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INTERNATIONAL JOURNAL
OF CURRENT RESEARCHInternational Journal of Current ResearchVol. 7, Issue, 01, pp.11907-11912, January, 2015

ISSN: 0975-833X

RESEARCH ARTICLE

THE POLYPHENOLS IN POMEGRANATE PLAY IMPORTANT ROLE IN EXHIBITING
ANTI-AGEING EFFECTS
Reethi Budanuru, Satyababu, M., *Sudeep, H.
H V. and Shyam Prasad, K.
Department of Biomedicinal Research, Vidya Herbs Pvt. Ltd, #101, Jigani II phase,
Bangalore 560078, Karnataka, India
BangaloreARTICLE INFOABSTRACT

Article History:

Pomegranate (Punica(
granatum L), in addition to its ancient historical uses, has been used in several
systems of medicine for a variety of ailments. Pomegranate juice is a polyphenol
polyphenol-rich juice with highantioxidant capacity. It is evident from the previous studies that pomegranate jjuice has been shown toexert significant antiatherogenic, antioxidant, anti-carcinogenic,
anti carcinogenic, and anti
anti-inflammatory effects. The
aim of the present study was to validate the skin care properties of P. granatum. The extract exhibitedhigh anti-elastaseanti
and collagenaseagenase activities at various concentrations indicating the significant anti
antiwrinkle effect (52.28 and 53.98 % respectively at 100 µg/ml concentration). Further, pomegranate
extract was highly effective in protecting the DNA from hydroxyl radical mediated scission. The insilico calculations predicted that lowest energy docked poses of phenolic compounds can interact withcatalysis dependent residues, thus making them possible catalytic inhibitors and of course
catalysis-dependent
physiologically active. This study concludes thatthat pomegranate can potentially be used as a readily
accessible source of skincare anti-ageing
anti ageing agents and demand the need for extensive research incosmeceutical applications.

Received 09th October, 2014
Received in revised form
15th November, 2014
Accepted 17th December, 2014
Published online 31st January, 2015

Key words:
Cosmeceutical,
Pomegranate,
Anti-wrinkle,
DNA damage, in silico

Copyright © 2015 Reethi Budanuru et al. This is an open access article distributed under the Creative Commons Attribution
ribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.

INTRODUCTION
Skin aging is a multisystem degenerative process that involves
the skin and the skin support system (Sjerobabski and Poduje,
Poduje2008). The process of skin aging may be divided as intrinsic
and extrinsic ageing (Jenkins, 2002; Schlotmann et al., 2001).
It may be caused by several factors, such as, UV irradiation,
stress, ROS generation or smoking. Acute exposure of humanskin to UV
V irradiation causes sunburn, inflammation, immune
suppression, and dermal connective tissue damage (Fisher
et al.,., 2002), and chronic exposure to UV over many years
disrupts the normal skin architecture and ultimately causes
photoaging and even skin cancer (Quan et al.,al 2009). Wrinkle
formation is a striking feature of photoaged skin and is causedby the degradation of collagen fibrils and gelatin fibers.
Pomegranate (Punica granatum L.) is a plant of family
Lythraceae cultivated in India,
ndia, Mediterranian countries,
Malaysia, tropical Africa and to some extent in United States(Viuda-Martos et al., 2010).
). The plant has been valued sinceancient times to treat many conditions such as diarrhea,
dysentery, hemorrhage, parasitic infections, ulcers andmicrobial infections (Lihua Zhang et al., 2010). Pomegranate
juice, which is rich in tannins, possesses anti-atherosclerotic,
antiantihypertensive, and potent anti-oxidativeoxidative characteristics*Corresponding author: Sudeep, H. V.
Department of Biomedicinal Research, Vidya Herbs Pvt. Ltd, #101,
Jigani II phase, Bangalore- 560078, Karnataka, India.

