vascular reactivity concerning orthosiphon stamineus benth

Hindawi Publishing CorporationInternational Journal of Vascular MedicineVolume 2013 Article ID 456852 8 pageshttpdxdoiorg1011552013456852

Research ArticleVascular Reactivity ConcerningOrthosiphon stamineus Benth-Mediated Antihypertensive inAortic Rings of Spontaneously Hypertensive Rats

Nurul Maizan Manshor1 Aidiahmad Dewa1 Mohd Zaini Asmawi2

Zhari Ismail3 Nadiah Razali1 and Zurina Hassan4

1 Department of Physiology School of Pharmaceutical Sciences Universiti Sains Malaysia 11800 Minden Penang Malaysia2 Department of Pharmacology School of Pharmaceutical Sciences Universiti Sains Malaysia 11800 Minden Penang Malaysia3 Department of Chemistry School of Pharmaceutical Sciences Universiti Sains Malaysia 11800 Minden Penang Malaysia4Centre for Drug Research Universiti Sains Malaysia 11800 Minden Penang Malaysia

Correspondence should be addressed to Nurul Maizan Manshor maizan84gmailcom

Received 24 April 2013 Accepted 5 June 2013

Academic Editor Mark Morasch

Copyright copy 2013 Nurul Maizan Manshor et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Orthosiphon stamineusBenth has been traditionally used to treat hypertensionThe study aimed to investigate the vascular reactivityof water extract (WOS) and water methanolic (1 1) extract (WMOS) of Orthosiphon stamineus Benth and AT

1receptors blocker

in the mechanisms of antihypertensive mediated by 1205721-adrenergic receptor and EDNO and PGI

2releases in the SHR aortic rings

SHR (230ndash280 g) were divided into four groups control WOS WMOS and losartan After being fed orally for 14 days theaorta was harvested and subjected to PE (10minus9 to 10minus5M) and ACh (10minus9 to 10minus5M) with and without L-NAME (100120583M) andindomethacin (10 120583M) respectively WOS WMOS and losartan significantly reduced the contractile responses to PE intactsuggesting the importance of endothelium in vasorelaxation Losartan significantly enhanced the ACh-induced vasorelaxation L-NAME significantly inhibited the ACh-induced relaxation in all groups Indomethacin enhanced ACh-induced vasorelaxation inWMOS Collectively Orthosiphon stamineus leaves extract reduced vasoconstriction responses by the alteration of 120572

1-adrenergic

and AT1receptors activitiesThe involvement of EDNO releases was clearly observed in this plant InWOS PGI

2releases might not

participate in the ACh-induced vasorelaxation However in WMOS enhancement of vasorelaxation possibly due to continuousrelease of PGI

2

1 Introduction

Orthosiphon stamineus Benth (synO aristatus (Bl) MiqOgrandiflorus Bold O spicatus (Thumb) Bak Lamiaceae) [1]or locally known as ldquoMisai Kucingrdquo leaves extracts have beenused as traditional medicine [2] and possess benefits suchas antidiabetic ability to increase plasma triglyceride andplasmaHDL-cholesterol concentrations [3] anti-lithiatic andhypouricemic effects [4 5] antifungal [6] and ability to treatkidney stone and urinary tract diseases [7ndash9] It has tradition-ally been used in Java for the treatment of hypertension anddiabetes [1] Hypertension has been reported to be associatedwith endothelium dysfunction in both human and animal

studies [10] Endothelium regulates vascular tone by releasingvasoconstrictors such as endothelins prostanoids and oxy-gen reactive species and vasodilators such as nitric oxide(NO) prostacyclin (PGI

2) and endothelial hyperpolarizing

factor (EDHF) These vasodilators were a great discovery byFurchgott and Zawadzki in 1980 [11] known as endotheliumderived relaxing factors (EDRF) It has been reported byPeach et al [12] that releases of EDRF caused vasorelaxanteffects of acetylcholine (ACh) which is dependent on thepresence of the endothelial cells [11 13]

Phenylephrine (PE) is a selective 1205721-adrenergic receptor

agonist that increases arterial blood pressure by periph-eral vasoconstriction 120572

1-Adrenergic receptors which exist

2 International Journal of Vascular Medicine

postsynaptically are G-protein-coupled receptors and thusactivation of cellular signaling is subsequent to the inter-action with a G-protein Activation of these receptors onvascular smooth muscle leads to vasoconstriction PE haspredominantly 120572

1-postjunctional receptors in ratrsquos aorta [14]

Since PE is a selective 1205721-adrenergic receptor agonist and

losartan is AT1receptor blocker there is possible relationship

between AT1and 120572

1receptors [15] In addition crosstalk

between AT1and 1205721receptors in the smoothmuscle of rabbit

aorta is endothelium dependent [16] It has been reportedthat MRC A isolated from Orthosiphon stamineus causescontinuous decreases in systolic blood pressure (SBP) andheart rate (HR) after subcutaneous administration in con-scious SHR [17] However studies on the antihypertensivemechanisms by Orthosiphon stamineus still remain unclearThe present study aimed to investigate the vascular reactivityof water extract (WOS) and water methanolic (1 1) extract(WMOS) of Orthosiphon stamineus Benth and AT

