INTRODUCTION:
During the last century chemical and pharmacological studies have been
performed on a lot of plant extracts in order to know their chemical
composition and confirm the indications of traditional medicine. Preparations
of Phyto medicine has been used for health maintenance since ancient times. The
Phytomedicines posses a lot of therapeutic uses. It is observed that most of
the biologically active phytoconstituents such as the flavonoids and terpenoids
are of highly polar nature or water soluble molecules. These highly water
soluble constituents are poorly absorbed due to their poor lipid solubility,
thus creating a hurdle to cross the highly lipid-rich biological membrane,
which finally results in poor bioavailability. Many approaches have been
developed for improving the bioavailability such as inclusion of solubility and
bioavailability enhancers, structural modification and entrapment with
lipophilic carriers.[1]
“Phytosomes” are
novel phytophospholipid carriers for herbal drug delivery. The term “Phyto “means
“Plant’’, while “some” means “cell”
like. Phytosomes are advanced microsphere or cell form of herbal product
that are better absorbed, utilized to produce better result than those produced
by conventional herbal extract. As standardized extracts which are shows, poor
bioavailability and limited their clinical utility. Prepared complexation with
certain other clinically useful nutrients substantially improved the
bioavailability of extracts. The
nutrients so helpful for enhancing the absorption of other nutrients are the
phospholipids. [2] Phytosome technology emerged in 1989. [3] Based
on a histochemical observation that certain polyphenols had strong bonding
affinity for phospholipids in their intact plant tissue. A group of Italian
researchers focused on polyphenol preparations known to be poorly bioavailable
when taken orally. These were typically mixtures of polyphenols extracted from
single plant species, and their conversion into phytosome forms markedly
increased their bioavailability.[4]
Most of
biologically active constituent of plant are polar or water soluble molecule
such as flavonoids, tannins, terpenoid etc. are poorly absorbed either due to
their large molecular size, multiple ring molecules that are too large to
absorb by simple diffusion or due to their poor lipid solubility, severely
limiting their ability to pass across the lipid rich biological membrane of
erythrocytes of small intestine.[5] Resulting poor bioavailability.[6]
The effectiveness of herbal product is depending on delivery and
effective level of the active compound.[5] The water soluble
phytoconstituent molecule can be converted in to lipid compatible molecular
complexes which are called Phytosomes. Phytosomes produce little cell because
of that valuable component of herbal extract are protected from destruction by
digestive secretion and gut bacteria. It has been also observed that the
separation and purification of various component of an extract may lead to a
partial loss of specific activity for purified compound. Phytosomes are
prepared by complexing the polyphenolic phytoconstituent in the ratio of 1:2 or
1:1 with phosphotidylcholine.[5] Phytosomes are obtained by reacting
soya phospholipids with selected botanical derivatives in an aprotic solvent on
the basis of their physical, chemical and spectroscopic characteristics. Their
sizes vary between 50 nm to a few hundred µm. These complexes can
be considered as novel entities. [7] Phytosomes are not liposome’s
structural. [8] Phytosomes is unit of few molecules bonded together
while liposome’s as an aggregate of many phospholipids molecules but without
specially bonding to them. [9] Chemical analysis indicates that in
phytosome usually a flavonoid or terpenoid that is polyphenolic molecule linked
with at least one phosphotidylcholine molecule. Bond is formed between these
two molecules creating a hybrid molecule. Phytosomes are better able to
transition from a hydrophilic environment into the lipid-friendly environment
of the enterocytes cell membrane. These highly lipid miscible hybrid bond is
better suited to merge in the lipid phase of the enterocytes outer cell
membrane and finally reaching to blood. [10]
As standardized extracts has, poor bioavailability often limited to
their clinical utility. By complexation with certain other clinically useful
nutrients the bioavailability of such extracts substantially improved. The
nutrients so helpful for enhancing the absorption of other nutrients are the
phospholipids. The enterocytes outer membrane has a lipid molecular bilayer
that consists largely of Phosphotidylcholine.[3] Phospholipids is a principle molecular
building block of cell membrane, making up the matrix into which fit a large
variety of proteins that are enzymes, transport proteins, receptors, and other
biological energy converters. In humans and other higher animals the
phospholipids are also employed as natural digestive aids and as carriers for
both fat-miscible and water miscible nutrients. They are miscible both in water
and in oil/ lipid environments, and are well absorbed orally.
