In vitro cytotoxic and in silico activity of piperine isolated from Piper nigrum fruits Linn

Abstract

Piper nigrum [Piperaceae], commonly known as black pepper is used as medicine fairly throughout the greater part of India and as a spice globally.
To isolate piperine and evaluate in vitro cytotoxic [antiproliferative] activity and in silico method.
Piperine was isolated from the fruits of P.nigrum. Piperine was characterized by UV,IR, 1 H-NMR, 13 C-NMR and Mass spectrum. Standardization of piperine was done also by HPTLC fingerprinting. In vitro cytotoxic activity was done using HeLa cell lines by MTT assay at different concentrations ranging from 20 to 100 μg/ml in triplicate and in silico docking studies using enzyme EGFR tyrosine kinase.
Fingerprinting of isolated piperine were done by HPTLC method. The IC 50 value was found to be 61.94 ± 0.054 μg/ml in in vitro cytotoxic activity in HeLa Cell lines. Piperine was subjected to molecular docking studies for the inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for inhibition of cancer cells. It has shown −7.6 kJ mol −1 binding and 7.06 kJ mol −1 docking energy with two hydrogen bonds.
piperine has shown to possess in vitro cytotoxic activity and in silico studies.

Full text

O R I G I N A L R E S E A R C H Open Access
In vitro cytotoxic and in silico activity of
piperine isolated from Piper nigrum fruits
Linn
Padmaa M. Paarakh
1*
, Dileep Chandra Sreeram
2
, Shruthi S. D
3
and Sujan P. S. Ganapathy
4
Abstract
Background: Piper nigrum [Piperaceae], commonly known as black pepper is used as medicine fairly throughout
the greater part of India and as a spice globally.
Purpose: To isolate piperine and evaluate in vitro cytotoxic [antiproliferative] activity and in silico method.
Methods: Piperine was isolated from the fruits of P.nigrum. Piperine was characterized by UV,IR,
1
H-NMR,
13
C-NMR
and Mass spectrum. Standardization of piperine was done also by HPTLC fingerprinting. In vitro cytotoxic activity
was done using HeLa cell lines by MTT assay at different concentrations ranging from 20 to 100 μg/ml in triplicate
and in silico docking studies using enzyme EGFR tyrosine kinase.
Results: Fingerprinting of isolated piperine were done by HPTLC method. The IC
50
value was found to be 61.94 ±
0.054 μg/ml in in vitro cytotoxic activity in HeLa Cell lines. Piperine was subjected to molecular docking studies for
the inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for inhibition of cancer cells. It has
shown −7.6 kJ mol
−1
binding and 7.06 kJ mol
−1
docking energy with two hydrogen bonds.
Conclusion: piperine has shown to possess in vitro cytotoxic activity and in silico studies.
Keywords: In vitro cytotoxic activity, In silico docking studies, Isolation, Piperine, Piper nigrum
Background
Cancer is one of the highest impacting diseases world-
wide with significant morbidity and mortality rates. The
current known therapies are based on radio and chemo-
therapies and although in many cases, the patients have
their health re-established, the treatment is very painful
since their immunological system is severely compro-
mised, because these procedures are not cells selective
[Leticia et al. 2013]. Substantial advances have been
made in understanding the key roles of receptor tyrosine
kinase (RTK) in the signalling pathways that govern fun-
damental cellular processes, such as proliferation, migra-
tion,metabolism, differentiation and survival. In the
normal cells RTK activity is tightly controlled. When they
are mutated or structurally altered, they become potent
oncoproteins which leads to abnormal activation of RTKs
in transformed cells has been shown to be causally in-
volved in the development and progression of many hu-
man cancers (Andreas et al. 2004; Wajapeyee et al. 2006).
Tyrosine kinases are an especially important target
because they play an important role in the modulation of
growth factor signaling. There are multiple types of
targeted therapies available, including monoclonal anti-
bodies, inhibitors of tyrosine kinases and antisense inhibi-
tors of growth factor receptors. But we have focussed only
on inhibitors of receptor tyrosine kinases. Tyrosine
kinases play a critical role in the modulation of growth
factor signaling. Activated forms of these enzymes can
cause increases in tumor cell proliferation and growth,
induce antiapoptotic effects and promote angiogenesis
and metastasis. In addition to activation by growth factors,
protein kinase activation by somatic mutation is a common
mechanism of tumor genesis. Ligand binding induces
dimerization of these receptor tyrosine kinases, resulting in
autophosphorylation of their cytoplasmic domains and acti-
vation of tyrosine kinase activity. Multiple cytoplasmic
* Correspondence: padmaparas@hotmail.com
1
Department of Pharmacognosy, The Oxford College of Pharmacy, 6/9, I
Cross, Begur Road, Hongasandra, Bangalore 560068, Karnataka, India
Full list of author information is available at the end of the article
© 2015 Paarakh 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.
