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R E S E A R C H A R T I C L E Open Access
Passiflora incarnata attenuation of
neuropathic allodynia and vulvodynia
apropos GABA-ergic and opioidergic
antinociceptive and behavioural mechanisms
Urooj Aman
1
, Fazal Subhan
1*
, Muhammad Shahid
1
, Shehla Akbar
1
, Nisar Ahmad
1
, Gowhar Ali
1
, Khwaja Fawad
1
and Robert D. E. Sewell
2
Abstract
Background: Passiflora incarnata is widely used as an anxiolytic and sedative due to its putative GABAergic
properties. Passiflora incarnata L. methanolic extract (PI-ME) was evaluated in an animal model of streptozotocin-
induced diabetic neuropathic allodynia and vulvodynia in rats along with antinociceptive, anxiolytic and sedative
activities in mice in order to examine possible underlying mechanisms.
Methods: PI-ME was tested preliminary for qualitative phytochemical analysis and then quantitatively by proximate
and GC-MS analysis. The antinociceptive property was evaluated using the abdominal constriction assay and hot
plate test. The anxiolytic activity was performed in a stair case model and sedative activity in an open field test. The
antagonistic activities were evaluated using naloxone and/or pentylenetetrazole (PTZ). PI-ME was evaluated for
prospective anti-allodynic and anti-vulvodynic properties in a rat model of streptozotocin induced neuropathic pain
using the static and dynamic testing paradigms of mechanical allodynia and vulvodynia.
Results: GC-MS analysis revealed that PI-ME contained predominant quantities of oleamide (9-octadecenamide),
palmitic acid (hexadecanoic acid) and 3-hydroxy-dodecanoic acid, among other active constituents. In the
abdominal constriction assay and hot plate test, PI-ME produced dose dependant, naloxone and pentylenetetrazole
reversible antinociception suggesting an involvement of opioidergic and GABAergic mechanisms. In the stair case
test, PI-ME at 200 mg/kg increased the number of steps climbed while at 600 mg/kg a significant decrease was
observed. The rearing incidence was diminished by PI-ME at all tested doses and in the open field test, PI-ME
decreased locomotor activity to an extent that was analagous to diazepam. The effects of PI-ME were antagonized
by PTZ in both the staircase and open field tests implicating GABAergic mechanisms in its anxiolytic and sedative
activities. In the streptozotocin-induced neuropathic nociceptive model, PI-ME (200 and 300 mg/kg) exhibited static
and dynamic anti-allodynic effects exemplified by an increase in paw withdrawal threshold and paw withdrawal
latency. PI-ME relieved only the dynamic component of vulvodynia by increasing flinching response latency.
Conclusions: These findings suggest that Passiflora incarnata might be useful for treating neuropathic pain. The
antinociceptive and behavioural findings inferring that its activity may stem from underlying opioidergic and
GABAergic mechanisms though a potential oleamide-sourced cannabimimetic involvement is also discussed.
Keywords: Passiflora incarnata, Allodynia, Vulvodynia, GABA receptors, Opioid receptors, Neuropathic pain
* Correspondence: fazal_subhan@upesh.edu.pk
1
Department of Pharmacy, University of Peshawar, Peshawar 25120, Khyber
Pakhtunkhwa, Pakistan
Full list of author information is available at the end of the article
© 2016 Aman et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77
DOI 10.1186/s12906-016-1048-6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Pain is an unpleasant sensory and emotional experience
associated with actual or potential tissue damage or de-
scribed in terms of such damage [1]. The phenomenon
of pain may be nociceptive or neuropathic in nature,
and caused by damage to non-neural or neuronal tissues
respectively [2, 3]. Neuropathic pain is a major cause of
morbidity and has a profound impact on patient well-
being. It involves the sensation of allodynia; a painful
sensation to a normally non-noxious stimulus and
hyperalgesia; an exaggerated pain response to a normally
noxious stimulus [4]. Neuropathic pain results from
various causes that affect the central nervous system in-
cluding multiple sclerosis, post stroke or spinal cord
pain. Alternatively, it may be associated with damage to
the peripheral nervous system, for instance, diabetic
neuropathy and trigeminal or post-herpetic neuralgia
[5]. Management of neuropathic pain poses an enor-
mous challenge due to the restricted efficacy of assorted
pharmacotherapies including both natural treatments
[6–8] and synthetic medicaments [9, 10] which are lim-
ited by the occurrence of side effects and the extent of
pain inhibition [11].
Passiflora incarnata L. (Additional file 1: Figure S1)
from the genus Passiflora (family: Passifloraceae) com-
monly known as Passion flower, is a fast growing peren-
nial vine widely spread in tropical and warm temperate
regions [12]. Phytochemical analysis of P. incarnata has
demonstrated that flavonoids constitute about 2.5 % of the
total phyto-constituents [13, 14] mainly present in the
leaves, the greatest concentration of flavonoid being
vitexin compared to the other species of its genus
[12, 15]. P. incarnata has been studied for its anal-
gesic [16], anxiolytic [17–20], anticonvulsant [21], an-
titussive [22], aphrodisiac [23], anti-asthmatic [24],
anti-diabetic and hypolipidemic properties [25] along
with efficacy in the treatment of cannabinoid [26],
morphine [27], nicotine [28] and alcohol dependence
[29]. Traditionally, P. incarnata has been used for
curing various ailments like anxiety, insomnia, convul-
sions, sexual dysfunction, cough and cancer [30] and
is well known in relieving neuropathic conditions
[12]. In this regard, an eye wipe test has been con-
ducted suggesting a potential application in relieving
trigeminal neuralgia [31]. Clinical investigations on P.
incarnata have indicated effectiveness in the treat-
ment of anxiety [32, 33], insomnia [34], opioid with-
drawal [35], attention deficit hyperactivity disorder
[36] and postmenopausal symptoms [37].