(Stowe, 2011); hence, it provides cardioprotective benefits(Basu and Penugonda, 2009).
). Pomegranate juice may havecancer-chemopreventive
chemopreventive as well as ccancer-chemotherapeutic
effects against prostate cancer, in humans ((Malik et al., 2005).
Pomegranate is a rich source of polyphenols and possesses anarray of compounds that have been attributed to thepharmacological activities such as anti
anti-tumor, astringent andantidiarrheal activities. The aim of this study was to investigate
the skin care properties of pomegranate juice extract through invitro and in silico approaches.

MATERIALS AND METHODS
Chemicals
All chemicals were obtained from Sigma Aldrich Ltd. (India)
unless otherwise stated.
Extraction of polyphenol rich fraction from P. granatum
100 ml of fresh P. granatum fruit juice was taken into aseparating funnel and extracted into 50 ml of ethyl acetate (LRgrade, Merck, India). The aqueous layer was washed twicewith ethyl acetate and the total organic fraction collected. At
the end of extraction, extracts were filt
filtered under vacuum
through a Whatman No. 1 filter paper and the process repeated11908Reethi Budanuru et al. The polyphenols in pomegranate play important role in exhibiting anti-ageing effects

until all soluble compounds had been extracted. The filtrates
obtained were concentrated in vacuo using a Rotavapor (Buchi
Flawil, Switzerland). The ethyl acetate extract was stored at 4
°C in air tight bottle until further use.
Elastase inhibition assay
The assay employed was based on methods in the literature(Kim et al., 2004). This assay was performed in 0.2 mM TrisHCL buffer (pH 8.0). Porcine pancreatic elastase (E.C.
3.4.21.36), was dissolved to make a 3.33 mg/mL stock solution
in sterile water. The substrate N-Succinyl-Ala-Ala-Ala-pnitroanilide (AAAPVN) was dissolved in buffer at 1.6 mM.
Different concentrations of PE were incubated with the
enzyme for 15 minutes before adding substrate to begin thereaction. The final reaction mixture (250 μL total volume)
contained buffer, 0.8 mM AAAPVN, 1 µg/mL pancreatic
elastase and various concentrations of PE. EGCG (1 mg/mL)
was used as a positive control. Negative controls were
performed using water. Absorbance values between 381 and402 nm (following pre-screen scans) were measured
immediately following addition of the substrate and thencontinuous for 20 minutes. The percent inhibition of elastase
was calculated as follows:
Enzyme inhibition (%) = [(ODcontrol- ODsample)/ ODcontrol] × 100,
Determination of anti-collagenase activity
The assay employed was based on spectrophotometric methods
reported in the literature (Van Wart and Steinbrink, 1981). The
assay was performed in 50 mM Tricine buffer (pH 7.5 with
400 mM NaCl and 10 mM CaCl2). Collagenase from
Clostridium histolyticum (ChC – EC.3.4.23.3) was dissolved inbuffer for use at an initial concentration of 0.8 units/mL
according to the supplier's activity data. The synthetic
substrate N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA)
was dissolved in Tricine buffer to 2 mM. Different
concentrations of PE were incubated with the enzyme in buffer
for 15 minutes before adding substrate to start the reaction.
The final reaction mixture (150 μL total volume) contained
Tricine buffer, 0.8 mM FALGPA, 0.1 units ChC and 20-100μg PE extract. Negative controls were performed with water.
Absorbance at 335 nm was measured immediately after adding
substrate and then continuous for 20 minutes. EGCG, 250 μM(0.114 mg/mL) was used as a positive control.
In vitro DNA protective activity
The experiment was conducted using Calf thymus DNA
following the method of Xican et al. (2013). Briefly, sample
was dissolved in ethanol at 1 mg/mL. 50 µl of different
concentrations of PE was then separately taken into mini tubes
followed by addition of 400 µL phosphate buffer (0.2 mol/L,
pH 7.4). Subsequently, 50 µL each of DNA sodium, H2O2
(50 mM/L), FeCl3 (3.2 mM/L) and Na2EDTA (1 mmol/L)
were added. The reaction was initiated by adding 50 µL
ascorbic acid (18 mmol/L) and the total volume of the reaction
mixture was adjusted to 800 µL with buffer. After incubation
in a water bath at 55 °C for 20 min, the reaction was
terminated by adding 250 µL TCA. The color was thendeveloped by addition of 150 µL of Thiobarbituric acid
(TBA)(0.4M/L in 1.25 % NaOH) and heating in an oven at 105°C for 15 min. The mixture was cooled and absorbance wasmeasured at 532 nm against the buffer (as blank). The percent
of protection against DNA damage is determined as follows:% Protective effect = (1 - A/A0) × 100
Where A0 is the absorbance of the mixture without sample;
A is the absorbance of the mixture with sample.
In silico docking studies
AutoDock tools was utilized to generate grids, calculate dockscore and evaluate the conformers of inhibitors bound in theactive site of enzymes elastase and collagenase as targets foranti-wrinkle activity. Automated docking is a graphical userinterface. AutoDock 4.2 was employed to get docking andbinding scores; which is implemented by Lamarckian genetic
algorithm method. The ligand molecules i.e., the phenolic
compounds from pomegranate juice (Figure 1), standard
molecule epigallocatechin gallate were designed and thestructure was analyzed using ACD/Chemsketch. The
PRODRG server was used to minimise energy of drug
compounds and 3D coordinates were prepared. The protein
structure files (PDB ID: 1GVK and 1CGL) were taken from
PDB and edited by removing the hetero atoms using Python
molecule viewer. The grid map was centred at particular
residues of the protein and was generated with AutoGrid. As
per genetic algorithm all the torsions were allowed to rotate
during docking. The Lamarckian genetic algorithm and thepseudo-Solis and Wets methods were applied forminimization, using default parameters (Rodriguez and
Infante, 2011).