1recep-

tors blocker in the mechanisms of antihypertensive effectsmediated by 120572

1-adrenergic receptor and prostacyclin (PGI

2)

and endothelium-derived nitric oxide (EDNO) releases in theSHR aortic rings

2 Materials and Methods

21 Preparation of Orthosiphon stamineus Leaves ExtractsVoucher specimen (no 11009) of the plant material wasdeposited at Herbal Room School of Pharmaceutical Sci-ences Universiti Sains Malaysia (USM) WMOS was pre-pared by having dried and ground Orthosiphon stamineusleaves extracted by a mixture of methanol water (1 1) usinga Soxhlet extractor for a period of 12 hours whereas prepara-tion ofWOS involved hotmaceration of the dried and groundOrthosiphon stamineus leaves at 50∘C for 6 hours and wasrepeated thrice Each extract was bulked and concentratedin a rotary evaporator under vacuum and then freeze-driedand kept in a freezer until used [18] WOS and WMOS werefreshly prepared in distilled water prior to the feeding of theanimals

22 Animals Male spontaneously hypertensive rats (SHR230ndash280 g) were housed in individual cages with free accessto foods and water and maintained at Animal Transit Facilityof School of Pharmaceutical Sciences USM All proceduresinvolving animals were conducted according to the ethicalguidelines by the Animal Ethics Committee USM The ani-mals were divided into four groups (1)WOS 1000mgkg (2)WMOS 1000mgkg (3) losartan 10mgkg and (4) control(vehicle) All animals were given daily treatment orally for 14days before being subjected to vascular reactivity studies

23 Drugs and Chemicals Phenylephrine hydrochloride(PE) acetylcholine (ACh) indomethacin and N120596-nitro-L-arginine methyl ester (L-NAME) were purchased fromSigma-Aldrich Germany while sodium chloride (NaCl)potassium chloride (KCl) potassium dihydrogen phosphate(KH2PO4) magnesium sulphate (MgSO

4sdot7H2O) glucose

sodium hydrogen carbonate (NaHCO3) and calcium chlo-

ride dehydrate (CaCl2sdotH2O) were purchased from RampM

Chem UK All drugs were freshly prepared in normal salineexcept indomethacin in 05 (wv) sodium carbonate priorto use

24 Vascular Reactivity Using Aortic Rings The rat was anes-thetized with sodium pentobarbital (60mgkg ip) A mid-line abdominal incision was performed to expose the aortaThe thoracic aorta was carefully isolated cleaned from theadherent fat and connective tissues and cut into 3ndash5mmrings The aortic rings were then suspended horizontally intissue chambers containing 10mLofKrebrsquos solution (mmolLNaCl 1186 KCl 48 CaCl

225 MgSO

4sdot7H2O 12 KH

2PO4

12 NaHCO3251 and glucose 110) The tissue-bath solution

was bubbled incessantly with 95O2and 5CO

2(carbogen)

at 37∘C Aortic rings were then allowed to equilibrate at anoptimal tension of 1 g for 45min Krebrsquos solution was replacedevery 15min and the tension was readjusted to 1 g whennecessary At the beginning of the experiment the presenceof intact endothelial cells was confirmed by precontractingthe tissues with PE (1 120583M) and followed by relaxation withACh (1120583M) Relaxation not less than 60 indicated thepresence of intact endothelial cells Responses were recordedisometrically via a force transducer (Grass FT03D) connectedto a computerized data acquisition system (PowerLab ADIn-struments Pty Ltd Australia) For vasoconstriction studythe concentration-response curves for PE (cumulative finalchamber concentration of 10minus9 to 10minus5M) were recordedThe contraction effects of PE were recorded in two differ-ent preparations intact and denude endothelium Denudeendothelium of aortic rings was obtained by gently rubbingthe intimal layer of the tissue with a blunt needle for a fewtimes The aortic rings were considered denuded when therewere less than 10 relaxations to ACh (1 120583M) precontractedwith PE (1 120583M)whereas in order to obtain the concentration-response curves of relaxation ACh (10minus9 to 10minus5M) wasadded cumulatively to the chamber at the plateau of the PE(1 120583M) precontracted aortic rings at 3-minute intervals Tofurther assess the involvement of EDNO and prostacyclin(PGI2) releases relaxations of aortic rings were performed

in WOS WMOS and losartan groups preincubated for 30minutes with L-NAME (100 120583M) a nonspecific NO syn-thase inhibitor and indomethacin (10 120583M) a nonselectivecyclooxygenase inhibitor respectively

25 Data Analysis All data are given as mean plusmn standarderror means (SEM) PE-induced contraction and ACh-induced relaxation were analysed using one-way ANOVAfollowed by Dunnettrsquos post hoc test whereas the effects ofACh-induced relaxation after preincubated by L-NAME andindomethacin were analysed using Studentrsquos t-test 119864max119877max and pD2 values were derived from nonlinear regressionanalysis All analyses were using the computer softwareGraphPad Prism 50 for Windows (GraphPad Software IncUSA) Values of 119875 lt 005 were considered statistically signif-icant

International Journal of Vascular Medicine 3

0

02

04

06

08

1

12

log [PE] (M)

PE-intact

Ten

sion

(g)

minus9 minus8 minus7 minus6 minus5

lowastlowastlowast

(a)

