Sains Malaysiana 47(10)(2018): 2421–2428

http://dx.doi.org/10.17576/jsm-2018-4710-18

Antihypertensive Effect of Piper sarmentosum in

L-NAME-Induced Hypertensive Rats
(Kesan Antihipertensi Piper sarmentosum pada Tikus Hipertensi Aruhan L-NAME)

NIK ALOESNISA NIK MOHD ALWI, ZAITON ZAKARIA, AMINUDDIN ABDUL HAMID KARIM,
NOR ANITA MEGAT MOHD NORDIN & AZIZAH UGUSMAN*

ABSTRACT
Hypertension is one of the risk factors for cardiovascular diseases and has been associated with about 13% of global deaths
worldwide. Oxidative stress and reduced nitric oxide (NO) bioavailability contribute to the development of endothelial
dysfunction and subsequently hypertension. Nɷ-nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibits NO synthesis;
leading to hypertension. Piper sarmentosum (PS) is an herb with antioxidant, antiatherosclerosis and antiinflammation
properties. PS also stimulated NO production by endothelial cells. The aim of this study was to determine the effects of
aqueous extract of Piper sarmentosum (AEPS) on blood pressure, oxidative stress and the level of nitric oxide in L-NAME-
induced hypertensive rats. Hypertension was induced by oral administration of L-NAME (100 mg/L) in drinking water for
four weeks. The rats were concurrently treated with AEPS by oral gavage in serial doses (125, 250 and 500 mg/kg/day).
Blood pressure was measured using non-invasive tail-cuff method at baseline and fortnightly thereafter. Serum level of
NO and an oxidative stress marker, malondialdehyde (MDA) were measured at baseline and at the end of treatment. The
results showed that treatment with three different doses of AEPS successfully reduced systolic blood pressure (p<0.001),
diastolic blood pressure (p<0.05) and mean arterial pressure (p<0.05) in L-NAME-induced hypertensive rats. Treatment
with AEPS also reduced MDA level (p<0.001) and increased serum NO (p<0.001) in L-NAME-induced hypertensive rats.
The findings showed that AEPS decreased blood pressure by protecting against oxidative stress and increasing NO in
L-NAME-induced hypertensive rats.

Keywords: Hypertension; nitric oxide; Nɷ-nitro-L-arginine methyl ester hydrochloride; oxidative stress; Piper sarmentosum

ABSTRAK
Hipertensi merupakan salah satu faktor risiko penyakit kardiovaskular dan ia dikaitkan dengan kira-kira 13% kematian
di seluruh dunia. Stres oksidatif dan pengurangan ketersediaan biologi nitrik oksida (NO) menyumbang kepada
terjadinya disfungsi endotelium yang seterusnya menyebabkan hipertensi. Nɷ-nitro-L-arginina metil ester hidroklorida
(L-NAME) merencat sintesis NO dan menyebabkan hipertensi. Piper sarmentosum (PS) adalah herba yang mempunyai
sifat antioksidan, antiaterosklerosis dan antiinflamasi. PS juga merangsang pengeluaran NO oleh sel endotelium. Tujuan
kajian ini adalah untuk menentukan kesan ekstrak akueus Piper sarmentosum (AEPS) terhadap tekanan darah, stres
oksidatif dan aras nitrik oksida dalam tikus hipertensi aruhan L-NAME. Hipertensi diaruh dengan pemberian L-NAME
(100 mg/L) secara oral di dalam air minuman selama empat minggu. Dalam masa yang sama, rawatan tikus dengan
AEPS turut diberi serentak melalui gavaj oral dalam dos bersiri (125, 250 dan 500 mg/kg/hari). Tekanan darah diukur
menggunakan kaedah kuf ekor tidak invasif sebelum rawatan dimulakan dan setiap dua minggu selepas itu. Aras serum
NO dan penanda stres oksidatif, malondialdehida (MDA) diukur sebelum uji kaji dimulakan dan selepas rawatan tamat.
Keputusan menunjukkan rawatan dengan tiga dos AEPS yang berbeza berjaya menurunkan tekanan darah sistolik (p<
0.001), tekanan darah diastolik (p<0.05) dan tekanan arteri purata (p<0.05) dalam tikus hipertensi aruhan L-NAME. AEPS
juga menurunkan aras MDA (p<0.01) dan meningkatkan aras NO (p<0.001) dalam serum tikus hipertensi aruhan L-NAME.
Keputusan yang diperoleh menunjukkan bahawa AEPS berupaya menurunkan tekanan darah dengan mengurangkan stres
oksidatif dan meningkatkan aras NO pada tikus hipertensi aruhan L-NAME.

