JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:147–155

https://doi.org/10.1007/s00764-023-00237-0

ORIGINAL RESEARCH PAPER

High‑performance thin‑layer chromatography method development

and validation for quantification of piperine in different extracts
of Piper longum L. and its antioxidant activity
Isha Gupta1 · Syeda Nashvia Adin1 · Mohd. Aqil2 · Mohd. Mujeeb1 · Mohd. Akhtar3

Received: 7 February 2023 / Accepted: 17 April 2023 / Published online: 13 June 2023
© Akadémiai Kiadó, Budapest, Hungary 2023

Abstract
A selective, simple, and sensitive high-performance thin-layer chromatography (HPTLC) method was developed and vali-
dated for identification and quantitative estimation of piperine in different extracts of Piper longum. A developing solvent,
acetone‒toluene (3:7, V/V) was employed to attain good separation of bands. Densitometric estimation was performed at
343 nm. Linearity was reckoned to be in the range of 0.2–1.0 μg per spot. Ethanolic extract of P. longum revealed a higher
content of piperine than dimethylformamide (DMF), acetone, and dimethyl sulphoxide (DMSO) extract during analysis.
The method was further validated for robustness, accuracy, precision, specificity, and linearity. Furthermore, the antioxidant
activity of P. longum extracts was scrutinized using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and the ethanolic
extract showed the highest antioxidant potential, with 55.26% inhibition.

Keywords Piper longum · High-performance thin-layer chromatography (HPTLC) · Piperine · Antioxidant activity

1 Introduction hepatoprotective, antiplatelet, analgesic, immunomodula-

Piper longum (Linn.) is one of the chief medicinal plants
of family Piperaceae (also known as Indian long pepper)
[1]. It is used as a principal herb in Ayurvedic and indig-
enous medicinal systems, e.g., trikatu, panchakola, etc.,
to treat diverse disorders [2, 3]. Owing to its multifaceted
medicinal properties, which include anticancer, antioxidant,
Isha Gupta and Syeda Nashvia Adin have contributed equally to this
work and should be considered as joint first authors.
* Mohd. Aqil
aqilmalik@yahoo.com
* Mohd. Mujeeb
mohdmujeeb72@gmail.com
1
Phytomedicine Laboratory, Department of Pharmacognosy
& Phytochemistry, School of Pharmaceutical Education &
Research, Jamia Hamdard, New Delhi 110062, India
2
Department of Pharmaceutics, School of Pharmaceutical
Education & Research, Jamia Hamdard, New Delhi 110062,
India
3 and antihypertensive activity [11‒15, 17‒23]. Owing to
Department of Pharmacology, School of Pharmaceutical
Education & Research, Jamia Hamdard (Deemed University),
New Delhi 110062, India
tory, hyperlipidemic, antidepressant, antifungal, antidia-
betic, antiinflammatory, antimicrobial, anti-amoebic, anti-
cardioprotective, and anti-obesity activities [4–7], the plant
is highly valued. In various Ayurvedic medicines, it is used
as the main component to cure tuberculosis and leprosy. It is
also used to treat cough, dyspnea, chronic fever, gout, rheu-
matic pain, cardiac, and spleen disorders [8]. Researchers
have identified diverse primary constituents from P. longum,
namely, pipermonaline, piperlongumine, diaeudesmin piper-
longuminine, piperundecalidine, sesamin, sylvatin, and pip-
erine, among which piperine is the chief bioactive phytocon-
stituent that gives pungency to P. longum [9, 10].
Piperine, an alkaloid with the formula (E,E)-1-[5-(1,3-
benzodioxol-5-yl)-1-oxo-2,4-pentdienyl] piperidine—(E,E)-
1-piperoylpiperidine, 1-piperoylpiperidine trans stereoiso-
mer—has been studied both in vitro and in vivo (Fig. 1)
[10]. Piperine possesses a multifaceted pharmacological
profile, which includes antioxidant, antitumor, analgesic,
antidiarrheal, hepatoprotective, antispasmodic, antifungal,
antimetastatic, antipyretic, anti-asthmatic, antiinflammatory,
anxiolytic, antibacterial, immunomodulatory, antimutagenic,
these properties, piperine can be considered as a propitious
candidate for quality control of P. longum in research and

