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Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: 2231-6876
FORMULATION AND CHARACTERIZATION OF EPIGALLOCATECHIN GALLATE
NANOPARTICLES
Ramkumar Ponnuraj1*
, Janakiraman K1
, Sivaraman Gopalakrishnan2
, Senthilnathan K2
, Meganathan
V2
, Saravanan P2
1
Annamalai University, Annamalai Nagar, Chidambaram, 608002, India.
2
apex Laboratories Private Limited, Alathur, Kanchipuram, 603110, India
Corresponding author
Ramkumar Ponnuraj
Department of Pharmacy,
Annamalai University,
Annamalai Nagar,
Chidambaram, 608002, India.
+91-9094204320, +91-9965592259
aadharshini@gmail.com
Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ARTICLE INFO ABSTRACT
Article history
Received 11/01/2015
Available online
30/01/2015
Keywords
Epigallocatechin Gallate,
Chitosan, Poloxamer 188,
Nanoparticles,
Bioavailability,
Ionic-Gelation.
Epigallocatechin gallate is a type of catechin has many therapeutic advantages, but its scope
is limited due to its poor bioavailability. Most of the ingested Epigallocatechin gallate
apparently does not get into the blood, since absorption takes place in the small gut and
substantial quantities pass from the small intestine to the large intestine, where it undergoes
further degradation by the action of local microbiota [1-4]. Chitosan, a polymer of linear
polysaccharide, enhances transport of drug across epithelial surfaces and is biocompatible and
biodegradable. The aim of this study is to formulate and characterize Epigallocatechin gallate
loaded Chitosan nanoparticles prepared by ionic-gelation method. This increases the
abosorption and bioavailaility of Epigallocatechin gallate. The resulting nanoparticles tend to
aggregate in biological fluid which can be minimized by addition of poloxamer 188. The
nanoparticles obtained were evaluated for percentage yield of drug, drug entrapment
efficiency, particle size and morphology using scanning electron microscopy (SEM),
compatibility studies using Fourier-Transform infrared spectroscopy (FTIR) and Differential
scanning calorimetry (DSC) and in vitro release kinetics. Among the four different ratios of
drug to polymer, 1:0.5 ratio showed high drug loading and encapsulation efficiency. The
resulting nanoparticles were spherical in shape with a smooth surface. The particle size range
was 197.84 ± 21.45 nm to 385.45 ± 15.87 nm. The prepared nanoparticles proved to be
promising dosage form of Epigallocatechin gallate, with improved bioavailability.
Please cite this article in press as Ramkumar Ponnuraj et al. Formulation And Characterization of Epigallocatechin Gallate
Nanoparticles. Indo American Journal of Pharm Research.2015:5(01).
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INTRODUCTION
Epigallocatechin gallate, [(2R, 3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate also
known as epigallocatechin-3-gallate is the ester of epigallocatechin and gallic acid and is a type of catechin. It has been the subject of
a number of studies investigating its potential use as a therapeutic for a broad range of disorders, which includes HIV [5-8], Cancer [9-
12], Chronic fatigue syndrome [13-15], Sjögren's syndrome [16], Endometriosis [17], Spinal muscular atrophy [18],
Neurodegeneration [19,20], Cannabinoid 1 receptor, CB1 receptor Activity [21], Periapical lesions [22], Cerebrovascular insult [23].
Epigallocatechin gallate is unstable in the gastrointestinal tract. It rapidly degrades in both acidic (pH below 2) and neutral
conditions. Encapsulating Epigallocatechin gallate into nanoparticles, significantly delayed its degradation in simulated digestive
fluids [24-27].
Nanoparticles are nanoscale shells made out of nontoxic polymer. They are vesicular systems made up of a polymeric
membrane which encapsulates drug core in nanoscale level [28,29]. These colloidal particles have a diameter in the range of 10 - 1000
nm. Nanoparticles have a myriad of uses.
Polymeric nanoparticles have been currently used as potential drug delivery system because of several advantages over the
other techniques, since the nano-sized structure of nanoparticles allows the permeation through cell membranes [30], which makes
them effective carriers of drug in biological systems, to achieve improved bioavailability of the drug [31]. Similarly, oral route is the
most accepted route of administration and hence the polymeric nanoparticles of the drug can be given by oral route.
Various methods have been proposed for the preparation of nanoparticles: Nanoprecipitation [32], Emulsion – Diffusion [33],
Double emulsification [34], Emulsification Coacervation [35] and recently, the encapsulation technique used in medicine.
Many natural polymers have gained importance in recent years, for their use in new drug delivery applications. The
dissolution rate of drugs from the formulations containing viscous carriers is generally sustained, due to the formation of gel layer on
the hydrated surfaces. We choose Chitosan polymer, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-
glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) derived by deacetylation of chitin [36]. It gained great
interest in pharmaceutical research because of its advantages like biodegradability, biocompatibility, non-toxicity, non-
immunogenicity and low cost [37]. Chitosan is insoluble in water, but can be dissolved in most of the organic acid solutions, at pH
less than 6.5 such as lactic acid, acetic acid, tartaric acid etc. [38].
Poloxamer is nonionic triblock copolymer composed of central hydrophobic chain of polyoxypropylene (poly(propylene
oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). It helps to reduce the aggregation of
nanoparticles and thereby stabilizing the nanoparticles.
The present study is aimed to increase the bioavailability of Epigallocatechin gallate, by developing nanoparticles using
Chitosan by ionic gelation method [39] and Poloxamer is used to reduce the aggregation of nanoparticles and thereby stabilizing the
same.
MATERIALS AND METHODS
Materials:
Epigallocatechin gallate (EGCG) was purchased from Novanat, China. Chitosan (CS) was obtained from Novamatrix,
Norway. Dimethyl sulfoxide (DMSO), Acetic acid, Sodium Tripolyphosphate (TPP), Diethyl ether, Trifluoroacetic acid and
Acetonitrile were procured from Sigma-Aldrich, USA. Poloxamer 188 was procured from BASF, Germany. Deionized water was
obtained from Millipore filtration system, USA.
Method of preparation of drug loaded nanoparticles:
Nanoparticles of EGCG is prepared using CS as a coating material by ionic gelation method, where the positively charged
amino group in the CS interacts with negatively charged TPP to form coacervates with a size in the range of nanometer.EGCG and CS
were weighed in different ratios (1:0.1, 1:0.2, 1:0.4 and 1:0.5). CS was dissolved separately in aqueous solution of Acetic acid (2%
v/v) at pH 5.5. Poloxamer 188 (Poloxamer 188/CS ratio: 5/1) was dissolved in the above solution. EGCG was dissolved separately in
Purified water (7.5% w/v of drug) and was added slowly to the above aqueous solution containing CS, while stirring under the
magnetic stirrer.
