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Original Article

Quantification of Punicalagin in Pomegranate Peels From

High‑performance Thin‑layer Chromatography
Pooja Gadkari, Sanjay J. Daharwal
Department of Pharmacy, University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur, Chhattisgarh, India

Abstract
Background: Punicalagin is the main phenolic compound present in pomegranate (Punica granatum), it possesses various kinds of activities
which is very essential as a dietary supplement, herbal supplements, or nutraceuticals are widely available in the market and are used clinically
for various therapeutic activities, in the recent years, especially in the pandemic period of COVID‑19. Hence, it is necessary to standardize
herbal medicines for quality control, quantitative analysis for purity, and routine analysis. The punicalagin shows potential antiviral activity
against the SARS‑COV‑2 virus, the literature review reveals that punicalagin is the area of interest during the recent research studies, and the
present work deals with the quantitative analysis of punicalagin from high‑performance thin‑layer chromatography (HPTLC) in marketed
herbal preparation and the in‑house preparation. Methods: The method development and quantitative analysis of punicalagin in pomegranate
are developed using the solvent system chloroform: ethyl acetate: formic acid (4:3:3 v/v/v), and the method is successfully developed.
Results: The punicalagin is quantified at 257 nm, acid (4:3:3). The content found in the various samples in PGGO is 3.207 mg, in PGBB is
1.257 mg, in PGNV is 1.743 mg, in PGWE is 807.6µg, in PGDF is 835.2 µg, and in in‑house is 867.2 µg of punicalagin, from 1 g of each
sample. Conclusion: The method was successfully developed, but there was no method developed for punicalagin in HPTLC, this is the novel
approach we have done, and the method can be used for routine analysis.

Keywords: Developed, method, phenolic, quantitative, solvent

Introduction part in nutrition and provide numerous health benefits for

Fruits are nature’s amazing gift given to society, they are
packed with essential nutrients, Phyto‑vitamins, minerals,
and fibers, there are many essential uses of fruits, they are
the absolute feast in our daily life and are considered to be a
unique profile for fighting against different kinds of diseases.[1]
The fruit pomegranate (Punica granatum) is a fruit‑bearing
deciduous shrub that belongs to the family – Lythraceae
subfamily – Punicoideae native from the areas of the Himalayas
in Northern India and cultivated all over the Mediterranean
region from ancient times.[2]
Pomegranate is a known source for its nutritional source
consists of hydrolyzable tannins. Phenolic acids, anthocyanins,
and organic acids, and capable for various health benefits
against diseases.[2]As there is an increase in the demand
for nutraceuticals, nutrition and infectious disease are
interrelated to each other, the nutrition affects the human
body’s development.[3] The herbal nutraceuticals play an
important role in the market, herbal products play a crucial
Access this article online
overall nutrition.[4]
Punicalagin
Punicalagin is a polyphenol with the highest molecular weight,
it is hydrolyzed in ellagic acid and has the highest antioxidant
capacity in pomegranate.[5] Punicalagin is a polyphenolic
ingredient isolated from pomegranate P. granatum. It is a
reversible and noncompetitive 3CL inhibitor and inhibits
SARS‑COV‑2 replication in vitro. It is an antihepatitis B virus
agent and has antioxidant, anti‑inflammatory, and anticancer
activity.[6]
Address for correspondence: Dr. Pooja Gadkari,
University Institute of Pharmacy, Pandit Ravishankar Shukla University,
Raipur, Chhattisgarh, India.
E‑mail: poojadeshpande0904@gmail.com
ORCID: Pooja Gadkari: http://orcid.org/0000‑0003‑0225‑5595
This is an open access journal, and articles are distributed under the terms of the Creative
Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix,
tweak, and build upon the work non‑commercially, as long as appropriate credit is given and
the new creations are licensed under the identical terms.