Phosphotidylcholine are complex molecules that are used in all known life forms
and responsible for formation of cell membrane. [11] They derived from soybean which is miscible both
in the water phase and in oil, lipid phases and water it is excellently
absorbed when taken orally.[12] Phospholipids are small lipid
molecule in which the glycerol is bonded only to two fatty acid instead of
three as in triglycerides, and the remaining sites occupied by a phosphate
group. The phytosome process has been applied to many popular herbal extract
including Ginkgo biloba, Grape seed, Milk thistle, Green tea, Quercetin, Curcumin, Herba Epidemic, Gallic acid and
Ginseng. The tannins flavonoid and terpenoid components of these herbal
extracts lend themselves quite well for the direct binding to
phosphotidylcholine. [9]
The aim of the present work is to develop phytosomes from the peels of
the pomegranate so that formulation having better bioavailability can be
developed.
Fig.1
Fruits and Peels of Pomegranate
MATERIAL AND METHODS:
Materials:
Phosphotidylcholine obtained as a gift sample by
Lipoid; Ludwigshafen, Germany.
Collection of
Plant:
The Fruit
were collected in the month of November to February from the surrounding areas
of Aurangabad District, Maharashtra, India. The plant material was identified
and authenticated by depositing the herbarium sheet of the plant specimens in
Botany Department, Dr. Babasaheb Ambedkar Marathawada University, Aurangabad, Maharashtra, India.
Under the voucher No: Botany/2012-13/81.Accession no.0562.
Preparation of the Plant Extract [13]:
Firstly fresh
pomegranates (500 gm) were obtained (in order to prepare fresh extraction) and
washed with distilled water then peeled and their edible portions were carefully
separated. The peels were air dried in a ventilated oven at 40°C for 48 hours.
Dried peels are coarsely powdered in an electric grinder and ground to a fine
powder and passed through a 24 mesh sieve and stored in plastic bags for the
next step. A 100 gm sample of powder was subjected for extraction in Soxhlet
extractor using petroleum ether for the removal of fatty content. The mark used
for extraction of phenolic content using 200 ml methanol (99.9%) in soxhlet
apparatus for 4-6 hrs i.e. 13-14 cycles. Then extract were dissolved in 70%
Methanol and Ethyl acetate in separating fennel shake for 30 min. separate the
organic layer, excess of ethyl acetate was removed with help of rotary
evaporator to produce a dry powder and
then subjected to preliminary chemical tests which revealed the presence of
polyphenol component in the plant extracts.
High Performance Liquid
Chromatography (HPLC) of extract:
Polyphenolic content of methanolic extract of
pomegranate peels was determined by using HPLC technique and compared with
standard.
Preparation of Phyto-Phospholipid complex by solvent
evaporation method [14]:
Different molar
concentration of Methanolic extract and Phosphatidylcholine (1:0.5, 1:1, 1:2)
was taken in the beaker. 20 ml methanol was added and placed beaker on magnetic
stirrer for 2 hours, then dried in vacuum oven at a temperature below 600C
till solvent was evaporated. Prepared phospholipids complex washed with 10 ml
N- Hexane with continuous stirring. The Flavonoid–phospholipid complex was
precipitated and the precipitate was filtered and dried under vacuum to remove
traces of solvents. The resultant phospholipids complex placed in an amber
coloured bottle, flushed with nitrogen and stored at room temperature.
Evaluation of Phyto
Phospholipid Complex:
IR Spectra [15]:
IR spectra of Extract-phospholipid complex were
obtained. The IR Spectrum was recorded using % Transmission mode with 4.0 cm-1
resolution and auto gain with HAPP-GENZEL apodization. IR was recorded using
detector 1(2.8mm/sec).
Differential scanning
Calorimetry (DSC) [16]:
The samples were
sealed in the aluminum crimp cell and heated at the speed of 100C/min
from 0 to 300◦C in nitrogen atmosphere (60 ml/min). The peak transition
onset temperature of Extract–phospholipid complex was noted.
Scanning Electron Microscope
(SEM) [17]:
Surface
morphology of complex was investigated by scanning electron microscope model
JSM5600 (JEOL).
Dissolution Study [18]:
In-vitro drug release of the
Phyto-phospholipid complex was conducted using dissolution test apparatus
USPXXIII at 50 rpm. The dissolution medium consisted of 900 ml of 6.8 phosphate
buffers, temperature maintained at 37±0.50c. Aliqouts
of 5 ml were withdrawn at predetermined time intervals and an equivalent amount
of fresh dissolution fluid equilibrated at the same temperature was replaced.
Aliquots withdrawn were filtered through 0. 45 µm Whatmann filter paper and diluted appropriately with vanillin sulphuric
acid. With absorbance measured on a UV- spectrophotometer at 228 nm.