Paarakh et al. In Silico Pharmacology (2015) 3:9
DOI 10.1186/s40203-015-0013-2
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

signaling pathways, including the Ras/Raf mitogen-activated
protein kinase pathway, the phosphoinositol 3’-kinase/Akt
pathway, the signal transducer and activator of transcription
3 pathway, the protein kinase C pathway, and scaffolding
proteins may then be activated (Schlessinger 2000; Bogdan
and Klambt 2001). Intracellular mediators in these pathways
transduce signals from membrane receptors through the
cytosol and into the nucleus, culminating in altered DNA
synthesis and cell division as well as effects on a variety of
biological processes, including cell growth, migration, dif-
ferentiation and death (Carpenter and Cohen 1990;
Blume-Jensen and Hunter 2001). Because all of these ef-
fects are initiated by receptor tyrosine kinase activation,
they are key targets for inhibitors.
The tyrosine kinase inhibitors compete with the ATP
binding site of the catalytic domain of several oncogenic
tyrosine kinases. They are orally active, small molecules
that have a favorable safety profile and can be easily
combined with other forms of chemotherapy or radi-
ation therapy. Several tyrosine kinase inhibitors (TKIs)
have been found to have effective antitumor activity and
have been approved or are in clinical trials. The inhibi-
tors used are imatinib mesylate, gefitinib, erlotinib, lapa-
tinib, canertinib, semaxinib, vatalanib, sorafenib,sutent
and leflunomide. TKIs are thus an important new class
of targeted therapy that interfere with specific cell sig-
naling pathways and thus allow target-specific therapy
for selected malignancies. Use of these targeted therapies
is not without limitations such as the development of re-
sistance and the lack of tumor response in the general
population. The availability of newer inhibitors and im-
proved patient selection will help overcome these prob-
lems in the future (Finley 2003).
The cost of treatment is very high and with lot of side
effects. In order to find new natural sources that are bio-
logically active substances from plants have acquired im-
mense attention. A number of studies have been carried
out on various plants, vegetables and fruits because they
are rich sources of phytoconstituents which prevent free
radical damage thereby reducing risk of chronic diseases
viz., cancer, cardiovascular diseases etc. This beneficial
role of plants has led to increase in the search for newer
plant based sources for the treatment of diseases like
cancer. One such plant is Piper nigrum.
Piper nigrum, commonly known as black pepper is
used as medicine fairly throughout the greater part of
India and considered as King of spice. The plant is re-
ported to possess antiapoptotic, antibacterial, anti-colon
toxin, antidepressant, antifungal, antidiarrhoeal, anti-
inflammatory, antimutagenic, anti-metastatic activity,
antioxidative, antispasmodic, antispermatogenic, antitu-
mor, antithyroid, gastric ailments, hepatoprotective, in-
secticidal activity, intermittent fever, larvicidal activity,
protection against diabetes induced oxidative stress,
analgesic,anti-inflammatory, anticonvulsant, antimalarial,
antifiliarial, and antifertility activities (Ahmad et al.
2012).
The chemopreventive effects of piperine against sev-
eral kinds of carcinogen, such as benzo(α)pyrene and
7,12-dimethyl benz(α)anthracene, show its potential as a
cancer preventive agent. Administration of piperine
(50 mg/kg or 100 mg/kg per day for 7 days) inhibits
solid tumor development in mice transplanted with sar-
coma 180 cells. A recent study has shown that piperine
inhibits breast stem cell self-renewal and does not cause
toxicity to differentiated cells. It has been demonstrated
that piperine induced apoptosis and increased the per-
centage of cells in G 2/M phase in 4 T1 cells and in-
duced K562 cells to differentiate into macrophages/
monocytes. Piperine also has very good antimetastatic
properties against lung metastasis induced by B16F-10
melanoma cells in mice (200 μM/kg) and suppresses
phorbol-12-myristate-13-acetate (PMA)-induced tumor
cell invasion (Lu et al. 2012).
