%), p.ccyopnyletnn (1,60%), o.lr*csene
(
10,50%), ar$ o-gathouli
Fncton
A
q
l;
ffi
(2,22%)
unknofln-l (4,3596), urknowrr2 (1,a4%), o-
guriunene (5.49%), oguaene (32.15%),
(fioJlfl o{ddn
d[fu
3
F+atdulene !zfi,h't. F
q&fpsone ('19,98%), gemacrsn&A
caFlodryleno (2,51%),
(7.1 7%) and
I
rcyclrllrno
rlen6 (9,r,1%),
oj*tu.ili
ahdrd
(4,60%)
Fncdon-3: w*no*n.l (11,?2%). unfnovn2 (6,13%), o-
gqunae
I
(16.06%),
ogrlar
lryldho
(lt,58tt),
(11,57!t), and otdnesene (6.17Y0)
Fnctior.2: unknown.l (1,69%). oguaene
2
rydtdhm (2J7tl,
q{atdroulert
(31,45%),
atlnosene
(15.16%),
{33,98%}. gonlacr0nsA(8,07%}, V-paHuJsne(3,91%}.
ild
onddofiahotrl(3,27%)
Frlcdo+l: ur*no*nl
I
g,ai€ne ({0,47%),
(8,87%.
qptUur
ogi4tmne (6,08%), o.
('l0p0tl}, ofatchodene
(5,52%), Fcaqoprry&mo {3,,l7%), otutneeone (t9,0?4).
ll
and
o-patdurlr abohd (5,97%)
Prdrouf O[ ogriurrne (t,61%], ograCIne (12,0{1(},
!.ydxllu. (dll.Al, ofeiod€ne (1,50%), p{atdtor1qB
PO
(1,96%), Fcaryophy,lenen (2,60%),
o{fncene
gfxrscrwteA( 1.904)and o-potdnuli alcdpl
fiS. (1). Isolate
lodistl04).
of
seyclrellene and the ottrer compounds
(CI,7S%).
(49,29961
of palchouli oil from fraction prcduct by fractional-vacuum d.istillation (pi-
3. RESULT
3.1. Fractionation and Identification of Seychellene
Fractionation of seychellene from patchouli oil was used
vacuum fractional distillation by piloDist-I04, Germany.
Identification of seychellene from fiaction-5, pcchouli oil,
and other fraction using GC-MS, as presented in Fig. (l)"
The fragmentation analysis of seychellen€ from fraction-S
was used GC-MS, is shown in Fig. (2). The analyses of GC-
MS vr.ith TIC retention time = 21.82 minutes. EI mass
spectrum analysis results with mlz: 204 (base), lg9, 175,
16l- 147, 133, 122, 107, 93, 79, 67, 55. Simularion of rhe
fr4gmentation pattem is presented in Fig. (2). The spectral
dda of the EI-MS analysis results in accordance wi&
l,Pr:n.q data seychellene [19]. The purity of seychellene
(CID519743) in fraction-S was 25.0-5%.
3.2. Selectivity ICe Value of Fraction-S to COX-U COX-2
ICsovalue of fraction-S (Seychelles 25.05o/o in the fraction-5) was calculated to the COX-Ovine test kit. The regression linier analyses to determine IC5q fraction-5 to COX-I
and COX-2, is shown in Fig (3) The IC5e value of fraction-
5 from COX-I
73.31FM.
was 73.47pM and from COX-2
3.3. The Aflect Fraction-S
to
COX-2 on Pre.Osteoblast Cell
Expression
COX-I
was
and
Expression of COX-I and COX-2 in the preosteoblast
cells in eight groups, i.e.: normal group (COX-ll C,OX-2 +
FITC), LPS group (LPS induction of l0 mg / mL + COX-I/
COX-2 + FITC), a group of fraction-S (concentration of
201601180 pM + COX-ll COX-} + FITC), and a group of
LPS_+ fraction-s (concentrdion of 20/ 601 180 pM + COXI I COX-} + FITC)) were incubate d for 24 hours for monitor-
ing using Confocal Single Laser Microscope
(CSLM).