Kata kunci: Hipertensi; nitrik oksida; Nɷ-nitro-L-arginina metil ester hidroklorida; Piper sarmentosum; stres oksidatif

INTRODUCTION (CVD) which accounted for 31% of global deaths (Mendis

Hypertension is defined as persistent elevation of systolic 2014). Hypertension is one of the risk factors for CVD and
blood pressure of 140 mmHg or greater and/or diastolic has been associated with about 17 million deaths annually.
BP of 90 mmHg or greater. According to World Health It contributes to kidney failure, stroke, heart disease and
Statistic, it was reported that non-communicable diseases premature mortality. In the early stage, hypertension is
(NCD) were mostly caused by cardiovascular diseases asymptomatic and many people go undiagnosed (WHO 2012).
2422

Endothelial dysfunction (ED) has been observed in Zainudin et al. 2013). Briefly, the leaves of PS were oven-
the early stage of hypertension and it is the commonest dried for 36 h at 50°C. Then the dried leaves were cut into
contributing factor to hypertension (Park & Park 2015). small pieces and crushed. 10 g of dried leaves were mixed
Several studies had suggested that oxidative stress with 900 mL of distilled water. Hot water extraction was
contributed to the development of hypertension through prepared by boiling the mixture at 80°C for 3 h. Then, the
nitric oxide (NO) inactivation (Baradaran et al. 2014; Sinha extract was concentrated and freeze-dried into powder
& Kumar Dabla 2015). Increased free radical production as form. It was stored at 4°C until use.
well as imbalance between the level of NO and endothelial
vasoconstrictors such as endoperoxides, endothelins EXPERIMENTAL ANIMALS
and thromboxane A lead to endothelial dysfunction and
hypertension (Davidge et al. 2015; Panth et al. 2016; This study had been approved by the Animal Ethics
Vanhoutte et al. 2017). Committee, Universiti Kebangsaan Malaysia (approval
Many methods to induce hypertension had been code: PP/FISIO/2011/AMINUDDIN/22-MARCH/360-JUNE-
used in animal models. Nω-nitro-L-arginine methyl ester 2011-JUNE-2012). Healthy adult male Wistar rats, aged 6
hydrochloride (L-NAME), an L-arginine analogue, acts to 8 weeks, weighing between 170-220 g were obtained
as a competitive inhibitor of non-specific nitric oxide from the Laboratory Animal Resource Unit, Universiti
synthases (NOS). L-NAME has been widely used to create Kebangsaan Malaysia. The rats were maintained in an
NO-deficient hypertensive model. It is well established air-conditioned room at 23 ± 3°C and were housed in
that inhibition of nitric oxide biosynthesis by in vivo individual cages with a 12-h light and 12-h dark cycle.
administration of L-NAME causes endothelial dysfunction The rats were provided with normal rat chow and clean
and vasoconstriction, hence leading to hypertension (Raja drinking water ad libitum. The rats were acclimatized for
