Journal of Functional Foods 19 (2015) 126140

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Potential of black pepper as a functional food for

treatment of airways disorders

Abdul Rehman a,b, Malik Hassan Mehmood a,*, Muhammad Haneef a,c,
Anwarul Hassan Gilani a, Maimoona Ilyas d, Bina Shaheen Siddiqui d,
Mansoor Ahmed c
a
Natural Products Research Division, Department of Biological & Biomedical Sciences, Aga Khan University,
Stadium Road, Karachi, Sindh 74800, Pakistan
b
Internal Medicine Section, Department of Medicine, Hamad Medical Corporation, P.O. Box 3050, Doha, Qatar
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi, Sindh 75270,
Pakistan
d
H.E.J. Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of
Karachi, Karachi, Sindh 75270, Pakistan

A R T I C L E I N F O A B S T R A C T

Article history: The potential of black pepper (Piper nigrum) to cause bronchodilation was examined using
Received 6 May 2015 in vivo and in vitro assays. HPLC fingerprint analysis of the crude extract of Piper nigrum (Pn.Cr)
Received in revised form 1 and its fractions showed piperine, piperidine, eugenol and catechin as plant constituents.
September 2015 In anaesthetized rats, Pn.Cr and piperine relieved carbachol (CCh)-induced bronchospasm.
Accepted 2 September 2015 In isolated guinea-pig trachea, Pn.Cr and piperine inhibited CCh and K+ (80 mM)-induced
Available online 25 September 2015 contractions, potentiated isoprenaline concentrationresponse curves (CRCs) and sup-
pressed Ca2+ CRCs. In guinea-pig atria, Pn.Cr and piperine showed stimulatory and inhibitory
Keywords: effects on rate and force of contraction. Its fractions showed similar activities with varied
Black pepper potency in the in vivo and in vitro assays. These results suggest that black pepper and pip-
Piper nigrum erine cause bronchodilation through dual inhibition of phosphodiesterase enzyme and Ca2+
Piperine influx, which substantiate its potential as a functional food ingredient for airway disorders.
Bronchodilator 2015 Elsevier Ltd. All rights reserved.
Phosphodiesterase inhibitor
Calcium channel blocker

tropical parts of Asia. In Pakistan, it grows mainly in North-

1. Introduction
Piper nigrum Linn. belongs to the family Piperaceae and its dried
unripe fruit is used commonly as black pepper. This peren-
nial shrub is indigenous to the Western coasts of India and
ern areas, such as Gilgit and Chitral valleys, and is vernacularly
known in Urdu as kali mirch (Usmanghani, Saeed, & Alam,
1997). Black pepper the dried unripe fruit of Piper nigrum is
used as a condiment in various cuisines worldwide and
is a part and parcel of every kitchen. In 2013, the global