Aaalysis of differences in all groups in fraction 5, is shorm
in Fig. (4). Fraction-5 showed a decreased expression of
COX-I and COX-2.
33. Selectivity to Binding Energr of Single and Ctuster of
Seychellene
to proteins) ligand single seychellene
- Docking (ligands
(CID519743)
to COX-I and COX-2 were useda rigid docking
4
Vol
C,.rrcnl Erzyme Inhibilion,20l1,
13, No.
I
Raharjo et
al
IB]
ot
.ffi
-o\
I
!
.>i
.l)
Itf:
a
I
"
. itl.
Clt
'ttrr f
'lt
-crt
-cat:
t
9t
aJ+'
NI
.Or
I
.crt
-/)
'h
ratr
fig.
ftiiltri,
(2). Fragmentation
f\
,h
C\A
7
lie . t!
*
of s€ychellene (A) anatysis (B) pauem
!o
t0
,0
lC-50
60
:
?0
13 41 uM
ta 30
3't tdyl
ta 50
,It {0
!a
t
c t0
a0
P 50
t0
f:09561
l0
10
0
C.50'73
60
l0
0
50
0
ls
150
Concadoalgl
ts:0"9!196
050rm$0
200
Omrrdoll*0
..a- COI.l.rrdr. ko.{Cl 05f Cr1S}
..i.C0l.l'tydrcE
n{Ct051E a3}
Fig. (3). Regression linier anatyses of IC5e fracrion-S to COX-I and COX-2
r30
II
t 2m
librml
a!
r lps
r50
.
l0
r f .s€odtt
rf .S.tffiril
r lPtt.5.l(lrM
rtPlF.$.mrM
I
$t,
o
(()x I
l
r"5.1{hdr,
rtP5s.9"lmlM
{()x/
liq {l). Eight groups of exprcssion COx-l ad COX-2 for fiaction-5 with nonnal, LpS, F-5-20, F-5{0, F-5-180, LPS-F-5-20. LPS-F-5{0,
LPS-F 5'180. and induced LPS r0 mg/ mL, Frrc coloron pre-osteoblast cells.
In Vitro od In silico: Selzaivirtcs ot'Seychellcne Conpound
Trbk
l.
C\crent Enryme Inhibitiorr,2OlT,
,bL
13, No.
I
5
Compleres etrer$/r protein ener$/, lisatrd eoergJi ard binding €ner$' shgle CID5I9743{OX-I1 COX-Z oompteres utd
cluster ClD5l9743+ther oompounds-COx-l1 COX-2 aompterg (GBMV Modet Sohent) were calculate discorrery studio
35 Clhnt softwarc.
Complcr energr
Ligend-C0X-l/ COX-2
No,
Proteln energr
Compleres
Ligend energr
Binding Energr
KkaU mol
I
cID5 19743-COX- 1-CIDs I 9743-CID5
19743
-ffiJW.6s
-60.121.94
201.51
-107.23
)
cIDst 9743-COX-2{ID5 t 9743-C1D5197 43
-6142l.El
-61514.4s
194.23
-l0l.6l
3.
cID519743-COX- l-CIDs 19743
-60.095.60
-60.137.95
133.?0
-91.36
4.
cIDs I 9743{OX -2-CID5
-61,4E9.98
-67,531.6
13122
-83.54
-60.t49.56
-60.137. I
l
60.76
-l
197 43
5
cID519743-COX-I
6.
cIDsI9743-COX-2
41-567.53
-61,560.02
60.76
{0.08
7
cID5 I 9743-COX- I -CIDI
071 52
-61.018.91
-60,052.63
127.69
-93.98
8.
cID5 19743-COX-2-CIDl07 ts2
-61520.31
-61,575.13
125.37
-70.57
9.
cIDl07l52-COX-1
-60,165.85
-6$214.N)
65.95
-78.40
10.