2010). one week before the experiment started.
Piper sarmentosum is a herb that had been reported
to have antioxidant activity (Hafizah et al. 2010). Toxicity EXPERIMENTAL DESIGN AND INDUCTION
studies on aqueous extract of PS showed that the extract is OF HYPERTENSION
safe for consumption (Mohd Zainudin et al. 2013). Previous Thirty six animals were divided into six groups with six
studies had reported various medicinal effects of PS such as animals in each group (n=6): control group; Aqueous
antihyperglycemia (Azlina et al. 2009), antiatherosclerosis extract Piper sarmentosum (AEPS) only group where the
(Amran et al. 2010), anticarcinogenesis (Ariffin et al. 2009) rats were given 500 mg/kg/day AEPS via oral gavage;
and antiinflammation (Ridtitid et al. 2007; Zakaria et al. L-NAME-induced hypertensive group was given 100 mg/L
2010). Apart from that, PS is also able to protect against L-NAME in drinking water and three groups of L-NAME-
glucocorticoid-induced osteoporosis (Mohamad Asri et induced-hypertensive rats with co-treatment of different
al. 2016) and paracetamol-induced oxidative liver injury doses of AEPS; 100 mg/L L-NAME and 125 mg/kg/day AEPS;
(Azlina et al. 2014). Previous studies had shown that PS is 100 mg/L L-NAME and 250 mg/kg/day AEPS; and 100 mg/L
able to reduce blood pressure in spontaneously hypertensive L-NAME and 500 mg/kg/day AEPS. The treatments were
rats (SHR) (Zainudin et al. 2015). Aqueous extract of PS is given for four weeks (Wheal et al. 2007). Group 2 was
able to stimulate NO production in human umbilical vein included in this study to investigate whether AEPS on its
endothelial cells (HUVECs) (Ugusman et al. 2012). This own could affect the parameters measured in this study. All
suggests that PS may regulate blood pressure via the NO the three doses of AEPS were adopted from previous study
pathway. Therefore, this study was designed to determine on anti-atherosclerosis effect of AEPS (Amran et al. 2010).
the effects of PS on blood pressure, oxidative stress and
NO level in L-NAME-induced hypertensive Wistar rats.
DETERMINATION OF BLOOD PRESSURE
Systolic blood pressure (SBP), diastolic blood pressure
MATERIALS AND METHODS (DBP) and mean arterial pressure (MAP) were measured
in non-anaesthetized rats by non-invasive tail-cuff CODA
PLANT MATERIALS blood pressure system (Kent Scientific Corporation, USA)
Fresh leaves of PS were collected in Selayang, Selangor, at baseline and every two weeks after starting treatment
Malaysia between January to February 2012 and were (Zainudin et al. 2015). At least five readings were recorded
identified by a plant taxonomist from the Medicinal Plant during each measurement. The maximum and minimum
Division, Forest Research Institute of Malaysia with plant values were discarded and the remaining three values were
identification number (PID) 170612-11. calculated as the average (Si & Liu 2008; Yang et al. 2008).

