Original Article

Preparation and characterization of standardized

pomegranate extract‑phospholipid complex as an
effective drug delivery tool
Amisha Kamlesh Vora,
Vaishali Y. Londhe1,
Nancy S. Pandita2
Departments of Pharmaceutical
Chemistry and 1Quality Assurance,
Shobhaben Pratapbhai Patel School
of Pharmacy and Technology
Management, SVKM’s NMIMS, 2School
of Science, SVKM’s NMIMS, Mumbai,
Maharashtra, India
J. Adv. Pharm. Technol. Res.
Abstract
Punicalagins, a pair of anomeric ellagitannins, present in Punica granatum (Pomegranates)
are known to possess excellent antioxidant activity in vitro, but poor oral bioavailability.
The reasons cited for poor bioavailability are their large molecular size, poor lipophilicity,
and degradation by colonic microflora into less active metabolites. The objective of the
present research work was to complex the standardized pomegranate extract (SPE) with
phospholipid to formulate standardized pomegranate extract‑phospholipid complex (SPEPC),
characterize it and check its permeability through an ex vivo everted gut sac experiment.
SPEPC was prepared by mixing SPE (30% punicalagins) and soya phosphatidylcholine (PC)
in 1:1 v/v mixture of methanol and dioxane and spray‑drying the mixture. The complex was
characterized by infrared spectroscopy, differential scanning calorimetry, X‑ray diffraction,
and scanning electron microscopy. It was evaluated for its octanol solubility, dissolution, and
permeability by everted the gut sac technique. The characterization methods confirmed the
formation of complex. Increased n‑octanol solubility of the complex proved its increased
lipophilicity. Dissolution studies revealed that the phospholipid covering may prevent the
punicalagins to be released in gastro‑intestinal tract, thus preventing their colonic microbial
degradation. SPEPC showed better apparent permeability than SPE in an everted gut sac
technique. Hence, it could be concluded that phospholipid complex of SPE may be of
potential use in increasing the permeability and hence the bioavailability of punicalagins.

Key words: Characterization, complexation, phospholipid, standardized pomegranate

extract

INTRODUCTION Ellagitannins are a class of polyphenols known to be

extremely effective through their antioxidant mechanisms
Punica granatum (Pomegranate) is a rich source of against cardiovascular disorders, cancers, and also wound
ellagitannins namely punicalagins and punicalins. [1] healing, owing to their antibacterial and antiviral activities.[2]
Among both the constituents, punicalagins [Figure 1a] are
Address for correspondence: considered to be the most potent antioxidant constituents
Ms. Amisha Kamlesh Vora, contributing to overall antioxidant property of the fruit.
Department of Pharmaceutical Chemistry, Shobhaben Punicalagins have also shown to be anti‑proliferative on
Pratapbhai Patel School of Pharmacy and Technology
Management, SVKM’s NMIMS, Mumbai ‑ 400 056, human oral, colon, and prostate tumor cells.[3] A recent study
Maharashtra, India. by Jean‑Gilles et al. shows inhibitory activity of punicalagins
E‑mail: amisha.vora@yahoo.com on type II collagen degradation in vitro which is a crucial
process in the development of arthritis.[4] Furthermore, they
Access this article online slow down the progression of neurodegenerative diseases
Quick Response Code: like Alzheimer’s and Parkinson’s disease by inhibiting the
Website: neuroinflammation in lipopolysaccharide‑activated rat
www.japtr.org primary microglia.[5] Despite their excellent activity in vitro,
their activity in vivo is limited due to their poor permeability
DOI: through bilipid layer of the gastrointestinal tract and also
10.4103/2231-4040.154542 degradation by colonic microflora to bioavailable but poor
antioxidant hydroxy‑6H‑dibenzopyran‑6‑one derivatives.[6]

75 Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2015 | Vol 6 | Issue 2
 Vora, et al.: Preparation and characterization of standardized pomegranate extract-phospholipid complex

Phospholipids [Figure 1b] play a major role as a carrier
for molecules requiring sustained release and stability
against microbial degradation in vivo. They form stable
and bioavailable amphiphilic drug lipid complexes with
reduced interfacial tension between the system and the
GI fluid; thereby facilitating the membrane permeability
of the drug. [7,8] Several other studies have indicated
the beneficial role of phospholipids in enhancing the
therapeutic efficacy of some bioactive phytoconstituents
having poor oral absorption such as naringenin, [9]
catechin,[10] embelin,[11] and extracts like green tea[12] and
grape seed.[13]
Percentage punicalagins entrapped
To determine the entrapment efficiency, SPEPC equivalent
to 50 mg of the SPE was shaken in 10 ml of water for 10 min.
Thereafter, the solution was centrifuged at 4000 rpm for
5 min, and the supernatant was analyzed for the content
of punicalagins using HPLC. The entrapped amount was
calculated as follows:
Percentage punicalagins entrapped
Total punicalagins added as SPE -
punicalagins unentrapped
= × 100
Total punicalagins added as SPE