* Corresponding author. Faculty of Health Sciences, Department of Biological & Biomedical Sciences, Aga Khan University, P.O. Box 3500,
Stadium Road, Karachi, Sindh 74800, Pakistan. Tel.: +92 21 3486 4465; fax: +92 21 3493 4294.
E-mail address: hassan.mehmood@aku.edu, malikhassan.mehmood@gmail.com (M.H. Mehmood).
Chemical compounds: Papaverine (PubChem CID: 4680); Piperidine (PubChem CID: 8082); Piperine (PubChem CID: 638024); Verapamil
(PubChem CID: 2520).
http://dx.doi.org/10.1016/j.jff.2015.09.006
1756-4646/ 2015 Elsevier Ltd. All rights reserved.
 Journal of Functional Foods 19 (2015) 126140
production of black pepper was reported to be nearly 355,000
tonnes (Anonymous, 2008). Due to its widespread consump-
tion, biochemical and pharmacologic properties of black pepper
have been investigated extensively (Butt et al., 2013).
Phytochemical studies on black pepper have determined the
presence of various minerals, vitamins (-carotenes, tocoph-
erols, ascorbic acid, thiamine, riboflavin and niacin),
polysaccharides (arabinose, rhamnose, galacturic acid), sterols
(-sitosterol, terpenoids, sesquiterpenes), fatty acids (linoleic
acid), volatile oils (camphenes, pinenes), alkaloids (piperine, pi-
peridine, piperolein, capsaicin, 2-dihydrocaspaicin), resins
(chavicin), organic acids (hexadecanoic acid, octadecanoic acid),
amides (pipnoohine, pipyahyine, guineensine, pipericide) and
various phenolic compounds (benzamides, gallic acid,
kaempferol, coumarins, quercetin) (Duke, 1992; Siddiqui et al.,
2004; 2005). Piperine, the active principle of black pepper, has
been found to possess anti-inflammatory (Mujumdar, Dhuley,
Deshmukh, Raman, & Naik, 1990), anti-convulsant (DHooge
et al., 1996), hypoglycaemic (Panda & Kar, 2003),
immunomodulatory (Sunila & Kuttan, 2004), anti-depressant
(Lee et al., 2005), vasomodulatory (Taqvi, Shah, & Gilani, 2008),
bioavailability-enhancing (Veda & Srinivasan, 2009), anti-
spasmodic (Mehmood & Gilani, 2010), anti-hyperlipidaemic
(Chen, Ma, Liang, Peng, & Zuo, 2011), insecticidal (Park, 2012),
anti-tumour (Do et al., 2013), cardioprotective (Dutta et al., 2014)
and anti-oxidant (Embuscado, 2015) properties. More re-
cently, novel compounds isolated from black peppercorns have
been found to possess larvicidal activity against Aides egypti,
the principle vector of dengue virus (Santiago, Alvero, &
Villaseor, 2015).
Besides being used as a spice, black pepper enjoys folk-
loric reputation for use in airway disorders including bronchitis
and asthma (Duke, Bogenschutz-Godwin, Du Celliar, & Duke,
2002; Kapoor, 1990; Usmanghani et al., 1997). Currently, no sci-
entific evidence is available to support its use in respiratory
disorders. We hypothesized that black pepper may contain
certain bioactive constituents that can cause bronchodilation.
As black pepper is consumed heavily throughout the world,
precipitation or relief of bronchospasm by its chemical con-
stituents can have important nutraceutical implications.
Moreover, as black pepper is an ingredient of numerous recipes
including soups, it may be an attractive option for the devel-
opment of functional foods and nutraceuticals. Therefore, in
the present study, we investigated the effects of black pepper
and its active principle piperine on tracheal tissues through
in vitro and in vivo experiments.
2. Materials and methods
2.1. Chemicals
Acetylcholine (ACh) perchlorate, carbachol (CCh), papaverine,
isoprenaline hydrochloride, histamine hydrochloride and
verapamil hydrochloride were purchased from Sigma Chemi-
cals Co. (St. Louis, MO, USA). Piperine, Tween80, thiopental
sodium and castor oil were obtained from Sigma-Aldrich Co.
(St. Louis, MO, USA), Scharlau Chemie S.A. (La Jota, Barcelona,
Spain), Abbott Laboratories (Karachi, Sindh, Pakistan) and KCL
Pharma (Karachi, Sindh, Pakistan). Chemicals used for making
127
physiological salt solutions were sodium chloride (BDH Labo-
ratory Supplies, Poole, England), potassium chloride (Sigma
Chemicals Co.), calcium chloride, glucose, magnesium chlo-
ride, magnesium sulphate, sodium bicarbonate, sodium
dihydrogen phosphate and potassium dihydrogen phosphate
(Merck, Darmstadt, Germany). All chemicals used were of the
analytical grade available and dissolved in distilled water, except
piperine, which was solubilized by Tween80.
2.2. Animals
SpragueDawley rats (200250 g) and guinea-pigs (350550 g)
of either sex were used in this study and housed at the Animal
House of the Aga Khan University, maintained at 2325 C.
Animals were given tap water ad libitum and a standard diet.
Guinea-pigs had free access to water, but food was with-
drawn 24 hours prior to the experiment. Experiments performed
on these animals complied with the rulings of the Institute of
Laboratory Animal Resources, Commission on Life Sciences,
National Research Council (1996) and were approved by the
Ethics Committee for Animal Care and Use (ECACU) at Aga Khan
University, Karachi, Sindh, Pakistan (approval number 42-
ECACU-BBS-14). This study is also the part of the PhD proposal
of Mr. Muhammad Hanif, approved by the Board of Advanced
Studies and Research (BASR/NO/02535/Pharm), University of
Karachi, Karachi, Pakistan.
2.3. Plant material
For the purpose of this study, dried fruits of Piper nigrum were
used, which are commonly known as kali mirch in Urdu
(native language) and black pepper in English. Botanical name
of this plant was further confirmed using an online resource
(http://www.theplantlist.org) on 25th January, 2014. Dried fruits
of Piper nigrum were bought from a local market (Jouria Bazaar)
in Karachi, Sindh, Pakistan and were authenticated by a quali-
fied botanist (Dr. Altaf A. Dasti, late) from the Institute of Pure
and Applied Biology, Bahauddin Zakariya University Multan,
Punjab, Pakistan. A sample voucher (Pn-F-06-07-72A) was sub-
mitted to the herbarium of Department of Biological and
Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
2.4. Preparation of crude extract and its fractionation
After cleaning of adulterant material, 450 g of fruits was crushed
and soaked in 3 L of 70% aqueous-methanol for 3 days with
occasional shaking. It was filtered through a muslin cloth and
then through a Whatman (Maidstone, UK) qualitative grade 1
filter paper (Williamson, Okpako, & Evans, 1996). This proce-
dure was repeated thrice and the combined filtrate was
evaporated on rotary evaporator (model RE-111, Buchi, Flawil,
Switzerland) to obtain the crude extract (Pn.Cr) yielding ap-
proximately 8.7%.
Activity-guided fractionation was carried out by using sol-
vents of increasing polarity to obtain organic and aqueous
fractions. Pn.Cr was dissolved in about 150 mL of distilled water
and petroleum ether was added to it with vigorous shaking.
This solution was taken in a separating funnel and the layers