PREPARATION OF AQUEOUS EXTRACT BLOOD SAMPLES COLLECTION

OF PIPER SARMENTOSUM Blood samples (2 mL) were obtained in the morning
Aqueous extract of Piper sarmentosum was prepared at baseline and at the end of four weeks treatment via
following the method as reported previously (Mohd retro orbital sinus puncture with the animal under the
 2423

combination anaesthesia of Zoletil, Xylazil and Ketamine RESULTS

and were collected in plain tubes. The blood samples were
centrifuged at 3000 rpm for 10 min to obtain the serum. EFFECT OF AEPS ON SYSTOLIC BLOOD PRESSURE
After the separation, the serum was aspirated and was
At baseline, there was no significant difference in SBP
frozen at -80°C until further experiments.
among the groups. Following two weeks of treatment,
L-NAME-induced rats had higher SBP (155.3 ± 2.14 mmHg)
DETERMINATION OF NITRIC OXIDE LEVEL compared to control rats (121.8 ± 0.60 mmHg) (p< 0.001).
Since nitric oxide is unstable in aqueous condition and Treatment of L-NAME-induced rats with all three doses of
has a short half-life, the level of its stable metabolites, AEPS (125, 250 and 500 mg/kg/day) successfully reduced
nitrite (NO2-) and nitrate (NO3-) was measured based on SBP to normal level (128.0 ± 4.37, 127.8 ± 5.19 and
Griess method using QuantiChromTM nitric oxide assay kit 122.17 ± 4.48 mmHg, respectively) (p< 0.001). At the
(BioAssay Systems, USA) according to the manufacturer’s end of four weeks treatment, the SBP of L-NAME-induced
instructions. Total NO2-/NO3- in the serum samples were rats were persistently high compared to control (172.3 ±
quantitated by measuring the optical density at 540 nm. 5.06 vs. 116.7 ± 1.52 mmHg, p< 0.001). Treatment with
all three doses of AEPS significantly attenuated L-NAME-
DETERMINATION OF PLASMA induced rise in SBP at the end of treatment (126.0 ± 5.2,
MALONDIALDEHYDE (MDA) 127.83 ± 3.79 and 129.67 ± 3.74 mmHg, respectively)
The lipid peroxidation indicator, plasma MDA was (p< 0.001). However, there was no significant difference
measured using thiobarbituric acid reactive substances in SBP between the three doses of AEPS following two and
(TBARS) assay as described previously (Borges et al. four weeks of treatment. In addition, administration of
2018). Total protein concentration in the serum was AEPS alone did not significantly alter the SBP compared
measured using protein biuret reaction (Pessoa et al. to control.
2017). Plasma MDA level was calculated based on the
following formula: EFFECT OF AEPS ON DIASTOLIC BLOOD PRESSURE
At baseline, there was no significant difference in DBP
OD sample × standard × Total volume
among the groups. Following two weeks of treatment,
OD standard concentration volume sample × protein (g)
L-NAME-induced rats had higher DBP (112.2 ± 3.25 mmHg)
compared to control rats (83.6 ± 1.23 mmHg) (p< 0.01).
Plasma level of MDA was expressed as nmol/g protein
Treatment of L-NAME-induced rats with three doses of
AEPS (125, 250 and 500 mg/kg/day) successfully reduced
STATISTICAL ANALYSIS DBP (94.0 ± 5.41, 87.0 ± 4.97 and 86.3 ± 8.2 mmHg) (p<
All statistical analysis was conducted using IBM SPSS 0.05). At the end of the four weeks treatment, the DBP of
Statistic version 22.0. The data were expressed as mean ± L-NAME-induced rats were persistently high compared to
SEM. SBP, DBP and MAP results were analysed using one- control (127.5 ± 3.93 vs. 78.8 ± 2.41 mmHg, p<0.01).
way analysis of variance (ANOVA) with Tukey post-hoc Treatment of L-NAME-induced rats with all three doses
test while serum nitric oxide and malondialdehyde level of AEPS significantly reduced DBP to normal level at the
were analysed using paired t-test. P< 0.05 was considered end of treatment (84.5 ± 4.38, 90.0 ± 2.44, and 86.3 ±
statistically significant. 4.19 mmHg, respectively) (p<0.05). However, there was

*p<0.001 versus L-NAME group within the same week, # p<0.001 versus pre-treatment group

FIGURE 1. Effect of AEPS on systolic blood pressure (SBP) in L-NAME induced hypertensive rats
2424

*p<0.05 versus L-NAME within the same week, # p<0.01 versus pre-treatment group
FIGURE 2. Effect of AEPS on diastolic blood pressure (DBP) in L-NAME induced hypertensive rats