0

02

04

06

08

1

12 PE-denude

Ten

sion

(g)

log [PE] (M)minus9 minus8 minus7 minus6 minus5

(b)

Figure 1 PE-induced contraction responses in intact (a) anddenude (b) aortic rings from SHR treated with control (◼) losartan(998771) andWOS (∙) andWMOS (Q) Responses to PE are expressed asdifference between absolute tension developed and baseline tensionValues are mean plusmn SEM of 5 to 8 SHRs in each group lowastDenotes 119875 lt005 compared to control analyzed by one-way ANOVA followed byDunnett post hoc test

3 Results

31 Vasoconstriction Effects of PE onAortic Rings Cumulativeadditions of PE (10minus9 to 10minus5M) produced a concentration-dependent contraction of aortic rings in all groups In PEintact WOS WMOS and losartan significantly decreased(119875 lt 005) the contractile responses as compared to controlwhereas in PE denude endothelium no significant changeswere obtained (Figure 1) Maximal contractile responses(119864max) in PE intact were significantly decreased in WOSWMOS and losartan (030 plusmn 006 033 plusmn 003 035 plusmn 003versus 090 plusmn 010) In contrast the 119864max of WMOS signif-icantly enhanced the contraction responses in PE denude(057 plusmn 002 versus 043 plusmn 004) The pD

2values from both

0

20

40

60

80

100

120

140

160

180

log [ACh] (M)

Rel

axat

ion

()


lowast

Figure 2 ACh-induced relaxation responses of aortic rings pre-contracted with PE (1120583M) in control (◼) losartan (998771) WOS (∙)and WMOS (Q) Responses to ACh are expressed as percentage ofrelaxation Values are mean plusmn SEM of 5 to 8 SHRs in each grouplowastDenotes 119875 lt 005 compared to control

PE intact and denude endothelium were unaltered as shownin Table 1

32 Vasorelaxant Effects of ACh Precontracted with PE on Aor-tic Rings ACh (10minus9 to 10minus5M) produced dose-dependentrelaxation in all groups in aortic rings precontracted with PE(1 120583M) Only losartan significantly enhanced (119875 lt 005) therelaxant effect of ACh as compared to control (119877max 11120 plusmn408 versus 7315 plusmn 303) Both extract groups did notsignificantly alter the vasorelaxant effects of ACh as shownin Figure 2 and Table 2

33 Effects of L-NAME on ACh-Induced Relaxation in AorticRings in WOS WMOS and Losartan Groups To assess thecontribution of EDNO the aortic rings were preincubatedwith L-NAME (100 120583M) a NO synthase inhibitor for 30min-utes ACh-induced relaxations in all groups were significantlyinhibited (119875 lt 005) by L-NAME as shown in Figure 3 119877maxand pD

2values were tabulated in Table 2

34 Effects of Indomethacin on ACh-Induced Relaxation inAortic Rings in WOS WMOS and Losartan Groups Toinvestigate the role of prostacyclin (PGI

2) releases the aor-

tic rings were preincubated with indomethacin (10120583M) aCOX inhibitor for 30 minutes Indomethacin significantlyreduced (119875 lt 005) the ACh-induced relaxations in losartanand in contrast significantly improved vasorelaxation inWMOS (Figure 4) 119877max and pD

2values were tabulated in

Table 2

35 Role of Intracellular and Extracellular Calcium Mobiliza-tion on the PE-Induced Contraction To assess the role ofintracellular and extracellular calcium mobilization the aor-tic rings were incubated in Ca2+-free medium containing


Table 1 Maximal contractile (119864max) responses and sensitivity (pD2) for PE intact- and PE denude-induced contractionrsquos in aortic rings

Group PE intact PE denude119864max (g) pD

2(minuslog EC50) 119864max (g) pD

2(minuslog EC50)

Control (vehicle) 090 plusmn 010 667 plusmn 031 043 plusmn 004 773 plusmn 032

Losartan (10mg kg) 035 plusmn 003lowast

684 plusmn 022 043 plusmn 002 839 plusmn 019

WOS (1000mgkg) 030 plusmn 006lowast


WMOS (1000mgkg) 033 plusmn 003lowast

724 plusmn 022 057 plusmn 002lowast

772 plusmn 010

Each value represents the mean plusmn SEM of 5 to 8 SHRs lowastDenotes 119875 lt 005 compared to control for each drug

Table 2 Maximal relaxant effects (119877max) and sensitivity (pD2) for ACh-induced relaxation in aortic rings in the absence and presence ofindomethacin and L-NAME

Treatment groupsACh ACh + indomethacin ACh + L-NAME

119877max( of relaxation) pD2 (minuslog EC50)

Rmax( of relaxation) pD2 (minuslog EC50)


Control 7315 plusmn 303 772 plusmn 015 9174 plusmn 239 733 plusmn 008 NI NILosartan(10mgkg) 11120 plusmn 408

798 plusmn 017 9020 plusmn 764lowast 731 plusmn 028 2955 plusmn 246

lowast673 plusmn 030

WOS(1000mgkg) 7642 plusmn 339 762 plusmn 017 9104 plusmn 307 726 plusmn 011 712 plusmn 135