were allowed to separate. The petroleum ether (upper) layer
was removed and petroleum ether was added to the remaining
128 Journal of Functional Foods 19 (2015) 126140
solution as before. Separation was done twice more and all the
petroleum ether layers so obtained were evaporated on a rotary
evaporator to obtain petroleum ether fraction (Pn.Pet). Chlo-
roform was then added to the remaining solution and the
chloroform layers were collected thrice. These were evapo-
rated together on a rotary evaporator to give the chloroform
fraction (Pn.CHCl3). The other (remaining) layer was again taken
into a separating funnel. Ethyl acetate was added into it, ethyl
acetate layer was separated and it was also evaporated in rotary
evaporator to give the ethyl acetate fraction (Pn.EtAc). The re-
maining lower layer was collected and evaporated to obtain
the aqueous fraction (Pn.Aq).
2.5. High performance liquid chromatography (HPLC)
fingerprints
HPLC analysis of Pn.Cr, its fractions and standard drugs (pip-
erine, piperidine, eugenol and catechin) was performed using
a Shimadzu (Kyoto, Japan) Prominence 20-AT equipped with
a pump (LC20AT), PDA detector (SPD-M20A) and Kromasil 100-
C18 column (Teknokroma; 8 m, 25 0.46 mm) with membrane
filter 0.45 m and syringe filter 0.45 m. Two milligrams (2 mg)
of each sample was dissolved in 2.0 mL of mobile phase and
the sample was sonicated for 30 min. Then, it was filtered
through a Millipore (0.45 m) filter prior to injection. The mobile
phase consisting of acetonitrile:0.1% acetic acid in water (94:06,
v/v) was delivered at a flow-rate of 0.1 mL/min, while column
temperature was maintained at 25 C. Ultraviolet detection was
performed at 310 nm.
2.6. Bronchodilator effect in anaesthetized rats
As described previously (Chaudhary et al., 2012), an intraperi-
toneal injection of thiopental sodium (80100 mg/kg) was used
to induce anaesthesia in SpragueDawley rats. After making
a small incision through the skin and subcutaneous tissues,
an endotracheal tube was introduced into the trachea. The tube
was secured in place with the help of silk ties. Mechanical ven-
tilation was initiated by means of a Miniature Ideal Pump
(Bioscience, Lincoln, NE) set at a respiratory rate of 70/min and
tidal volume of 8 mL/kg. Changes in airway resistance were re-
corded by a pressure transducer connected to a PowerLab
console via a Bridge amplifier (Quad Bridge Amp, ML112). Digital
recordings were saved in a computer system using LabChart
version 8.0 (ADInstrument, Bella Vista, Australia). Intrave-
nous access was established through a cannula passed into the
jugular vein. CCh, in a dose of 100 g/kg, was used to induce
bronchoconstriction and changes in airway resistance were re-
corded. In order to evaluate the effects of test extracts and
drugs, they were injected intravenously 5 min before admin-
istering CCh. Changes in airway resistance were again recorded
and compared with baseline readings (control). Experiments
were repeated 48 times and averaged readings were used to
plot bar graphs.
2.7. In vitro experiments
2.7.1. Isolated guinea-pig tracheal preparations
Using a previously described method (Shah & Gilani, 2010),
guinea-pigs were sacrificed by dislocating their cervical spine
and their trachea was then dissected out. Tracheal rings of
510 mm thickness were prepared and placed in a tissue bath
assembly containing 10 mL of physiologic Krebs solution at
37 C bubbled with carbogen. A fixed preload of 1 g was applied
to the tracheal rings and they were allowed to equilibrate in
this state for about half an hour. ACh and histamine were then
used to produce contraction of tissues until reproducible re-
sponses were recorded. CCh was added to the tissues to produce
sustained contraction and thereafter, test extracts or com-
pounds were added in a cumulative fashion to detect the
presence of bronchodilator effects. Verapamil and papaver-
ine were also used to relax CCh-induced contractions and
served as controls.
In order to construct isoprenaline concentrationresponse
curves (CRCs), CCh was used to induce sustained contraction
of tracheal tissue. Thereafter, increasing doses of isoprena-
line were added in a cumulative fashion until tissue relaxed
to its baseline level. This process was repeated by pre-treating
tissues with test extracts or drugs. CRCs were then obtained
by plotting isoprenaline concentration (X-axis) against per-
centage of contraction (Y-axis) to obtain a logarithmic curve.
Substances which cause a leftward shift in the isoprenaline
CRCs potentiate cyclic 3,5-adenosine monophosphate (cAMP)-
mediated smooth muscle relaxation (Shah & Gilani, 2010).
For constructing Ca2+ CRCs, tissues were initially stabi-
lized in normal Krebs solution and, subsequently, they were
placed in a Ca2+-free Krebs solution for 30 min. As Ca2+-free
Krebs solution contained ethylenediaminetetraacetic acid (EDTA
0.1 mM), Ca2+ ions were chelated and removed completely from
the tissues. Following this, tissues were incubated for 30 min
in a K+-rich, Ca2+-free Krebs solution. Ca2+ was added to the
tissues in a cumulative fashion and concentration-dependent
responses were recorded. When the control Ca2+ CRCs were
found super-imposable (usually after two cycles), tissues were
pre-treated with test extracts or compounds for 60 min. CRCs
of Ca2+ were reconstructed in the presence of different con-
centrations of test material. Substances which cause non-
parallel shift with suppression of the maximal response (Emax)
of Ca2+ CRCs possess Ca2+-antagonist like properties (Mehmood,
Rehman, Rehman, & Gilani, 2013).
2.7.2. Spontaneously beating isolated guinea-pig atria
As established previously (Shah & Gilani, 2010), guinea-pigs were
sacrificed by cervical dislocation and their right atrium was dis-
sected out. After cleaning off adipose tissue, atria were mounted
in individual 15 mL tissue baths filled with Krebs solution main-
tained at 32 C and bubbled with carbogen (mixture of 95% O2
and 5% CO2). Under these conditions, atria beat spontane-
ously due to the presence of sinoatrial node (pacemaker cells).
A preload of 1 g was applied to the tissues and they were
allowed to stabilize for 30 min. Thereafter, control responses
of isoprenaline (1 M) were obtained until tissue responses
became uniform. Increasing doses of test extracts or com-
pounds were then added in a cumulative fashion. Contraction
of atrial tissues was recorded by BioScience transducers con-
nected to a PowerLab station. Rate and force of contraction were
determined by using LabChart version 8.0 (ADInstrument, Bella
Vista, Australia). This arrangement allowed assessment of effects
of test drugs on spontaneous beating of guinea-pig atrium.
 Journal of Functional Foods 19 (2015) 126140 129