Aqueous solution of sodium tripolyphosphate (TPP) (CS/TPP ratio: 1/0.1) was added in drops to the above solution; under
magnetic stirring for 3 hrs at a speed of 1000 RPM (Remi Magnetic Stirrer MLH, Chennai) resulted in cross linking of CS and TPP, as
depicted in Fig 1 and produced the nanoparticles. After 3 hrs, nanoparticles formed were recovered by centrifugation (REMI cooling
centrifuge, Chennai) at 3000 rpm for 30 min. The recovered nanoparticles were washed using diethyl ether and lyophilized.
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Fig 1: Schematic of nanoparticle formation process by ionic gelation process. Chitosan forms a cross linking with Sodium
Tripolyphosphate.
Determination of nanoparticle yield, drug content and entrapment efficiency:
The yield of nanoparticle (%) was calculated using the equation (1) below:
Drug Content and Entrapment Efficiency were determined using equation (2) and (3) respectively.
Nanoparticles equivalent to 250mg of drug were dissolved in 50mL mixture of Trifluoroacetic acid, Acetonitrile and Milli-Q
Water in a ratio of 0.1:65.0:34.9. The solution was further diluted with the same mixture of solvents. Drug quantification was
performed using HPLC (Waters HPLC system equipped with a quaternary pump, SIL-10AD auto injector, CTO-10A column oven
and UV detector units) with a Sunfire™ column (C8 250mm x 4.6 mm, 5 μm).
The chromatographic conditions were injection volume = 50 µL, flow rate = 2.0 mL min-1
mobile phase = 0.1/65.0/34.9 of
Trifluoroacetic acid/Acetonitrile/Milli-Q Water (pH 2.40). UV detection was performed at 210 nm.
All samples were measured in triplicate. The method was validated in terms of linearity, precision, accuracy, limit of
detection (LOD), and limit of quantification (LOQ). The results were expressed as mean ± standard deviation.
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Particle characterization:
Particle size, polydispersity index and zeta potential of EGCG nanoparticles were measured using Malvern Zetasizer
(Malvern Instruments, UK), by dynamic light scattering and Electrophoretic light scattering principle. The particle size analysis was
performed at a scattering angle of 90°C, at room temperature. The diameter was averaged from three parallel measurements and
expressed as mean ± standard deviation.
SEM was used to examine the particle surface morphology and shape. The nanoparticles were coated in Sputter coater, with a
thin layer of gold, in an argon gas environment (Cressington 108 Auto sputter coater, UK). Photographs were taken using FEI Quanta
FEG 200, (FEI, USA), at room temperature.
Differential scanning calorimetry (DSC) interpretation:
Thermograms of the EGCG and its nanoparticles were recorded on MettlerSTAR SW 9.01 instrument (Mettler Toledo,
Switzerland). The analysis was carried out in nitrogen atmosphere, by heating 2 to 3 mg of sample on an aluminum crimp pan, at
heating rate of 10°C/min. The runs were made in triplicate.
Fourier Transform Infrared Radiation Measurements (FT-IR):
FT-IR analysis was carried out for pure drug and nanoparticles obtained, by mixing with Potassium Bromide (KBr), by pellet
method, after a baseline correction was made with dried potassium bromide, to confirm the compatibility. The pellet was prepared to a
pressure of about 5 x 106
Pa, in an evacuated die, to produce a clear transparent disc of diameter 2 cm and thickness of 0.2 cm. The
spectra were recorded at room temperature on Fourier transform spectrometer (Perkin-Elmer, USA) from 4000 cm-1
to 400 cm-1
.
Powder X-Ray diffraction (PXRD) study:
Crystallinity study was carried out by comparing XRD spectrum of drug with nanoparticles using Siemens Diffractometer
D5000 (Siemens, Germany), to check peak of drug in individual state and in nanoparticles. The data was recorded at 2θ range of 10°C
to 60°C at time of 0.5 sec. The relative intensity I/I0 and inter-planar distance (d) corresponding to 2θ value were reported and
compared.
In vitro release study:
The in vitro release study of EGCG loaded CS nanoparticles having highest entrapment efficiency was carried out using USP
dissolution apparatus type II (paddle method). Nanoparticles equivalent to 250 mg of EGCG were loaded using 900 ml of citric acid
buffer (pH 4, where the sink conditions could be easily maintained), at a rotating speed of 50 rpm and 37 C ± 0.5 C. Samples were
collected at specific time intervals. 2 ml of aliquot was collected during each sampling point and it was replaced with an equal volume
of fresh buffer.
The amount of drug release was determined by HPLC method, as described under the determination of drug loading in the
nanoparticles.
The in vitro release data was analyzed using various kinetic models which include, the zero-order kinetic model [40], the
first-order kinetic model [41], the Higuchi model [42], the Hixson-Crowell model [43], the Korsmeyer-Peppas model [44], the Makoid
Banakar model, [45] the Peppas Sahlin model, [46] the Weibull model, [47] the Baker Lonsdale model [48] the Hopfenberg model,
[49] the Probit model [50,51] the Logistic model [52] the Quadratic model [53,54] the Gompertz model [55].
The model which gives the highest correlation coefficient (R2
) is considered as the best fit of release data.
RESULTS AND DISCUSSION
Determination of nanoparticle yield, drug content and entrapment efficiency:
The nanoparticle yield was high with increase in drug to polymer ratio. Similarly, the drug content and the entrapment
efficiency increase with the increase in drug to polymer ratio (Table 1). As depicted in Fig 2, it is observed that increasing the amount
of polymer results in better drug entrapment. From the entrapment efficiency, 1:0.5 ratio showed better yield, compared to the other
three ratios. The nanoparticle yield increases with increase in drug entrapment. Similarly, the low drug to polymer ratio of 1:0.1
reflects with low entrapment efficiency.
Table 1: Nanoparticle yield, drug content and entrapment efficiency of different batches of nanoparticles prepared.
