African Journal of Pharmacy and Pharmacology Vol. 6(6), pp.

376-379, 15 February, 2012

Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.362
ISSN 1996-0816 ©2012 Academic Journals

Full Length Research Paper

The in vivo antihypertensive effects of standardized

methanol extracts of Orthosiphon stamineus on
spontaneous hypertensive rats: A preliminary study
Nurul Alia Azizan1, Rashidi Ahmad2*, Khaleed Mohamed1, Mohd Zikri Ahmad2 and
Zaini Asmawi3
1
Advanced Medical and Dental Institute, Bertam, Penang, Malaysia.
2
School of Medical Sciences, Health Campus USM, Kelantan, Malaysia.
3
School of Pharmaceuticals Sciences, Main Campus, Penang, Malaysia.
Accepted 20 January, 2012

The purpose of this randomized control trial study was to investigate the effect of oral Orthosiphon
stamineus (OS) on the systolic blood pressure of spontaneous hypertensive rats (SHR). The rats were
divided randomly into five different groups of three rats each; group 1 received irbesartan (IR) drug (20
mg/kg) served as positive control group, group 2 received distilled water (control group) and group 3, 4
and 5 were treated with 250, 500 and 1000 mg/kg of OS extracts, respectively. Systolic blood pressure
(SBP) was measured prior to and 2 weeks after intervention. There was a significant mean reduction of
blood pressure among the intervention group. The efficacy of OS as antihypertensive is comparable
with irbesartan 20 mg/kg.

Key words: Orthosiphon stamineus, systolic blood pressure, spontaneous hypertensive rats.

INTRODUCTION

Hypertension is among the common diseases, reported
to have affected millions worldwide. World Health
Organization (2003) estimated that this disease may
cause 4.5% of current global disease burden in many
developing as well as developed countries. According to
the Malaysia Third National Health and Morbidity Survey
(2006), hypertension ranked the first place out of ten
diseases that causes hospitalization (Norzila et al., 2009).
Currently, the prevalence of hypertension is estimated to
have 4.8 million individuals in Malaysia especially aged
30 years old and above (Norzila et al., 2009) .
In order to treat hypertension, there are many classes
of synthetic drugs available in the pharmaceutical
industry, such as diuretics, beta blockers, calcium
channels blockers (CCBs), angiotensin-II receptor
blockers (ARBs) and angiotensin converting enzyme
inhibitors (ACEIs). On the other hand, depending on the
*Corresponding author. E-mail: shidee_ahmad@yahoo.com.
Tel: +60139383889. Fax: +6097673219.
synthetic drugs therapy to reduce blood pressure are
very costly (WHO, 2003). The main concern of synthetic
antihypertensive agents is undesirable side effects, such
as depression, delirium, potassium deficiency, sexual
dysfunction, insomnia, angioedema, cough and fetal
abnormalities (Syed Sulaiman et al., 2009; Seok-Koo et
al., 2009; Norzila et al., 2009).
Instead of conventional treatment, people nowadays
are more interested in the usage of dietary herbs and
medicinal plants as an alternative management for
hypertension. This is because of its minor side effects
and has well therapeutically performance (Maghrani et
al., 2005). In Malaysia, one of the local herbs which is
known as Orthosiphon stamineus (OS) is believed to
have antihypertensive properties (Indubala and Lein Teik,
2000, Yam et al., 2000; Sriplang et al., 2007; Hossain et
al., 2008; Sahib et al., 2009; Adam et al., 2009; Chun-
Hoong et al., 2010; Muhammad et al., 2011).
The purpose of this study was to investigate the effect
of two weeks oral OS on the blood pressure (BP) of
spontaneous hypertensive rats (SHR). We hypothesized
that OS administration at certain dosage has significant

