International Journal of Applied Research in Natural Products

Vol. 3 (2), pp. 22-27, June-July 2010

Available online http://www.healthy-synergies.com
2010 Healthy Synergies Publications

Original Article
Antihypertensive Effect of an Aqueous Extract of
Passiflora nepalensis Wall
Patel SS1*, Verma NK1, Ravi V1, Gauthaman K1, Soni N2
1
2

Department of Pharmacology, Himalayan Pharmacy Institute, Majhitar, East Sikkim-737136,

Cipla Limited, Kumrek, Rangpo, East Sikkim-737132, India.

Summary: The present study was designed to investigate the antihypertensive effect of an aqueous extract of the
whole plant of Passiflora nepalensis wall (APN) in renal hypertensive rats. Hypertension in experimental animals
was induced by renal ischemia and reperfusion (IR). The blood pressure, pulse pressure and heart rate fell dosedependently in renal hypertensive and normotensive rats after intravenous administration of 80, 160 and 320 mg/kg
APN, suggesting that APN possesses antihypertensive, hypotensive and negative chronotropic effects. Renal IR
significantly increased the levels of thio-barbituric acid reactive substances and reduced the levels of superoxide
dismutase, catalase, and glutathione peroxidase (p<0.001 versus control) in kidney, which was altered after
intravenous administration of 160 and 320 mg/kg of APN, suggesting that APN protect kidney from IR injury
(p<0.001 versus renal IR). Histological evaluation showed that renal IR increased, whereas APN decreased the focal
glomerular necrosis, degeneration of tubular epithelium, necrosis in tubular epithelium, interstitial inflammatory
infiltration, and congestion of blood vessels.
Industrial relevance: Herbal medicines are getting more importance in the treatment of high blood pressure
because the modern synthetic medicines have side effects. A large proportion of the Indian population for their
physical and psychological health needs depend on traditional system of medicine. Medicinal plants have become
the focus of intense study in term of conservation as to whether their traditional uses are supported by actual
pharmacological effects or merely based on folklore. Herbal medicines are free from side effects and less costly
when compared to synthetic drugs. The present study will help the industry to produce herbal drugs with fewer side
effects, which are affordable and more effective in the treatment of hypertension.
Keywords: Mean arterial pressure; Passiflora; Renal hypertension; Superoxide dismutase

Introduction
High blood pressure is a prevalent risk factor for cardiovascular disease, affecting >72 million peoples in the
United State and >1 billion peoples worldwide (Rosamond et al., 2007). In the United States, based on results of the
National Health and Nutrition Examination Survey (NHANES), 28.7% (age-adjusted prevalence) of U.S. adults or
~58.4 million individuals have hypertension. Hypertension prevalence is 33.5% in non-Hispanic blacks, 28.9% in
non-Hispanic whites, and 20.7% in Mexican Americans. The burden of hypertension increases with age, and among
individuals aged 60, hypertension prevalence is 65.5%. The prevalence of hypertension and stroke mortality rates
is higher in the Southeastern United States than in another region (Kotchen, 2008). The Seventh Report of the Joint
National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7)
______________________
*Corresponding Author:
Tel: +91-3592-246462
E-mail: white_lotus4941@yahoo.com