cIDl07l52-cox-2
-60.560.97
-61.561.17
65.95
-73.47
1.89
Note: CIDs19?43 = sydrell6n6; CIDIf/152 = alphrguiem
*
npsl3?${ox.rfi0ar152
$dEila'-
il
clDSr 9741{OX:iCDs197G
fi*o&rrl
eltllBrild
ltr;L iacryc31- !t.:{i.d,Br :'
i*
{3
s**
et
,
CEt0?152-COX"'
6."x
H{igfft!:$3ilEilrl
cto5l97.$clox.'re
5t
*dry-p.'r36s11o1
,Bft'(i
ir&
-lt
*il6!
fl ofi gr&cox. r.ct03l
9ai34r0s$il*
g{t r/=
Iis-.(s)'. linding eners/ and 2D-interaction active sit€ single/ cluser scychetlere compourds with CoX-l protein rcceptor by Discovery
Studio 3.5 software.
6
Caneat Enqme
Inhibbion,20l7,l,ol
13, No. 1
Rahafio
*a
E&
BP
flrUcr*g''lt-cr*i*;i
3
6
"-,
cl61s?43.cox.2{Dtoii52 n
d aI
a
mlgl4t&X.t€polgr.3
srtoterrq.rUhr'qF
t*
rF
,1
._lr,:r.i,'-.,.
: trj-.r,
t1
ffi 1$il*Cor-2486rsrtclElcT.3 *
t
Ilhri";iLrfitr:erc)'
:&*"Hl'"-*rg
&
and 2D-interaction active site single,/ cluser scyctreltene
compounds with
cox-2
prcrein rcceptor by Discovery
0.rt
o,:6
O.ltl
0.rl
0.1
o.8
0.(b
o.{x
0"(}2
o
{.0,
tal|b rrd (lr*.r rrycr!.[...
Fig (7)' Selectivil-r"'s to binding
(OO9f 97at)
energy single and cluster seychellene/ alpha-guaiene with
pare with selectivity to IC5e seychellene (fiaction-5).
coX
complexes by Discovery studio 3.5, com-
ln
)fitm
aitl In silico: Selectivtias
o./
Hex 8.0 software and 2D-visualization analys€s by Discovery Studio 3.5 software. We were also docking cluster seychellene-seychellene and alpha-guaiene (CIDl07l52)seychellene to COX-I and COX-2, as shown io Figs. (5 and
6). Discovery Studio 3.5 software was also used to simulatr
complex of single/ cluster seychellene (CIDs19743) interacting through COX-I and COX-2 wi& MM-GBMV model
solvent aod calculations ofbinding energy using tie equation
AG = G*r6*- [Gr,6"a + Gfig-d], are presented in Tabte I and
Figs. (5 and 6). Setectivity
Fig. (7).
of binding energy is sho$,n in
4. DISCUSSION
Patchouli plants collected from differeqt cultivation rcgions and harvested at different times showed differenc€s in
their votatile oil compositions [20]. Generally, steam or fractional distillation is used ro obtain patchouli oil, although
these processes cause the tremal degradation of a few of the
chemical constituents present in the oil. Patchouli oit consists of rich sesquibrpenoid, mainlv the patchouli alcohol
(pa[choulol) and a tricyclic sesquiteryenoid, such as alphapatchouli alcohot, alpha-bulnesene, alpha-guaiene, seychellene and also the otfier sesquiterpenoid (alpha-patchoulene,
beta-palchoulene, beta-caryophyllene, gemacrcne-A, alphagu{unene) [l9,23]. The standard ISO 3757:2002 pa,lchouli
oil, the major constituent of patchouli oil were alphapdchouli alcohol (30-35%), alpha-bulnesene (13-2to/o) ard,
alpha-guaiene (11-16%) [211. In the res€arch, the GC-MS
analysis of patchouli oil compounds consist alpha-patchouli
alcohol (49.29W, alpha-bulnesene (20.79%), alpha-guaiene
(12.64%), seychelle[e (4.78oh\, and the other components.