Piperine is a potent inhibitor of NF-κB, c-Fos, cAMP re-
sponse element-binding (CREB), activated transcription fac-
tor 2 (ATF-2), among others. It suppresses PMA-induced
MMP-9 expression via the inhibition of PKCα/extracellular
signal-regulated kinase (ERK) 1/2 and reduction of NF-κB/
AP-1 activation. Remarkably, piperine also inhibits the
functions of P-glycoprotein (P-gp) and CYP3A4, which not
only affects drug metabolism but also re-sensitizes multi-
drug resistant (MDR) cancer cells. Piperine increases the
therapeutic efficacy of docetaxel in a xenograft model with-
out inducing more adverse effects on the treated mice by
inhibiting CYP3A4, one of the main metabolizing enzymes
of docetaxel. The tyrosine kinases inhibitor activity of piper-
ineisnotstudiedtilldate.Theaimofthepresentstudyis
to isolate piperine from dried fruits of Piper nigrum and
perform in silico activity and in vitro MTT assay to prove
its cytotoxic activity.
Methods
Plant material
The dried fruits of Piper nigrum (Piperaceae) were col-
lected, identified and authenticated by Dr Shiddamallayya
N at National Ayurveda Dietetics Research Institute,
Bengaluru, Karnataka. A voucher specimen was deposited
in the Herbarium of Department of Pharmacognosy, The
Oxford College of Pharmacy, Bangalore. The fruits were
dried under normal environmental conditions. The dried
fruits were powdered and stored in a closed container for
further use.
Drugs and chemicals
DMEM medium (GIBCO), heat-inactivated fetal bovine
serum (FBS), trypsin, ethylene-diaminetetraacetic acid
(EDTA),PBS and MTT were purchased from Hi media
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and Sigma Chemicals. All chemicals and reagents used
in this study were at least of analytical grade.
Extraction and isolation procedure
The dried fruits of P.nigrum (150 g) was macerated
with glacial acetic acid (6 × 300 ml) for 5 min each
time. Filter and pooled acetic acid layer was mixed
with equal volume of water. Extract with chloroform
3 times and combined chloroform was washed with
10 % sodium carbonate and water. Chloroform layer
was dried over anhydrous sodium sulphate and
concentrated to dryness at 60 °C. The residue was
dissolved in minimum quantity of chloroform,add di-
ethyl ether which resulted in separation of needle
shaped crystals of crude piperine. The crude needles
were repeatedly crystallized as above to give shinning
yellow needles of piperine (0.35 g).
Characterization of piperine
The structure of Piperine was characterized by UV,
IR, NMR and Mass spectrum. HPTLC fingerprinting
was done to confirm the presence and purity of
Piperine.
Chromatographic finger printing of the dried fruits of P.
nigrum using piperine
Weigh 2 g of coarsely powdered drug and transfer to a
250-ml conical flask. Extract with 50 ml of methanol by
refluxing for about 20 min and filter. Repeat the process
4–5 times till the raw material is completely exhausted
or till the extract is colourless. Combine the extracts and
concentrate to a volume of about 100 ml, cool to room
temperature. Use the solution for TLC profiling. Stand-
ard solution was prepared by dissolving 10 mg of Piper-
ine in 100 ml of methanol. Solvent system used was
Hexane: Ethyl acetate (5:3). Apply 20 μloftestsolution
and 5 μl of standard solution separately on a precoated
Fig. 1 Strucuture of the compound piperine
Fig. 2 HPTLC profile of standard and isolated piperine and extract of
Piper nigrum dried fruits at 254 nm. 1: Piperine standard; 2: Isolated
Piperine; 3: P. nigrum extract
Fig. 3 HPTLC profile of standard and isolated piperine and extract of
Piper nigrum dried fruits at 366 nm. 1: Piperine standard; 2: Isolated
Piperine; 3: P. nigrum extract
Paarakh et al. In Silico Pharmacology (2015) 3:9 Page 3 of 7
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silica gel 60F
254
TLC plate (E. Merck) of uniform thickness
(0.2 mm). Develop the plate in the solvent system till
the solvent rises to a distance of 8 cm. Visualization
was done after spraying with anisaldehyde-sulphuric
acid reagent and followed by heating at 105 °C for
5–10 min. The R
f
valueandcolouroftheresolved
bands were noted.
In vitro cytotoxic activity using HeLA cell lines by MTT
assay
Cell culture
HeLa cell line was maintained in DMEM medium
(GIBCO) supplemented with 10 % (v/v) heat-inactivated
fetal bovine serum (FBS) and 1 % antibiotic solution
(penicillin 100U/ml and streptomycin 100 μg/ml) at 37 °C
in a humidified atmosphere of 95 % air/5 % CO
2
.The
medium was changed every second day, and cells were
subcultured when confluency reach to 95 % by 0.25 %
trypsin containing 0.02 % ethylene-diaminetetraacetic acid
(EDTA) in PBS for 3 min at 37 °C.