Antihypertensive Effect of Passiflora nepalensis

recommends a blood pressure treatment goal of <140/90 mmHg for most patients and <130/80 mmHg for patients
with diabetes mellitus (DM) or kidney disease (Cheng, 2008). When determined by these criteria, hypertension
affects 20% to 30% of the adult population in most developed countries, and its prevalence appears to increase with
the age of the patient (Brown et al., 2000; Pardell et al., 2000; Schoen, 2000). Cardiovascular diseases have emerged
as an important health problem in India. High blood pressure is a major risk factor and better control can lead to
prevention of 300,000 of the 1.5 million annual deaths from cardiovascular diseases in India. Epidemiological
studies demonstrate that prevalence of hypertension is increasing rapidly among Indian urban populations and using
the current definitions more than two-fifths of the Indian urban adult population has hypertension. The prevalence is
lower in rural populations, but is increasing (Gupta et al., 2009).
The genus Passiflora consists of 500 species which are mostly found in warm and tropical regions. Passiflora
comes from Latin word "Passio" that was first time discovered by Spanish discoverers in 1529 and was described as
a symbol for "Passion of Christ" (Kinghorn, 2001; Dhawan et al., 2004). Passiflora was used widely in traditional
medicine in East India, Mexico, Netherland, South America, Italia and Argentina. One of species of this genus
named as Passiflora nepalensis (Passifloraceae) is more popular than its other species in Eastern India. Passiflora
nepalensis is used in folklore medicine for treating hypertension and inflammation (Patel, 2009; Patel et al., 2009).
Passiflora contains several compounds including alkaloids, phenols, glycosyl flavonoids and cyanogenic compounds
(Dhawan et al., 2004; Patel, 2009; Patel et al., 2009). Hence the present study was designed to evaluate the
antihypertensive activity of APN in acute renal hypertensive and normotensive rats.
Materials and Methods
Animals: Male Sprague-Dawley rats, weighing 300 to 350 g, were used for the experiment. They were housed in
standard environmental condition like, ambient temperature (25o 1oC), relative humidity (555%) and 12/12h light
dark cycle. Animals had free access to standard pellet diet and water ad libitum. All animal experiments were
carried out in accordance with the guidelines of CPCSEA. The institute animal ethical committee has given approval
for conducting animal experiments (HPI/08/60/IAEC/0060).
Preparation of Plant extract: The whole plant of Passiflora nepalensis were collected in the month of October
from the Eastern part of India (Sikkim Himalayas). The Herbarium Specimen (No- 168) of plant was deposited in
the department of Pharmacognosy and identified from Himalayan Pharmacy Institute, Majhitar. The shade-dried
powder of whole plant extracted in a Soxhlet extractor with water.
Acute toxicity study: The acute toxicity study of aqueous extract of whole plant of Passiflora nepalensis (APN)
was performed (Turner, 1965; Verappan et al., 2007). The dead animals obtained from primary screening studies,
LD50 value determination experiments, & the acute studies were subjected to post mortem studies. The external
appearance of the dead animals, the appearance of the viscera, heart, lungs, stomach, intestine, liver, kidney, spleen
& brain were carefully noted and any apparent and significant features or differences from the normal was recorded.
Induction of experimental hypertension and Sample preparation: Male Sprague-Dawley rats, weighing 300-350
g were used. The animals were anesthetized by intraperitoneal injection of 100 mg/kg hexobarbital sodium. PVCcoated Dieffenbach clip was placed onto the left hilum of the kidney. The renal artery was occluded for 3.54 h
(ischemia) following the surgery; the animals than anesthetized by intraperitoneal injection of 3040 mg/kg
pentobarbital sodium. To measure hemodynamic parameters, the cannula in the carotid artery was connected to a
pressure transducer (ML224-Quad Bridge Amplifier, ADInstrument, Australia) and Powerlab data acquisition
system (8/30 LabChart, ADInstrument, Australia). For administration of the test compound, a jugular vein was
cannulated. After 3.54 h the renal arterial clip was removed (reperfusion). This leads to a rise in blood pressure as a
consequence of elevated plasma renin level. Within 15 min a stable hypertension is achieved (Vogel et al., 2002).