Formulation trial code Drug to polymer ratio
Nanoparticle yield^
(% ± SD)
Drug Content^
(% ± SD)
Entrapment Efficiency^
(% ± SD)
FT-001 to FT-003 1:0.1 45.95 ± 9.14 27.70 ± 8.75 14.00 ± 4.58
FT-004 to FT-006 1:0.2 60.66 ± 7.00 53.65 ± 8.47 39.33 ± 3.79
FT-007 to FT-009 1:0.4 76.39 ± 4.34 58.25 ± 3.38 64.00 ± 3.61
FT-010 to FT-012 1:0.5 95.05 ± 3.25 62.25 ± 1.67 91.67 ± 2.08
Note: Values are mean of three consecutive trials with its Standard Deviation (SD)
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Fig 2: Comparative results of nanoparticle yield, drug content and entrapment efficiency. Trials taken with various ratios of
Epigallocatechin gallate and Chitosan.
Particle characterization:
The results indicate that the particle size increases with increase in concentration of CS. It was observed that the particle size
of the EGCG loaded nanoparticles were between 197.84 ± 21.45 nm to 385.45 ± 15.87 nm.
Monodisperse samples have lower Polydispersity Index value, whereas higher values of Polydispersity Index indicate a wider
particle size distribution, due to aggregation. The usual range of Polydispersity Index values is 0 to 0.05 for monodisperse standard,
0.05 to 0.08 for nearly monodisperse, 0.08 to 0.7 for mid range polydispersity and >0.7 for highly polydisperse. The nanoparticles
formulated have Polydispersity Index of 0.219 ± 0.014 to 0.502 ± 0.065, as shown in Table 2 indicate mid range polydispersity. The
addition of Poloxamer 188 aids in reducing the aggregation of nanoparticles, which in turn was confirmed by the low Polydispersity
Index of nanoparticles.
Zeta potential of prepared nanoparticles was found to be in the range of +21.4 ± 2.8 mV to +45.5 ± 1.2 mV. It was found that
higher the zeta potential, lesser will be the particle aggregation, due to electric repulsion. The stability will be high, if the Zeta
Potential is high.
Table 2: Average Particle Size, Polydispersity index and Zeta Potential of nanoparticles prepared.
Drug to polymer ratio Average Particle Size (nm)
Polydispersity index
(Đ)
Zeta Potential
(mV)
1:0.1 197.84 ± 21.45 0.502 ± 0.065 +21.4 ± 2.8
1:0.2 323.51 ± 18.58 0.413 ± 0.054 +25.6 ± 1.9
1:0.4 369.87 ± 22.15 0.310 ± 0.024 +35.9 ± 1.7
1:0.5 385.45 ± 15.87 0.219 ± 0.014 +45.5 ± 1.2
The microscopic imaging of SEM shown in the Fig 3, infers the surface morphology of EGCG nanoparticles: almost spherical surface,
smooth and discrete nanoparticles.
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Fig 3: Scanning Electron Microscopic imaging. Epigallocatechin gallate nanoparticles observed under the scanning electron
microscope.
Determination of purity by Differential scanning colorimetry (DSC)
DSC thermograms of pure EGCG (Fig 4) showed a small endothermic peak at 227.47°C, which closely corresponds to its
melting point.
Fig 4: DSC thermogram. The endothermic and exothermic peak of Epigallocatechin gallate obtained using DSC.
DSC thermogram of CS (Fig 5) show a broad endothermic peak centered at about 93.45°C. This peak is attributed to the loss
of water associated with the hydrophilic groups of the polymer, due to evaporation. This peak was observed even after drying the
sample and keeping in the desiccator, before the analysis.
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Fig 5: DSC thermogram. The endothermic and exothermic peak of Chitosan obtained using DSC.
The exothermic peak, which appears in the temperature about 290°C, corresponds to the decomposition of the polymer. The
thermal degradation is due to the saccharide rings’ dehydration, depolymerization, decomposition of deacetylated and acetylated
chitosan units.
After heating to 150°C and holding for a minute, followed by cooling to 40°C, the moisture was removed and a second
heating run was done, where it was found that the endothermic peak was not observed, which confirmed that the peak at around
93.45°C was due to water content in the sample. An exothermic peak was also observed due to degradation of chitosan as mentioned
earlier.
The EGCG nanoparticles showed an endothermic peak at 227.45°C. The slight shift in the peak of nanoparticles was due to
the incorporation of CS polymer. The results shown in Fig 6 clearly indicate the compatibility between drug and polymer. There were
no interactions observed in the obtained nanoparticles.
Fig 6: DSC thermogram. The endothermic and exothermic peak of Epigallocatechin gallate nanoparticles obtained using DSC.
Determination of purity by Fourier transforms infrared spectroscopy:
The FT-IR spectrum of EGCG (Fig 7) revealed the characteristic peaks of 3482.81cm-1
and 3357.46cm-1
for O-H group
attached to the aromatic ring, 1691.27cm-1
and 1616.06cm-1
strong for C=O group that links the trihydroxybenzoate group and
chroman group, 1447.31cm-1
for C-H group present in the Chroman ring, 1348.00cm-1
, 1222.65cm-1
for O-C=O group, 1148.40cm-1
and 1097.30cm-1
for O-H group, 1041.37cm-1
for C-O-C group which links the Chromane ring and trihydroxy benzoate ring,
853.35cm-1
, 825.38cm-1
for C-H group in the aromatic ring, 788.74cm-1
for the three neighboring aromatic C-H group in Chroman
ring.
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Fig 7: FTIR spectrum. Finger print region between 4000cm-1
to 400cm-1
of Epigallocatechin gallate.
The FT-IR spectrum of CS (Fig 8) revealed the characteristic peaks of 3407.13 cm-1 for amine N-H symmetric vibration,
2915.54 cm-1 and 2887.54 cm-1 for C-H stretching, 1645.65 cm-1 for C-C stretching, 1558.25 cm-1 for amino group vibration,
1158.19 cm-1 and 905.53 cm-1 corresponded to the saccharide structure of chitosan. The broad peak at 1050.91 cm-1 indicated C-O
stretching vibration.
Fig 8: FTIR spectrum. Finger print region between 4000cm-1
to 400cm-1
of Chitosan.
The FT-IR spectrum of the final nanoparticles (Fig 9) revealed the presence of characteristic peaks of EGCG with a
negligible shift, which included 3487.54cm-1
and 3365.57cm-1
for O-H group attached to the aromatic ring, 1683.71cm-1
and
1606.65cm-1
strong for C=O group that linked the trihydroxybenzoate group and chroman group, 1451.24cm-1
for C-H group present
in the Chroman ring, 1332.46cm-1
, 1218.57cm-1
for O-C=O group, 1054.54cm-1
for C-O-C group which linked the Chromane ring and
trihydroxy benzoate ring, 794.04cm-1
for the three neighboring aromatic C-H group in Chroman ring.