RESULTS
The anti-wrinkle effect of Pomegranate juice extract wasinvestigated in vitro. Results of anti-elastase activity of PE arepresented in Figure 2A as percentage inhibition. The extract
presented a strong inhibition of elastase at various
concentrations. Interestingly, at all tested concentrations theextract showed better activity than the standard EGCG. The
activity of PE was nearly one fold higher than EGCG with amaximum of 52.28 % at 100 µg/ml and IC50 value of 32.37µg/ml. In the present study, appreciable anti-collagenase
activity was found at tested concentrations of PE (Figure 2B)
comparable to standard EGCG. This could be explained by thepresence of pharmacologically active phytochemicals in
pomegranate (Figure 1).
Further, in this study, the protective effect of PE on hydroxyl
radical induced DNA scission was demonstrated. The DNA
scission induced by hydroxyl radical was efficiently protectedby various concentrations of PE (20-100 µg/ml). Interestingly,
the activity was better than the standard BHA at all theconcentrations (Figure 3). At low concentration of 10 µg/ml
itself, PE exerted 60.2 % activity. The extract exhibited morethan 75 % of protection at 100 µg/ml. The docking results
showed compounds with more favorable interactions with thetargets and also indicated that some of the compounds present
certain structural motifs that could make them form extensive
Van der Waals interactions and hydrogen bonding with targets.

11909International Journal of Current Research, Vol. 7, Issue, 01, pp.11907-11912, January, 2015
OH
OH

HO
OH
HO
O

O

OH

O

OH

O

O

O

OH

O

HO

O

OH
OH
O

HO
O

O

OH
O

O

O
HO

O

HO

O

O

OH

O

O
HO

HO

OH

O

OH
O

O

HO
OH

OH

HO

OH

O
OH HO

HO

OH
H

HO

OH

O

Punicalagin

OH

Ellagic acid

Corilagin
OH HO

HO

OH
H

O

OH

O

OH
O

O
HO

OH

HO

OH

O
O

HO

HO

O

OH

HO

O

O

O

O

O
OH
H

HO

O

OH

Brevifolin COOH

Punicalin
OH

OH

OH
O

HO

OH

O
HO

O

O
O
OH
OH

OH

O

ApigeninOH

O

Luteolin 4-O-glucopyranoside

Fig 1. Structures of docked polyphenols discussed in this workA

B

Fig 2. Anti-wrinkle
wrinkle effects of P. granatum juice extract. Values expressed as mean±SD (n=3).
(A) Inhibition of elastase activity (B) Collagenase inhibitory effect of PE

The orientation of polyphenols in the catalytic sites of elastaseand collagenase indicated profound interaction in the form ofprotein-ligand hydrogen bond formation (Table 1 & 2).