WMOS(1000mgkg) 7956 plusmn 297 724 plusmn 012 9637 plusmn 633

lowast732 plusmn 024 NI NI

Values are mean plusmn SEM of 5 to 8 SHRs in each group Denotes 119875 lt 005 compared to control and lowastdenotes 119875 lt 005 compared to ACh without inhibitorsNI not identified by nonlinear regression analysis by GraphPad Prism 50

01mM EGTA Under this condition PE induced tran-sient contraction mainly from sarcoplasmic reticulum Inendothelium-denuded aortic rings a transient contractileresponse in Ca2+-free medium was elicited by 10minus6M PE Asecond contraction known as sustained contraction was theninduced again by PEThe percentage contractile responses toPE were significantly reduced (119875 lt 005) in losartan (3069 plusmn441) and WOS (2535 plusmn 161) as compared to control(4869 plusmn 759) in response to PE in Ca2+-free medium(Figure 5)When the same procedure was repeated in normalCa2+-containing medium which contained 25mM CaCl

2

no significant difference was seen in the treatment groups

4 Discussion

The present study demonstrated that in intact endotheliumthe contractile response to phenylephrine (PE) a selectiveagonist for a

1-adrenergic receptor was significantly lowered

in SHR treated with WOS and WMOS as compared to con-trol No significant change was seen in denude endotheliumThese results showed that 14-day oral treatment of WOSand WMOS affected the 120572

1-adrenergic receptors activities

in this preparation The use of PE as a vasoconstrictor inthe present study because the ratrsquos aorta has predominantly1205721-postjunctional receptors [14] Furthermore Griffith et al

[19] and Martin et al [20] demonstrated that suppressionof constrictor responses to several agonists such as PE inthe intact vascular endothelium may be due continuouslybasal release of endothelium-derived relaxing factor (EDRF)from endothelial cells As seen in the present study WOSandWMOS inhibited the contraction induced by PE as com-parable to losartan There were studies found that possible

crosstalk between AT1and 1205721-adrenoceptors existed [21ndash23]

FurthermoreMaeso et al [24] reported that losartan reducedvasoconstrictor responses to PE in SHR aortic rings viaendogenous Angiotensin II (AngII) acting on AT

1receptors

Activation of AT1receptors results in increasing systolic

blood pressure (SBP) blood vessels growth and associatedvascular smoothmuscle cells (vsmc) apoptosis [25] Blockadeof AT

1receptors which inhibit the effects of AngII may

promote good prognosis in pathological conditions such ashypertension and to inhibit vasoconstriction [26] Hence wemay suggest that (1) Orthosiphon stamineus leaves extractsexert their antihypertensive effects by blunting the increase ofblood pressure in SHR and (2)Orthosiphon stamineus leavesextracts may play their role in reducing vasoconstrictionsimilar as AT

1receptor blocker WOS and WMOS may

possibly possess antihypertensive properties and exert similareffects through these interactions

Our results showed that the presence of endothelium isvery important in vasorelaxation It is likely that contributionby the variable EDRF such as NO prostacyclin and EDHFcaused vascular smooth muscle cells to relax The necessaryendothelial cells for the relaxation by acetylcholine (ACh) tobe occurred have been discovered since 1980 by Furchgottand Zawadzki They demonstrated that loss of endotheliumby rubbing the intimal surface of aorta caused no relaxationinduced by ACh ACh acts on muscarinic receptors of thesecells thus stimulates substances that caused relaxation ofvascular smooth muscle cells In the present study aorticrings from bothWOS andWMOS showed essentially similarrelaxant effects with control by dose-response manner toACh (10minus9 to 10minus5M) It may speculate that both extractsgiven orally did not alter the endothelium of the rats thus


0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(c)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(d)

Figure 3 ACh-induced relaxation responses of aortic rings precontracted with PE (1120583M) in the presence of L-NAME (100120583M) in control(vehicle) (a) losartan (10mgkg) (b) WOS (1000mgkg) (c) andWMOS (1000mgkg) (d) Responses to ACh are expressed as percentage ofrelaxation Values are mean plusmn SEM of 5 to 6 SHRs in each group lowastDenotes 119875 lt 005 compared to ACh without L-NAME (◼)

the aortic rings isolated from these rats showed no effectto the ACh-induced relaxation In contrast losartan showedsignificantly greater relaxation It is plausible that blockade ofAT1receptor further enhanced the relaxation caused by ACh

Furthermore Schiffrin and Touyz [27] demonstrated thatlosartan enhanced the endothelium-dependent relaxation toACh in SHR aortic rings In this preparation the relaxation toACh was probably due to the production or release of EDRF[12] and the endothelial vasorelaxant factors derived fromcyclooxygenase (COX) pathways (prostacyclin PGI

2released

from endothelial cells)WOS showed similar result of ACh-induced relaxation

after preincubation with indomethacin This similar effecthas been reported by Luscher and Vanhoutte [28] HoweverWMOS improved vasorelaxation to ACh after blockade ofCOXpathways In this case there was plausibility because thevasodilator PGI

2was continuously released as indicated by

its tonic effects on platelet cyclic adenosine monophosphate(cAMP) [29] In contrast losartan significantly reducedthe ACh-induced relaxations which may be due to theattenuation of PGI