n‑octanol solubility
Hence, the aim of the present study was to formulate and
The enhancement in lipid solubility due to complexation,
characterize the phospholipid complexes of standardized
solubility of the SPE and the SPEPC were determined in
pomegranate extract (SPE). Considering the additive and
n‑octanol by shake flask method.
synergistic effect of various constituents,[3,14] the whole
extract standardized to the content of punicalagins was used
Scanning electron microscopy
for formulation rather than only punicalagins.
To determine the surface morphology of the complex,
scanning electron microscopy (SEM) was performed
MATERIALS AND METHODS at IIT, Mumbai using scanning electron microscope
(JSM‑7600F).
Standardized pomegranate extract containing 30%
punicalagins was prepared by a method previously
Fourier transform infrared spectroscopy
described.[14] Soya phosphatidylcholine (LECIVAS70) (PC)
Fourier transform infrared spectroscopy (FT‑IR) spectra
was a gift sample from VAV Life SCIENCES Pvt. Ltd.,
of the SPE, PC, and SPEPC, were taken (Perkin Elmer
Mumbai. Punicalagins standard was purchased from
FT‑IR‑Spectrum RX 1) in the transmission mode with the
Sigma‑Aldrich, India. All other chemicals were of analytical
wave number region 4000–500 cm−1.
grade.
Differential scanning calorimetry
Standardized pomegranate extract‑phospholipid
Thermograms of SPE, PC, Physical mixture of SPE and
complex (SPEPC) prepared using 2:1 molar concentrations
PC and SPEPC were recorded on a differential calorimeter
of PC and punicalagins (calculated in terms of amount of
(SIECKO, SII Nano Technology, Japan). The thermal
SPE required) was stirred in a mixture of equal volumes of
behavior was studied by heating 2 mg of each individual
dioxane and methanol kept in a 250 ml conical flask for 4 h at
sample in a covered sample pan under nitrogen gas flow
room temperature. The mixture was spray‑dried, collected,
over the temperature range 25–250°C with a heating rate
and stored in a vacuum desiccator until further use.[15]
of 10°C/min.
Calibration curve for punicalagins
X‑ray powder diffraction
Working standards of 25–500 μg/ml were prepared by
The crystalline state of drugs in the different samples
diluting the stock solution (1000 μg/ml) in a mobile phase
was evaluated by X‑ray powder diffraction (XRPD) (D‑8
of 1% formic acid and acetonitrile. The analysis was carried
Advance Bruker, USA). The X‑ray generator was operated
out using a previously validated high‑performance liquid
at 40 kV tube voltages and 40 mA of tube current, using the
chromatography (HPLC) (Perkin Elmer Series 200 EP DAD)
Kα lines of copper as the radiation source. The scanning
method[16] [Table 1].
Table 1: Gradient elution program for the analysis
of punicalagins by HPLC
Time in min A (%) B (%)
0 5 95
35 35 65
38 80 20
40 80 20
a b 45 5 95
A: Acetonitrile, B: 1% HCOOH, Flow rate: 1 ml/min, detection wavelength: 254 nm.
Figure 1: Chemical structure of punicalagins (a) and Column: Kromasil 100‑5C18 250×4.6 mm E55950. HPLC: High performance liquid
phosphatidylcholine (b) chromatography

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 Vora, et al.: Preparation and characterization of standardized pomegranate extract-phospholipid complex