MTT Assay
The MTT assay was carried out as described previously to
measure cell viability (Rahman et al. 2011). Ten thousand
cells in 100 μl of DMEM media were seeded in the wells of
a 96-well plate. After 24 h, existing media was removed
and 100 μl of various concentrations of compound [20–
100 μg/ml] were added and incubated for 48 h at 37 °C in
aCO
2
incubator. Control cells were supplemented with
0.05 % DMSO vehicle. At the 48th hour of incubation,
MTT (3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium
bromide- supplied from Sigma, 10 μlof5mg/ml)was
added to the plate. The contents of the plate were pipetted
out carefully, the formazan crystals formed were dissolved
in 100 μl of DMSO, and the absorbance was measured at
550 nm in a microplate reader (Tecan, Infinite F200 Pro).
Experiments were performed in triplicate [3 times x 3 wells
each time/group] and the results were expressed as mean
of percentage inhibition. A graph of the concentration ver-
sus percentage growth inhibition was plotted, and the con-
centration at which 50 % cell death occurred was
considered as the IC
50
value. Before adding MTT, bright
field images (Olympus 1 × 81, cellSens Dimension soft-
ware) were taken for visualizing the cell death.
In silico activity: molecular docking studies
The three dimensional structure of target protein EGFR
tyrosine kinase (PDB ID:2J5F) was downloaded from
Fig. 4 HPTLC profile of standard and isolated piperine and extract of
Piper nigrum dried fruits after dervatization. 1: Piperine standard;
2: Isolated Piperine; 3: P. nigrum extract
Fig. 5 Cytotoxic activity of piperine showing cell death, a-control; b-treated
Paarakh et al. In Silico Pharmacology (2015) 3:9 Page 4 of 7
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PDB (www.rcsb.org/pdb) structural database. This file
was then opened in SPDB viewer edited by removing the
heteroatoms, adding C terminal oxygen. The active
pockets on target protein molecule were found out using
CASTp server (Binkowski et al. 2003). The ligands were
drawn using ChemDraw Ultra 6.0 and assigned with
proper 2D orientation (ChemOffice package). 3D coordi-
nates were prepared using PRODRG server (Ghose and
Crippen 1987). Autodock V3.0 was used to perform Au-
tomated Molecular Docking in AMD Athlon (TM) 2 × 2
215 at 2.70 GHz, with 1.75 GB of RAM. AutoDock 3.0
was compiled and run under Microsoft Windows XP
Fig. 6 3D structure of EGFR tyrosine kinase from PDB (a); Interacting amino acids as predicted from the ligplot (b); Enfolding of piperine in the
active pocket (c)
Table 1 Molecular docking results of piperine with EGFR tyrosine kinase
Molecule Binding energy Docking energy Inhibitory constant Intermol energy H-bonds Bonding
PR −7.6 7.06 2.69e-006 −8.22 2 PR::DRG:OAD:TK:A:PRO699:O
PR::DRG:OAB:TK:A:ARG831:HH12
Paarakh et al. In Silico Pharmacology (2015) 3:9 Page 5 of 7
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service pack 3. For docking, grid map is required in
AutoDock, the size of the grid box was set at 102,
126 and 118 Å (R, G, and B) and grid center −58.865,
−8.115, −24.556 for x, y and z-coordinates. All torsions
were allowed to rotate during docking. The Lamarckian
genetic algorithm and the pseudo-Solis and Wets methods
were applied for minimization, using default parameters
(Morris et al. 1998).
Results and Discussions
The characterization of the piperine (Fig. 1)
Physical data
Piperine is orange needle shaped crystals, mp 132 °C; lit
mp 131–132 °C. Soluble in chloroform and methanol;
insoluble in water.
Molecular formula: C
17
H
19
NO
3;
Molecular weight:
285.342.
Spectral data
UV-VIS spectrum shows absorption at 340 nm. The UV-
VIS spectrum indicates the presence of chromophoric
system in the molecule. The IR spectrum showed shows
peaks corresponding to the functional groups present in
piperine.
1
H NMR spectrum (DMSO-d
6
-300 MHz): The
signal at δ1.55–1.71 (6H, m), 3.52 (2H, s), 3.63 (2H, s),
5.98 (2H,s,methylene dioxy-H), 6.44 (1H, d), 6.76 (1H,
dd), 6.77 (1H,d), 6.89 (1H, dd), 6.98 (1H, d, aromatic-H),
7.40 (1H, ddd). The δvalues were comparable with that
of reported
1
H NMR piperine (Sakpakdeejaroen and
Arunporn 2009).