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Fig 9: FTIR spectrum. Finger print region between 4000cm-1
to 400cm-1
of Epigallocatechin gallate nanoparticles.
Hence from the above spectrum, it is clear that there were no incompatibilities present with the any of the added excipients.
Determination of crystallinity by X-ray diffraction study:
PXRD was used to determine the crystallinity of compounds. Polymorphic changes of drug was an important factor, which
might affect the dissolution rate and in turn bioavailability. Crystallinity of the drug and the nanoparticles was determined. The XRD
pattern (Fig 10) of pure drug showed a broad diffraction peaks at 2θ, as the drug was amorphous. Similarly nanoparticles also did not
show any sharp peak at 2θ.
(A) (B)
Fig 10: XRD Study (A) Epigallocatechin gallate showing no sharp peak (B) Epigallocatechin gallate
nanoparticles showing no sharp peak.
In-vitro release studies and release profile:
EGCG nanoparticles prepared with CS in a ratio of 1:0.5 (FT010, FT011 and FT012) were taken for dissolution, since they
had the highest entrapment efficiency, when compared to other ratios. As shown in Fig 11, nanoparticles showed an initial burst
release of around 20% EGCG in the first hour. This initial rapid release, characterized as ―burst effect‖ was due to the fact that some
amounts of EGCG were localized, on the surface of nanoparticles, by adsorption, which could be released easily by diffusion. After
this initial burst effect, a slower sustained release occurred throughout the dissolution period.
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Fig 11: Dissolution profile of Epigallocatechin gallate Nanoparticles. Dissolution profile of nanoparticles prepared using 1:0.5
ratio of Epigallocatechin gallate and Chitosan in citric acid buffer solution (pH 4).
Table 3 demonstrates correlation values (R2) and release parameters determined from the results of model fitting of the
release profiles. According to the correlation values, they fitted to Makoid Banakar, Peppas-Sahlin, Korsmeyer-Peppas and Higuchi
models, which indicated that EGCG was released by diffusion. Moreover, the Korsmeyer-Peppas release model (high correlation
values) exponent, , which was about 0.4932, showed the characteristics of anomalous kinetics (non-Fickian), suggesting that more
than one mechanism, might be involved in release kinetics, referring to the combination of diffusion and erosion based drug release
mechanism. This mechanism could result from an increased plasticization at the relaxing boundary. The release data of the Hixson-
Crowell model suggested that there was no change in the surface area as a function of time.
Table 3: Mathematical Models and Correlation values (R2
) based on release data.
S No Mathematical Model Correlation Value (R2
)
1 Makoid Banakar 0.9981
2 Peppas-Sahlin 0.9979
3 Korsmeyer Peppas 0.9971 (n=0.4932)
4 Higuchi 0.9970
5 Weibull 0.9921
6 Baker Lonsdale 0.9605
7 First Order 0.9479
8 Hopfenberg 0.9479
9 Probit 0.9379
10 Logistic 0.9359
11 Quadratic 0.9044
12 Hixson Crowell 0.9054
13 Gompertz 0.8925
14 Zero Order 0.5683
CONCLUSION
EGCG loaded CS nanoparticles were successfully prepared by ionic gelation method, in four different ratios 1:0.1, 1:0.2,
1:0.4 & 1:0.5. According to the efficiency of yield and entrapment, 1:0.5 ratio showed better yield, compared to the other three ratios.
The entrapment efficiency was found in the range of 14.00 ± 4.58% to 91.67 ± 2.08%. Average size of prepared nanoparticles varied
from 197.84 ± 21.45 nm to 385.45 ± 15.87 nm, with a polydispersity index in the range of 0.502 ± 0.065 to 0.219 ± 0.014. As the
amount of polymer increased, the size of the nanoparticles also increased. It was found that the inclusion of poloxamer 188 increased
the zeta potential, due to which the particle aggregation was less and hence, the stability of nanoparticles was high. DSC and FTIR
completely suggested the drug to polymer compatibility. In-vitro release study showed sustained release of drug from 1:0.5 ratio of
polymer, upto 24 hours following diffusion and swelling mechanism. From the present study, it is concluded that EGCG loaded CS
nanoparticles is an effective carrier for the design of controlled drug delivery of EGCG. The increase in absorption and bioavailability
of EGCG nanoparticles has to be further researched.
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ACKNOWLEDGMENTS
This work was carried out in apex laboratories, as a part of a project investigating the production of nanoparticle systems
using antilipidemic drugs, for effective treatment. The instruments and infrastructure used to conduct the above experiment was
facilitated by apex laboratories.
Abbreviations
1. % - Percentage
2. °C - Degree Centigrade
3. cm - Centimeter
4. HIV- Human Immuno Virus
5. HPLC - High Performance Liquid Chromatography
6. mg - milligram
7. min - minute
8. ml - milliliter
9. mm - millimeter
10. mV - millivolts
11. nm - nanometer
12. Pa - Pascal
13. rpm - Revolutions per minute
14. SD - Standard Deviation
15. sec - second
16. UK - United Kingdom
17. USA - United States of America
18. UV - Ultra Violet
19. v/v - volume by volume
20. w/v - weight by volume
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EGCG Nanoparticles Characterization

  • 1. www.iajpr.com Page387 Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: 2231-6876 FORMULATION AND CHARACTERIZATION OF EPIGALLOCATECHIN GALLATE NANOPARTICLES Ramkumar Ponnuraj1* , Janakiraman K1 , Sivaraman Gopalakrishnan2 , Senthilnathan K2 , Meganathan V2 , Saravanan P2 1 Annamalai University, Annamalai Nagar, Chidambaram, 608002, India. 2 apex Laboratories Private Limited, Alathur, Kanchipuram, 603110, India Corresponding author Ramkumar Ponnuraj Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, 608002, India. +91-9094204320, +91-9965592259 aadharshini@gmail.com Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE INFO ABSTRACT Article history Received 11/01/2015 Available online 30/01/2015 Keywords Epigallocatechin Gallate, Chitosan, Poloxamer 188, Nanoparticles, Bioavailability, Ionic-Gelation. Epigallocatechin gallate is a type of catechin has many therapeutic advantages, but its scope is limited due to its poor bioavailability. Most of the ingested Epigallocatechin gallate apparently does not get into the blood, since absorption takes place in the small gut and substantial quantities pass from the small intestine to the large intestine, where it undergoes further degradation by the action of local microbiota [1-4]. Chitosan, a polymer of linear polysaccharide, enhances transport of drug across epithelial surfaces and is biocompatible and biodegradable. The aim of this study is to formulate and characterize Epigallocatechin gallate loaded Chitosan nanoparticles prepared by ionic-gelation method. This increases the abosorption and bioavailaility of Epigallocatechin gallate. The resulting nanoparticles tend to aggregate in biological fluid which can be minimized by addition of poloxamer 188. The nanoparticles obtained were evaluated for percentage yield of drug, drug entrapment efficiency, particle size and morphology using scanning electron microscopy (SEM), compatibility studies using Fourier-Transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC) and in vitro release kinetics. Among the four different ratios of drug to polymer, 1:0.5 ratio showed high drug loading and encapsulation efficiency. The resulting nanoparticles were spherical in shape with a smooth surface. The particle size range was 197.84 ± 21.45 nm to 385.45 ± 15.87 nm. The prepared nanoparticles proved to be promising dosage form of Epigallocatechin gallate, with improved bioavailability. Please cite this article in press as Ramkumar Ponnuraj et al. Formulation And Characterization of Epigallocatechin Gallate Nanoparticles. Indo American Journal of Pharm Research.2015:5(01).