Fig. 1 High performance liquid chromatography fingerprints of (A) catechin, (B) eugenol, (C) piperidine, and (D) piperine,
which are four standard bioactive compounds present in unripe fruits of P. nigrum. Peaks identified in figure indicate
retention time (in min) of respective compounds.

2.8. Statistical analysis
All data expressed are mean standard error of mean (S.E.M.,
n = number of experiments). Inhibitory effects are expressed
as the median inhibitory concentrations (IC50) with 95% con-
fidence intervals (CI). Concentration-dependent inhibitory curves,
isoprenaline CRCs and Ca2+ CRCs were analyzed by applying
non-linear regression using GraphPad Prism program, version
6.0 (GraphPAD, San Diego, CA, USA) and compared by applying
two-way analysis of variance (ANOVA) followed by Bonferroni
post-test correction. For in vivo bronchodilator effect, one-way
ANOVA followed by Dunnetts test and/or Students t-test were
used, while stimulatory effect of test drugs on atrial prepara-
tions was analyzed using one-tailed, Students t-test. A p-value
of less than 0.05 was considered statistically significant.
P. nigrum (Pn.Cr) revealed the presence of all four compounds
in the crude extract as illustrated in Fig. 2A. Quantitative analy-
sis of Pn.Cr revealed that catechin and piperidine were present
in the highest concentration, followed by eugenol and piper-
ine (see Fig. S1). Moreover, piperine was detected in petroleum
ether fraction (Pn.Pet) (Fig. 2E), while piperidine was detected
in aqueous (Pn.Aq) (Fig. 2B) and ethyl acetate (Pn.EtAc) (Fig. 2D)
fractions. Eugenol and catechin were both present in Pn.Aq
(Fig. 2B) as well as Pn.EtAc (Fig. 2D). An unidentified com-
pound with a Rt of 22.268 min was detected in Pn.Cr and in
all of its fractions, most notably in chloroform fraction (Pn.CHCl3)
(Fig. 2C). Based on previously published literature, this was likely
a phenolic compound (Scott, Jensen, Philogne, & Arnason,
2008). Several unidentified minor compounds were also
noted in HPLC fingerprints of Pn.Cr and its various fractions
(Fig. 2).

3. Results 3.2. Bronchodilator effects of Pn.Cr, piperine and various

3.1. HPLC fingerprint analysis
HPLC fingerprints of four standard bioactive compounds were
obtained viz. catechin (Fig. 1A), eugenol (Fig. 1B), piperidine
(Fig. 1C) and piperine (Fig. 1D). The retention times (Rt) for each
of these compounds were 2.573, 3.073, 2.244 and 7.057 min re-
spectively. HPLC fingerprint analysis of the crude extract of
fractions in anaesthetized rats
Pn.Cr caused dose-dependent relaxation of CCh-induced bron-
chospasm in anaesthetized rats (Fig. 3A). At doses of 10, 30 and
100 mg/kg, Pn.Cr caused 10 1.7, 21.7 2 and 37.3 2.9% re-
laxation of CCh-induced bronchoconstriction, respectively.
Piperine also caused relaxation of CCh-induced broncho-
spasm at dose range of 10100 mg/kg with a maximum
130 Journal of Functional Foods 19 (2015) 126140

Fig. 2 High performance liquid chromatography fingerprints of (A) the crude extract, and (B) aqueous, (C) chloroform, (D)
ethyl acetate, and (E) petroleum ether fractions of P. nigrum. Labelled peaks represent the presence of multiple compounds
in the crude extract and its various fractions. Symbols indicate the probable presence of the pure compounds of P. nigrum
as shown in Fig. 1.

relaxation of 26.7 4.4% at the highest tested dose of 100 mg/
kg (Fig. 3B), indicating lesser efficacy compared to that of parent
crude extract. Aminophylline used as a positive control also
caused bronchodilator effect (Fig. 3C).
When different fractions were tested for their bronchodi-
lator effect, they caused dose-dependent relaxation of CCh-
induced bronchoconstriction at 10100 mg/kg. Pn.Pet (Fig. 3G)
was the most efficacious fraction as it caused 60.3 3.5% re-
laxation of CCh-induced bronchoconstriction at highest tested
dose (100 mg/kg). Pn.Aq (Fig. 3D) was the second most potent
fraction as it caused 40.8 5.9% relaxation of CCh-induced
bronchoconstriction at 100 mg/kg. In a similar fashion, at
highest dose of 100 mg/kg, Pn.CHCl3 (Fig. 3E) and Pn.EtAc (Fig. 3F)
caused 21.4 3.6 and 24 4.4% relaxation, respectively.
3.3. In vitro experiments
3.3.1. Effects of Pn.Cr, piperine and various fractions on CCh
and K+ (80 mM)-induced contractions in guinea-pig tracheal
preparations
Fig. 4 illustrates the inhibitory effects of Pn.Cr, piperine, pa-
paverine and verapamil on CCh and K+ (80 mM)-induced
contractions of isolated guinea-pig tracheal strips. The respective
IC50 values for Pn.Cr, piperine, papaverine and verapamil against
CCh-induced contractions were 0.24 mg/mL (0.190.32, 95% CI,
n = 4), 23.82 M (17.9229.19, n = 6), 0.29 M (0.230.35, n = 5)
and 0.3 M (0.260.33, n = 4). When tested against K+ (80 mM)-
induced contractions, Pn.Cr (Fig. 4A) and piperine (Fig. 4B)
caused inhibition with respective IC50 values 0.28 mg/mL (0.24
0.33, n = 5) and 30.4 M (24.7837.23, n = 4), while showed similar
potency as seen against CCh contractions. Papaverine (Fig. 4C)
also showed a similar pattern of inhibition against CCh and
K+ (80 mM)-induced contractions with respective IC50 values
of 0.29 M (0.250.36, n = 4) and 0.34 M (0.300.41, 95% CI, n = 6).
In contrast, verapamil (Fig. 4D) was more potent against K+
(80 mM)-induced contractions (p < 0.001) with the respective
IC50 value of 0.09 M (0.060.13, n = 5) compared to its effect
against CCh [0.43 M (0.290.52, n = 4)].
Among fractions of the crude extract of P. nigrum, Pn.Aq
(Fig. 5A) and Pn.CHCl3 (Fig. 5B) inhibited CCh-induced contrac-
tions in a fashion similar to that of the parent extract (Fig. 4A)
with respective IC50 values of 0.28 mg/mL (0.240.31, n = 4) and
0.27 mg/mL (0.230.30, n = 6). These fractions also equally in-
hibited K+ (80 mM)-induced contractions with IC50 values of
0.25 mg/mL (0.210.28, n = 7) and 0.24 mg/mL (0.190.28, n = 5),
respectively. On the other hand, Pn.EtAc (Fig. 5C) inhibited K+
 Journal of Functional Foods 19 (2015) 126140 131