Neuropathic pain results from a cascade of neurobio-
logical events that induces electrical hyperexcitability in
somatosensory conduction pathways and results in
hyperesthesia, dysesthesia, hyperalgesia, paresthesia or
allodynia [38]. Currently, the most common choices of
therapy for neuropathic pain are tricyclic antidepressants
and anticonvulsants [39, 40]. However, these therapies
are only partially effective and are usually accompanied
by a variety of side effects [41]. The use of complemen-
tary and alternative medicine has been shown to pro-
duce some beneficial effects in the management of
painful neuropathy [42] and several herbal medicines ex-
hibit promise in different types of experimentally in-
duced neuropathic pain models [6, 8, 43–45]. Thus there
is some scope for new herbal medicines to combat neuro-
pathic pain syndromes [46]. The present study was there-
fore designed to evaluate the ameliorative effect of P.
incarnata methanolic extract (PI-ME) in an animal model
of streptozotocin-induced diabetic neuropathic allodynia
and vulvodynia [47] in rodents. Additionally, PI-ME in-
duced antinociceptive, anxiolytic and sedative activities
were also investigated using naloxone and pentylenetetra-
zole (PTZ) to probe its possible underlying mechanisms.
Methods
Chemicals
Morphine (Punjab Drug House, Lahore, Pakistan), diclo-
fenac sodium (≥98 %, Continental Chemicals Company
Pvt. Ltd. Pakistan), naloxone (98 %, Hangzhou Uniwise
International Co., Ltd, China), gabapentin (99 %, MKB
Pharmaceuticals Pvt Ltd Peshawar, Pakistan), diazepam
(Valium 10 mg/ 2 ml, Roche, Pakistan), pentylenetetra-
zole (≥98 %, Sigma Aldrich, UK) , streptozotocin (≥98 %,
Sigma Aldrich, UK) and commercial grade methanol
(Haq Chemicals Ltd Peshawar, Pakistan).
Preparation of Passiflora incarnata methanolic extract
P. incarnata whole plant was collected from the botanical
garden of the Department of Pharmacy, University of
Peshawar. It was authenticated by Prof. Dr. Mohammad
Ibrar of the Department of Botany, University of Peshawar
and a specimen was deposited in the herbarium with a vou-
cher number 20062 (PUP). The aerial parts were separated,
shade dried, and coarsely powdered (1000 g). It was macer-
ated for 7 days with commercial grade methanol (5 L). The
extract was filtered and concentrated under reduced pres-
sure at 60 °C in a rotary evaporator until a semisolid extract
containing no methanol was obtained (yield: 31.20 %).
Phytochemical analysis
PI-ME was preliminary evaluated by qualitative phyto-
chemical analysis [47] and was further screened by quanti-
tative analysis of flavonoids, alkaloids, saponins and tannins
[48, 49]. It was also subjected to gas chromatography/mass
spectrometry (GC/MS) analysis which was carried out on a
6890 N Agilent gas chromatograph coupled to a JMS
600 H JEOL mass spectrometer. The compound mixture
was separated on a fused silica capillary SPBI column,
30 m × 0.32 mm, 0.25 μm film thickness, in a temperature
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 2 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
programfrom50to256°Cwitharateof4°C/minwith
2 min hold. The injector was at 260 °C and the flow rate of
the carrier gas, helium was 1 mL/min. The EI mode of the
JMS 600 H JEOL mass spectrometer had an ionization volt-
age of 70 eV, electron emission of 100 μA, ion source
temperature of 250 °C and analyzer temperature of 250 °C.
Samples were injected manually in split mode and the total
elution time was 90 min. MS scanning was performed from
m/z 85 to 390. Identification of the active constituents was
based on the computer evaluation of mass spectra of the
sample through NIST-based AMDIS (automated mass
spectral deconvolution and identification software), direct
comparison of peaks and retention times with those of
standard compounds as well as by following the character-
istic fragmentation patterns of the mass spectra of particu-
lar classes of compounds.
Animals
BALB/c mice (18–26 g) and female Sprague Dawley rats
(150-200 g) maintained in a 12 h light/dark cycle at 22
± 2 °C were used in the experiments. Food and water
were provided ad libitum. Experiments on animals were
performed in accordance with the UK Animals (Scientific
Procedures) Act 1986 and according to the rules and
ethics set forth by the Ethical Committee of the Department
of Pharmacy, University of Peshawar. Approval for this
study was granted with the registration number: 06/EC-14/
Pharm (dated: April 06, 2014). The animal control groups
used in experiments were given normal saline which was
also the vehicle for all the drugs administered throughout
all the experiments.
Abdominal constriction assay
BALB/c mice (18–22 g, n= 8 mice per group) of either
sex were injected with 0.6 % acetic acid (10 mL/kg, i.p)
to induce an abdominal constriction response [50, 51].
In the abdominal constriction assay, the mean incidence
of constrictions expressed as % protection across all
experiments was normalized relative to untreated con-
trols. PI-ME (150, 200 and 250 mg/kg, p.o), morphine
(5 mg/kg, i.p) or diclofenac (50 mg/kg, i.p) were
administered 30 min before acetic acid injection. In
the opioid antagonism study, the animals were pre-
treated with naloxone (0.5 mg/kg, s.c), 5 min before
acetic acid administration. Percentage protection was
calculated as:
%Protection ¼1–Number of abdominal constrictions after
treatment=Number of abdominal constrictions
of untreated control100
Hot plate test
BALB/c mice (18–22 g, n= 8 mice per group) of either
sex were pretested for their response latencies on a hot
plate (Harvard apparatus, USA) maintained at 54.0 ±
0.1 °C. The response end-point was signified by hind
limb flick, lick or jumping at which point animals were
immediately removed from the thermal nociceptive
stimulus in order to avoid any tissue damage or possibil-
ity of subsequent hyperalgesia. A cut-off time of 30 s
was imposed such that if they did not respond within
this latency period then they were immediately removed
from the hot plate stimulus [51]. Thirty minutes after
pretesting, the animals were administered PI-ME (100,
150, 200 mg/kg; p.o), morphine (5 mg/kg; i.p) or diclofe-
nac (50 mg/kg, i.p). In the antagonism studies, naloxone
(1.0 mg/kg, s.c) or PTZ (10 mg/kg, i.p) were administered
10 or 30 min respectively before treatment and the animal
response latencies were measured at 30, 60, 90 and
120 min. The percentage antinociception was calculated as:
%Antinociception ¼Test latency –control latencyðÞ=
Cut‐off time –control latencyðÞ100
Anxiolytic activity (Staircase test)
BALB/c mice (18–24 g, n= 8 mice per group) of either
sex were administered PI-ME (200, 400 and 600 mg/kg,
p.o) or diazepam (2 mg/kg, i.p). In the drug combination
experiments, PTZ (10 mg/kg, i.p) was administered
30 min prior to drug treatment. The number of rears
and steps climbed by each animal was observed for
3 min using the staircase apparatus and the methods de-
scribed by Simiand and coworkers [52]. A step was con-
sidered to be climbed only if the criterion was met
whereby an animal placed all four paws on the step.