13
C NMR spectrum (CD
3
OD −300 MHz): the signals
at δ24.66, 25.66,26.67, 43.25, 46.93, 101.24, 105.71,108.
46,120.15,122.41,125.41,131.07,138.14,142.39,148.11,148.
21 and 165.43. The δvalues were comparable with that of
reported
13
C NMR piperine (Avijit et al. 1984).
The HPTLC fingerprinting confirmed the presence of
Piperine (Figs. 2, 3 and 4). A grey coloured band was ob-
served at (R
f
0.37) corresponding to piperine is visible in
both the test and standard solution tracks under UV at
254 nm,366 nm and after derivatization.
In vitro cytotoxic activity on HeLa cell lines
The MTT values obtained demonstrated that piperine
has cytotoxic effect as the IC
50
value was found to be
61.94 ± 0.054 μg/ml. Microscopy images representing the
cell death caused by the compounds are as seen in Fig. 5.
It is very clear that it is cytotoxic agent when compared
to control cell with vehicle alone.
In silico molecular docking studies
The tyrosine kinase receptors have multidomain extra-
cellular Ligands for specific Ligand, a signal pass trans-
membrane hydrophobic helix and tyrosine kinase
domain. The receptor tyrosine kinases are not only cell
surfaces transmembrane receptors, but are also enzymes
having kinase activity (Bari et al. 2012). In cancer, angio-
genesis is an important step in which new capillaries de-
velop for supplying a vasculature to provide nutrient and
removing waste material. So tyrosine kinase inhibitor as
an anti-angiogenic agent is new cancer therapy. Devel-
oping natural drugs and prodrugs as inhibitor is today’s
trend. Low molecular weight substances, which inhibit
tyrosine kinase phosphorylation block signaling pathway,
initiated in the extracellular part of receptor (Paul and
Manlay 2002). Since, the type I receptor tyrosine kinase
is a major regulator of several distinct and diverse cellu-
lar pathways we have evaluated it as a target.
Piperine was taken and docked to get the best con-
former. Results were compared for the binding energy,
docking energy and number of hydrogen bonds formed.
According to the docking results (Table 1), it has binding
energy of −7.6 kJ mol
−1
with two hydrogen bonds formed.
Molecular docking with EGFR tyrosine kinase domain
revealed that, our compound has inhibitory capability and
thereby exhibiting interactions with one or the other
amino acids in the active pockets as shown in Fig. 6. The
topology of the active site of EGFR tyrosine kinase was
similar in all synthesized molecules, which is lined by
interacting amino acids as predicted from the ligplot
(Fig. 6). In in vitro studies the molecule emerged to be ac-
tive against the cell line used in inhibiting the cell growth.
Conclusion
Piperine has shown to possess in vitro cytotoxic activity
and in silico studies. The IC
50
value was found to be
61.94 ± 0.054 μg/ml and in silico studies, it has more
number of hydrogen bonds with minimum binding and
docking energy and may be considered as inhibitor of
EGFR tyrosine kinase. More experiments are required to
understand the exact mechanism by which the cells are
affected. It is important to correlate the structure of
these compounds with their biological effect, which will
be valuable to propose new lead compounds with better
cytotoxic potential.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
Dr. PMP and Mr. DCS carried out the isolation and characterization of
Piperine, Dr. SSD carried out in vitro MTT assay and Dr. SPSG carried out in
silico activity. All authors have read and approved the final manuscript.
Acknowledgement
The authors are grateful to The Chairman and Executive Director, Children’s
Education Society and Department of Pharmacognosy, The Oxford College
of Pharmacy, Bangalore, for providing the facilities for carrying out the entire
experiment.
Author details
1
Department of Pharmacognosy, The Oxford College of Pharmacy, 6/9, I
Cross, Begur Road, Hongasandra, Bangalore 560068, Karnataka, India.
2
R&D
Paarakh et al. In Silico Pharmacology (2015) 3:9 Page 6 of 7
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[Phytochemistry], Natural Remedies Private limited, Bangalore, India.
3
Microbiology and Cell Biology Department, Indian Institute of Science,
Bangalore, Karnataka, India.
4
Research and Development Centre, Olive
LifeSciences Pvt. Ltd., Nelamangala, Bangalore 562123Karnataka, India.
Received: 29 July 2015 Accepted: 20 October 2015
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