Fig. 3 Dose-dependent effects of (A) the crude extract of Piper nigrum (Pn.Cr), (B) piperine, (C) aminophylline, and (D)
aqueous (Pn.Aq), (E) chloroform (Pn.CHCl3), (F) ethyl acetate (Pn.EtAc), and (G) petroleum ether (Pn.Pet) fractions of P. nigrum
on CCh (carbachol, 100 g/kg)-induced bronchoconstriction in anaesthetized SpragueDawley rats. Bars represent
mean standard error of mean (number of observations = 48). *, ** and *** represent p-values of less than 0.05, 0.01 and
0.001 respectively, while ns (not significant) indicates a p-value of more than 0.05, for comparison of effect of respective test
materials on CCh-induced bronchoconstriction. ### represents p-value of less than 0.001 for comparison of CCh-induced
bronchoconstriction vs. baseline. p-Values obtained by one-way analysis of variance followed by Dunnetts test.
132 Journal of Functional Foods 19 (2015) 126140

Fig. 4 Concentration-dependent inhibitory effects of (A) the crude extract of P. nigrum (Pn.Cr), (B) piperine, (C) papaverine,
and (D) verapamil against CCh (carbachol, 1 M) or K+ (80 mM)-induced contractions in isolated guinea-pig tracheal
preparations. Symbols represent mean standard error of mean (number of observations = 46). *** and ns represent
p-values of less than 0.001 and more than 0.05 for comparison of the inhibitory effects of respective test material on K+
(80 mM) vs. CCh (1 M)-induced contractions. These p-values were obtained using two-way analysis of variance followed by
Bonferroni post-test correction.

(80 mM)-induced contractions more potently (p < 0.01) with IC50
value of 0.2 mg/mL (0.130.26, n = 5) as compared to its effect
against CCh [0.53 mg/mL (0.310.77, n = 6)]. This pattern was
similar to that of verapamil (Fig. 4D) and different to that of
Pn.Cr (Fig. 4A). Furthermore, Pn.Pet inhibited CCh and K+
(80 mM)-induced contractions more potently (Fig. 5D) as com-
pared to Pn.Cr (Fig. 4A) with resultant IC 50 values of
0.24 mg/mL (0.20.29, n = 4) and 0.22 mg/mL (0.180.27, n = 5),
respectively.
3.3.2. Effects of Pn.Cr, piperine and various fractions on
isoprenaline and Ca2+ CRCs in guinea-pig tracheal preparations
To elucidate the mechanism of tracheal smooth muscle re-
laxation, CRCs of isoprenaline and Ca2+ were constructed in the
absence and presence of progressively higher concentrations
of Pn.Cr, piperine, papaverine and verapamil (Fig. 6). At low con-
centrations, Pn.Cr (0.01, 0.03 mg/mL) and piperine (0.3, 1 M)
caused a leftward shift in the CRCs of isoprenaline CRCs (Fig. 6A
and B), similar to the effect caused by papaverine (Fig. 6C). In
contrast, verapamil (Fig. 6D) did not show any appreciable effect
in the CRCs of isoprenaline. Respective IC50 values of isoprena-
line in the absence and presence of Pn.Cr, piperine, papaverine
and verapamil are given in Table 1. When tested for the pres-
ence of Ca2+ antagonist like activity, Pn.Cr (0.1 and 0.3 mg/mL)
and piperine (3 and 10 M) caused a non-parallel rightward shift
in the CRCs of Ca2+ with suppression of the maximum re-
sponse (Fig. 6E and F), similar to activity pattern observed with
papaverine and verapamil (Fig. 6G and H). The respective Emax
values of Ca2+ CRCs in the absence and presence of Pn.Cr and
piperine are given in Table 1.
When isoprenaline and Ca2+ CRCs were constructed in the
absence and presence of various fractions, Pn.Aq (Fig. 7A) and
Pn.Pet (Fig. 7D) caused leftward shifts in isoprenaline CRCs,
similar to that caused by papaverine (Fig. 6C). Pn.CHCl3 (Fig. 7B)
caused a minimal leftward shift in the CRCs of isoprenaline
at tested concentrations of 0.01 and 0.03 mg/mL, while Pn.EtAc
 Journal of Functional Foods 19 (2015) 126140 133

Fig. 5 Concentration-dependent inhibitory effects of (A) aqueous (Pn.Aq), (B) chloroform (Pn.CHCl3), (C) ethyl acetate
(Pn.EtAc), and (D) petroleum ether (Pt.Pet) fractions of P. nigrum on CCh (carbachol, 1 M) or K+ (80 mM)-induced contractions
in isolated guinea-pig tracheal preparations. Symbols represent mean standard error of mean (number of
observations = 47). ** and ns represent p-values of less than 0.01 and more than 0.05 respectively for comparison of the
inhibitory effects of respective fractions on K+ (80 mM) vs. CCh-induced contractions. These p-values were obtained using
two-way analysis of variance with Bonferroni post-test correction.