2production which was compensated for

by the enhanced release of another vasodilator for examplenitric oxide (NO) In this point of view we might suggestthat WMOS and blockade of AT

1receptors modulate the

derived endothelial vasorelaxant factors such as PGI2from

COX pathwaysThe release of NO by endothelial cells to vascular smooth

muscle cells causes vasorelaxation NO to play a vital role inthe maintenance of vascular tone [30] In order to assess thecontribution ofNO releases in the vasorelaxant effects elicitedby Orthosiphon stamineus leaves extracts we preincubatedthe rat aortic rings with L-NAME (100120583M) NO synthaseinhibitor Our study showed that the ACh-induced vasore-laxation in all treatment groups reduced significantly after


0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(c)

0102030405060708090

100110120130

log [ACh] (M)

lowast

Rela

xatio

n (

)


(d)

Figure 4 ACh-induced relaxation responses of aortic rings precontracted with PE (1120583M) in the presence of indomethacin (10120583M) in control(vehicle) (a) losartan (10mgkg) (b) WOS (1000mgkg) (c) andWMOS (1000mgkg) (d) Responses to ACh are expressed as percentage ofrelaxation Values are mean plusmn SEM of 5 to 6 SHRs in each group lowastDenotes 119875 lt 005 compared to ACh without indomethacin (◼)

inhibition of NO synthase pathways From the present datait could be clearly proven that NO synthase pathways wereinvolved in the vasorelaxation in the SHR

In view of the present study it is plausible that the vasore-laxant activities produced by Orthosiphon stamineus extractsmay take place in the vascular smooth muscle cells Toinvestigate the effects of both extracts and losartan on the roleintracellular Ca2+ on the contractility of the vascular smoothmuscle cells of the aortic ringsmedia absent of andwith Ca2+were used Significantly reduced contraction response to PEin denude aortic rings observed in the WOS and losartanwere possibly due to inhibition of intracellular Ca2+ releasefrom the sarcoplasmic reticulum at the level of vascularsmoothmuscle cells Decreased intracellular Ca2+ concentra-tion and increased myosin light chain phosphatase activity

may had caused the smooth muscle to undergo weaker vas-cular contractility Also inhibition of receptor- and voltage-operated Ca2+ channels in the plasma membrane reducedCa2+ influx may contribute as well [31]

5 Summary

In conclusion our studies showed that water extract (WOS)and water methanolic (1 1) extract (WMOS) ofOrthosiphonstamineus Benth leaves promote antihypertensive effectsby reducing vasoconstriction through the alteration of1205721minusadrenergic and AT

1receptors activities Vasorelaxant

effects of bothWOS andWMOSmay possibly involvemainlythe release of EDNO In WOS PGI

2releases might not be

participated in the ACh-induced vasorelaxation However


0

20

40

60

80

Group

Con

trac

tion

()

Control Losartan WOS WMOS

Ca2+-containing medium

(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)

Figure 5 Histograms showing themean of the response induced by 10minus6MPEwithout endothelium inCa2+-containingmediumor Ca2+-freemedium Results are expressed as mean plusmn SEM of 6 experiment sets lowast119875 lt 005 compared to control

in WMOS enhancement of vasorelaxation might be due tothe fact that vasodilator PGI

2is continuously released as

indicated by its tonic effects on platelet cAMP In additionWOS inhibited the contraction of aortic rings induced by PEimplying that WOS inhibits the release of intracellular Ca2+andor blocks ROCC

Acknowledgments

This study was supported financially by the Universiti SainsMalaysia Fellowship Institut Pengajian Siswazah (IPS) Grad-uate Fund fromUniversiti SainsMalaysia (the correcpondingauthor was a recipient of the fellowship) and Fundamen-tal Research Grant Scheme (FRGS 203PFarmasi61711142)from the Ministry of Sciences and Technology (MOSTI)Malaysia The authors wish to thank Mr Roseli HassanMadame Noor Hafizoh Saidan Madame Nurul Hasnida MdYusoff Miss Farah Wahida Suhaimi Mr Mohd Shahidyand Mr Muhammad Ammar Rifqi for their kind help andsupport

Conflict of Interests

The authors declare that they have no conflict of interests

References

[1] S Awale Y Tezuka A H Banskota S Shimoji K Taira andS Kadota ldquoNorstaminane- and isopimarane-type diterpenes ofOrthosiphon stamineus fromOkinawardquoTetrahedron vol 58 no27 pp 5503ndash5512 2002

[2] C Wiart ldquoOrthosiphon stamineus Benthrdquo in Medicinal Plantsof Southeast Asia F K Wong Ed pp 264ndash265 Prentice HallKuala Lumpur Malaysia 2002

[3] K Sriplang S Adisakwattana A Rungsipipat and S Yibchok-anun ldquoEffects of Orthosiphon stamineus aqueous extract onplasma glucose concentration and lipid profile in normal and

streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 109 no 3 pp 510ndash514 2007

[4] G A Schut and J H Zwaving ldquoPharmacological investigationof some lipophilic flavonoids from Orthosiphon aristatusrdquoFitoterapia vol 64 no 2 pp 99ndash102 1993