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Fig. 1  The structure of piperine
development areas. Piperine is mostly found and isolated
from P. longum, Piper nigrum, and Piper officinarum [16].
The various analytical methods available for piperine analy-
sis are ultraviolet‒visible (UV‒VIS) spectroscopy [25, 30],
high-performance liquid chromatography (HPLC) [31, 32], gas
chromatography‒flame-ionization detection (GC‒FID) [33],
and high-performance thin-layer chromatography (HPTLC)
[34, 35], among which, thin-layer chromatography (TLC) has
long been used to determine the content, purity, and identity of
herbal medicines. For quality control analysis of phytodrugs,
chromatographic fingerprinting analysis is used as a requisite
approach for phytobioactive constituents [24–29].
A literature survey revealed that various HPTLC meth-
ods for piperine in plant extracts, herbal formulations, and
mixtures have been reported, with solvent systems compris-
ing toluene‒ethyl acetate (7:3, V/V), acetone‒hexane (3:2,
V/V), and petroleum ether‒acetone (6.5:3.5, V/V). However,
these methods exhibit severe limitations such as a lack of
sensitivity and poor resolution and reproducibility [36–43].
Therefore, our current work aimed to develop and validate
a simple, economical, rapid, specific, precise, and sensitive
HPTLC method with high reproducibility and good resolu-
tion for the quantification and identification of piperine in
P. longum. The in vitro antioxidant activity of piperine in
different extracts of P. longum was also determined using the
1,1-diphenyl-2-picrylhydrazyl (DPPH) method.
2 Experimental
2.1 Collection and authentication of the plant
material
The fruits of P. longum were acquired from the Herbal Gar-
den, Jamia Hamdard, Hamdard Nagar, New Delhi, India.
Authentication of the fruits was made by a taxonomist and
the voucher specimen was preserved for further reference.
2.2 Chemicals and instruments
Standard piperine was acquired from Sigma-Aldrich (St.
Louis, MO, USA). Toluene, acetone, and methanol were pro-
cured from S.D. Fine Chem Ltd. (Mumbai, India). Precoated
HPTLC plates 60 ­F254 were purchased from Merck (Mum-
bai, India). A CAMAG (Muttenz, Switzerland) HPTLC
system was used equipped with Linomat 5 applicator, TLC
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JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:147–155
Scanner 3, which is operated through winCATS software
(version 1.2.0), using a Hamilton syringe (100 μL; Bonaduz,
Switzerland).
2.3 Preparation of plant material
The fruits were thoroughly cleaned to eliminate all for-
eign materials and dust particles, and washed under run-
ning water. Further, they were air dried and powdered. The
extraction process was carried out in a reflux apparatus (hot
solvent extraction method) using the following solvents:
acetone, ethanol, dimethylformamide (DMF), and dimethyl
sulphoxide (DMSO) of varying polarity at 50 °C for 1 h,
with a solvent-to-drug ratio of 10 mL/g. The plant residue
was filtered after extraction, and dried under a low-pressure
vacuum using a rotary evaporator.
2.4 HPTLC fingerprint analysis
2.4.1 Standard stock solutions
Standard stock solution of piperine (1 mg/mL) was prepared
by taking 2 mg of piperine in 2 mL of HPLC methanol in a
tube. This was stored in refrigerator until analysis.
2.4.2 Mobile phase system development
Different solvent systems: ethyl acetate‒toluene (3:7,
V/V), ethyl acetate‒formic acid‒toluene (2:1:7, V/V),
chloroform‒toluene (3:7, V/V), chloroform‒toluene‒
formic acid (2:7:1, V/V), acetone‒toluene (3:7, V/V), and
acetone‒toluene‒formic acid (2:7:1, V/V) were tested
for good separation of bands. The mobile phase system
comprising acetone‒toluene (3:7, V/V) was chosen on the
basis of resolution.
2.4.3 Sample application and chromatogram development
The standard piperine solution of different concentra-
tions (0.2–1.0 μg/band) and sample solutions (different
extracts of P. longum) were applied (6 μL) on an ­F 254
silica gel HPTLC plate with a microsyringe, with a band
width of 6 mm each using the CAMAG instrument. Plate
development was performed in the CAMAG twin-trough
development chamber with the mobile phase comprising
toluene‒acetone (7:3, V/V). The development chamber
was saturated for 30 min with the mobile phase and the
length of chromatographic run was 85 mm. After devel-
opment, the plate was dried. Visualization and scanning
of the plate were done using a CAMAG HPTLC Scanner
JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:147–155
3, and the wavelength was selected based on piperine