Locomotor activity
BALB/c mice (18–26 g, n= 6 mice per group) of either
sex were administered with PI-ME (200, 400 and
600 mg/kg, p.o) or diazepam (4 mg/kg, i.p). In the drug
combination experiments, PTZ (10 mg/kg, i.p) was ad-
ministered 30 min prior to drug treatment. Thirty min
later, the animals were placed in the recording apparatus
with a floor area of 50 × 40 cm divided into four equal
quadrants by lines. The number of lines crossed by each
animal was recorded for 30 min using a digital camera
(Cat’s Eye IR IP Camera, Taiwan) [53].
Streptozotocin induced neuropathic pain
Induction of mechanical allodynia and vulvodynia
Female Sprague Dawley rats (150–200 g, n= 6 rats
per group) food withdrawn for 16 h were administered
streptozotocin (50 mg/kg, i.p) and food was immediately
provided. On the 5
th
day, animals exhibiting random
blood glucose levels greater than 270 mg/dl were in-
cluded in the study [54]. Body weights and blood glucose
were measured at specified time periods. The bedding
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 3 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
material was frequently changed to avoid any infection
due to excessive urination. On the 29
th
day post strepto-
zotocin administration, animals were transferred to wire
mesh cages and acclimatized for 15–45 min. They were
then assessed for mechanical allodynia or vulvodynia be-
fore and after PI-ME or standard gabapentin administra-
tion using the von Frey up-down method [55].
Treatment schedule
Animals were divided into five groups. Group I received
normal saline and served as control. Group II remained
as the streptozotocin positive control group. Group III
received a single intraperitoneal dose of gabapentin
(75 mg/kg) and served as the standard. Group IV and V
were treated with PI-ME at doses of 200 and 300 mg/kg
respectively. The therapeutic doses of PI-ME for evalu-
ation in neuropathic pain were selected on the basis of
its analgesic, anxiolytic, locomotor and respective antag-
onistic activities.
Assessment of static and dynamic allodynia
Static allodynia was assessed using a series of von Frey fila-
ments (0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15 g), starting with a
2.0 g force applied perpendicularly to the plantar surface of
the right hind paw for 5 s or until the animal displayed a
withdrawal response (lifting of the paw). Animals respond-
ing to 3.63 g force or below were included in the study and
15 g was selected as the cut-off force [54].
Dynamic allodynia was assessed by lightly stroking the
plantar surface of the hind paw with a cotton bud. Lift-
ing or licking the paw was considered as a withdrawal
response and the time taken to show a withdrawal reac-
tion was considered as the paw withdrawal latency
(PWL). Animals responding to the cotton bud within 8 s
were included in the study and 15 s was selected as the
cut off time [54].
Assessment of static and dynamic vulvodynia
Static vulvodynia was assessed by shaving the anogenital
area including the mons pubis. A series of von Frey fila-
ments (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1 g), were
applied perpendicularly to the mucous membrane of the
anogenital region for 4 s starting with a 0.04 g force,
until a flinching response occurred. Animals responding
to a 0.16 g force or below were included in the study
and a 1.0 g force was selected as the cut-off force [56].
Dynamic vulvodynia was assessed by lightly brushing a
cotton bud over the mucous membrane of the anogeni-
tal region for 10 s or until a flinching response occurred.
Animals showing a flinching response within 5 s were
included in the study and 10 s was selected as the cut-
off time [56].
Statistical analysis
Data were expressed as mean ± SEM. Statistical compari-
sons were carried out by one way ANOVA followed by
Dunnett’s, Bonferroni or Tukey’s multiple comparison tests
where appropriate using GraphPad Prism 5 (GraphPad
Software Inc. San Diego CA, USA). Statistical significance
was deduced at P≤0.05.
Results
Phytochemical analysis of Passiflora incarnata
Preliminary qualitative analysis of PI-ME disclosed the
presence of flavonoids, alkaloids, carbohydrates, tannins,
glycosides, fixed oils and saponins (Table 1). Subsequent
more detailed quantitative analysis revealed the presence
of flavonoids (72 %), saponins (10 %) and alkaloids
(13.4 %) in PI-ME. The major compounds obtained from
GC-MS analysis of PI-ME included: 9-Octadecenamide
(Oleamide) (C
18
H
35
NO, MW: 281), n-Hexadecanoic acid
(Palmitic acid) (C
16
H
32
O
2
, MW: 256), dodecanoic acid,
3-hydroxy- (C
12
H
24
O
3
, MW: 216), 4H-Pyran-4-one, 2,3-
dihydro-3,5-dihydroxy-6-methyl- (C
6
H
8
O
4
, MW: 144),
vitamin-E (C
29
H
50
O
2
, MW: 430), cis,cis,cis-7,10,13-Hexa-
decatrienal (C
16
H
26
O, MW: 234), β-Sitosterol (C
29
H
50
O,
MW: 414), 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol,
(3β,5Z,7E)- (C
27
H
44
O
3
, MW: 416), pregnane-3,11,20,21-
tetrol, cyclic 20,21-(butyl boronate), (3α,5β,11β,20R)-
(C
25
H
43
BO
4
, MW: 418), ethyl 9-hexadecenoate (C
18
H
34
O
2
,
MW: 282), stigmasterol (C
29
H
48
O, MW: 412), octade-
canoic acid (C
18
H
36
O
2
, MW: 284), 2H-1-Benzopyran-
6-ol, 3,4-dihydro-2,8-dimethyl-2- (4,8,12-trimethyltri-
decyl)-, [2R-[2R*(4R*,8R*)]]- (C
27
H
46
O
2
, MW: 402),
and phytol (C
20
H
40
O, MW: 296) among other import-
ant constituents (Table 2 and Fig. 1).