The test substance was then administered by intravenous injection at doses of 80, 160, and 320 mg/kg to renal
hypertensive and normotensive rats. Hemodynamic parameters were monitored continuously. After measurement of
blood pressure, the left kidney was removed. Frozen tissue samples of the kidney were weighed and homogenized
(1:10, wt/vol) in 100 mmol/L phosphate buffer (pH 7.4) containing 0.05% sodium azide in an ice bath. The
homogenate was centrifuged at 5000 g for 10 min. The supernatant was frozen at -78C in aliquots until used for
biochemical assays. The protein content of the supernatant was determined by the Bradford method (Bradford,
1976). Thiobarbituric acid reactive substances (TBARS) levels in the tissues were determined by the method
described by Okhawa (Okhawa et al., 1979). Glutathione peroxidase (GSH-Px) was estimated by the method of
Ellman (Ellman, 1959). Superoxide dismutase (SOD) levels in the tissues were determined by the method described
by Kakkar (Kakkar et al., 1984). Catalase (CAT) level was estimated by the method described by Aebi (Aebi, 1974).
Histopathology: Kidneys were removed from rats at the end of the experimental period and were cut into two
sagittal sections. Renal tissues were fixed in 10% buffered-formaldehyde at room temperature. After dehydration
using graded ethanol, pieces of kidney were embedded in paraffin, cut in fine (5 m) sections, and mounted on glass
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Patel et al
slides. Sections were then deparaffinized with xylene, counterstained with hematoxylin and eosin, and viewed under
a light microscope at X200.
Statistical analysis: All the data were expressed as means SEM (n=6). The means of the different groups were
compared using one-way analysis of variance followed by Tukey tests.
Results and Discussion
The dead animals obtained from the acute toxicity experiments usually presented with their respiratory arrest and
convulsion. Apart from this characteristic observation, no other significant observation deviant from the normal was
seen in these dead animals. The LD50 value of APN was calculated as1621.81 mg/kg. The mean arterial pressure
(MAP), pulse pressure, systolic and diastolic blood pressure, and heart rate obtained in normotensive rats before and
after APN treatment are reported in Table 1. Reduction in MAP, pulse and blood pressure in normotensive rats is a
direct indication of hypotensive effect (Mojiminiyi et al., 2007). MAP, pulse and blood pressure, and heart rate
raised significantly in renal hypertensive rats (p<0.001 versus control), Whereas MAP, blood pressure, pulse
pressure and heart rate fell dose-dependently in renal hypertensive rats after intravenous administration of 80, 160
and 320 mg/kg APN, suggesting that APN possesses antihypertensive and negative chronotropic effects (p<0.001
versus IR; p<0.01 versus IR group), reported in Table 2. In rats, renal hypertension is induced by clamping the left
renal artery. After reopening of the vessel, accumulated renin is released into circulation. The protease renin
catalyzes the first and rate-limiting step in the formation of angiotensin II leading to acute hypertension (Vogel et
al., 2002). Blockade of renin angiotensin system is one of the important mechanisms for antihypertensive effect in
this regards (Kotchen, 2008). Renal IR significantly increased the levels of thio-barbituric acid reactive substances
and reduced the levels of superoxide dismutase, catalase, and glutathione peroxidase (p<0.001 versus control) in
kidney, which were altered significantly after intravenous administration of 160 and 320 mg/kg of APN, suggesting
that APN protect kidney from IR injury (p<0.001 versus renal IR; p<0.01 versus IR group), are reported in Table 3.
The APN increased the GSH-Px activity in kidney. GSH-Px is an enzyme which prevents the generation of
hydrogen peroxide and alkyl hydroperoxides in association with GSH and GSH-reductase, as well as the generation
of more harmful metabolites such as the hydroxyl radical (Therond et al., 2000; Parodi, 2007). The primary
reactive oxygen species O2- radical is depleted by a dismutation reaction catalyzed by SOD and the less reactive
H2O2 is produced. However, O2- can further interact with other
Table 1. Effect of APN on mean arterial pressure, pulse pressure, systolic BP, diastolic BP, and heart rate
Treatment