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Website:
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How to cite this article: Gadkari P, Daharwal SJ. Quantification of
punicalagin in pomegranate peels from high-performance thin-layer
chromatography. Biomed Biotechnol Res J 2022;6:586-90.
DOI:
10.4103/bbrj.bbrj_312_22 Submitted: 01‑Jul‑2022; Revised: 16‑Oct‑2022;
Accepted: 20‑Nov‑2022; Published: 15-Dec-2022.

586 © 2022 Biomedical and Biotechnology Research Journal (BBRJ) | Published by Wolters Kluwer - Medknow
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Gadkari and Daharwal: Punica granatum
Method and Materials
Chemicals and reagents
Punicalagin was purchased from MedChem Express (USA).
High‑performance thin‑layer chromatography (HPTLC)
plates (20 cm × 10 cm, precoated silica gel aluminum plates 60
F254 (0.25 mm) were procured from E. Merck (Darmstadt, Germany).
High‑performance thin‑layer chromatography (HPLC) grade
methanol, chloroform, ethyl acetate, and formic acid were used.
All other ingredients were of analytical grade. Double‑distilled
water was used for all experiments.
Plant material
The plant and fruit part of the pomegranate was collected from
the botanical garden of the Department of Biotechnology,
Pandit Ravishankar Shukla University, Raipur, Chhattisgarh.
Authentication of the plant was done by the Botanical Survey
of India, Allahabad (U.P).
Preparation of sample solution
Standard stock solution of punicalagin
4.5 mg of punicalagin is accurately weighed transferred in
10 ml of volumetric flask and dissolved in methanol. The
solution is then diluted up to the mark (10 ml) to make the
solution of (0.450 µg/ml) concentration (stock solution).
Quantification of punicalagin
The methanolic extract (1.0–7.0 µl) was applied seven times
and densitogram were obtained under the same conditions as
of standard, and area under the peak under that of standard is
calculated. Figures 4-7 show the quantification of punicalagin
in various samples.
Preparation of methanolic sample solution
Marketed preparation
The five marketed preparation of pomegranates from different
companies have randomly selected from the local market
for study and then compared with in‑house preparation. The
marketed products from 1 to 5 as PGGO, PGBB, PGNV,
PGWE, PGDF, and the in‑house preparation are coded as
PGIH.
Sample solution
Weighed 1gm of pomegranate peel powder preparation (in‑house
preparation) transferred in 10 ml volumetric flask then the
powder is dissolved in methanol, then the solution is diluted
up to the mark (10 ml), to make the solution 100mg/ml.
The solution is then filtered through membrane filter paper.
The same procedure is being followed for five marketed
preparations to make the solution of 1000 ug/ml.
Standard marker solution preparation
Stock solutions of punicalagin were prepared by dissolving
0.45 mg of punicalagin in 10 ml of methanol.
High‑performance thin‑layer chromatography analysis
Instrumentation and chromatographic conditions – The
instruments and chromatographic conditions used are as
follows:
Biomedical and Biotechnology Research Journal ¦ Volume 6 ¦ Issue 4 ¦ October-December 2022
A. HPTLC System – Linomat 5 applicator, Camag 3 Scanner,
and Twin Trough Chamber
B. Stationary Phase – Precoated Silica Gel G 60 F254,
aluminum sheets (20 × 10), 0.2 mm thickness
C. Mobile phase – Chloroform: ethyl acetate: formic
acid (4:3:3 v/v/v)
D. HPTLC Chamber – Twin trough (20 × 10) CAMAG
E. Chamber saturation time – 20 min
F. Temperature – Room temperature
G. Slit Dimension – 6 mm × 0.45 mm
H. Detection wavelength – 254 nm–450 nm
I. Scanning speed – 20mm/s
J. Derivatizing agent.
HPTLC method development of punicalagin in pomegranate
peels – The 4.5 mg of punicalagin standard is prepared in
methanol, the calibration curve is measured at 257 nm [Figure 1],
and the silica gel G 60 F254 (20 × 10) plates are used with solvent
system chloroform: ethyl acetate: formic acid (4:3:3 v/v/v) as
a solvent system. The sample was spotted on precoated TLC
plates using Linomat 5 applicator, the ascending mode is being
used for the development of thin‑layer chromatography, TLC
plates were developed from solvent front position 70.00 mm.
The plates were air‑dried and then scanned at 254 nm and
366 nm, the content and Rf values were then compared with
the marketed and in‑house preparations.
Results
High‑performance thin‑layer chromatography
Method optimization
HPTLC conditions were optimized in terms of the selection
of mobile phase, absorption maxima, and slit dimensions.
Solvent systems in different combinations were tried.
The absorption spectrum of punicalagin was obtained at
257 nm. The chromatogram was obtained for the standard
compound punicalagin and the five marketed compounds from
wavelengths 254 nm and 366 nm, Figures 3 and 4. Punicalagin
was identified by the Rf and the peak purity [Table 1]. The
linearity of the method for the quantification of punicalagin
was obtained at the concentration of 1.0 µl to 7.0 µl [Figure 1].
The concentration with a statistically acceptable value of R2
is 99.84 %
Figure 1: Standard calibration curve of punicalagin, standard marker,
and linear graph of punicalagin shows the calibration range, reference,
and samples
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Gadkari and Daharwal: Punica granatum
There are various contents of punicalagin in different samples
of in‑house and marketed preparation the band of punicalagin
is not visible with the naked eyes but the band is visible in
the area under the curve graphs as shown in Figures 5‑7. The
wavelength of punicalagin shows at 257 nm as shown in
Figures 6 and 8.
Quantification of phenolic compound punicalagin from
pomegranate peels
A quantitative HPTLC method has been developed
with Rf values for the determination of punicalagin in
pomegranate peels. The six‑point linear calibration curves
of punicalagin [Figure 1] and the chromatogram [Figure 2]
indicating the method has good sensitivity. The specificity
of the method has been evaluated by comparing the Rf
values for the standard and plant sample under the same
condition. Ten microliter of the sample was applied in
each sample. The concentration of each sample found is
indicated in Table 2. All the concentrations are found from
1 g of the sample.
Figure 2: Chromatogram of punicalagin
Figure 4: The chromatogram obtained at 366 nm for standard punicalagin
with the in‑house herbal preparation and five marketed pomegranate
peel products
588 Biomedical and Biotechnology Research Journal ¦ Volume 6 ¦ Issue 4 ¦ October-December 2022
Discussion
There has been a great increase in interest in food industries and
food products such as dietary supplements and nutraceuticals,
there are various marketed food products/food supplements of
pomegranate available in the market such as fresh juice, jelly,
jams, candies, peel powder, face creams, and various other
products formulated with pomegranate fruit part.[7]
The chemical constituents present in pomegranate include
polyphenols, anthocyanins, and Vitamin C provides nutritional
and therapeutic effect, apart from this it also contains acids,
sugars, vitamins, polysaccharide, polyphenols, minerals,
glucose, and fructose are the main sugars in pomegranate,
for acids citric acid, tartaric acid, fumaric acid, succinic acid,
and ascorbic acid are the main organic acids of pomegranate.
Phenolic compounds – Pomegranate peels are a rich source
of polyphenolic compounds in which punicalagin A and
Figure 3: The chromatogram obtained at 254 nm for standard punicalagin
with in‑house herbal preparation and five marketed products
Figure 5: The chromatogram obtained at visible region R white for
standard punicalagin with the in‑house preparation and five marketed
pomegranate peel products
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Gadkari and Daharwal: Punica granatum