  • 2. www.iajpr.com Page388 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 INTRODUCTION Epigallocatechin gallate, [(2R, 3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate also known as epigallocatechin-3-gallate is the ester of epigallocatechin and gallic acid and is a type of catechin. It has been the subject of a number of studies investigating its potential use as a therapeutic for a broad range of disorders, which includes HIV [5-8], Cancer [9- 12], Chronic fatigue syndrome [13-15], Sjögren's syndrome [16], Endometriosis [17], Spinal muscular atrophy [18], Neurodegeneration [19,20], Cannabinoid 1 receptor, CB1 receptor Activity [21], Periapical lesions [22], Cerebrovascular insult [23]. Epigallocatechin gallate is unstable in the gastrointestinal tract. It rapidly degrades in both acidic (pH below 2) and neutral conditions. Encapsulating Epigallocatechin gallate into nanoparticles, significantly delayed its degradation in simulated digestive fluids [24-27]. Nanoparticles are nanoscale shells made out of nontoxic polymer. They are vesicular systems made up of a polymeric membrane which encapsulates drug core in nanoscale level [28,29]. These colloidal particles have a diameter in the range of 10 - 1000 nm. Nanoparticles have a myriad of uses. Polymeric nanoparticles have been currently used as potential drug delivery system because of several advantages over the other techniques, since the nano-sized structure of nanoparticles allows the permeation through cell membranes [30], which makes them effective carriers of drug in biological systems, to achieve improved bioavailability of the drug [31]. Similarly, oral route is the most accepted route of administration and hence the polymeric nanoparticles of the drug can be given by oral route. Various methods have been proposed for the preparation of nanoparticles: Nanoprecipitation [32], Emulsion – Diffusion [33], Double emulsification [34], Emulsification Coacervation [35] and recently, the encapsulation technique used in medicine. Many natural polymers have gained importance in recent years, for their use in new drug delivery applications. The dissolution rate of drugs from the formulations containing viscous carriers is generally sustained, due to the formation of gel layer on the hydrated surfaces. We choose Chitosan polymer, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D- glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) derived by deacetylation of chitin [36]. It gained great interest in pharmaceutical research because of its advantages like biodegradability, biocompatibility, non-toxicity, non- immunogenicity and low cost [37]. Chitosan is insoluble in water, but can be dissolved in most of the organic acid solutions, at pH less than 6.5 such as lactic acid, acetic acid, tartaric acid etc. [38]. Poloxamer is nonionic triblock copolymer composed of central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). It helps to reduce the aggregation of nanoparticles and thereby stabilizing the nanoparticles. The present study is aimed to increase the bioavailability of Epigallocatechin gallate, by developing nanoparticles using Chitosan by ionic gelation method [39] and Poloxamer is used to reduce the aggregation of nanoparticles and thereby stabilizing the same. MATERIALS AND METHODS Materials: Epigallocatechin gallate (EGCG) was purchased from Novanat, China. Chitosan (CS) was obtained from Novamatrix, Norway. Dimethyl sulfoxide (DMSO), Acetic acid, Sodium Tripolyphosphate (TPP), Diethyl ether, Trifluoroacetic acid and Acetonitrile were procured from Sigma-Aldrich, USA. Poloxamer 188 was procured from BASF, Germany. Deionized water was obtained from Millipore filtration system, USA. Method of preparation of drug loaded nanoparticles: Nanoparticles of EGCG is prepared using CS as a coating material by ionic gelation method, where the positively charged amino group in the CS interacts with negatively charged TPP to form coacervates with a size in the range of nanometer.EGCG and CS were weighed in different ratios (1:0.1, 1:0.2, 1:0.4 and 1:0.5). CS was dissolved separately in aqueous solution of Acetic acid (2% v/v) at pH 5.5. Poloxamer 188 (Poloxamer 188/CS ratio: 5/1) was dissolved in the above solution. EGCG was dissolved separately in Purified water (7.5% w/v of drug) and was added slowly to the above aqueous solution containing CS, while stirring under the magnetic stirrer. Aqueous solution of sodium tripolyphosphate (TPP) (CS/TPP ratio: 1/0.1) was added in drops to the above solution; under magnetic stirring for 3 hrs at a speed of 1000 RPM (Remi Magnetic Stirrer MLH, Chennai) resulted in cross linking of CS and TPP, as depicted in Fig 1 and produced the nanoparticles. After 3 hrs, nanoparticles formed were recovered by centrifugation (REMI cooling centrifuge, Chennai) at 3000 rpm for 30 min. The recovered nanoparticles were washed using diethyl ether and lyophilized.
  • 3. www.iajpr.com Page389 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 1: Schematic of nanoparticle formation process by ionic gelation process. Chitosan forms a cross linking with Sodium Tripolyphosphate. Determination of nanoparticle yield, drug content and entrapment efficiency: The yield of nanoparticle (%) was calculated using the equation (1) below: Drug Content and Entrapment Efficiency were determined using equation (2) and (3) respectively. Nanoparticles equivalent to 250mg of drug were dissolved in 50mL mixture of Trifluoroacetic acid, Acetonitrile and Milli-Q Water in a ratio of 0.1:65.0:34.9. The solution was further diluted with the same mixture of solvents. Drug quantification was performed using HPLC (Waters HPLC system equipped with a quaternary pump, SIL-10AD auto injector, CTO-10A column oven and UV detector units) with a Sunfire™ column (C8 250mm x 4.6 mm, 5 μm). The chromatographic conditions were injection volume = 50 µL, flow rate = 2.0 mL min-1 mobile phase = 0.1/65.0/34.9 of Trifluoroacetic acid/Acetonitrile/Milli-Q Water (pH 2.40). UV detection was performed at 210 nm. All samples were measured in triplicate. The method was validated in terms of linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). The results were expressed as mean ± standard deviation.