[5] O M Arafat S Y Tham A Sadikun I Zhari P J Haughtonand M Z Asmawi ldquoStudies on diuretic and hypouricemiceffects of Orthosiphon stamineus methanol extracts in ratsrdquoJournal of Ethnopharmacology vol 118 no 3 pp 354ndash360 2008

[6] MAHossain Z Ismail A Rahman and S C Kang ldquoChemicalcomposition and anti-fungal properties of the essential oilsand crude extracts of Orthosiphon stamineus Benthrdquo IndustrialCrops and Products vol 27 no 3 pp 328ndash334 2008

[7] D D Doan N H Nguyen H K Doan et al ldquoStudies onthe individual and combined diuretic effects of four Viet-namese traditional herbal remedies (Zea mays Imperata cylin-drica Plantago major and Orthosiphon stamineus)rdquo Journal ofEthnopharmacology vol 36 no 3 pp 225ndash231 1992

[8] Y Tezuka P Stampoulis A H Banskota et al ldquoConstituentsof the Vietnamese medicinal plant Orthosiphon stamineusrdquoChemical and Pharmaceutical Bulletin vol 48 no 11 pp 1711ndash1719 2000

[9] S Awale Y Tezuka A H Banskota K Kouda K M Tun andS Kadota ldquoFive novel highly oxygenated diterpenes of Ortho-siphon stamineus from Myanmarrdquo Journal of Natural Productsvol 64 no 5 pp 592ndash596 2001

[10] S Taddei A Virdis L Ghiadoni G Salvetti and A SalvettildquoEndothelial dysfunction in hypertensionrdquo Journal of Nephrol-ogy vol 13 no 3 pp 205ndash210 2000

[11] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980

[12] M J Peach A L Loeb H A Singer and J Saye ldquoEndothelium-derived vascular relaxing factorrdquo Hypertension vol 7 no 311pp 94ndash100 1985

[13] R F Furchgott J V Zawadzki and P D Cherry ldquoRole ofendothelium in the vasodilator response to acetylcholinerdquo in


Vasodilatation P M Vanhoutte and I Leusen Eds pp 49ndash66Raven Press New York NY USA 1981

[14] LAlosachie andTGodfraind ldquoThemodulatory role of vascularendothelium in the interaction of agonists and antagonists with120572-adrenoceptors in the rat aortardquo British Journal of Pharmacol-ogy vol 95 no 2 pp 619ndash629 1988

[15] M H Abdulla M A Sattar N A Abdullah et al ldquoInhibitionof Ang II and renal sympathetic nerve influence dopamine-andisoprenaline-induced renal haemodynamic changes in normalWistar-Kyoto and spontaneously hypertensive ratsrdquo Autonomicand Autacoid Pharmacology vol 28 no 4 pp 95ndash101 2008

[16] S Jerez P De Bruno Marıa and C Alfredo ldquoCross talkbetween angiotensin II and alpha 1 adrenegic receptors in rabbitaortarole of endotheliumrdquo Journal of Cardiovascular Pharma-cology vol 43 no 3 pp 402ndash409 2004

[17] T Matsubara T Bohgaki M Watarai H Suzuki K Ohashiand H Shibuya ldquoAntihypertensive actions of methylripari-ochromene A from Orthosiphon aristatus an Indonesian tradi-tional medicinal plantrdquo Biological and Pharmaceutical Bulletinvol 22 no 10 pp 1083ndash1088 1999

[18] M J A Siddiqui and Z Ismail ldquoSimultaneous analysis of bioac-tive markers from Orthosiphon stamineus benth leaves extractsby reverse phase high performance liquid chromatographyrdquoTropical Journal of Pharmaceutical Research vol 10 no 1 pp97ndash103 2011

[19] T M Griffith D H Edwards and M J Lewis ldquoThe nature ofendothelium-derived vascular relaxant factorrdquoNature vol 308no 5960 pp 645ndash647 1984

[20] W Martin R F Furchgott G M Villani and D JothianandanldquoDepression of contractile responses in rat aorta by sponta-neously released endothelium-derived relaxing factorrdquo Journalof Pharmacology and Experimental Therapeutics vol 237 no 2pp 529ndash538 1986

[21] M H Abdulla M A Sattar I M Salman O Z Ameer NA Abdullah and E J Johns ldquoThe interaction between renin-angiotensin and sympathetic systems in the renal vasculature ofWistar-Kyoto ratsrdquoAsian Journal of Pharmaceutical andClinicalResearch vol 2 no 2 pp 1ndash5 2009

[22] M H Abdulla M A Sattar N A Abdullah M A H Khan HH AbdAllah and E J Johns ldquoChronic treatment with losartanand carvedilol differentially modulates renal vascular responsesto sympathomimetics compared to treatment with individualagents in normalWistar Kyoto and spontaneously hypertensiveratsrdquo European Journal of Pharmacology vol 612 no 1ndash3 pp69ndash74 2009

[23] M H Abdulla M A Sattar N A Abdullah M A H Khan KR L Anand Swarup and E J Johns ldquoThe effect of losartan andcarvedilol on vasopressor responses to adrenergic agonists andangiotensin II in the systemic circulation of Sprague DawleyratsrdquoAutonomic and Autacoid Pharmacology vol 31 no 1-2 pp13ndash20 2011