Table 1: Rf values of all samples from starting position to the end position of the chromatogram, the table also shows
the peak area of the samples
Track Samples Start position Start height Maximum position Maximum height End position End height Area
1 PGGO 0.834 Rf 0.0000 AU 0.885 Rf 0.1130 AU 0.937 Rf 0.0002 AU 0.00555 AU
2 PGBB 0.831 Rf 0.0000 AU 0.887 Rf 0.0814 AU 0.931 Rf 0.0000 AU 0.00326 AU
3 PGNV 0.827 Rf 0.0000 AU 0.885 Rf 0.0806 AU 0.934 Rf 0.0000 AU 0.00383 AU
4 PGWE 0.827 Rf 0.0000 AU 0.881 Rf 0.0642 AU 0.921 Rf 0.0000 AU 0.00273 AU
5 PGDF 0.827 Rf 0.0000 AU 0.885 Rf 0.0581 AU 0.929 Rf 0.0000 AU 0.00276 AU
6 In‑house preparation 0.832 Rf 0.0000 AU 0.887 Rf 0.0637 AU 0.931 Rf 0.0000 AU 0.00280 AU
7 Punicalagin 0.840 Rf 0.0000 AU 0.890 Rf 0.0579 AU 0.926 Rf 0.0000 AU 0.00235 AU
8 Punicalagin 0.831 Rf 0.0003 AU 0.894 Rf 0.0649 AU 0.932 Rf 0.0000 AU 0.00289 AU
10 Punicalagin 0.827 Rf 0.0000 AU 0.884 Rf 0.1075 AU 0.939 Rf 0.0005 AU 0.00551 AU