(Fig. 7C) did not show any effect on isoprenaline CRCs. Re-
spective IC50 values of isoprenaline in the absence and presence
of various fractions of Pn.Cr are given in Table 1. At slightly
higher concentrations, all fractions of Pn.Cr (Fig. 7EH) caused
a non-parallel rightward shift in the CRCs of Ca2+ with sup-
pression of the maximum response, similar to papaverine
(Fig. 6G) and verapamil (Fig. 6H). Respective Emax values of Ca2+
CRCs in the absence and presence of various fractions of
P. nigrum are given in Table 1.
3.3.3. Effects of Pn.Cr, piperine and various fractions on
spontaneously beating guinea-pig atrial preparations
When tested on spontaneously beating guinea-pig atria, Pn.Cr
and piperine (Fig. 8A and B) caused mild acceleration in the
rate and force of contraction at initial lower tested concen-
trations followed by inhibition in both parameters at relatively
higher tested concentrations. These effects of Pn.Cr and pip-
erine were similar to that of papaverine (Fig. 8C) while were
unlike that of verapamil (Fig. 8D).
When various fractions of Pn.Cr were tested, Pn.Aq (Fig. 8E)
and Pn.Pet (Fig. 8H) were found to dual stimulatory and in-
hibitory effects on atrial rate and force of contractions, similar
to their parent extract (Fig. 8A), piperine (Fig. 8B) and papav-
erine (Fig. 8C). On the other hand, Pn.CHCl3 (Fig. 8F) and Pn.EtAc
(Fig. 8G) exhibited only inhibitory effects on atrial rate and force
of contractions, similar to verapamil (Fig. 8D).
4. Discussion
On account of its reputed use in bronchitis and asthma
(Duke, Bogenschutz-Godwin, Du Celliar, & Duke, 2002;
134 Journal of Functional Foods 19 (2015) 126140

Fig. 6 Concentrationresponse curves of (AD) isoprenaline-mediated relaxation of CCh (carbachol, 1 M)-induced

contractions, and (EH) Ca2+-induced contractions, in isolated guinea-pig tracheal preparations in the presence and absence
of (A and E) the crude extract of P. nigrum (Pn.Cr), (B and F) piperine, (C and G) papaverine, and (D and H) verapamil. Symbols
represent mean standard error of mean (number of observations = 38). *, ** and *** represent p-values of less than 0.05,
0.01 and 0.001 respectively, while ns represents p-value of more than 0.05, showing comparison of the effect of different
concentrations of test materials with respective control curves. All p-values were obtained using two-way analysis of
variance followed by Bonferroni post-test correction.
 Journal of Functional Foods 19 (2015) 126140 135

Table 1 Median inhibitory concentrations of isoprenaline inhibitory curves (IIC50) on carbachol (1 M)-induced
contractions and the maximal response of Ca2+ (Emax) at highest tested concentration in the absence and presence of
different concentrations of the crude extract of P. nigrum (Pn.Cr), piperine and various fractions of Pn.Cr, in isolated
guinea-pig tracheal tissues.
Group Concentration IIC50 (95% CI, p-Value* Concentration Emax of Ca2+ (%) p-Value
n = 38) M SEM (n = 48)
Control 1.26 (1.111.44) 100 0
Pn.Cr 0.01 mg/mL 0.33 (0.260.39) <0.001 0.1 mg/mL 69.12 5.1 <0.001
0.03 mg/mL 0.11 (0.090.14) <0.001 0.3 mg/mL 44.37 4.72 <0.001
Control 0.34 (0.280.4) 100 0
Piperine 0.3 M 0.17 (0.140.2) <0.01 3 M 85 2.89 <0.05
1 M 0.04 (0.030.06) <0.001 10 M 61.67 4.41 <0.01
Control 0.57 (0.460.71) 100 0
Pn.Aq 0.01 mg/mL 0.23 (0.190.28) <0.001 0.1 mg/mL 88.67 4.1 <0.05
0.03 mg/mL 0.09 (0.070.11) <0.001 0.3 mg/mL 75.17 6.19 <0.05
Control 0.58 (0.460.72) 100 0
Pn.CHCl3 0.01 mg/mL 0.31 (0.230.39) ns 0.1 mg/mL 88.33 4.41 <0.05
0.03 mg/mL 0.18 (0.150.21) <0.05 0.3 mg/mL 68.33 4.91 <0.01
Control 0.76 (0.670.86) 100 0
Pn.EtAc 0.01 mg/mL 0.59 (0.520.66) ns 0.03 mg/mL 80 5.77 <0.05
0.03 mg/mL 0.44 (0.380.49) ns 0.1 mg/mL 55.67 5.61 <0.01
Control 0.84 (0.661.08) 100 0
Pn.Pet 0.003 mg/mL 0.24 (0.190.29) <0.001 0.03 mg/mL 81.67 6.01 <0.05
0.01 mg/mL 0.09 (0.070.11) <0.001 0.1 mg/mL 62.5 6.29 <0.01
CI = confidence interval; ns = not significant (p > 0.05); Pn.Aq, Pn.CHCl3, Pn.EtAc, Pn.Pet = aqueous, chloroform, ethyl acetate, and petroleum ether
fractions of P. nigrum respectively; Pn.Cr = crude extract of P. nigrum; SEM = standard error of mean.
* p-Value obtained by comparing concentrationresponse curves of isoprenaline in the absence (control) and presence of test drug or extract
using two-way analysis of variance (ANOVA) followed by Bonferronis post-test correction.

p-Value obtained by comparing concentrationresponse curves of Ca2+ in the absence (control) and presence of test drug or extract using
two-way analysis of variance (ANOVA) followed by Bonferronis post-test correction.