Antinociceptive activity of Passiflora incarnata
Abdominal constriction assay (tonic visceral chemically-
induced nociception)
A significant attenuation (F
(5,42)
= 91.99, P< 0.001) of
acetic acid incited abdominal constriction was produced
by PI-ME at doses of 200 mg/kg (P< 0.01) and 250 mg/
kg (P< 0.001) compared to saline control. Similarly, a
significant increase (P< 0.001) in the percentage protec-
tion against abdominal constriction was observed with
diclofenac (50 mg/kg) and morphine (5 mg/kg) (Fig. 2a).
Naloxone (0.5 mg/kg) (F
(9,70)
= 44.75, P< 0.001) signifi-
cantly reversed the antinociceptive activity of morphine
(P< 0.001) and PI-ME (200 and 250 mg/kg) (P< 0.05)
but not that of diclofenac (50 mg/kg) as shown in
Fig. 2b.
Hot plate test (acute phasic thermal nociception)
In the hot plate test, 30 min after drug administration
(F
(5,42)
=200.2,P< 0.001) a marked increase in percentage
antinociception was observed with morphine (5 mg/kg)
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(P< 0.001), diclofenac (50 mg/kg) (P< 0.05) and PI-ME at
adoseof200mg/kg(P< 0.05). After 60 min (F
(5,42)
=
55.36, P< 0.001), the increase in percentage response was
less significant (P< 0.05) for morphine whilst it was highly
significant (P< 0.001) for PI-ME (150 and 200 mg/kg), the
activity being retained in the latter case up to 90 min
(F
(5,42)
=36.61, P< 0.001, not shown). However, after
120 min (F
(5,42)
=4.352, P< 0.01) it was only PI-ME at
doses of 150 mg/kg (P< 0.05) and 200 mg/kg (P<0.01)
that afforded protection against thermal nociception
(Fig. 3). Naloxone (1.0 mg/kg) (F
(7,56)
=46.60, P<0.001)
reduced the % antinociceptive effect of both morphine
(P< 0.001) and PI-ME (150 and 200 mg/kg) (P<0.01)
(Fig. 4a). Pentylenetetrazole (10 mg/kg) (F
(7,56)
= 35.91,
P< 0.001) by way of contrast, significantly reduced
the antinociceptive effect of PI-ME only at the
150 mg/kg dose (P< 0.05) (Fig. 4b).
Anxiolytic-like activity of Passiflora incarnata
In the staircase test, there was a substantial increase in
the number of steps climbed (F
(4,25)
= 21.04, P< 0.001) in
response to both diazepam (2 mg/kg, P< 0.001) and PI-
ME (200 mg/kg, P< 0.05) versus the animal control
group treated with saline vehicle. However, at the high-
est dose (600 mg/kg) the passiflora extract significantly
reduced (P< 0.05) the number of steps climbed in com-
parison with the controls (Fig. 5a). In contrast, the num-
ber of rears (F
(4,25)
= 5.403, P< 0.01) was inhibited not
only by treatment with diazepam (P< 0.01) but also by
all three doses of PI-ME (200 and 400 mg/kg, P< 0.05;
600 mg/kg, P< 0.01) in comparison with the saline ve-
hicle controls (Fig. 5b). The post hoc test revealed that
there was no significant effect of pentylenetetrazole
(10 mg/kg) by itself on step climbing nor was there any
modification of the stair climbing responses when it was
administered in combination with diazepam or PI-ME
(Fig. 6a). However, it did reverse the decrement in rears
initiated by PI-ME (200, 400 and 600 mg/kg) and actu-
ally augmented (P< 0.05) the overall rearing incidence
(F
(9,50)
= 6.497, P< 0.001) as shown in Fig. 6b.
Sedative activity of Passiflora incarnata
Locomotor activity
In the locomotor activity study, there was a pronounced
reduction in cage line crossing instigated by both (F
(4,25)
=
15.39, P< 0.001) diazepam (4.0 mg/kg, P< 0.001) and PI-
ME at 400 mg/kg (P< 0.01) and 600 mg/kg (P<0.001)
though there was no significant motoric decline at the
lowest PI-ME dose (200 mg/kg, P> 0.05) (Fig. 7a). Penty-
lenetetrazole (10 mg/kg) (F
(7,40)
=26.88,P<0.001)blocked
(P< 0.05) the reduced locomotor effect of PI-ME (400
and 600 mg/kg) by increasing the incidence of line
crossing but it did not modify the diazepam loco-
motor regression (Fig. 7b).
Effect of Passiflora incarnata on mechanical allodynia and
vulvodynia
Animals administered a single streptozotocin (50 mg/kg)
treatment developed both static and dynamic allodynia
in their hind paws when tested 29 days later (Fig. 8).