Figure 7: Punicalagin bands in all samples at wavelength 257nm which

Figure 6: The graph showing peak areas of punicalagin from code
number 7–13 and it also shows content of punicalagin in marketed and
in‑house preparation
punicalagin B possess key phenolic activity, pomegranates
are also rich in flavonoids, catechins, punicalin, gallic acid,
and ellagic acid and other flavonoids and hydrolyzable tannins
which are responsible for antioxidant activity.
Punicalagin is an important phenolic compound present in
pomegranate peels, among the different bioactive constituents
showed that the peel possesses the highest content of phenolic
compounds as compared to other parts. The secondary
metabolites present in the peel part like phenolic compounds
include (gallic acid ellagic acid, and caffeic acid), flavonoids such
as catechin, gallocatechin, and epicatechin, and hydrolyzable
tannins like punicalagin[8] according to some in vitro and in vivo
studies, these compounds possess various kinds of biological
and health benefits such as antioxidants, anti‑inflammatory,
antimutagenic, anticarcinogenic, and antihypertensive benefits,
not only that their presence includes various other treatments
such as cardiovascular problems, diabetes, and obesity.[9]
There are various herbal products available in the market but
the quantification and identification of the concentration in a
particular species are necessary for quality and purity purposes.
Among various mobile phase solvent systems, we have tried the
mobile phase consisting of chloroform: ethyl acetate: formic
were not visible in HPTLC plate, bands are visible at wavelength 257 nm.
HPTLC: High‑performance thin‑layer chromatography
acid (4:3:3)[10] demonstrated for the best resolution between
other peaks of the extract. The content variation found in the
various samples in PGGO is 3.207 mg, in PGBB is 1.257 mg, in
PGNV is 1.743 mg, in PGWE is 807.6µg, in PGDF is 835.2 µg,
and in in‑house is 867.2 µg of punicalagin in each sample. The
variation in the content of punicalagin in various samples is due
to different factors such as climatic regions, time of collection,
and mode of drying; these variables may impact the daily dose
and may impact health.
As there is an increase in the demand for herbal/herbs, common
people use them as self‑medication to cure common disorders,
the content variation in an active constituent of marketed
herbal/Ayurvedic products represents a major challenge to
controlling the quality of herbal medicines for quality control,
safety, efficacy of herbal marketed products the qualitative and
quantitative analysis are the useful techniques that are related
to the marker compound/active constituent which is present
in the plant.[8]
Punicalagin contains high phenolic contents constituents
present in the pomegranate peels. The chromatographic
fingerprinting technique is the most sophisticated and reliable
technique found in the quality control of herbal medicines,
it is found to be a realistic and practical method for quality
control of herbal medicines. Although there are developed

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Gadkari and Daharwal: Punica granatum

developed for HPTLC in the quantification of punicalagin

in pomegranate peels. The developed method was found
to be suitable for rapid screening of punicalagin. The data
obtained can be reproducible and economical and useful for
routine analysis, the developed method can be helpful for the
quantification of punicalagin in the marketed samples, and
it helps as a source of detection and quality of the product.
Punicalagin is available in the marketed as well as in the
in‑house preparation. The developed method can be useful
in routine analysis in the pharmaceutical industry for the
quantification of any medicinal and herbal product.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.