Usmanghani et al., 1997), the extract was tested for its pos-
sible bronchodilator effect in isolated guinea-pig tracheal strips,
where it relaxed both CCh- and K + (80 mM)-induced
contractions with similar potency. Papaverine, a known in-
hibitor of phosphodiesterase (PDE) and Ca2+-influx (Pch &
Kukovetz, 1971), also caused a similar pattern of inhibitory effect
with comparable potency against CCh and K+ (80 mM)-in-
duced contractions. On the other hand, verapamil, a standard
Ca2+-channel antagonist (Lee & Tsien, 1983), was more potent
against K+ (80 mM)-induced contractions than CCh-induced con-
tractions. The presence of papaverine-like constituent(s) was
confirmed when pre-treatment of the tissue with extract po-
tentiated isoprenaline-mediated smooth muscle relaxation.
Beta ()-Adrenoceptor agonists (like isoprenaline) and PDE
inhibitors (like papaverine) are known to produce
bronchodilation via an increase in intracellular level of cAMP
but through different target sites. -Adrenoceptor agonists ac-
tivate adenylyl cyclase via -subunit of G-protein coupled
receptors, which leads to degradation of adenosine triphos-
phate (ATP) to cAMP (Johnson, 2006). Likewise, PDE inhibitors
directly reduce degradation of cAMP by inhibiting the enzyme
PDE (Fredholm, Brodin, & Strandberg, 1979). Increase in cAMP
causes relaxation of smooth muscles and stimulation of cardiac
muscles (Morgan, Perreault, & Morgan, 1991). Keeping this in
view, pre-treatment of guinea-pig tracheal tissues with Pn.Cr
most likely resulted in inhibition of PDE due to the presence
of certain papaverine-like constituents within the crude extract.
This caused an increase in intracellular concentration of cAMP
and therefore, effect of isoprenaline on such tissues was po-
tentiated. Another possibility could be elaboration of nitric oxide
due to induction of the enzyme nitric oxide synthase (NOS) in
the myenteric plexus. NOS produces nitric oxide by deami-
nating L-arginine to L-citrulline. Nitric oxide, being a soluble
neurotransmitter and second messenger, can diffuse into
smooth muscle cells and stimulate guanylyl cyclase (Harvey
& Ferrier, 2011). In turn, guanylyl cyclase can produce cyclic
5-guanosine monophosphate (cGMP) from guanosine triphos-
phate (GTP), which causes activation of protein kinase G.
Consequently, phosphorylation of Ca2+ channels by protein
kinase G leads to decreased entry of Ca2+, reduced binding of
Ca2+ to calmodulin, inhibition of myosin light-chain kinase and
relaxation of smooth muscle (Somlyo & Somlyo, 2003).
In an effort to identify the papaverine-like constituents
within Pn.Cr, activity-directed fractionation was performed.
Pn.Aq, Pn.CHCl3, Pn.EtAc and Pn.Pet were tested separately for
their ability to inhibit CCh-induced and K+ (80 mM)-induced
contractions. Pn.Pet was found to be the most potent frac-
tion suggesting that papaverine-like constituent(s) were
concentrated in petroleum ether fraction due to their organic
and polar nature.
To identify the chemical constituents responsible for
bronchodilation, we performed HPLC analysis of Pn.Cr and its
fractions. The results demonstrated the presence of piperine,
piperidine, eugenol and catechin within the crude extract and
varied distribution in its respective fractions. Several other un-
identified compounds were also present in the crude extract,
chloroform and petroleum ether fractions. Some of these com-
pounds may account for the smooth muscle relaxant effects
of Pn.Cr observed in our study. Specifically, catechin has been
reported to have bronchodilator effects mediated through
136 Journal of Functional Foods 19 (2015) 126140

Fig. 7 Concentrationresponse curves of (AD) isoprenaline-mediated relaxation of CCh (carbachol, 1 M)-induced

contractions, and (EH) Ca2+-induced contractions, in the presence and absence of (A and E) aqueous (Pn.Aq), (B and F)
chloroform (Pn.CHCl3), (C and G) ethyl-acetate (Pn.EtAc) and (D and H) petroleum ether (Pn.Pet) fractions of P. nigrum in
isolated guinea-pig tracheal preparations. Symbols represent mean standard error of mean (number of observations = 4
7). *, ** and *** represent p-values of less than 0.05, 0.01 and 0.001 respectively, while ns represents p-value of more than
0.05, for comparison of the effect of different concentrations of test materials with respective control curves. Statistical test
applied for performing all comparisons was two-way analysis of variance followed by Bonferroni post-test correction.
 Journal of Functional Foods 19 (2015) 126140 137