Brevifolin COOH showed better inhibition of enzyme elastasein comparison to other compounds, with binding energy of 8.28 kJmol-1 and forming four hydrogen bonds with active

11910Reethi Budanuru et al. The polyphenols in pomegranate play important role in exhibiting anti-ageing
anti ageing effects

pocket residues; Leu130, Arg230, Val163, Cys182 (Figure
4A). After comparative docking analysis it was learnt that theactive principles of PE extract more efficiently inhibited the
enzyme collagenase than elastase. Among the tested
molecules, Luteolin 4-O-glucopyranoside exhibited top posewith low binding energy (-8.85 kJmol-11) and forming 6hydrogen bonds with active pocket residues; Thr241, Ser243,
Gln250, Asp245, Leu248 (Figure 4B).

DISCUSSION
PE exerted its anti-wrinkle
wrinkle effect by the inhibition of key
enzymes involved in ageing. Elastase, a member of thechymotrypsin family of proteases, is responsible primarily forthe breakdown of elastin, an insoluble elastic fibrous protein
that, together withith collagen, determines the mechanicalproperties of connective tissue (Antonicelli et al., 2007).

Fig 3. Protective effect of P. granatum juice extract on non-site
non site specific hydroxyl radical mediated 22-deoxy-d-ribose degradation

A

B

Fig 4. (A) Interaction of Brevifolin COOH with active pocket residues of Elastase; (B) Binding of Luteolin 44-O-glucopyranoside withactive site of Collagenase
Table 1. In silico docking score of polyphenols from P. granatum and EGCG with Elastase
Molecule
Punicalagin
Ellagic acid
Corilagin
Brevifolin COOH
Punicalin
Apigenin
Luteolin 4-O-glucopyranoside
EGCG (Std)Binding Energy(kJ/mol)
-7.25
3.16

Ligand Efficiency(kJ/mol)
-0.33
0.51

Inhibitory constant
(µM)
4.82
18.98

H-BondInteraction

3
6

-8.28
-7.44
-7.25
-9.84

-0.39
-0.37
-0.23
-0.3

857.37
3.52
4.88
61.17

4
2
4
6

Cys182, Asn178
Asn178, Asp164, Val163,
Tyr165, Cys182
Leu130, Arg230, Val163, Cys182
Met180, Tyr165
Met180, Cys182, Leu130, Asn178
Met180, Ile129, Asp164, Tyr165, Leu130,
Asn178

Table 2. In silico docking score of polyphenols from P. granatum and EGCG with Collagenase
Molecule
Punicalagin
Ellagic acid
Corilagin
Brevifolin COOH
Punicalin
Apigenin
Luteolin 4-O-glucopyranoside
EGCG (Std)Binding Energy(kJ/mol)
-4.48
-7.46
-7.73
-7.28
-8.13
-7.04
-8.85
-8.41

Ligand Efficiency(kJ/mol)
-0.06
-0.34
-0.17
-0.35
-0.15
-0.35
-0.29
-0.25

Inhibitory constant(µM)
522.07
3.4
2.17
4.61
1.11
6.95
328.4
689.45

H-BondInteraction

4
6
6
5
4
3
6
6

Asp231, Ser243
Gln247, Ser243, Arg214, Ile232
Gln247, Ser243, Leu248, Asp245
Arg214, Gln247, Thr241, Ile232
Arg214, Gln247, Thr241,
Thr241, Ser243, Asp245, Gln247
Thr241, Ser243, Gln250, Asp245, Leu248
Asp245, Ser243, Ile232, Gln247

11911

International Journal of Current Research, Vol. 7, Issue, 01, pp.11907-11912, January, 2015