[24] R Maeso J Navarro-Cid R Munoz-Garcıa et al ldquoLosartanreduces phenylephrine constrictor response in aortic ringsfrom spontaneously hypertensive rats role of nitric oxide andangiotensin II type 2 receptorsrdquoHypertension vol 28 no 6 pp967ndash972 1996

[25] Q N Diep J-S Li and E L Schiffrin ldquoIn vivo study of AT1

andAT1angiotensin receptors in apoptosis in rat blood vesselsrdquo

Hypertension vol 34 no 4 pp 617ndash624 1999[26] T Unger ldquoThe role of the renin-angiotensin system in the

development of cardiovascular diseaserdquo American Journal ofCardiology vol 89 no 2 supplement 1 pp 3ndash9 2002

[27] E L Schiffrin and R M Touyz ldquoFrom bedside to bench to bed-side role of renin-angiotensin-aldosterone system in remodel-ing of resistance arteries in hypertensionrdquo American Journal ofPhysiology vol 287 no 2 pp H435ndashH446 2004

[28] T F Luscher and P M Vanhoutte ldquoEndothelium-dependentcontractions to acetylcholine in the aorta of the spontaneouslyhypertensive ratrdquoHypertension vol 8 no 4 pp 344ndash348 1986

[29] U Pohl C Nolte A Bunse M Eigenthaler and U Wal-ter ldquoEndothelium-dependent phosphorylation of vasodilator-stimulated protein in platelets during coronary passagerdquo Amer-ican Journal of Physiology vol 266 no 2 pp H606ndashH612 1994

[30] S Moncada R M J Palmer and E A Higgs ldquoNitric oxidephysiology pathophysiology and pharmacologyrdquo Pharmaco-logical Reviews vol 43 no 2 pp 109ndash142 1991

[31] R CWebb ldquoSmoothmuscle contraction and relaxationrdquoAmer-ican Journal of Physiology vol 27 no 1ndash4 pp 201ndash206 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


postsynaptically are G-protein-coupled receptors and thusactivation of cellular signaling is subsequent to the inter-action with a G-protein Activation of these receptors onvascular smooth muscle leads to vasoconstriction PE haspredominantly 120572

1-postjunctional receptors in ratrsquos aorta [14]

Since PE is a selective 1205721-adrenergic receptor agonist and

losartan is AT1receptor blocker there is possible relationship

between AT1and 120572

1receptors [15] In addition crosstalk

between AT1and 1205721receptors in the smoothmuscle of rabbit

aorta is endothelium dependent [16] It has been reportedthat MRC A isolated from Orthosiphon stamineus causescontinuous decreases in systolic blood pressure (SBP) andheart rate (HR) after subcutaneous administration in con-scious SHR [17] However studies on the antihypertensivemechanisms by Orthosiphon stamineus still remain unclearThe present study aimed to investigate the vascular reactivityof water extract (WOS) and water methanolic (1 1) extract(WMOS) of Orthosiphon stamineus Benth and AT

1recep-

tors blocker in the mechanisms of antihypertensive effectsmediated by 120572

1-adrenergic receptor and prostacyclin (PGI

2)

and endothelium-derived nitric oxide (EDNO) releases in theSHR aortic rings

2 Materials and Methods

21 Preparation of Orthosiphon stamineus Leaves ExtractsVoucher specimen (no 11009) of the plant material wasdeposited at Herbal Room School of Pharmaceutical Sci-ences Universiti Sains Malaysia (USM) WMOS was pre-pared by having dried and ground Orthosiphon stamineusleaves extracted by a mixture of methanol water (1 1) usinga Soxhlet extractor for a period of 12 hours whereas prepara-tion ofWOS involved hotmaceration of the dried and groundOrthosiphon stamineus leaves at 50∘C for 6 hours and wasrepeated thrice Each extract was bulked and concentratedin a rotary evaporator under vacuum and then freeze-driedand kept in a freezer until used [18] WOS and WMOS werefreshly prepared in distilled water prior to the feeding of theanimals

22 Animals Male spontaneously hypertensive rats (SHR230ndash280 g) were housed in individual cages with free accessto foods and water and maintained at Animal Transit Facilityof School of Pharmaceutical Sciences USM All proceduresinvolving animals were conducted according to the ethicalguidelines by the Animal Ethics Committee USM The ani-mals were divided into four groups (1)WOS 1000mgkg (2)WMOS 1000mgkg (3) losartan 10mgkg and (4) control(vehicle) All animals were given daily treatment orally for 14days before being subjected to vascular reactivity studies

23 Drugs and Chemicals Phenylephrine hydrochloride(PE) acetylcholine (ACh) indomethacin and N120596-nitro-L-arginine methyl ester (L-NAME) were purchased fromSigma-Aldrich Germany while sodium chloride (NaCl)potassium chloride (KCl) potassium dihydrogen phosphate(KH2PO4) magnesium sulphate (MgSO

4sdot7H2O) glucose

sodium hydrogen carbonate (NaHCO3) and calcium chlo-

ride dehydrate (CaCl2sdotH2O) were purchased from RampM

Chem UK All drugs were freshly prepared in normal salineexcept indomethacin in 05 (wv) sodium carbonate priorto use