spectra.
2.5 Validation
The proposed HPTLC method was validated for the fol-
lowing parameters: sensitivity, robustness, accuracy, lin-
earity, specificity, and precision, in accordance with the
International Council for Harmonisation (ICH) guide-
lines. For assessing linearity, a working standard solu-
tion of 1 mg/mL was prepared by taking 2 mg of piperine
in 2 mL of methanol. This was then used to acquire the
linear correlation among the 0.2‒1.0 μg/bands. Different
volumes (0.2‒1.0 μL) of the above solution were applied
on the HPTLC plate. A linear regression analysis was
employed for comparing the peak areas of piperine with
their respective concentration. Sensitivity was determined
by scrutinizing the limit of detection (LOD) and limit of
quantification (LOQ). A calibration plot was constructed
between the amount of piperine and peak area (response),
and the regression coefficient with regression equation was
attained. Standard deviation of the peak area (response)
was measured, and LOQ and LOD were calculated using
the following equation:
LOQ = 10 × 𝜎∕S
LOD = 3 × 𝜎∕S,
where σ is the standard deviation of peak area and S is the
slope of the calibration plot.
Precision was assessed by evaluating inter-day and
intra-day precisions. Relative standard deviation (RSD)
was assessed by analyzing the working solution of piperine
in the range of 0.2‒1.0 μg/bands in three replicates on the
same day for intra-day precision, and on three concsecu-
tive days for inter-day precision. Accuracy was assessed
by spiking samples at three different concentrations, 50%,
75%, and 100% with the standard to obtain the target con-
centration in three replicates. For each sample, percent-
age recovery was estimated. Specificity was assessed by
running standards and samples under suitable chroma-
tographic conditions and comparing their peak purities
at different levels. Robustness was assessed by varying
method parameters such as room temperature, saturation
time, volume of chloroform in the mobile phase, and the
scanning wavelength.
2.6 Determination of antioxidant activity
Diverse extracts of P. longum were evaluated for antioxi-
dant activity using the DPPH method at 517 nm using a UV
149
spectrophotometer. To different tubes, 20, 40, 60, 80, and
100 μg/mL of extracts and standard were added, followed by
methanol (5 mL) and 1 mM DPPH (0.5 mL). A blank solu-
tion was prepared comprising methanol (5 mL) and 1 mM
DPPH (0.5 mL). The tubes were then incubated for 30 min
at room temperature. The antioxidant activities of diverse
extracts of P. longum were estimated using the formula:
%Scavenging activity
Absorbance of blank solution − absorbance of test
= × 100.
Absorbance of blank solution
3 Results and discussion
Piperine is an alkaloid, which is insoluble in water and
soluble in solvents such as ethanol, acetone, DMSO, and
DMF. Hence, these nonpolar solvents were used for piper-
ine extraction using the reflux technique [22]. The different
extracts of P. longum were loaded on the HPTLC plate and
developed using the optimized solvent system. Since piper-
ine is nonpolar, various reported solvent systems comprising
toluene‒ethyl acetate (7:3, V/V), acetone‒hexane (3:2, V/V),
and petroleum ether‒acetone (6.5:3.5, V/V) were utilized
for the development of HPTLC plate but they showed poor
resolution [36–38]. Therefore, ethyl acetate was replaced
with acetone and chloroform owing to its more nonpolar
nature, and the different solvent systems were tested for good
resolution.
3.1 Mobile phase system
Diverse solvent systems, namely, ethyl acetate‒toluene (3:7,
V/V), ethyl acetate‒toluene‒formic acid (2:7:1, V/V), chlo-
roform‒toluene (3:7, V/V), chloroform‒toluene‒formic
acid (2:7:1, V/V), acetone‒toluene (3:7, V/V), and acetone‒
toluene‒formic acid (2:7:1, V/V) were tested to attain sharp
symmetric peaks. The mobile phase with acetone‒toluene
(3:7, V/V) provided the best results with good resolution
(Table 1).
3.2 Development and validation of the HPTLC
method
A HPTLC method was developed (Fig. 2) and validated
to quantify and identify piperine in diverse extracts of
P. longum (Table 2). The TLC densitogram of standard
solutions of piperine and different extracts of P. longum are
shown in Fig. 3 at 343 nm based on piperine spectra, and
ICH guidelines were used for validation of the following
parameters.
Linearity was assessed by determining the standard
plot between the piperine concentration in μg/band against
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150 JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:147–155