mean reduction of BP of SHR.
MATERIALS AND METHODS
Study design
This randomized controlled trial (RCT) was completed at the
Laboratory Animal Research Unit (LARU), Health Campus,
Universiti Sains Malaysia Kubang Kerian Kelantan. Animal ethics
approval was obtained from the Animal Ethic Committee of
Universiti Sains Malaysia in December 2010 (Ref USM/ Animal
Ethics Approval/2010/ (63) (257)).
Source of the materials
Magnetic stirrer hot plate (Stuart Scientific®), weighing balance,
weighing boat, spatula, 100 ml cylindrical measurement, vortex,
250 ml beaker, Scott tissue roll, aluminium foil, parafilm, urine
container, 15 and 50 ml conical tubes, filter paper, desiccators
chamber, 16 inches oral gavage needle, 1 3 and 5 ml syringes, IITC
Model 179 Blood Pressure Analyzer (Life Science California, USA),
Blood Chemical Analyser, plain tubes (non- EDTA tubes), needles
32 G and 21 G, rats pellets Gold Coin, beddings, warm chamber,
restrainers, OS powdered extract, irbesartan drugs (Aprovel®),
distilled water, Ketamine, Xylazine, drinking bottles, cages, 70%
alcohol, gloves, mask, gauze, Dettol® soap, disposable lab coat,
autoclave bags, biohazard bags, cotton wool and alcohol swap.
Plant material and extraction
About 265 g of standardized methanol extracts of OS was provided
by professor Zaini Asmawi from School of Pharmaceutical
Sciences, Universiti Sains Malaysia. Herb Farm of Perlis provided
OS leaves. OS leaves were extracted with 50% methanol using a
maceration method (200 g of dried leaves in 2 L of 50% ethanol at
55°C for 24 h, 2 cycles). The extract was then concentrated and
freeze-dried, resulting in a yield of dried leaf (Mohamed et al.,
2011).
Selection of animals
Fifteen (15) adult male SHR (12 weeks old) weighing between 150
and 250 g were used in this preliminary study. An animal model of
hypertension; SHR is available where hypertension gets
established by the age of 12 weeks (Inuwa et al., 2005). SHR were
obtained from Laboratory Animal Research Unit (LARU), Health
Campus, Universiti Sains Malaysia and were acclimatized to
laboratory conditions for 7 days prior to the experiments. The rats
were randomly housed into three rats per cage for fourteen days
with access to food (standard rat diet Gold Coin) and water ad
libitum. They were maintained on a 12 h light and 12 h dark cycle
during the study. The handling and use of animals were in
accordance with the institutional guidelines.
Experimental animals
The rats were randomly divided into five different groups of three
rats each (n = 5 × 3) and were treated as follows: group 1 (n = 3)
received irbesartan drug (20 mg/kg body weight) and served as
positive control group, group 2 (n = 3) received distilled water and
served as negative control group and treatment groups (groups 3, 4
and 5) were treated with OS extract at three different doses,
Azizan et al. 377
respectively which are 250 mg/kg body weight (n = 3), 500 mg/kg
body weight (n = 3) and 1000 mg/kg body weight (n = 3).
The rats were given the drug solutions (OS or irbesartan) or vehicle
(distilled water) by oral gavage in a volume of 10 ml/kg body weight
once a day during two weeks. The influences of circadian rhythms
were avoided by starting all the experiments at 10 a.m. immediately
after administration (Maghrani et al., 2005; Norazmir and Ayub,
2010). Then, on day 15, after blood collection from cardiac puncture
procedure, the carbon dioxide gas was use to sacrifice the rats.
Systolic blood pressure (SBP) measurement
SBP was measured in rats by the tail cuff method before starting
the experiments (D0) and after oral administration; 2 weeks (D14).
Before the measurement, the rats were kept at 38°C for 10 min to
detect the pulsations of the tail artery. Blood pressure was taken
with rats under conscious conditions. Three measurements were
taken to get the average value for SBP. During acclimatisation, the
rats were placed in the restrainers for 10 to 15 min at 30°C daily to
ensure that the rats are comfortable with the restrainers, tail cuff
detector and warming chamber (Maghrani et al., 2005; Norazmir
and Ayub, 2010).
Statistical analysis
Statistical analysis was performed using the Statistical Package for
Social Sciences (SPSS) 12.0 software for Windows. Results were
expressed as mean and standard deviation (SD) of three
observations in each group. Student’s t-test was used to compare
the means (pre and post intervention). Differences were considered
statistically significant at P < 0.05.
RESULT
The mean of SBP prior to and after intervention in each
group were as shown in Table 1. We noted that all
studied groups achieved mean SBP of less than 140
mmHg (or less than mean SBP pre treatment) except the
control group (group 2).
The mean SBP of SHR who received OS treatment
achieved less than 120 mmHg despite elevated dosages.
Interestingly, group 3 who received the least OS dose
(250 mg/kg) had remarkable reduction of mean SBP than
the other groups.
Student’s T-test revealed there was a significant mean
reduction of SBP in all treated studied groups ( P≤ 0.05).
Confidence interval (CI) of all groups, except the control
group were more than 1. Group 1 had the widest CI and
group 4 had the narrowest CI. These results suggested
that group 4 had better clinical significance. In other
word, 500 mg of OS administration has statistic and
clinical significant as anti-hypertensive agent for SHR
(Table 2).
DISCUSSION
O. stamineus Benth. is known as Cat’s Whiskers or
“misai kucing”, and it is from the Lamiaceae family. It also
has other synonyms, such as Java tea, rau meo, cay bac
378 Afr. J. Pharm. Pharmacol.