Hence, there was a substantial decrease (P< 0.001) in
PWT and PWL in comparison with saline treated ani-
mals. One hour after PI-ME dosing in STZ-pretreated
animals on the test day, there was an ensuing increase in
PWT (F
(4,25)
= 31.41, P< 0.001) and PWL (F
(4,25)
= 20.25,
P< 0.001) observed for PI-ME at doses of 200 mg/kg
Table 1 Preliminary qualitative phytochemical analysis of Passiflora incarnata methanolic extract (PI-ME)
Sample Test Observation Result
1. Aqueous solution of PI-ME + 10 % ammonium hydroxide solution Appearance of yellow
coloration
Flavonoids present
2. A portion of PI-ME + few drops of Wagner’s reagent Reddish brown
precipitate
Alkaloids present
3. A small volume of PI-ME + 1–2 drops of Mayer’s reagent Creamy or white
precipitate
Alkaloids present
4. 0.5 ml PI-ME + 0.5 ml benedict’s reagent →mixed and boiled for 2 min Characteristic colored
precipitate
Carbohydrates
present
5. 1 ml PI-ME + 1 ml Barfoed’s reagent →boiled for 2 min Red precipitate Carbohydrates
present
6. 50 mg PI-ME + 5 ml distilled water + small amount of 5 % ferric chloride solution Intense green
coloration
Tannins and phenolic
compounds present
7. 50 mg PI-ME + conc. HCL →heated on water bath for 2 h →resultant hydrolysate filtered →
2 ml hydrolysate + 3 ml chloroform →chloroform layer separated out + 10 % ammonia
solution
Pink coloration Glycosides present
8. A small amount of PI-ME →compressed between two pieces of filter paper Formation of oil spot
on filter paper
Fixed oils present
9. 50 mg PI-ME + 20 ml distilled water →shaken for 15 min Formation of 2 cm
thick layer of foam
Saponins present
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(P< 0.001, P<0.01)and300mg/kg(P<0.001). Simi-
larly, 2 h following treatment with PI-ME on the test
day in the STZ-pretreated group, there was a sizeable
increase in PWT (F
(4,25)
= 17.92, P< 0.001) noted at
doses of 200 mg/kg (P< 0.01) and 300 mg/kg (P<
0.001) of PI-ME. However, at the 2 h test, PWL
(F
(4,25)
= 59.63, P< 0.001) was increased only by the
300 mg/kg (P< 0.001) rather than the 200 mg/kg PI-
ME dose. Gabapentin (75 mg/kg) administered as a
positive control, also generated an alleviation of
mechanical allodynia by elevating (P< 0.001) PWT
and PWL compared to the 29-day streptozotocin
alone pretreated animals at both the 1 and 2 h testing
times (Fig. 8).
The animal group pretreated with streptozotocin by it-
self 29 days earlier expressed mechanical static and dy-
namic vulvodynia (P< 0.001) compared to the saline
vehicle treated controls on the test day (Fig. 9). It was
notable that PI-ME (200 and 300 mg/kg) did not modify
the diminished FRT (streptozotocin induced static vul-
vodynia) at either the 1 h (F
(4,25)
= 49.85, P< 0.001) or
2h(F
(4,25)
= 17.12, P< 0.001) test day readings. However,
there was a significant increase in streptozotocin-
shortened FRL (dynamic vulvodynia) within 1 h (F
(4,25)
= 27.38, P< 0.001) and 2 h (F
(4,25)
= 10.08, P< 0.001) of
PI-ME treatment at 200 mg/kg (P< 0.001, P< 0.05) and
300 mg/kg (P< 0.001, P< 0.01). The single test day posi-
tive control dose of gabapentin (75 mg/kg) alleviated
both mechanical static and dynamic vulvodynia at the 1
and 2 h readings as evidenced by significant increases in
FRT (P< 0.001, P< 0.01) and FRL (P< 0.001) versus the
streptozotocin alone pretreated animals.
Discussion
The antinociceptive activity of P. incarnata methanolic
extract (PI-ME) was evaluated in mice using the hot
Table 2 GC/MS analysis of Passiflora incarnata methanolic extract
Chemical constituent Formula Molecular weight R.T. (min) Percent abundance
10-Undecen-1-al, 2-methyl- C
12
H
22
O 182 8.465 0.377
1,3-Pentanediamine C
5
H
14
N
2
102 8.809 0.353
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C
6
H
8
O
4
144 10.27 5.477
1-Pentanol, 2-methyl-, acetate C
8
H
16
O
2
144 10.83 0.621
1,2,6-Hexanetriol C
6
H
14
O
3
134 11.10 0.623
4-Cyclopropylcarbonyloxytridecane C
17
H
32
O
2
268 11.32 0.664
5-Cyclopropylcarbonyloxypentadecane C
19
H
36
O
2
296 11.60 0.796
9-Tetradecen-1-ol, acetate, (E)- C
16
H
30
O
2
254 12.87 0.488
trans-2-undecenoic acid C
11
H
20
O
2
184 15.98 0.438
Dodecanoic acid, 3-hydroxy- C
12
H
24
O
3
216 15.99 13.64
4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol C
10
H
12
O
3
180 18.64 0.393
d-Mannose C
6
H
12
O
6
180 18.86 0.378
7-Methyl-Z-tetradecen-1-ol acetate C
17
H
32
O
2
268 19.34 0.395
l-Gala-l-ido-octose C
8
H
16
O
8
240 19.54 0.406
n-Hexadecanoic acid; (Palmitic acid) C
16
H
32
O
2
256 23.68 21.86
Phytol C
20
H
40
O 296 30.20 1.004
9-Hexadecyn-1-ol C
16
H
30
O 238 31.33 0.956
cis,cis,cis-7,10,13-Hexadecatrienal C
16
H
26
O 234 31.81 2.175
Octadecanoic acid C
18
H
36
O
2
284 33.09 1.209
9-Octadecenamide, (Z)-; (Oleamide) C
18
H
35
NO 281 42.11 33.52
9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3β,5Z,7E)- C
27
H
44
O
3
416 42.79 1.762
Pregnane-3,11,20,21-tetrol, cyclic 20,21-(butyl boronate), (3α,5β,11β,20R)- C
25
H
43
BO
4
418 42.98 1.422
2H-1-Benzopyran-6-ol, 3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-,
[2R-[2R*(4R*,8R*)]]-
C
27
H
46
O
2
402 43.69 1.033
Ethyl 9-hexadecenoate C
18
H
34
O
2
282 45.78 1.390
Vitamin E C
29
H
50
O
2
430 45.99 2.579
Stigmasterol C
29
H
48
O 412 48.01 1.229
β-Sitosterol C
29
H
50
O 414 49.08 1.776
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 6 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
plate test which is suitable for assessing centrally medi-
ated acute phasic nociception [57] and the acetic acid in-
duced abdominal constriction assay for tonic visceral
nociception [58, 59]. Mice were selected as the species
of choice in these specific tests because they are mani-
festly sensitive not only to opioid mediated effects but
also to coexistent non-steroidal anti-inflammatory drug