angle ranged from 1 to 60 of 2θ in step scan mode (step
with 10/min).
Dissolution study
In vitro dissolution studies for SPE and SPEPC were
performed in triplicate using USP XXIII dissolution
apparatus (8 station dissolution apparatus Campbell
Electronics DR8) at 100 rpm and 37°C. An accurately
weighed 100 mg of SPE and an amount of SPEPC
equivalent to 100 mg of SPE was introduced into 900 ml
of simulated gastric fluid (SGF) without pepsin and the
aliquots were withdrawn at the end of 10, 30, 60, and
120 min, replacing the media each time to maintain
the sink conditions. Withdrawn samples were filtered
through 0.45 μ membrane, filtered and analyzed by
HPLC. Similar study was done to check the dissolution
pattern of SPE and SPEPC simulated intestinal fluid (SIF)
to simulate the intestinal pH for a period of 8 h.
Everted gut sac technique
Permeability of punicalagins from SPE and SPEPC
was assessed by everted gut sac experiments. The
experiment was performed as per the protocol approved
by Institutional Animal Ethics Committee (CPCSEA/
IAEC/SPTM P‑38/2013). Male Albino rat (Wistar strain)
weighing 220 g was sacrificed by cervical dislocation,
the abdomen was opened by a midline incision and
the intestine was carefully maneuvered to identify the
ileocecal junction. A 7 cm segment of the intestine was
excised. The intestinal segment was transferred to a petri
dish containing Kreb’s medium, cleaned and then gently
everted using a glass rod. This tissue was then used for
permeability experiments. Appropriate volumes of the
fluid were withdrawn from the serosal compartment
at 5, 10, 15, 30, 45, 60, 75, and 120 min and analyzed by
HPLC for the content of punicalagins.
RESULTS AND DISCUSSION
Formulation of standardized pomegranate
extract‑phospholipid complex
A simple and reproducible method was used to obtain
SPEPC in the form of dry powder with a yield of 75% w/w.
Percentage punicalagins entrapped
Entrapment efficiency (percent loading) of punicalagins in
SPEPC as estimated by HPLC was found to be 99.7%w/w
(Data not shown) [Figure 2].
n‑octanol solubility
Standardized pomegranate extract is poorly soluble in
n‑octanol, but its complexation with phospholipid increased
the solubility of punicalagins in n‑octanol from 0.005 to
0.26 mg/ml (Data not shown).
Scanning electron microscopy
Scanning electron micrographs of the complex are shown
in Figure 3. Unlike irregular shape of SPE, its phospholipid
complex was found to be spherical and coated with the layer
of phospholipid. The average particle size of the complex
was found to be 50 µm.
Infra‑red spectroscopy
The formation of the complex was confirmed by FT‑IR
spectroscopy [Figure 4]. FT‑IR spectra of SPEPC showed
changes in the positions of the peaks as compared to SPE
and PC. The SPE showed the characteristic O‑H stretching
vibration of phenolic groups at 3294.22 cm−1 [Figure 4a].
PC showed sharp characteristic C‑H stretching vibration
peaks (of fatty acid chain) at 2924.11 cm−1 and 2853.67 cm−1
and C = O stretching vibration at 1739.44 cm−1 [Figure 4b].
However, the FT‑IR spectra of SPEPC showed a very broad
peak at 3371 cm−1 suggesting that possible interaction had
occurred at –OH groups of polyphenols present in the

The apparent permeability (Papp) value was calculated

according to the following equation:
dQ 1
P app = *
dT A(C0)
Papp: Apparent permeability co‑efficient.
dQ/dT: The cumulative amount of drug (Q) appearing in
the acceptor (serosal) compartment as a function of time,
and was obtained from the slope of the linear portion of the
amount transported‑versus‑time plot;
A: Surface area of the intestine (cm2) (0.18 cm as radius);
C0: The initial concentration of drug in the donor
compartment (μg/ml).

Statistical analysis Figure 2: High-performance liquid chromatography chromatogram

All the analyses were done in triplicates, and results were of α and β-punicalagin; RT of α-punicalagin 10.51 min, RT of
expressed as mean values and standard deviation. β-punicalagin 12.84 min

77 Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2015 | Vol 6 | Issue 2
 Vora, et al.: Preparation and characterization of standardized pomegranate extract-phospholipid complex

extract. Similarly, in the region of 1200 cm−1–960 cm−1, the X‑ray powder diffraction studies
region corresponding to the phosphate group in SPEPC X‑ray diffraction (XRD) analysis is a powerful tool to
showed variation as compared to PC. This confirmed the analyze the crystallinity of powders. The X‑ray diffraction
formation of hydrogen bonding between the phosphate pattern of SPE [Figure 6a] showed two sharp peaks showing
group of PC and phenolic groups of the constituents of partially crystalline characteristics whereas PC [Figure 6b]
SPE [Figure 4c]. Further, peaks due to C‑H stretching in PC showed amorphous character showing almost flat broad
were also present in the IR spectra of SPEPC, confirming peaks. The broad peak of PC masked the sharp peaks of
the presence of fatty acid chain in the complex. SPE in case of physical mixture of SPE and PC [Figure 6c].
The XRD pattern of SPEPC exhibited single broad peak
Differential scanning calorimetry studies signifying amorphous character.
Standardized pomegranate extract showed a peak at
128.7°C [Figure 5a] while PC shows two peaks, one at Dissolution study
34.5°C, and the other one at 188°C [Figure 5b]. The physical To study the effect of complexation on the dissolution profile
mixture of SPE and PC shows two peaks, one corresponding of SPE, dissolution study of SPE and SPEPC complex was
to the endothermic peak of PC at 34.5°C and other ones
in the range of 138–143°C [Figure 5c]. This shows that
partial interaction may have taken place between the
SPE and PC when at 34.5°C, the PC melts and being in
the liquid state interacts with the SPE. The thermogram
of SPEPC [Figure 5d] shows disappearance of peaks
at 34.5°C, 188°C (corresponding to PC), disappearance
of peak at 128.7°C (corresponding to SPE) and appearance
a
of sharp endothermic peaks in a completely different range
of 172–179°C. This confirms the interaction between SPE
and PC and formation of complex which is different from
the starting materials, SPE and PC.