Figure 8: Punicalagin and all samples superimposed at the wavelength

257 nm
References
1. Adams LS, Seeram NP, Aggarwal BB, Takada Y, Sand D, Heber D.
Pomegranate juice, total pomegranate ellagitannins, and punicalagin
Table 2: The content of punicalagin from the marketed suppress inflammatory cell signaling in colon cancer cells. J Agric Food
Chem 2006;54:980‑5.
samples and in‑house preparation found from 1 g of 2. Nuncio‑Jáuregui N, Calín‑Sánchez Á, Vázquez‑Araújo L,
sample Pérez‑López AJ, Frutos‑Fernández MJ, Carbonell‑Barrachina ÁA.
Processing pomegranates for juice and impact on bioactive components.
Samples (marketed and inhouse) Sample weight (1 g)
In: Processing and Impact on Active Components in Food. New York,
PGGO 3.207 mg NY, USA: Academic Press; 2015. p. 629‑36.
PGBB 1.257 mg 3. Farhadi S, Ovchinnikov RS. The relationship between nutrition and
PGNV 1.743 mg infectious diseases: A review. Biomed Biotechnol Res J (BBRJ)
PGWE 807.6 µg 2018;2:168.
4. Bishnoi S, Mudgil D. Current concepts and prospects of herbal
PGDF 835.2 µg
nutraceutical. In: Handbook of Nutraceuticals and Natural Products:
In–house 867.2 µg Biological, Medicinal, and Nutritional Properties and Applications.
Vol. 1. 2022. p. 189‑204. Available from: https://onlinelibrary.wiley.
com/doi/chapter-epub/10.1002/9781119746843.ch10.
analytical methods for punicalagin in high‑performance liquid 5. Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG,
et al. In vitro antiproliferative, apoptotic and antioxidant activities
chromatography, thin layer chromatography, UPLC, and liquid of punicalagin, ellagic acid and a total pomegranate tannin extract
chromatography–mass spectrometry. are enhanced in combination with other polyphenols as found in
pomegranate juice. J Nutr Biochem 2005;16:360‑7.
The HPTLC is the routine analytical technique for the 6. Du R, Cooper L, Chen Z, Lee H, Rong L, Cui Q. Discovery of chebulagic
quantification of plant active constituents which is quick, acid and punicalagin as novel allosteric inhibitors of SARS‑CoV‑2 3CL.
accurate, reliable, efficient, and time‑consuming. However, the Antiviral Res 2021;190:105075.
review of the literature reveals that there is no method developed 7. Al‑Maiman SA, Ahmad D. Changes in physical and chemical properties
during pomegranate (Punicagranatum L.) fruit maturation. Food Chem
in HPTLC for punicalagin, in the present work, a densitometric
2002;76:437‑41.
HPTLC method has been developed for quantification of 8. Kadam PV, Yadav KN, Bhingare CL, Patil MJ. Standardization
punicalagin in pomegranate peels marketed herbal products and quantification of curcumin from Curcuma longa extract using
and its comparison with in‑house herbal preparation. UV visible spectroscopy and HPLC. J Pharmacogn Phytochem
2018;7:1913‑8.
9. Kandylis P, Kokkinomagoulos E. Food applications and potential health
Conclusion benefits of pomegranate and its derivatives. Foods 2020;9:122.
10. Government of India, Ministry of Health and Family Welfare,
A simple method was developed for the quantification of Department of Ayush. Ayurvedic Pharmacopoeia of India. Part 1. Vol.
punicalagin in pomegranate peel. In‑house preparation and II. Ghaziabad , India : Pharmacopoeia Commission For Indian Medicine
the marketed herbal preparation, as there is no method was & Homoeopathy; 2016. p. 32‑3.

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