(NSAID) activity [51]. What is more, accumulating evi-
dence indicates that GABAergic transmission plays a
pivotal role in the inhibitory regulation of the nocicep-
tive process, and the murine abdominal constriction
assay as well as the hot plate test both detect dose
dependent GABA agonist antinociception in this species
[60, 61]. In both tests, diclofenac as a standard anti-
inflammatory analgesic and PI-ME produced antinocicep-
tive activity consistent with previous studies [16, 31, 62]. It
was notable that the antinociceptive effect of PI-ME was
reversed by the opioid- and GABA
A
- receptor antagonists,
naloxone and pentylenetetrazole (PTZ) respectively, sug-
gesting an involvement of opioidergic and GABAergic
mechanisms in the mediation of the antinociception. Opi-
oid agonists decrease pain transmission by activating de-
scending nerve fibers from the periaqueductal gray and
raphe nuclei supraspinally and also by inhibition of affer-
ent nerve transmission by binding to pre- and postsynap-
tic opioid receptors within the spinal cord dorsal horn
[63]. Furthermore, GABAergic neurons and receptors that
are intercalated within the spinal cord and higher brain
pathways are important for the origination, transmission,
and modification of pain impulses in such a way that alter-
ation of GABA transmission yields antinociception [64]. P.
incarnata has been shown to modulate the activity of
GABAergic and opioid systems [21] to produce central
analgesic activity as evaluated by a reduced duration of
paw licking in neurogenic and inflammatory nociceptive
phases in the formalin test [31]. Due to a prevalence of
GABA as a non-α-amino acid constituent of P. incarnata
extract [65], several of its pharmacological effects have
been ascribed to mediation via the GABA system. These
include not only affinity for GABA
A
but also GABA
B
re-
ceptors in addition to modification of GABA uptake [66].
The antinociceptive effects of both GABA
A
and GABA
B
receptor agonists are known to involve activation or inhib-
ition of other neurotransmitter or neuromodulator path-
ways [64] and it is evident that central GABAergic
systems are involved in opioid-mediated analgesia [67].
Thus, it is possible that administration of GABA receptor
agonists in combination with other agents may yield
GABA receptor-related therapies for the treatment of
acute and chronic pain [64].
The anxiolytic-like activity of PI-ME was assessed by
the incidence of rears or steps climbed in the stair case
test. The extract at a dose of 200 mg/kg significantly in-
creased the number of climbed steps, although at a
Fig 1 MS chromatogram of Passiflora incarnata methanolic extract
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 7 of 17
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Fig. 2 aAntinociceptive activity of Passiflora incarnata (PI-ME) in the mouse abdominal constriction assay. ***P< 0.001, **P< 0.01 compared to
saline vehicle control (ANOVA followed by Dunnett’spost hoc test), (n= 8 mice per group). bEffect of naloxone (NXL-0.5) on the antinociceptive
activity of PI-ME in the mouse abdominal constriction assay. *P< 0.05, ***P< 0.001 compared to morphine (MOR-5), diclofenac (DIC-50) or PI-ME
(ANOVA followed by Bonferroni’s multiple comparison post hoc test), (n= 8 mice per group)
Fig. 3 Antinociceptive activity of Passiflora incarnata (PI-ME) in the mouse hot plate test. *P< 0.05, **P< 0.01, ***P< 0.001 compared to saline
vehicle control (ANOVA followed by Dunnett’spost hoc test), (n= 8 mice per group)
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 8 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
higher dose (600 mg/kg) it decreased this parameter.
Similarly, the frequency of rears was diminished by the
extract at all three doses tested and this outcome was
blocked by PTZ. Anxiolytic-like activity has been shown
to be associated with an increase in the number of steps
climbed by mice whilst sedative activity is thought to be
linked to a decrease in the frequency of rears [18] and
this is the very reason why this paradigm was chosen in
this species to evaluate P. incarnarta. Other studies have
attributed an increased rearing incidence to an anxiety-
like behavior and a decreased number of steps climbed
to a sedative effect [68]. In conjunction with this, anxio-
lytic activity has been coupled with lower doses while
sedative effects have been related to higher doses of
plant extracts or reference drugs [69]. In this context,
PI-ME displayed an anxiolytic-like effect at 200 mg/kg,
while at 600 mg/kg it exhibited sedative activity. This
was also confirmed in the open field test where it was
observed that PI-ME decreased the number of lines
crossed at doses of 400 and 600 mg/kg comparable to
that of diazepam and these findings concur with the lit-
erature [17–19, 70]. Since PTZ reversed the anxiolytic-
like and sedative actions of PI-ME, underlying GABA
mediated mechanisms may well be implicated. In a se-
lection of studies, the sedative and anxiolytic properties
of P. incarnata have been attributed to benzodiazepine
and GABA receptor mediated biochemical processes in
the body [18, 19, 71, 72], binding to GABA
A/B
sites and
Fig. 4 aEffect of naloxone (NXL-1); or bpentylenetetrazole (PTZ-10) on the antinociceptive effect of Passiflora incarnata (PI-ME) in the mouse hot
plate test. *P< 0.05, **P< 0.01, ***P< 0.001 compared to morphine (MOR-5) or PI-ME (150 or 200 mg/kg) (ANOVA followed by Bonferroni’s mul-
tiple comparison post hoc test), (n= 8 mice per group)
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 9 of 17
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inhibition of GABA uptake being of particular conse-
quence [66].