no significant difference in DBP between the three doses the end of treatment (97.0 ± 3.44, 101.2 ± 1.86 and 100.5
of AEPS following two and four weeks of treatment. In ± 2.71 mmHg, respectively) (p<0.05). However, there was
addition, administration of AEPS alone did not significantly no significant difference in MAP between the three doses
alter the DBP compared to control. of AEPS following two and four weeks of treatment. In
addition, administration of AEPS alone did not significantly
EFFECT OF AEPS ON MEAN ARTERIAL BLOOD PRESSURE
alter the MAP compared to control.
The changes in MAP were in accordance to changes in SBP
and DBP. At baseline, there was no significant difference in EFFECTS OF AEPS ON SERUM NO
MAP among the groups. Following two weeks of treatment, L-NAME induction caused reduction of serum NO compared
L-NAME-induced rats had higher MAP (126.0 ± 2.45 mmHg) to its pre-induction level (4.5 ± 1.92 vs 40.7 ± 6.26
as compared to control rats (96.7 ± 0.88 mmHg) (p<0.05). μM, p<0.05) as well as compared to control (p<0.001).
Treatment of L-NAME-induced rats with all three doses of Treatment with all three doses of AEPS (125, 250, 500 mg/
AEPS successfully reduced MAP (104.7 ± 4.97, 98.8 ± 3.93 kg/day) increased serum NO compared to L-NAME group
and 97.8 ± 6.89 mmHg) (p<0.05). At the end of the four (p<0.001) with the values of 56.33 ± 9.15, 80.88 ± 8.55
weeks treatment, the MAP of L-NAME-induced rats were and 75.02 ± 8.46 μM, respectively. Apart from that, there
persistently high compared to control (142.0 ± 4.49 vs. 91.2 was significant difference in the level of NO before and
± 1.78 mmHg, p< 0.05). Treatment of L-NAME-induced rats after treatment in AEPS, L-NAME + AEPS 250 mg/kg/day and
with all three doses of AEPS significantly reduced MAP at L-NAME + AEPS 500 mg/kg/day groups (p<0.05).

*p<0.05 vs L-NAME within the same week, # p<0.05 vs pre-treatment group

FIGURE 3. Effect of AEPS on mean arterial pressure (MAP) in L-NAME induced hypertensive rats
 2425

*p<0.001 versus L-NAME, # p<0.05 versus pre-treatment

FIGURE 4. Effect of AEPS on serum NO in L-NAME- induced-hypertensive rats

EFFECT OF AEPS ON SERUM MDA contributes to EH (Arora et al. 2009). Increased in free
Four weeks following L-NAME induction, serum MDA was radical generation inactivates prostacyclin and NO, hence
increased compared to its pre-induction level (65.59 ± causing half-life of prostacyclin and NO to be decreased.
5.46 vs. 23.81 ± 4.33 nmol/g protein, p<0.01) as well as This situation may lead to increase in peripheral vascular
compared to control (p<0.05). Treatment with all three resistance and subsequently hypertension (Kumar &
doses of AEPS (125, 250, 500 mg/kg/day) reduced serum Das 1993). Basal production of NO was reduced in
MDA level compared to L-NAME group (p<0.05) with the spontaneously hypertensive rats (SHR) (Dohi et al. 1990).
values of 22.70 ± 3.63, 16.57 ± 4.64 and 25.15 ± 11.39 The endothelium-dependant vasodilator responses
nmol/g protein, respectively. were attenuated in patients who suffered from essential
hypertension which was mainly contributed to reduced
bioactivity of NO (Panza et al. 1990).
DISCUSSION The main objective in this study was to evaluate whether
Hypertension is categorized into primary or essential the high blood pressure in NO-deficient hypertensive
hypertension (EH) and secondary hypertension. More than rats induced by L-NAME, an L-arginine analogue that
90% of hypertensive patients have EH whereby the cause inhibits nitric oxide synthases could be improved by
is still unclear. Current studies suggest that NO deficiency AEPS treatment. In the present study, the administration