a b c
Figure 3: Scanning electron microscopy of standardized pomegranate Figure 4: IR spectra of standardized pomegranate extract (a),
extract (a) and standardized pomegranate extract-phospholipid phosphatidylcholine (b) and standardized pomegranate extract-
complex (b) phospholipid complex (c)

a b

c d
Figure 5: Differential scanning calorimetry thermograms of standardized pomegranate extract (SPE) (a), phosphatidylcholine (PC) (b), physical
mixture of SPE and PC (c) and standardized pomegranate extract-phospholipid complex (d)
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 Vora, et al.: Preparation and characterization of standardized pomegranate extract-phospholipid complex

done in SGF (without pepsin) and SIF. Dissolution profile
in SGF shows that in case of SPE, the entire water soluble
punicalagins was released in the dissolution media in first
10 min [Figure 7a]. However, in case of the complex SPEPC,
the release was reduced to 3.04% even at the end of 2 h
indicating that 96.96% of punicalagins remained confined in
the PC bilayers. Similar observations were made in SIF wherein
94.36% was found to be entrapped at the end of 8 h [Figure 7b].
Everted gut sac technique
The everted gut sac technique is a reliable and predictive
in vitro method to study the permeability of various
molecules and their formulation across the intestinal
membrane. The Papp values for SPE and SPEPC by this
technique were found to be 60 and 135 nm/s. This showed
that the apparent permeability of SPE increased almost two
times on complexation with PC.
DISCUSSION
For the facilitating interaction between the SPE and PC, it
is necessary that both these components are soluble in the
d
same solvent system to form the homogenous mixture.
PC being highly lipophilic was insoluble in water as
well as methanol in which SPE was found to be soluble.
Dioxane being safe nonpolar solvent and miscible with
methanol was used to solubilize PC. Using equal volumes
of methanol‑dioxane, solution of SPE and PC mixture was
spray dried to obtain herbosomes in the form of dry powder.
The SEM micrographs showed spherical shape of the
complex in contrast with irregular shape of SPE suggesting
that the layer of PC enveloped the constituents of SPE to
form spherical cell‑like structures. The IR spectroscopy
confirmed the formation of hydrogen bond between the
polyphenolic components of SPE and the choline and
phosphate group of PC. Similarly, the differential scanning
calorimetry [Figure 5] thermogram of the complex showed
a markedly different profile of the complex than that of the
individual components and the physical mixture. Even, the
XRD study revealed amorphous character of the SPEPC
ascribed to the entrapment in lipid bilayers. Further, the
HPLC analysis indicated that the complexation showed an
almost complete entrapment resulting in almost 50 times
enhancement of n‑octanol solubility of punicalagins. This
increase in lipophilicity of punicalagins also contributes to
the increased permeability (by 2 times) in vivo and enhanced
bioavailability across the lipid‑rich biomembranes as
established by an everted gut sac experiment.

Comparative dissolution study of SPE and SPEPC in

SGF (without pepsin) and SIF over a period of 2 and 8 h
c demonstrated the protective nature of phospholipids.
Hence, it can be assumed that SPEPC may prevent
microbial degradation in the gut, thereby increasing the
a
bioavailability.
b
Therefore, it can be concluded that the phospholipid
complexes can be an effective technique to improve the
bioavailability of phytoconstituents across gastrointestinal
Figure 6: X-ray diffraction of standardized pomegranate extract membranes. Similar strategy can be explored for other
(SPE) (a), phosphatidylcholin (PC) (b), physical mixture of SPE and improving the in vivo biological activity of bioactive
PC (c) and SPE-PC (d) phytoconstituents which show excellent in vitro activity.

a b
Figure 7: Dissolution study of standardized pomegranate extract and standardized pomegranate extract-phospholipid complex in simulated
gastric fluid (without pepsin) (a) and in simulated intestinal fluid (b)

79 Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2015 | Vol 6 | Issue 2
 Vora, et al.: Preparation and characterization of standardized pomegranate extract-phospholipid complex

Thus, complexation with phospholipid can be a value added
drug delivery system to overcome this problem.
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How to cite this article: Vora AK, Londhe VY, Pandita NS.
Preparation and characterization of standardized pomegranate
extract-phospholipid complex as an effective drug delivery tool. J
Adv Pharm Technol Res 2015;6:75-80.
Source of Support: Nil, Conflict of Interest: Nil.

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