The modulatory effect of P. incarnata on GABAergic
and opioid systems may provide some insight into its
beneficial effect in various painful neuropathic condi-
tions. Neuropathy induced hypersensitivity is known to
involve disruption of tonic GABAergic transmission [73]
and GABA agonists and metabolic inhibitors have been
shown to be effective in various neuropathic nociceptive
models [74–76]. Neuropathic pain has been reported to
be refractory to opioids [77, 78]. However, several studies
have shown that neuropathic pain can be attenuated by
morphine and other μ-opioid receptor agonists [79–81]
and these reports suggest that local μ-opioid receptors on
the terminals of uninjured primary afferent nociceptive
neurons are an essential target for alleviating mechanical
allodynia. In the current study we have evaluated the
methanolic extract of P. incarnata in a novel streptozoto-
cin induced diabetic animal model of neuropathic pain
established exclusively in rats [56]. The results showed
that PI-ME (200 and 300 mg/kg) induced mechanical
anti-allodynic activity exemplified by an increase in paw
withdrawal threshold (PWT) and paw withdrawal latency
(PWL) 1 and 2 h post treatment. Similarly, PI-ME also re-
lieved dynamic vulvodynia by increasing the flinching re-
sponse latency (FRL) although the extract was devoid of
activity on the static component of vulvodynia. The inten-
sity of the PI-ME dynamic anti-vulvodynia response was
comparable to that of gabapentin which was used as a ref-
erence drug due to the fact that it has proven pain reliev-
ing effects in various neuropathic pain models [54].
Gabapentin also exhibits an established propensity to alle-
viate both static and dynamic components of allodynia
and vulvodynia [56] and the current study corroborates
this assertion. The present findings also indicate that the
behavioural and antinociceptive effects of PI-ME involve
GABAergic and opioidergic mechanisms because they
were reversed by PTZ and naloxone respectively. Conse-
quently, it might be inferred that analogous processes are
implicated in PI-ME anti-allodynic/vulvodynic activity and
this requires a direct focus of further study. In relation to
this notion, Ingale and Kasture [31] suggested that opioi-
dergic as well as the nicotinic cholinergic system are in-
volved in the central analgesic activity of butanolic P.
incarnata extract in the eye wipe test. This paradigm is
used to study trigeminal pain because corneal nociceptive
receptors have a large representation in the trigeminal
ganglion through the ophthalmic branch of the tri-
geminal nerve [82]. Moreover, it has been suggested
from radioligand binding studies that it is very un-
likely that P. incarnata acts via the benzodiazepine-site of
the GABA
A
-receptor [66]. In this connection, it has been
postulated that GABA
A
α1-sparing benzodiazepine-site li-
gands might constitute a class of analgesics suitable for
the treatment of chronic pain syndromes [83]. Further-
more, there is considerable evidence implicating an im-
portant role for diminished GABAergic tone in the
development of central sensitization and hyperalgesia in
neuropathic pain models [84–86].
The phytochemical screening results of our study ver-
ify the presence of a preponderance of flavonoids as well
as alkaloids in P. incarnata as described elsewhere
[25, 87, 88]. Flavonoids are reported to be the major
phytoconstituents of P. incarnata and include chrysin,
vitexin, isovitexin, orientin, isoorientin, apigenin and
kampferol [14, 30, 89]. These polyphenolic metabo-
lites may play a role in the neuropharmacological ac-
tivity of several plants [90–92] including P. incarnata
[18, 93, 94]. Additionally, flavonoids have been reported
to elicit an analgesic effect through opioid [95] as well as
GABAergic systems [96] and have a beneficial role in
relieving neuropathic pain conditions [97–99]. Some
flavonoids like quercetin have also been identified in
P. incarnata extract [100] and are believed to be ef-
fective in diabetes mellitus induced peripheral neur-
opathy [101, 102] the activity being mediated through
an opioidergic mechanism [103]. The GCMS analysis
Fig. 5 Effect of Passiflora incarnata (PI-ME), and diazepam (2 mg/kg)
on athe number of steps climbed in the staircase test and bthe
incidence of rears in mice. *P< 0.05, **P< 0.01, ***P< 0.001 compared
to saline vehicle control (ANOVA followed by Dunnett’spost hoc test),
(n= 8 mice per group)
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 10 of 17
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in this study revealed that P. incarnata contains a pre-
dominance of the fatty acid amide 9-octadecenamide (also
known as oleamide), which has hypnotic, analgesic, and
anxiolytic actions [104]. Many of oleamide’s behavioural
effects stem from its activity on various receptor systems
including GABA
A
[105–107], 5HT
1A
,5HT
2A
,5HT
2C
,
5HT
7
[108–110], G-proteins [111], voltage gated sodium
channels [107, 112] and CB
1
receptors [113]. In this re-
spect, oleamide enhances GABA receptor activity and spe-
cifically potentiates the peak chloride current when applied
with GABA to benzodiazepine-sensitive GABA
A
receptors
[106]. The cannabimimetic action of oleamide resulting
from its agonist action at CB
1
receptors [110, 113] gives
rise to cannabinoid antagonist reversible antinociception
which is also sensitive to blockade by the GABA
A
antagonist bicuculline [104]. It has been posited that en-
dogenous fatty acid derivatives such as oleamide interact
with endocannabinoids like anandamide in the modulation
of pain sensitivity [114] which may well contribute to the
inhibitory effect of P. incarnata on allodynia and vulvody-
nia observed in this study.
Other important constituents present in P. incarnata
include hexadecanoic acid (palmitic acid), 3-hydroxy-
dodecanoic acid, 2,3-dihydro-3,5-dihydroxy-6-methyl-
4H-Pyran-4-one, and vitamin-E, that have strong anti-
oxidant and neuroprotective activities and/or modulate
the GABAergic system [115–119].