24 Vascular Reactivity Using Aortic Rings The rat was anes-thetized with sodium pentobarbital (60mgkg ip) A mid-line abdominal incision was performed to expose the aortaThe thoracic aorta was carefully isolated cleaned from theadherent fat and connective tissues and cut into 3ndash5mmrings The aortic rings were then suspended horizontally intissue chambers containing 10mLofKrebrsquos solution (mmolLNaCl 1186 KCl 48 CaCl

225 MgSO

4sdot7H2O 12 KH

2PO4

12 NaHCO3251 and glucose 110) The tissue-bath solution

was bubbled incessantly with 95O2and 5CO

2(carbogen)

at 37∘C Aortic rings were then allowed to equilibrate at anoptimal tension of 1 g for 45min Krebrsquos solution was replacedevery 15min and the tension was readjusted to 1 g whennecessary At the beginning of the experiment the presenceof intact endothelial cells was confirmed by precontractingthe tissues with PE (1 120583M) and followed by relaxation withACh (1120583M) Relaxation not less than 60 indicated thepresence of intact endothelial cells Responses were recordedisometrically via a force transducer (Grass FT03D) connectedto a computerized data acquisition system (PowerLab ADIn-struments Pty Ltd Australia) For vasoconstriction studythe concentration-response curves for PE (cumulative finalchamber concentration of 10minus9 to 10minus5M) were recordedThe contraction effects of PE were recorded in two differ-ent preparations intact and denude endothelium Denudeendothelium of aortic rings was obtained by gently rubbingthe intimal layer of the tissue with a blunt needle for a fewtimes The aortic rings were considered denuded when therewere less than 10 relaxations to ACh (1 120583M) precontractedwith PE (1 120583M)whereas in order to obtain the concentration-response curves of relaxation ACh (10minus9 to 10minus5M) wasadded cumulatively to the chamber at the plateau of the PE(1 120583M) precontracted aortic rings at 3-minute intervals Tofurther assess the involvement of EDNO and prostacyclin(PGI2) releases relaxations of aortic rings were performed

in WOS WMOS and losartan groups preincubated for 30minutes with L-NAME (100 120583M) a nonspecific NO syn-thase inhibitor and indomethacin (10 120583M) a nonselectivecyclooxygenase inhibitor respectively

25 Data Analysis All data are given as mean plusmn standarderror means (SEM) PE-induced contraction and ACh-induced relaxation were analysed using one-way ANOVAfollowed by Dunnettrsquos post hoc test whereas the effects ofACh-induced relaxation after preincubated by L-NAME andindomethacin were analysed using Studentrsquos t-test 119864max119877max and pD2 values were derived from nonlinear regressionanalysis All analyses were using the computer softwareGraphPad Prism 50 for Windows (GraphPad Software IncUSA) Values of 119875 lt 005 were considered statistically signif-icant


0

02

04

06

08

1

12

log [PE] (M)

PE-intact

Ten

sion

(g)


lowastlowastlowast

(a)

0

02

04

06

08

1

12 PE-denude

Ten

sion

(g)


(b)


3 Results


2values from both

0

20

40

60

80

100

120

140

160

180

log [ACh] (M)

Rel

axat

ion

()


lowast










Table 2






2(minuslog EC50)








772 plusmn 010
















2


4 Discussion









1receptors









0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(c)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(d)




2released












0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(c)

0102030405060708090

100110120130

log [ACh] (M)

lowast

Rela

xatio

n (

)


(d)





5 Summary







0

20

40

60

80

Group

Con

trac

tion

()



(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

log [PE] (M)

PE-intact

Ten

sion

(g)


lowastlowastlowast

(a)

0

02

04

06

08

1

12 PE-denude

Ten

sion

(g)


(b)


3 Results


2values from both

0

20

40

60

80

100

120

140

160

180

log [ACh] (M)

Rel

axat

ion

()


lowast










Table 2






2(minuslog EC50)








772 plusmn 010
















2


4 Discussion









1receptors









0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(c)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(d)




2released












0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(c)

0102030405060708090

100110120130

log [ACh] (M)

lowast

Rela

xatio

n (

)


(d)





5 Summary







0

20

40

60

80

Group

Con

trac

tion

()



(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2(minuslog EC50)








772 plusmn 010
















2


4 Discussion









1receptors









0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(c)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(d)




2released












0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(c)

0102030405060708090

100110120130

log [ACh] (M)

lowast

Rela

xatio

n (

)


(d)





5 Summary







0

20

40

60

80

Group

Con

trac

tion

()



(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)

lowast


(c)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(d)




2released












0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(c)

0102030405060708090

100110120130

log [ACh] (M)

lowast

Rela

xatio

n (

)


(d)





5 Summary







0

20

40

60

80

Group

Con

trac

tion

()



(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(a)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


lowast

(b)

0102030405060708090

100110120130

log [ACh] (M)

Rela

xatio

n (

)


(c)

0102030405060708090

100110120130

log [ACh] (M)

lowast

Rela

xatio

n (

)


(d)





5 Summary







0

20

40

60

80

Group

Con

trac

tion

()



(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

Group

Con

trac

tion

()



(a)

0

20

40

60

80

Group

Con

trac

tion

()


Ca2+-free medium

lowast

lowast

(b)





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















vascular reactivity concerning orthosiphon stamineus benth

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