*p<0.001 vs L-NAME; # p<0.05 vs L-NAME; + p<0.01 vs pre-treatment

FIGURE 5. Effect of AEPS on serum MDA level in L-NAME induced-hypertensive rats

2426

of L-NAME in drinking water had induced hypertension (Nox4) expressions in oxidative stress-induced HUVECs.
in rats which concurred with previous studies (Raja Nox4 is the predominant enzyme for ROS production
2010). Concomitant treatment with serial doses of AEPS in endothelial cells (Ugusman et al. 2010). Several
in the L-NAME-induced hypertensive rats had significantly chemical compounds with antioxidant activities found in
attenuated the hypertension. PS are polyphenols, vitamins C and E, carotenes, tannins,
Nitric oxide produced by endothelial nitric oxide xanthophylls, flavonoids and amides (Hussain et al. 2015).
synthase (e NOS ) is the major source of circulating Polyphenols had been shown to reduce blood pressure in
NO. L-NAME interferes with the activity of e NOS, thus NO-deficient model of hypertension (Bernátová et al. 2002;
reducing NO production (Raja 2010). Since NO is a Rodrigo et al. 2016). Since this study used crude extract
potent vasodilator, decreased NO production may impair of PS and not its isolated bioactive components, this study
endothelial-dependent vasodilation, causing increased was unable to specify the active compound responsible
peripheral resistance and blood pressure (Park & Park for the antihypertensive effect. However, it is suggested
2015). Previous study had proven that PS increased NO level that the antihypertensive effect of PS in NO-deficient
by stimulating eNOS expression and activity in HUVECs model of hypertension observed in this study is due to its
(Ugusman et al. 2010). Therefore, increased level of NO polyphenols content.
in L-NAME-induced hypertensive rats treated with AEPS
could be due to increase in the activity of eNOS. A study CONCLUSION
by Ugusman et al. (2014) had found that a flavonoid,
rutin, one of the compound in Piper sarmentosum, may AEPS reduces blood pressure in L-NAME -induced
improve endothelial function by augmenting NO production hypertensive rats and the antihypertensive effect may be
in HUVEC. Another recent study on PS showed that the partly mediated by increased NO and reduced oxidative
extract was able to reduce blood pressure by increasing stress. Our findings suggest that AEPS has the potential
NO production in SHR (Zainudin et al. 2015). to be developed as a therapeutic agent for hypertension.
Apart from inhibiting NO synthesis, L-NAME induces However, further studies using isolated bioactive
hypertension by causing oxidative stress. L-NAME is components from AEPS are required in order to support the
responsible to cause imbalance in renin-angiotensin therapeutic potential of AEPS for hypertension.
system (RAS) whereby it increased the expression of
angiotensin II and also caused renal dysfunction (Rincon ACKNOWLEDGEMENTS
et al. 2015). The excessive production of angiotensin
This study was funded by UKMMC Fundamental Research
II leads to increase vascular superoxide (O2-) formation
Fund (FF-174-2011). The authors express their sincere
through increased expression of NADPH-dependent oxidase
thanks to the staff of Physiology Department; Norizam
in aortic smooth muscle cells (Tsai et al. 2016). The
Salamt, Fadzilah Mohd Suratman, Zanariyah Asmawi,
excessive O2- react rapidly with NO to form peroxynitrite
Musmarlina Omar, Aini Farzana Zulkefli and Kamariah
(ONOO−). Peroxynitrite is a strong pro-oxidant molecule
Othman for the technical assistance.
which causes lipid peroxidation and tissue injury (Hogg
et al. 2017). In addition, hypertension itself leads to
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Zainudin, M.M., Zakaria, Z. & Nordin, N.A.M.M. 2015. The use Aminuddin Abdul Hamid Karim
of Piper sarmentosum leaves aqueous extract (Kadukmy™) Physiology Unit
as antihypertensive agent in spontaneous hypertensive rats. Universiti Pertahanan Nasional Malaysia
BMC Complementary and Alternative Medicine 15: 54. 57000 Kuala Lumpur, Federal Territory
Zakaria, Z., Patahuddin, H., Mohamad, A., Israf, D. & Sulaiman, Malaysia
M. 2010. In vivo anti-nociceptive and anti-inflammatory
activities of the aqueous extract of the leaves of Piper Nik Aloesnisa Nik Mohd Alwi
sarmentosum. Journal of Ethnopharmacology 128: 42-48. Basic Science and Oral Biology Unit
School of Dental Sciences
Universiti Sains Malaysia
Nik Aloesnisa Nik Mohd Alwi, Zaiton Zakaria, Nor Anita Megat 16150 Kubang Kerian, Kelantan Darul Naim
Mohd Nordin & Azizah Ugusman* Malaysia
Department of Physiology, Faculty of Medicine
Universiti Kebangsaan Malaysia Medical Centre *Corresponding author; email: [email protected]
Jalan Yaacob Latiff, Bandar Tun Razak, Cheras
56000 Kuala Lumpur, Federal Territory Received: 25 March 2018
Malaysia Accepted: 6 June 2018