According to many researchers, alpha-patchouli alcohol and
alpha-patchoulene are the rnajor constituerts which regulate
and control patchouli oit quality. Some other minor sesquiterpenoid, caryophvtlene, pogostol, alpha-, beta-, gammaand delta-patchoulene, seychellene, cyclo-seychellene, alpha- and beta-bulnesene, alpha- and beta-guaiene and norpatahoulercl are also reporied in patchouli oil [20]. The
characteristics patchouli oil compounds from TrenggalekEast Java had a major component, including alpha-patchouli
alcohol, alpha-bulnesene, alpha-guaiene and ssychellene,
respoctively
Currcn Enqrne Inhihition 2Ol7,l'ol
Sefcheuene Con?ound
.
Seychellene structure has a different sttucture with alphaguaiene and alpha-guaiene or tlte ca.lled stuctrre isomers.
The properties in Seychelles, Cr:Hz.r; rveight molecule =
204.351 [g/Mol]; XlogP = 5.1 u9,231. Seychellene was one
of the secondary metabolites found in plants of Waldheimia
glabru (Dearc.) regal (Nepat) potential as an antioxidant
[24] and also present in the essential oil (9.9%) of lndiaq
Ya]e'ja,] (Valeriana jdtamansi Jones) [25'1. Ideftificatioo of
seychellene was detemined by GC-MS analyscs with RTXWa,.( column, d€tector MS and Wiley8 Libmr),. Data EI-MS
of saychellene (Faction-5) as a member of sesquiterpenoid
were showed decre asing peak similar with Wilev8 Librarv.
According to Silverste; i2005) decreasing peak mlz = i4
was showrl drat the compound as saturated hydrocarbon
group. itrcluding sesquiterpenoid group. The fiagmetrlation
patem of this compound was simulated as fotlow: start€d
wilh termiDation of these compounds tirough the release of
CH3 radicals in the peak molecutar ion (M'), so that the peak
ll,
No.
1
1
rn/z = (M+-15) = 189. Furthermore, the peak (m/z =189) occurs reduction of mlz = 14 to obtain peak mlz = 175, thert
release the C2H{ thus obtained peak m/z = 133. The peak m/z
= 189 split again by releasrng C5HT to obtain peak I22. The
peak m/z = 107 was obtained through C2H2 release of peak
ntlz = 133. Furthermore, the peak m/z 133 was broken with
the release of C2H4 to produce a peak mlz = 79. The peak
m/z = 79 underyo breakdon'n by releasilg C2H2 to obtain
peak mlz = 53. Data of EI-MS spaat"a was fairly accorded
*ith experimental results reportod in literdure (seychellene)
[9]. The purity of seychellene (CID519743) from fraction-5
was 25.05o/o. The other components of a fraction-S were alpha-patchoulene (28.04%), alpha-guaiene (21.02%), and
alpha-bulnesene (10.50%). These compounds Fedicted the
effect IC5q value and the expression ofCOX-l/ COX-2.
In this research, IC5e of &action-5 (seychellene 25.057o)
from COX-I was 73.41pM and from COX-2 was 73.3IUM.
If &e calculated of value logaritlmic (raro tcto COX-2/
COX-I) obtajnable to the value -59 x l0{, it's the nrean
suggested ron seleative COX inhibitor. It's similar, such as
aspiin also had the ability as COX non-selective inhibitor
[26]. It is aiso supported by the observation ofthe influeace
&action-5 to the expression of COX-I/ COX-2 on preosteoblast cell with eight different groups wcre used in this
research: nomal, LPS induction, &roe differellt concentrations of fracrion-s (20, 50, and 180 pM) and LPS togcthor
with three different cpncentrations of fraction-S. The measurement results of tle inteoslty of the expression of COX-I
and COX-2 were statisticall] (SPSS 15.0) showed significant
differences b€tween trealment and bet$,een conc€ntrations
groups (p> 0.05). The analysis of frmtions-5 \ras candidate
reduce the er