The modulation of GABAergic and/or opioidergic sys-
tems by P. incarnata reported in this study may consti-
tute a proportion of the mechanisms implicated in the
Fig. 6 Effect of pentylenetetrazole (PTZ-10) on diazepam (DIZ-2) or Passiflora incarnata (PI-ME, 200 and 600 mg/kg) with respect to athe number
of steps climbed in the staircase test and bthe incidence of rears in mice. *P< 0.05 compared to PI-ME alone (200, 400 or 200 mg/kg) (ANOVA
followed by Dunnett’spost hoc test), (n= 8 mice per group)
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 11 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 7 aEffect of diazepam and Passiflora incarnata (PI-ME) on mouse locomotor activity. **P< 0.01, ***P< 0.001 compared to saline vehicle
control (ANOVA followed by Dunnett’spost hoc test), (n= 6 mice per group). bEffect of pentylenetetrazole (PTZ-10) on the mouse locomotor
activity induced by Passiflora incarnata (PI-ME 400 and 600 mg/kg) or diazepam (DIZ-4). *P< 0.05 compared to PI-ME (400 or 600 mg/kg) alone
(ANOVA followed by Bonferroni’s multiple comparison post hoc test), (n= 6 mice per group)
Fig. 8 Effect of Passiflora incarnata (PI-ME) and gabapentin on astatic or bdynamic allodynia, at 1 or 2 h post-treatment times in female rats on
day 29 in a streptozotocin (STZ) induced neuropathic pain model.
###
P< 0.001 compared to saline control, *P< 0.01, **P< 0.05, ***P< 0.001
compared to streptozotocin alone treated animals, (n= 6 rats per group)
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 12 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
amelioration of diabetic neuropathy. Additional pro-
cesses however, like a cannabimimetic action [110, 113,
114] cannot be ignored inasmuch as P. incarnata ex-
hibits antihyperglycemic and hypolipidemic activities in
streptozotocin induced diabetes mellitus [25] which
would otherwise lead to neuropathic allodynia and
vulvodynia [56]. Hyperglycemia and dyslipidaemia driven
oxidative stress is a major contributor to reduced nerve
function [120, 121] and diabetes mellitus is a major
cause of peripheral neuropathy, commonly manifested
as distal symmetrical polyneuropathy [122]. Further-
more, diabetes mellitus has been reported to be linked
Fig. 9 Effect of Passiflora incarnata (PI-ME) and gabapentin on astatic or bdynamic vulvodynia at 1 or 2 h post-treatment times in female
rats on day 29 in the streptozotocin (STZ) induced neuropathic pain model.
###
P< 0.001 compared to saline, *P< 0.01, **P< 0.05, ***P< 0.001
compared to streptozotocin alone treated animals, (n= 6 rats per group)
Fig. 10 Scheme summarizing the anti-allodynic/anti-vulvodynic properties of Passiflora incarnarta plus it’s antinociceptive, anxiolytic-like and
higher dose sedative activities
Aman et al. BMC Complementary and Alternative Medicine (2016) 16:77 Page 13 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
with vulvodynia either as an isolated symptom or as part
of a constellation of other neuropathic abnormalities.
Such neuropathic morbidity has been termed ‘diabetic
vulvopathy’and it profoundly affects patient’s quality of
life and management needs in order to address the phys-
ical, psychological and relationship problems caused by
the pain [123]. Our study showed that the methanolic ex-
tract of P. incarnata significantly alleviated only the dy-
namic component of vulvodynia which has been reported
more likely to be provoked by contact with clothing
among other triggers [124] and the cotton swab test is
usually used to localize painful areas in vulvodynia [125].
Conclusion
In conclusion, the methanolic extract of P. incarnata
possesses peripheral and central phasic as well as tonic
antinociceptive activity mediated through modulation of
GABA
A
and opioid receptors (GABAergic and opioider-
gic mechanisms shown in Fig. 10) which are disclosed
by their naloxone and PTZ reversibility. The findings
also manifest anxiolytic-like and higher dose sedative ac-
tivity of the extract, resulting from GABAergic stimula-
tion as indicated by their sensitivity to PTZ inhibition.
The extract also exhibited significant mechanical anti-
allodynic and dynamic anti-vulvodynic effects (Fig. 10)
that may be attributable at least in part to the oleamide
content and a cannabinoid-like action [110, 113, 114].
The outcomes from our study advocate an effectiveness
of P. incarnata in the treatment of various neuropathic
pain conditions. However, further studies are warranted
in order to determine a more precise association be-
tween the active constituents responsible for the anal-
gesic, anxiolytic and sedative effects of P. incarnata as
well as the specific molecular mechanisms underlying its
actions on allodynia and vulvodynia.
Additional file
Additional file 1: Passiflora incarnata plant, grown in the botanical
garden of the Department of Pharmacy, University of Peshawar,
Pakistan. (DOCX 637 kb)
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
FS initiated the idea and guided the research group as supervisor in
planning and conducting experiments throughout the research project. UA
conducted the experiments and carried out calculations and statistical
analysis. She also prepared the initial draft of the manuscript. MS helped in
the analysis and interpretation of data as well as in preparing the final
version of the manuscript. SA provided her help during pharmacological
experiments throughout the study. NA assisted in the extraction of plant
material and other pharmacological activities. GA helped in the neuropathic
pain related experiments. KF attributed materials during pharmacological
screening of the plant extract. RDES guided the research group and
interpreted the results as well as critically revised the manuscript for
important intellectual content. All authors read and approved the final
manuscript.
Author details
1
Department of Pharmacy, University of Peshawar, Peshawar 25120, Khyber
Pakhtunkhwa, Pakistan.
2
Cardiff School of Pharmacy and Pharmaceutical
Sciences, Cardiff University, Cardiff CF10 3NU, UK.
Received: 15 August 2015 Accepted: 11 February 2016
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