  • 4. www.iajpr.com Page390 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Particle characterization: Particle size, polydispersity index and zeta potential of EGCG nanoparticles were measured using Malvern Zetasizer (Malvern Instruments, UK), by dynamic light scattering and Electrophoretic light scattering principle. The particle size analysis was performed at a scattering angle of 90°C, at room temperature. The diameter was averaged from three parallel measurements and expressed as mean ± standard deviation. SEM was used to examine the particle surface morphology and shape. The nanoparticles were coated in Sputter coater, with a thin layer of gold, in an argon gas environment (Cressington 108 Auto sputter coater, UK). Photographs were taken using FEI Quanta FEG 200, (FEI, USA), at room temperature. Differential scanning calorimetry (DSC) interpretation: Thermograms of the EGCG and its nanoparticles were recorded on MettlerSTAR SW 9.01 instrument (Mettler Toledo, Switzerland). The analysis was carried out in nitrogen atmosphere, by heating 2 to 3 mg of sample on an aluminum crimp pan, at heating rate of 10°C/min. The runs were made in triplicate. Fourier Transform Infrared Radiation Measurements (FT-IR): FT-IR analysis was carried out for pure drug and nanoparticles obtained, by mixing with Potassium Bromide (KBr), by pellet method, after a baseline correction was made with dried potassium bromide, to confirm the compatibility. The pellet was prepared to a pressure of about 5 x 106 Pa, in an evacuated die, to produce a clear transparent disc of diameter 2 cm and thickness of 0.2 cm. The spectra were recorded at room temperature on Fourier transform spectrometer (Perkin-Elmer, USA) from 4000 cm-1 to 400 cm-1 . Powder X-Ray diffraction (PXRD) study: Crystallinity study was carried out by comparing XRD spectrum of drug with nanoparticles using Siemens Diffractometer D5000 (Siemens, Germany), to check peak of drug in individual state and in nanoparticles. The data was recorded at 2θ range of 10°C to 60°C at time of 0.5 sec. The relative intensity I/I0 and inter-planar distance (d) corresponding to 2θ value were reported and compared. In vitro release study: The in vitro release study of EGCG loaded CS nanoparticles having highest entrapment efficiency was carried out using USP dissolution apparatus type II (paddle method). Nanoparticles equivalent to 250 mg of EGCG were loaded using 900 ml of citric acid buffer (pH 4, where the sink conditions could be easily maintained), at a rotating speed of 50 rpm and 37 C ± 0.5 C. Samples were collected at specific time intervals. 2 ml of aliquot was collected during each sampling point and it was replaced with an equal volume of fresh buffer. The amount of drug release was determined by HPLC method, as described under the determination of drug loading in the nanoparticles. The in vitro release data was analyzed using various kinetic models which include, the zero-order kinetic model [40], the first-order kinetic model [41], the Higuchi model [42], the Hixson-Crowell model [43], the Korsmeyer-Peppas model [44], the Makoid Banakar model, [45] the Peppas Sahlin model, [46] the Weibull model, [47] the Baker Lonsdale model [48] the Hopfenberg model, [49] the Probit model [50,51] the Logistic model [52] the Quadratic model [53,54] the Gompertz model [55]. The model which gives the highest correlation coefficient (R2 ) is considered as the best fit of release data. RESULTS AND DISCUSSION Determination of nanoparticle yield, drug content and entrapment efficiency: The nanoparticle yield was high with increase in drug to polymer ratio. Similarly, the drug content and the entrapment efficiency increase with the increase in drug to polymer ratio (Table 1). As depicted in Fig 2, it is observed that increasing the amount of polymer results in better drug entrapment. From the entrapment efficiency, 1:0.5 ratio showed better yield, compared to the other three ratios. The nanoparticle yield increases with increase in drug entrapment. Similarly, the low drug to polymer ratio of 1:0.1 reflects with low entrapment efficiency. Table 1: Nanoparticle yield, drug content and entrapment efficiency of different batches of nanoparticles prepared. Formulation trial code Drug to polymer ratio Nanoparticle yield^ (% ± SD) Drug Content^ (% ± SD) Entrapment Efficiency^ (% ± SD) FT-001 to FT-003 1:0.1 45.95 ± 9.14 27.70 ± 8.75 14.00 ± 4.58 FT-004 to FT-006 1:0.2 60.66 ± 7.00 53.65 ± 8.47 39.33 ± 3.79 FT-007 to FT-009 1:0.4 76.39 ± 4.34 58.25 ± 3.38 64.00 ± 3.61 FT-010 to FT-012 1:0.5 95.05 ± 3.25 62.25 ± 1.67 91.67 ± 2.08 Note: Values are mean of three consecutive trials with its Standard Deviation (SD)
  • 5. www.iajpr.com Page391 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 2: Comparative results of nanoparticle yield, drug content and entrapment efficiency. Trials taken with various ratios of Epigallocatechin gallate and Chitosan. Particle characterization: The results indicate that the particle size increases with increase in concentration of CS. It was observed that the particle size of the EGCG loaded nanoparticles were between 197.84 ± 21.45 nm to 385.45 ± 15.87 nm. Monodisperse samples have lower Polydispersity Index value, whereas higher values of Polydispersity Index indicate a wider particle size distribution, due to aggregation. The usual range of Polydispersity Index values is 0 to 0.05 for monodisperse standard, 0.05 to 0.08 for nearly monodisperse, 0.08 to 0.7 for mid range polydispersity and >0.7 for highly polydisperse. The nanoparticles formulated have Polydispersity Index of 0.219 ± 0.014 to 0.502 ± 0.065, as shown in Table 2 indicate mid range polydispersity. The addition of Poloxamer 188 aids in reducing the aggregation of nanoparticles, which in turn was confirmed by the low Polydispersity Index of nanoparticles. Zeta potential of prepared nanoparticles was found to be in the range of +21.4 ± 2.8 mV to +45.5 ± 1.2 mV. It was found that higher the zeta potential, lesser will be the particle aggregation, due to electric repulsion. The stability will be high, if the Zeta Potential is high. Table 2: Average Particle Size, Polydispersity index and Zeta Potential of nanoparticles prepared. Drug to polymer ratio Average Particle Size (nm) Polydispersity index (Đ) Zeta Potential (mV) 1:0.1 197.84 ± 21.45 0.502 ± 0.065 +21.4 ± 2.8 1:0.2 323.51 ± 18.58 0.413 ± 0.054 +25.6 ± 1.9 1:0.4 369.87 ± 22.15 0.310 ± 0.024 +35.9 ± 1.7 1:0.5 385.45 ± 15.87 0.219 ± 0.014 +45.5 ± 1.2 The microscopic imaging of SEM shown in the Fig 3, infers the surface morphology of EGCG nanoparticles: almost spherical surface, smooth and discrete nanoparticles.