Fig. 8 Concentration-dependent excitatory and inhibitory effects of (A) the crude extract of P. nigrum (Pn.Cr), (B) piperine,
(C) aminophylline, and (D) aqueous (Pn.Aq), (D) chloroform (Pn.CHCl3), (E) ethyl acetate (Pn.EtAc), and (F) petroleum ether
(Pn.Pet) fractions on rate and force of contractions of isolated spontaneously beating guinea-pig atria. Symbols represent
mean standard error of mean (number of observations = 38). *, ** and *** represent p-values of less than 0.05, 0.01 and
0.001 respectively, while ns represents p-value of more than 0.05, for comparison of stimulatory effect of respective test
materials against baseline using one-tailed, paired t-test.
138 Journal of Functional Foods 19 (2015) 126140
Ca2+-channel blockade (Ghayur, Khan, & Gilani, 2007). More-
over, certain piperidine derivatives have also been evaluated
for their potential to relax smooth muscles (Saeed, Saify, Gilani,
& Iqbal, 1998). However, piperine has not been reported pre-
viously to have bronchodilator effects. As piperine has structural
homology to piperidine and was a predominant constituent
of Pn.Cr and Pn.Pet, we tested it against CCh- and K+ (80 mM)-
induced contractions. The effect of piperine on these
contractions was found to be similar to that of papaverine and
unlike that of verapamil. To further confirm the presence of
papaverine-like activity in piperine, we constructed isoprena-
line CRCs by incubating guinea-pig tracheal tissues with small
concentrations of piperine. Potentiation of the effect of iso-
prenaline on such tissues confirmed the ability of piperine to
inhibit PDE in such tissues.
Ability of piperine to block voltage-gated calcium (Ca2+v)
channels (Taqvi, Shah, & Gilani, 2008) and stimulate opioid re-
ceptors (Mehmood & Gilani, 2010) has already been reported.
In the present study, we have reported for the first time its
ability to inhibit PDE enzyme. It is interesting to note here that
piperine exhibits different activities at different concentra-
tions. In our study, we found that PDE inhibition is most
pronounced at 330 M concentration in guinea-pig tracheal
tissues. On the other hand, in a study by Taqvi, Shah & Gilani
(2008), Ca2+ channel blockade was most prominent at 30
300 M in bovine coronary arterial preparations. Likewise,
stimulation of opioid receptors by piperine also occurs chiefly
at 3001000 M (Mehmood & Gilani, 2010).
P. nigrum has folkloric reputation as an aphrodisiac (Nadkarni,
1982). Commercially available PDE inhibitors, such as sildenafil,
are known to enhance erection and are widely used for the
treatment of erectile dysfunction (Terrett, Bell, Brown, & Ellis,
1996). In this study, we have demonstrated the ability of pip-
erine and P. nigrum to inhibit PDE enzyme. This may partly
account for its folkloric reputation in this regard. Further-
more, PDE inhibitors, such as theophylline and caffeine, are also
known to have positive chronotropic, inotropic and dromo-
tropic effects (Weishaar, Kobylarz-Singer, Steffen, & Kaplan,
1987). In our study, piperine was found to have positive chro-
notropic and inotropic effects at low concentrations, followed
by negative chronotropic and inotropic effects at higher con-
centrations. These observations were consistent with inhibition
of PDE enzyme at low concentrations and blockade of Ca2+v
channels at higher concentrations. Again, this may provide a
scientific basis for the folkloric reputation of black pepper as
a cardiac stimulant (Duke, Bogenschutz-Godwin, Du Celliar, &
Duke, 2002). Tachycardia is a prominent side effect of com-
mercially available PDE inhibitors, which limits their use as
bronchodilators, especially in patients with ischaemic heart
disease (Salpeter, Ormiston, & Salpeter, 2004). The ability of
P. nigrum to inhibit PDE enzyme at low doses and block Ca2+v
channels at high doses implies that its bronchodilator effects
would persist at high doses without producing significant
tachycardia.
The use of 2-Adrenoceptor agonists and PDE inhibitors for
the treatment of asthma and chronic obstructive pulmonary
disease is well-established (Sears et al., 1990). Interestingly, Ca2+-
channel blockers have also been found to be useful in this
condition (Barnes, 1983). The ability of piperine to inhibit PDE
and block Ca2+v channels suggests that black pepper may be
potentially useful for the treatment of asthma. Moreover, as
mentioned previously, piperine and Pn.Cr can also stimulate
opioid receptors (Mehmood & Gilani, 2010). This can explain
the anti-tussive properties of black pepper and its traditional
use in persistent cough (Duke, Bogenschutz-Godwin, Du Celliar,
& Duke, 2002).
In our study, bronchodilation caused by black pepper in an-
aesthetized SpragueDawley rats was the most pronounced at
100 mg/kg. Based on standardized conversion formulae
(Reagan-Shaw, Nihal, & Ahmad, 2008), the equivalent dose of
black pepper for a 60-kg man can be calculated to lie between
0.6 and 6 g. Given that black pepper is edible and consumed
worldwide, such a low dose of black pepper is very unlikely
to cause toxicity. Based on these results, black pepper can be
an attractive candidate for the development of functional foods
and nutraceuticals particularly for disorders of airway hyper-
activity Interestingly, piperine, known to be the main active
principle, was not more effective or potent than the parent
crude extract, which suggest that either compound also con-
tributes towards bronchodilator activity or synergistic
interaction of different chemicals present in pepper occurs.
5. Conclusion
Black pepper and its principle alkaloid, piperine, possess
bronchodilatory activity possibly mediated through dual in-
hibition of PDE enzyme and Ca2+ influx, thus providing an
evidence to the folk medicinal function of black pepper in bron-
chitis and asthma. The data presented also highlight therapeutic
potential of black pepper and piperine as promising candi-
dates for the development of nutraceuticals and functional food
ingredients to treat airways disorders.
Acknowledgements
The study was carried out during elective rotation of Mr. Mu-
hammad Hanif, research volunteer enrolled in PhD (BASR/NO/
02535/Pharm) at Department of Pharmaceutical Chemistry,
University of Karachi, Karachi, working under Co-supervision
of Dr. Malik Hassan Mehmood at Department of Biological and
Biomedical Sciences (BBS) with partial support from Depart-
mental Faculty Support Fund awarded to Dr. Malik Hassan
Mehmood, while the rest of the work of this project has been
completed using Research Module Project Funds awarded to
Dr. Malik Hassan Mehmood, as Principal Investigator (PI), and
Dr. Abdul Rehman, as co-PI at Department of BBS, Aga Khan
University, Karachi.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.jff.2015.09.006.
 Journal of Functional Foods 19 (2015) 126140 139

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