Elastin, due to its unique elastic recoil properties, is vital forgiving elasticity to arteries, lungs, ligaments and skin (Kimet al., 2004; Baylac and Racine, 2004). (Melzig et al., 2001;
Siedle et al., 2002) Elastases can cleave elastin as well as
having a broad substrate portfolio including ability to cleave
collagen, fibronectin and other ECM proteins (Melzig et al.
2001; Siedle et al., 2002). During the recent past a number ofstudies have investigated the interactions between elastase andits inhibitors (Kim et al., 2007; Masuda et al., 2009; Thring etal., 2009). Because of the presence of phenolics, medicinal
plant extracts exhibit promising inhibitory effect on elastaseactivity and hence may be good choices for cosmetic purposes
because of their relatively low incidence of side effects. In thepresent study, due to its high phytochemical profile, thepomegranate extract exhibited intense activity.
Matrix Metalloproteinases (MMPs) are part of a group oftransmembrane zinc containing endopeptidases which include
collagenases
andgelatinases.
Collagenases
aremetalloproteinases capable of cleaving other molecules foundwithin the cell for example collagenase-2 (MMP-8) can cleave
aggrecan, elastin, fibronectin, gelatine and laminin as well as
collagen (Raffetto and Khalil, 2008). Collagenase cleaves the
X-gly bond of collagen and also synthetic peptides that containthe sequence -Pro-X-Gly-Pro where X is almost any aminoacid provided that the amino terminus is blocked (Van Wartand Steinbrink, 1981). Plant secondary metabolites and whole
extracts have been widely investigated and found to have anticollagenase activity. Plants contain a vast variety ofcompounds such as polyphenols such as phenolic acids,
flavanoids, tannins etc. which attribute to their anti-ageing andanti-oxidant effects. Polyphenols isolated from herbal extracts
such as green tea, Aloe vera (Aloe barbadensis), Persimmon(Diospyros kaki) have profound collagenolytic activity(Barrantes and Guinea, 2003; Lee et al., 1999). In agreementwith the previous reports, PE had shown better anticollagenase activity indicating the possible mechanism of its
action.
UV exposure causes physical changes to the skin due toalterations that occur in the connective tissue via the formationof lipid peroxides, cell contents and enzymes (Lee et al., 2006)
and reactive oxygen species (ROS) (Benaiges et al., 1998).
When ROS are overproduced, redox-active transition metal
ions such as iron(II) or copper(II) can cause severe oxidative
stress and thus damage tissues and the cellular biomolecules
such as DNA, protein, lipid and carbohydrate constituents
within (Kaur et al., 2006). In our study, a dose dependent
increase in the protective effect of PE against hydroxyl radical
mediated DNA scission reaching upto 89 % was observed.
Interestingly, the activity was much higher than the standard
BHA. This could be attributed to the synergistic effect ofpolyphenols in the extract. These results clearly indicate that
pomegranate phytoconstituents are effective in scavenging thehydroxyl radicals and give protection against the hydroxyl
radical mediated complications such as DNA damage.
This work also presents the molecular docking of polyphenols
from pomegranate with the key metabolic enzymes responsible
for skin wrinkle formation; with a view of obtaining structural
motifs that preferentially interact with these molecules. The

major polyphenols present in pomegranate were analyzed fortheir interaction with elastase and collagenase in silico. The
docked conformations clearly reflected the inhibitory role ofthese polyphenols in PE. Several studies have been reported on
docking studies of polyphenols from plants against major
therapeutic targets as applications to rational drug design(Smith et al., 2004).
Conclusion
To meet the demand for cosmetic preparations with
antiwrinkle properties, many cosmetics companies have beendeveloping elastase inhibitors because of their potential asactive agents. The present study was carried out at Vidya
Herbs Pvt Ltd. in order to scientifically validate the skin care
properties of pomegranate. The pomegranate extract was
highly effective in inhibiting the major enzymes involved inageing. The cosmeceutical application of pomegranate wasfurther supported by the in silico interaction studies and the
DNA protection by free radical scavenging. On the basis of theresults, it is suggested that pomegranate can be used as acomprehensive source of cosmeceuticals for the maintenanceof healthy skin.

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