  • 6. www.iajpr.com Page392 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 3: Scanning Electron Microscopic imaging. Epigallocatechin gallate nanoparticles observed under the scanning electron microscope. Determination of purity by Differential scanning colorimetry (DSC) DSC thermograms of pure EGCG (Fig 4) showed a small endothermic peak at 227.47°C, which closely corresponds to its melting point. Fig 4: DSC thermogram. The endothermic and exothermic peak of Epigallocatechin gallate obtained using DSC. DSC thermogram of CS (Fig 5) show a broad endothermic peak centered at about 93.45°C. This peak is attributed to the loss of water associated with the hydrophilic groups of the polymer, due to evaporation. This peak was observed even after drying the sample and keeping in the desiccator, before the analysis.
  • 7. www.iajpr.com Page393 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 5: DSC thermogram. The endothermic and exothermic peak of Chitosan obtained using DSC. The exothermic peak, which appears in the temperature about 290°C, corresponds to the decomposition of the polymer. The thermal degradation is due to the saccharide rings’ dehydration, depolymerization, decomposition of deacetylated and acetylated chitosan units. After heating to 150°C and holding for a minute, followed by cooling to 40°C, the moisture was removed and a second heating run was done, where it was found that the endothermic peak was not observed, which confirmed that the peak at around 93.45°C was due to water content in the sample. An exothermic peak was also observed due to degradation of chitosan as mentioned earlier. The EGCG nanoparticles showed an endothermic peak at 227.45°C. The slight shift in the peak of nanoparticles was due to the incorporation of CS polymer. The results shown in Fig 6 clearly indicate the compatibility between drug and polymer. There were no interactions observed in the obtained nanoparticles. Fig 6: DSC thermogram. The endothermic and exothermic peak of Epigallocatechin gallate nanoparticles obtained using DSC. Determination of purity by Fourier transforms infrared spectroscopy: The FT-IR spectrum of EGCG (Fig 7) revealed the characteristic peaks of 3482.81cm-1 and 3357.46cm-1 for O-H group attached to the aromatic ring, 1691.27cm-1 and 1616.06cm-1 strong for C=O group that links the trihydroxybenzoate group and chroman group, 1447.31cm-1 for C-H group present in the Chroman ring, 1348.00cm-1 , 1222.65cm-1 for O-C=O group, 1148.40cm-1 and 1097.30cm-1 for O-H group, 1041.37cm-1 for C-O-C group which links the Chromane ring and trihydroxy benzoate ring, 853.35cm-1 , 825.38cm-1 for C-H group in the aromatic ring, 788.74cm-1 for the three neighboring aromatic C-H group in Chroman ring.
  • 8. www.iajpr.com Page394 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 7: FTIR spectrum. Finger print region between 4000cm-1 to 400cm-1 of Epigallocatechin gallate. The FT-IR spectrum of CS (Fig 8) revealed the characteristic peaks of 3407.13 cm-1 for amine N-H symmetric vibration, 2915.54 cm-1 and 2887.54 cm-1 for C-H stretching, 1645.65 cm-1 for C-C stretching, 1558.25 cm-1 for amino group vibration, 1158.19 cm-1 and 905.53 cm-1 corresponded to the saccharide structure of chitosan. The broad peak at 1050.91 cm-1 indicated C-O stretching vibration. Fig 8: FTIR spectrum. Finger print region between 4000cm-1 to 400cm-1 of Chitosan. The FT-IR spectrum of the final nanoparticles (Fig 9) revealed the presence of characteristic peaks of EGCG with a negligible shift, which included 3487.54cm-1 and 3365.57cm-1 for O-H group attached to the aromatic ring, 1683.71cm-1 and 1606.65cm-1 strong for C=O group that linked the trihydroxybenzoate group and chroman group, 1451.24cm-1 for C-H group present in the Chroman ring, 1332.46cm-1 , 1218.57cm-1 for O-C=O group, 1054.54cm-1 for C-O-C group which linked the Chromane ring and trihydroxy benzoate ring, 794.04cm-1 for the three neighboring aromatic C-H group in Chroman ring.
  • 9. www.iajpr.com Page395 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 9: FTIR spectrum. Finger print region between 4000cm-1 to 400cm-1 of Epigallocatechin gallate nanoparticles. Hence from the above spectrum, it is clear that there were no incompatibilities present with the any of the added excipients. Determination of crystallinity by X-ray diffraction study: PXRD was used to determine the crystallinity of compounds. Polymorphic changes of drug was an important factor, which might affect the dissolution rate and in turn bioavailability. Crystallinity of the drug and the nanoparticles was determined. The XRD pattern (Fig 10) of pure drug showed a broad diffraction peaks at 2θ, as the drug was amorphous. Similarly nanoparticles also did not show any sharp peak at 2θ. (A) (B) Fig 10: XRD Study (A) Epigallocatechin gallate showing no sharp peak (B) Epigallocatechin gallate nanoparticles showing no sharp peak. In-vitro release studies and release profile: EGCG nanoparticles prepared with CS in a ratio of 1:0.5 (FT010, FT011 and FT012) were taken for dissolution, since they had the highest entrapment efficiency, when compared to other ratios. As shown in Fig 11, nanoparticles showed an initial burst release of around 20% EGCG in the first hour. This initial rapid release, characterized as ―burst effect‖ was due to the fact that some amounts of EGCG were localized, on the surface of nanoparticles, by adsorption, which could be released easily by diffusion. After this initial burst effect, a slower sustained release occurred throughout the dissolution period.
