Journal of Advanced Pharmaceutical Technology & Research

: 2015  |  Volume : 6  |  Issue : 2  |  Page : 75--80

Preparation and characterization of standardized pomegranate extract-phospholipid complex as an effective drug delivery tool

Amisha Kamlesh Vora1, Vaishali Y Londhe2, Nancy S Pandita3,  
1 Department of Pharmaceutical Chemistry, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM's NMIMS, Mumbai, Maharashtra, India
2 Quality Assurance, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM's NMIMS, Mumbai, Maharashtra, India
3 School of Science, SVKM's NMIMS, Mumbai, Maharashtra, India

Correspondence Address:
Amisha Kamlesh Vora
Department of Pharmaceutical Chemistry, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKMSQs NMIMS, Mumbai - 400 056, Maharashtra


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.

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

How to cite this URL:
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 [serial online] 2015 [cited 2023 Mar 27 ];6:75-80
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Punica granatum (Pomegranate) is a rich source of ellagitannins namely punicalagins and punicalins. [1] Ellagitannins are a class of polyphenols known to be extremely effective through their antioxidant mechanisms against cardiovascular disorders, cancers, and also wound healing, owing to their antibacterial and antiviral activities. [2] Among both the constituents, punicalagins [Figure 1]a are considered to be the most potent antioxidant constituents contributing to overall antioxidant property of the fruit. Punicalagins have also shown to be anti-proliferative on human oral, colon, and prostate tumor cells. [3] A recent study by Jean-Gilles et al. shows inhibitory activity of punicalagins on type II collagen degradation in vitro which is a crucial process in the development of arthritis. [4] Furthermore, they slow down the progression of neurodegenerative diseases like Alzheimer's and Parkinson's disease by inhibiting the neuroinflammation in lipopolysaccharide-activated rat primary microglia. [5] Despite their excellent activity in vitro, their activity in vivo is limited due to their poor permeability through bilipid layer of the gastrointestinal tract and also degradation by colonic microflora to bioavailable but poor antioxidant hydroxy-6H-dibenzopyran-6-one derivatives. [6]{Figure 1}

Phospholipids [Figure 1]b 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]

Hence, the aim of the present study was to formulate and characterize the phospholipid complexes of standardized pomegranate extract (SPE). Considering the additive and synergistic effect of various constituents, [3],[14] the whole extract standardized to the content of punicalagins was used for formulation rather than only punicalagins.


Standardized pomegranate extract containing 30% punicalagins was prepared by a method previously described. [14] Soya phosphatidylcholine (LECIVAS70) (PC) was a gift sample from VAV Life SCIENCES Pvt. Ltd., Mumbai. Punicalagins standard was purchased from Sigma-Aldrich, India. All other chemicals were of analytical grade.

Standardized pomegranate extract-phospholipid complex (SPEPC) prepared using 2:1 molar concentrations of PC and punicalagins (calculated in terms of amount of SPE required) was stirred in a mixture of equal volumes of dioxane and methanol kept in a 250 ml conical flask for 4 h at room temperature. The mixture was spray-dried, collected, and stored in a vacuum desiccator until further use. [15]

Calibration curve for punicalagins

Working standards of 25-500 μg/ml were prepared by diluting the stock solution (1000 μg/ml) in a mobile phase of 1% formic acid and acetonitrile. The analysis was carried out using a previously validated high-performance liquid chromatography (HPLC) (Perkin Elmer Series 200 EP DAD) method [16] [Table 1].{Table 1}

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:


n-octanol solubility

The enhancement in lipid solubility due to complexation, solubility of the SPE and the SPEPC were determined in n-octanol by shake flask method.

Scanning electron microscopy

To determine the surface morphology of the complex, scanning electron microscopy (SEM) was performed at IIT, Mumbai using scanning electron microscope (JSM-7600F).

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) spectra of the SPE, PC, and SPEPC, were taken (Perkin Elmer FT-IR-Spectrum RX 1) in the transmission mode with the wave number region 4000-500 cm−1 .

Differential scanning calorimetry

Thermograms of SPE, PC, Physical mixture of SPE and PC and SPEPC were recorded on a differential calorimeter (SIECKO, SII Nano Technology, Japan). The thermal behavior was studied by heating 2 mg of each individual sample in a covered sample pan under nitrogen gas flow over the temperature range 25-250°C with a heating rate of 10°C/min.

X-ray powder diffraction

The crystalline state of drugs in the different samples was evaluated by X-ray powder diffraction (XRPD) (D-8 Advance Bruker, USA). The X-ray generator was operated at 40 kV tube voltages and 40 mA of tube current, using the Kα lines of copper as the radiation source. The scanning angle ranged from 1 to 60 of 2è 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.

The apparent permeability (Papp) value was calculated according to the following equation:


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 (cm 2 ) (0.18 cm as radius);

C0: The initial concentration of drug in the donor compartment (μg/ml).

Statistical analysis

All the analyses were done in triplicates, and results were expressed as mean values and standard deviation.


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].{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.{Figure 3}

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 4]a. 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 4]b. 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 extract. Similarly, in the region of 1200 cm−1 -960 cm−1 , the region corresponding to the phosphate group in SPEPC showed variation as compared to PC. This confirmed the formation of hydrogen bonding between the phosphate group of PC and phenolic groups of the constituents of SPE [Figure 4]c. Further, peaks due to C-H stretching in PC were also present in the IR spectra of SPEPC, confirming the presence of fatty aid chain in the complex.{Figure 4}

Differential scanning calorimetry studies

Standardized pomegranate extract showed a peak at 128.7°C [Figure 5]a while PC shows two peaks, one at 34.5°C, and the other one at 188°C [Figure 5]b. The physical mixture of SPE and PC shows two peaks, one corresponding to the endothermic peak of PC at 34.5°C and other ones in the range of 138-143°C [Figure 5]c. 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 5]d 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 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.{Figure 5}

X-ray powder diffraction studies

X-ray diffraction (XRD) analysis is a powerful tool to analyze the crystallinity of powders. The X-ray diffraction pattern of SPE [Figure 6]a showed two sharp peaks showing partially crystalline characteristics whereas PC [Figure 6]b showed amorphous character showing almost flat broad peaks. The broad peak of PC masked the sharp peaks of SPE in case of physical mixture of SPE and PC [Figure 6]c. The XRD pattern of SPEPC exhibited single broad peak signifying amorphous character.{Figure 6}

Dissolution study

To study the effect of complexation on the dissolution profile of SPE, dissolution study of SPE and SPEPC complex was 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 7]a. 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 7]b.{Figure 7}

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.


For the facilitating interaction between the SPE and PC, it is necessary that both these components are soluble in the 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 demonstrated the protective nature of phospholipids. Hence, it can be assumed that SPEPC may prevent microbial degradation in the gut, thereby increasing the bioavailability.

Therefore, it can be concluded that the phospholipid complexes can be an effective technique to improve the bioavailability of phytoconstituents across gastrointestinal membranes. Similar strategy can be explored for other improving the in vivo biological activity of bioactive phytoconstituents which show excellent in vitro activity. Thus, complexation with phospholipid can be a value added drug delivery system to overcome this problem.


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