MAP
Pulse Pressure
Systolic BP
(mm Hg)
(mm Hg)
(mm Hg)
Normotensive rats
96.70.63
31.70.41
117.60.64
Normotensive+ APN 80 mg/kg
92.91.25
29.30.30
98.30.36*
Normotensive+ APN 160 mg/kg
55.80.52*
17.60.55*
76.30.86*
Normotensive+ APN 320 mg/kg
48.190.64*
64.81.1*
15.40.59
*p<0.001 versus control; p<0.01 versus control; p<0.05 versus control

Diastolic BP
(mm Hg)
95.30.45
86.20.40*
64.81.6*
57.80.56*

Heart Rate
(beats/ min)
439.31.02
436.10.94
428.30.42*
427.50.61*

Table 2. Effect of APN on mean arterial pressure, pulse pressure, systolic BP, diastolic BP, and heart rate
Treatment

MAP
Pulse Pressure
Systolic BP
(mm Hg)
(mm Hg)
(mm Hg)
Normotensive rats
97.40.36
31.80.24
119.591.04
Renal IR
158.90.55
43.40.55
143.10.30

Renal IR + APN 80 mg/kg

138.20.68*
41.00.04
136.50.50*
Renal IR + APN 160 mg/kg
133.51.06*
36.30.35*
127.70.49*
Renal IR + APN 320 mg/kg
113.21.39*
36.20.36*
122.70.50*

p<0.001 versus control; *p<0.001 versus IR; p<0.01 versus IR group

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Diastolic BP
(mm Hg)
97.30.39
112.60.29
107.90.88*
99.70.76*
99.20.43*

Heart Rate
(beats/ min)
438.30.66
446.80.70
443.50.76
442.10.40*
441.80.40*

Antihypertensive Effect of Passiflora nepalensis

Table 3. Results of the biochemical analysis in kidney
Treatment

TBARS
SOD
CAT
(nmol/mg-protein)
(IU/mg-protein)
(IU/mg-protein)
Control
21.620.21
6.440.08
0.220.00
Renal IR
28.360.47
3.520.04
0.180.00
Renal IR + APN 80 mg/kg
27.960.42
4.020.19
0.190.00
Renal IR + APN 160 mg/kg
24.670.47*
5.530.23*
0.220.00*
Renal IR + APN 320 mg/kg
24.110.45*
5.450.13*
0.220.00*

p<0.001 versus control; * p<0.001 versus IR group; p<0.01 versus IR group

GSH-Px
(IU/mg-protein)
3.150.06
2.630.06
2.700.07
3.010.08
3.030.04

Figure 1. Effect of APN on renal morphology: (A) Control rat kidney showed normal structure. (B) Renal IR group
showed Focal glomerular necrosis, degeneration of tubular epithelium, necrosis in tubular epithelium, interstitial
inflammatory infiltration, and congestion of blood vessels. (C) APN 80mg/kg + renal IR could not relieve the
damage. (D) APN 160 mg/kg showed a preservation of tissue histology. (E) APN 320 mg/kg showed near the
normal histological characteristics.
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Patel et al
molecules to generate secondary reactive species, either by directly or prevalently through enzyme- or metalcatalyzed processes (Valko et al., 2006). H2O2 is further converted to H2O and O2 by the enzymes CAT and GSHPx, and this reaction prevents the formation of the highly reactive OH- radicals. In addition, GSH-Px simultaneously
consumes reduced glutathione to give oxidized tripeptide glutathione in this reaction (Valko et al., 2006). In our
study, the levels of SOD, GSH-Px and CAT were elevated significantly in kidney, and the dose level of 160 and 320
mg/kg of APN were found most effective. Renovascular hypertension has become increasingly recognized as an
important cause of clinically atypical hypertension and chronic kidney disease, the latter by virtue of renal ischemia.
Renovascular hypertension is the clinical consequence of renin-angiotensin-aldosterone activation. Renal artery
occlusion creates ischemia, which triggers the release of renin and a secondary elevation in blood pressure.
Hyperreninemia promotes conversion of angiotensin I to angiotensin II, causing severe vasoconstriction and
aldosterone release followed by hypertension. APN ameliorate renal oxidation by stimulation of endogenous
antioxidant enzymes and thus attenuate renovascular hypertension (Schmidt et al., 2009). Histological evaluation
showed that renal IR increased, whereas APN decreased the focal glomerular necrosis, degeneration of tubular
epithelium, necrosis in tubular epithelium, interstitial inflammatory infiltration, and congestion of blood vessels,
reported in figure 1.
In conclusion, APN showed antihypertensive and negative chronotropic effects may be
due to blockade of renin angiotensin system/or renal antioxidant effects. Therefore further studies may carry-out to
prove the potential of this plant. The plant is becoming the endangered species now so more work can be done on
agricultural and climatic conditions to grow this plant.
Acknowledgement
Author(s) acknowledge to AICTE, New Delhi, for their financial support to carry-out research work on Passiflora
nepalensis and renal hypertension.
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