  • 10. www.iajpr.com Page396 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 Fig 11: Dissolution profile of Epigallocatechin gallate Nanoparticles. Dissolution profile of nanoparticles prepared using 1:0.5 ratio of Epigallocatechin gallate and Chitosan in citric acid buffer solution (pH 4). Table 3 demonstrates correlation values (R2) and release parameters determined from the results of model fitting of the release profiles. According to the correlation values, they fitted to Makoid Banakar, Peppas-Sahlin, Korsmeyer-Peppas and Higuchi models, which indicated that EGCG was released by diffusion. Moreover, the Korsmeyer-Peppas release model (high correlation values) exponent, , which was about 0.4932, showed the characteristics of anomalous kinetics (non-Fickian), suggesting that more than one mechanism, might be involved in release kinetics, referring to the combination of diffusion and erosion based drug release mechanism. This mechanism could result from an increased plasticization at the relaxing boundary. The release data of the Hixson- Crowell model suggested that there was no change in the surface area as a function of time. Table 3: Mathematical Models and Correlation values (R2 ) based on release data. S No Mathematical Model Correlation Value (R2 ) 1 Makoid Banakar 0.9981 2 Peppas-Sahlin 0.9979 3 Korsmeyer Peppas 0.9971 (n=0.4932) 4 Higuchi 0.9970 5 Weibull 0.9921 6 Baker Lonsdale 0.9605 7 First Order 0.9479 8 Hopfenberg 0.9479 9 Probit 0.9379 10 Logistic 0.9359 11 Quadratic 0.9044 12 Hixson Crowell 0.9054 13 Gompertz 0.8925 14 Zero Order 0.5683 CONCLUSION EGCG loaded CS nanoparticles were successfully prepared by ionic gelation method, in four different ratios 1:0.1, 1:0.2, 1:0.4 & 1:0.5. According to the efficiency of yield and entrapment, 1:0.5 ratio showed better yield, compared to the other three ratios. The entrapment efficiency was found in the range of 14.00 ± 4.58% to 91.67 ± 2.08%. Average size of prepared nanoparticles varied from 197.84 ± 21.45 nm to 385.45 ± 15.87 nm, with a polydispersity index in the range of 0.502 ± 0.065 to 0.219 ± 0.014. As the amount of polymer increased, the size of the nanoparticles also increased. It was found that the inclusion of poloxamer 188 increased the zeta potential, due to which the particle aggregation was less and hence, the stability of nanoparticles was high. DSC and FTIR completely suggested the drug to polymer compatibility. In-vitro release study showed sustained release of drug from 1:0.5 ratio of polymer, upto 24 hours following diffusion and swelling mechanism. From the present study, it is concluded that EGCG loaded CS nanoparticles is an effective carrier for the design of controlled drug delivery of EGCG. The increase in absorption and bioavailability of EGCG nanoparticles has to be further researched.
  • 11. www.iajpr.com Page397 Vol 5, Issue 01, 2015. Ramkumar Ponnuraj et al. ISSN NO: 2231-6876 ACKNOWLEDGMENTS This work was carried out in apex laboratories, as a part of a project investigating the production of nanoparticle systems using antilipidemic drugs, for effective treatment. The instruments and infrastructure used to conduct the above experiment was facilitated by apex laboratories. Abbreviations 1. % - Percentage 2. °C - Degree Centigrade 3. cm - Centimeter 4. HIV- Human Immuno Virus 5. HPLC - High Performance Liquid Chromatography 6. mg - milligram 7. min - minute 8. ml - milliliter 9. mm - millimeter 10. mV - millivolts 11. nm - nanometer 12. Pa - Pascal 13. rpm - Revolutions per minute 14. SD - Standard Deviation 15. sec - second 16. UK - United Kingdom 17. USA - United States of America 18. UV - Ultra Violet 19. v/v - volume by volume 20. w/v - weight by volume REFFERENCES [1]. Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, Lambert G, Mohr S, Yang CS. Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomark Prev. 2002; 11:1025–1032. [2]. Auger C, Mullen W, Hara Y, Crozier A. Bioavailability of polyphenon E flavan-3-ols in humans with an ileostomy. J Nutr.2008; 138:1535S–1542S. [3]. Stalmach A, Troufflard S, Serafini M, Crozier A. Absorption, metabolism and excretion of Choladi green tea flavan-3-ols by humans. Mol Nutr Food Res.2009; 53:S44–S53. [4]. Roowi S, Stalmach A, Mullen W, Lean ME, Edwards CA, Crozier A. Green tea flavan-3-ols: Colonic degradation and urinary excretion of catabolites by humans. J Agric Food Chem. 2010; 58:1296–1304. [5]. Williamson MP, McCormick TG, Nance CL, Shearer WT (December 2006). "Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy". The Journal of Allergy and Clinical Immunology 118 (6): 1369–74. [6]. Hamza A, Zhan CG (February 2006). "How can (-)-epigallocatechin gallate from green tea prevent HIV-1 infection? Mechanistic insights from computational modeling and the implication for rational design of anti-HIV-1 entry inhibitors". The Journal of Physical Chemistry. B 110 (6): 2910–7. [7]. Yamaguchi K, Honda M, Ikigai H, Hara Y, Shimamura T (January 2002). "Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1)". Antiviral Research 53 (1): 19–34. [8]. Nance CL, Shearer WT (November 2003). "Is green tea good for HIV-1 infection?". The Journal of Allergy and Clinical Immunology 112 (5): 851–3. [9]. Chung MY, et al. Molecular mechanisms of chemopreventive phytochemicals against gastroenterological cancer development World J Gastroenterol. 2013 Feb 21;19(7):984-93. [10]. Connors SK, et al New insights into the mechanisms of green tea catechins in the chemoprevention of prostate cancer Nutr Cancer. 2012;64 [11]. Landis-Piwowar K, et al Novel epigallocatechin gallate analogs as potential anticancer agents: a patent review (2009 - present) Expert Opin Ther Pat. 2013 Feb;23(2):189-202. [12]. Chen D, et al. EGCG, green tea polyphenols and their synthetic analogs and prodrugs for human cancer prevention and treatment Adv Clin Chem. 2011;53:155-77. [13]. Sachdeva, A K; Kuhad, A; Chopra, K (2011). "Epigallocatechin gallate ameliorates behavioral and biochemical deficits in rat model of load-induced chronic fatigue syndrome". Brain Research Bulletin 86 (3–4): 165–72. [14]. Sachdeva, A K; Kuhad, A; Tiwari, V; Arora, V; Chopra, K (2010). "Protective effect of epigallocatechin gallate in murine water-immersion stress model of chronic fatigue syndrome". Basic & Clinical Pharmacology & Toxicology106 (6): 490–96. [15]. Sachdeva, A K; Kuhad, A; Tiwari, V; Chopra, K (2009). "Epigallocatechin gallate ameliorates chronic fatigue syndrome in mice: behavioral and biochemical evidence".Behavioural Brain Research 205 (2): 414–20.
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