Table 1. A distribution of mean SBP in each study group prior to and after 2 weeks of OS treatment.

Mean SBP ± SD
Group Before intervention After intervention
(Day 0) (Day 14)
Group 1 (20 mg/kg Irbesartan) 152.33 ± 2.08 110.00 ± 14.18
Group 2 (distilled water) 144.67 ± 18.58 144.67 ± 16.74
Group 3 (250 mg/kg OS) 149.00 ± 2.65 108.00 ± 7.94
Group 4 (500 mg/kg OS) 146.00 ± 7.81 119.33 ± 10.79
Group 5 (1000 mg/kg OS) 153.67 ± 11.02 116.00 ± 15.10

Table 2. The significance of antihypertensive effect of OS among SHR (Student’s T-test).

Mean of SBP difference t-statistic

Group **P-value
(95% CI*) (df)
Group 1 42.33 (1.95, 82.72) 4.51 (2) 0.046
Group 2 0.00 (-6.57, 6.57) 0.00 (2) 1.000
Group 3 41.00 (21.13, 60.87) 8.88 (2) 0.012
Group 4 26.67 (17.94, 35.39) 13.15 (2) 0.006
Group 5 37.67 (2.42, 72.91) 4.60 (2) 0.044
*CI: confidence interval; **P-value is less than 0.05 in groups 1,3,4 and 5.

(Thailand) and moustaches de chat (French). OS is a
native plant of Southeast Asia, a herbaceous shrub or
perennial herb is 0.3 to 1 m high. The leaves are simple
with sized 3 cm wide and 6 cm long. The flowers are
white or purple in colour and also have stamens that
exceed more than 2 cm from the corolla (Noraida, 2005;
Adam et al., 2009).
OS extraction contains several active chemical
compounds which have therapeutics effects and health
benefits. Tezuka et al. (2000) identified terpenoids
(diterpenes and triterpenes), polyphenol (lipophilic
flavonoids and phenolic acids) and sterols. Muhammad et
al. (2011) identified flavonoids (sinensetin, eupatorin),
caffeic acid and rosmarinic acid same with other studies.
This polyphenols extracts exhibited significant free
radical-scavenging activity and also is believed to
improve general health, treatment of kidney diseases,
bladder inflammation, gout, fever and diabetes (Akowuah
et al., 2005). Awale et al. (2002, 2003) isolated
secoorthosiphols A to C and siphonols A to E that have
an ability to inhibit nitric oxide action that is activated by
specific proinflammatory agents, such as endotoxins,
tumor necrosis factor (TNF), interferon-gamma (IFN-g)
and interleukin-1 (IL-1). Recently, Hossain et al. (2008)
reported that monoterpenes and sesquiterpenes were the
major phytochemicals from the extract of OS leaves and
stems by gas chromatography-mass spectrometry/mass
spectrometry (GC-MS/MS)-based determination.
Our study revealed that OS has antihypertensive effect
and the effect can be seen within 2 weeks of treatment.
We summarized the OS effects to blood pressure as
follows:
1. OS at 250, 500 and 1000 mg/kg have significant
antihypertensive effect.
2. OS at 250 mg/kg is the optimum antihypertensive.
3. OS at above 250 mg/kg does not further reduce the
SBP.
4. The efficacious of OS as antihypertensive is
comparable with irbesartan 20 mg/kg.
OS at 500 mg/kg has better statistic and clinical signi-
ficant. The possible mechanisms are OS extract increase
urine flow and urinary sodium excretion (Olah et al.,
2003; Arafat et al., 2008; Adam et al., 2009). According to
Yuliana et al. (2009), OS appears to influence the activity
of adenosine A (1) receptor antagonists to increase the
volume of urinary excretion (diuresis) and to reduce uric
acid level in serum.
To strengthen the current study and to ensure a
significant results for future related research we provide
some recommendations.
1. We advocate the continuation of similar study with a
calculation of sample size.
2. We advocate two different species of animals to
compare any changes of blood pressure level between
nomotensive and hypertensive rats.
3. We advocate two different methods of blood pressure
measurement, that is, direct (tail cuff method) and indirect

measurement (cannulation).
Conclusion
In vivo, OS has antihypertensive potential in SHR and its
efficacious is comparable to modern recent
antihypertensive agent.
ACKNOWLEDGEMENTS
The authors are thankful to all staffs of Advanced Medical
and Dental Institute of USM, School of Pharmaceuticals
Sciences, USM Penang, Hospital Universiti Sains
Malaysia, Department of Emergency Medicine,
Department of Physiology and Laboratory Animal
Research of USM Health Campus for their valuable
support to complete this study.
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