Table 1  Optimized HPTLC conditions for the analysis of piperine spiking samples at three different concentrations, 50%,
HPTLC instrument CAMAG Linomat 5
75%, and 100% with the standard to get the target concen-
tration in three replicates. Percentage recovery for each
Stationary phase Precoated plate of HPTLC sample was estimated and is summarized in Table 4. Spec-
with silica gel 60 ­F254
ificity was assessed by comparing the peak purity of the
Mobile phase Toluene–acetone (7:3,
piperine standard and sample spectra at three levels: peak
V/V)
start, peak end, and peak apex (Figs. 5 and 6). The cor-
Observed RF values Piperine (0.65)
relation coefficient was determined as 0.9908. Robustness
Band width 6 mm
was assessed by varying the method parameters such as
Saturation time 30 min
room temperature, saturation time, volume of chloroform
Solvent front distance 85 mm
in mobile phase, and scanning wavelength. No significant
Plate activation time 15 min
differences were found on varying the above conditions.
Detection lamp Deuterium
Detection wavelength 343 nm
Scanning rate 20 mm/s

the peak area shown in Fig. 4. Linear regression analysis

was employed and the regression equation was attained
from the standard plot, which revealed the high correla- Table 2  Piperine content determined by the developed HPTLC‒den-
sitometric method in the samples
tion between the peak area and concentration of piperine,
with a small RSD value (Table 3). LOD and LOQ were Sample no. Sample HPTLC pip-
measured and found to be 2.87 and 9.57 ng/band, respec- erine (mg/g)
tively. The precision of the proposed HPTLC method was 1 E1 30.57
assessed by evaluating the inter-day and intra-day preci- 2 E2 23.98
sion. Working solutions of piperine betwixt the range of 3 E3 26.96
0.2‒1.0 μg/band were analyzed in three replicates on the 4 E4 22.76
same day to determine the intra-day precision, and on
three consecutive days to determine inter-day precision E1 ethanolic extract of Piper longum, E2 acetonic extract of
Piper longum, E3 DMSO extract of Piper longum, and E4 DMF
(results presented in Table 3). Accuracy was assessed by extract of Piper longum

Fig. 2  Developed HPTLC plate (a) at λ 254 nm, and b at λ 366 nm. Piper longum; lane E3: DMSO extract of Piper longum;; and Lane
Lanes S1‒S5: piperine standard (0.2–1.0 μg/band); lane E1: E4: DMF extract of Piper longum
ethanolic extract of Piper longum; lane E2: acetonic extract of

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JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:147–155 151

Fig. 3  HPTLC densitograms of standard piperine (a), ethanolic extract of Piper longum (b), acetonic extract of Piper longum (c), DMSO extract
of Piper longum (d), and DMF extract of Piper longum (e)

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Fig. 4  Calibration plot for

piperine at λ 343 nm

Table 3  Regression data of piperine and intra-day and inter-day precision of the HPTLC method developed for Piper longum
Parameters Value piperine

Linearity range 0.2–1.0 μg/spot

Detection wavelength 343 nm
Regression coefficient Y = 5392 + 14.67x
Correlation coefficient (R2) 0.9908
LOD 2.87 ng/spot
LOQ 9.57 ng/spot
RF value 0.65
% Recovery of piperine 99.54%
Intra-day precision (n = 3)
Compounds Amount (μg/band) Area Mean area ± SD %RSD

Piperine 0.2 7526.94 7530.31 3.54 0.05

0.4 12,011.87 12,016.62 5.09 0.04
0.6 14,669.51 14,673.50 4.26 0.03
0.8 17,124.97 17,128.32 3.52 0.02
1.0 19,641.20 19,645.4 4.41 0.02
Mean %RSD 0.032
Inter-day precision (n = 3)
Compounds Amount (μg/band) Area Mean area ± SD %RSD

Piperine 0.2 7546.82 7550.27 3.59 0.05

0.4 12,030.26 12,036.08 5.87 0.05
0.6 14,685.59 14,692.20 6.24 0.04
0.8 17,144.23 17,148.07 3.88 0.02
1.0 19,671.50 19,675.5 4.27 0.02
Mean %RSD 0.036

3.3 Determination of the antioxidant activity
The antioxidant activity of P. longum extracts in different
solvents was evaluated using the DPPH method [29]. Test
samples were assessed based on utilization of DPPH free
radicals in test samples to give purple color. The antioxidant
activity of the P. longum extracts in different solvents was
compared with the standard vitamin C.
The ethanolic extract of P. longum displayed the maxi-
mum scavenging activity at a concentration of 100 mg/mL,
i.e., 55.26% compared with P. longum extracts in DMF,
acetone, and DMSO, which were determined to be 45.23%,

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JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:147–155 153

Table 4  Recovery studies Standard Amount of Amount of piperine Amount after spiking Amount recov- SD %RSD
ethanol extract standard added ered (% mean)
used

Piperine 0.9 μg/band 50% (0.5 μg/band) 1.39 μg/band 99.35 0.23 0.15
0.9 μg/band 75% (0.75 μg/band) 1.64 μg/band 99.50 0.18 0.08
Mean 0.9 μg/band 100% (1.0 μg/band) 1.89 μg/band 99.77 0.17 0.08
99.54

Fig. 5  3D overlaid spectra of

HPTLC densitograms of dif-
ferent extracts of Piper longum
and standard piperine at 343 nm

Fig. 6  Overlaid spectra of

standard piperine and sample in
the range 200‒400 nm

32.65%, and 49.28%, respectively. Vitamin C displayed extract revealed the maximum activity in comparison with
88.15% DPPH scavenging activity. other extracts. It can be concluded that antioxidant activity
Figure 7 shows the antioxidant activity of extract aug- of ethanolic extract of P. longum could be ascribable to the
ments on increasing the extract concentration, and ethanolic presence of alkaloids, flavanoids, and phenolic compounds.

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154
Fig. 7  Dose-dependent scaveng-
ing of DPPH radicals by the dif-
ferent extracts of Piper longum
compared with the standard
drug ascorbic acid. Each value
represents mean ± SD (n = 3)
4 Conclusions
A selective, simple, cost-effective, time-saving, and sensitive
HPTLC method with high reproducibility and good resolu-
tion was developed for the identification and quantitative
estimation of piperine in P. longum, which may be employed
as a quality control tool for the routine analysis of piperine
in polyherbal formulations and diverse plant extracts. Also,
the DPPH scavenging activity revealed that the P. longum
ethanolic extract has a higher antioxidant activity than other
extracts, and further characterization of ethanolic extract
by other researchers could be beneficial in identifying new
therapeutic entities.
Acknowledgements The authors are thankful to the Head, Department
of Pharmacognosy and Phytochemistry, School of Pharmaceutical
Research & Education, Jamia Hamdard, New Delhi, India, for provid-
ing the necessary research facilities to carry out this study.
Funding This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declarations
Conflict of interest The authors declare that they have no conflicts of
interest.
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