Ex-vivo absorption study
using everted small intestine sac method [18]:
Intestine of
goat (2–2.5 inch piece) was taken, washed, and freed of intestinal contents and
everted using a glass rod. One end of the intestine was fastened using thread
while a thread-tied cannula was fitted at another end, and kept in PBS solution
(pH 7.4). Two flasks were taken, one of which was filled with 50 ml phosphate
buffer saline containing methanolic extract (A) and another one with the 50 ml
of phosphate buffer saline containing Methanolic extract and phospholipid
complex (B); 2.0 ml of mammalian ringer’s solution was injected in each of the
two intestine pieces and immersed in the separate flasks containing Methanolic
extract (A) and Methanolic extract and phospholipid complex (B) solutions.
After the specified time interval the serosal fluid of each intestine fragment
was assayed for drug content using a UV spectrophotometer by taking absorbance
at 204 nm.
RESULT AND DISCUSSION:
Chemical test for Methanolic Extract:
Phytochemical
investigation of methanolic extract showed the presence of tannins, flavonoids
and other Phenolic compounds.
High Performance Liquid Chromatography:
HPLC was carried out for the
qualitative estimation of ellagic acid present in the methanolic extract of
pomegranate peels.
Fig 2. HPLC
chromatogram of Pomegranate peel extract
Preparation of Phytosomal complex:
Methanolic
extract-phospholipids complex was prepared to improve the lipophilic properties
of Methanolic extract. The complex with different ratios of phospholipids and
extract such as 0.5, 1, 2 was prepared. The results showed that when the ratio
was lower than 1, the stability of the Phyto–phospholipids complex was worse.
Stable Phytosomal complex was obtained with Phyto-phospholipids complex with a
1:1 and 1:2 ratios of ingredients. Practical yield was high with ratio 1:2. The
obtained complex was used for the subsequent structural analysis.
Table 1.
Percent practical yield of extract phospholipid complex
|
S.N.
|
Ingredients
|
Quantity(mg)
(concentration)
|
% yield
|
|
1
|
Methanolic extract
Phosphotidylcholine
|
834.56
380.45
|
80.65
|
|
2
|
Methanolic extract
Phosphotidylcholine
|
834.56
760.90
|
84.26
|
|
3
|
Methanolic extract
Phosphotidylcholine
|
834.56
1521.8
|
87.26
|
Differential Scanning Calorimetry (DSC) of
the complex:
The DSC
thermograms of phospholipids, Methanolic extract,
their physical mixture and phospholipids complex were shown in Fig. 4. Phospholipids show two different
kinds of endothermal peaks, and the first (74.85°C) end thermal peak appears
mild, it was considered that the formation of this peak was due to hot movements
of phospholipids molecule polarity parts. However, the second endothermal peak
at 190.6◦C appears sharp-pointed; it was considered that owing to the
transition from gel state to liquid crystal state, the carbon–hydrogen chain in
phospholipids perhaps happened to be melt, isomerous or the crystal changes methanolic extract is not pure, so it shows abroad
endothermal peak, and its beginning melting point at 136.5◦C.
Physical mixture of and phospholipids
shows that there are two endothermal peaks, and the former is 28.8◦C, the
same with the onset temperature of phospholipids complex; another is 136.5◦C,
the same with the onset temperature of
Methanolic extract. It was
considered that when the temperature was increased, phospholipids were melt and
drugs were dissolved in the phospholipids and partly formed phospholipids
complex, which could be explained through the theory of preparation by melt-out
method. DSC of phospholipids complex shows the endothermal peaks of drug and
phospholipid are disappeared and the phase transition temperature is lower than
the phase transition temperature of phospholipids.
After the
combination of methanolic extract and the
phospholipids molecule polarity parts, the carbon–hydrogen chain in
phospholipids could turn freely and enwrap the phospholipids molecule polarity
parts, which made the sequence decrease between phospholipids aliphatic
hydrocarbon chains, made the second endothermal peak of phospholipids disappear
and depressed the phase transition temperature.
a) Methanolic extract
b)
Phosphatidylcholine
c) Physical mixture of Extract– phospholipids
d) Extract-phospholipid complex
Fig. 4 DSC Thermograms of a) Methanolic
extract b) Phosphatidylcholine c) Physical mixture of
Extract– phospholipids d)
Extract-phospholipid complex
Scanning Electron Microscopy (SEM)
The
surface morphology of phospholipids complex as examined by SEM is Phospholipids
complex were made up of phospholipids and drugs and appeared spherical shape when at 200 x magnification, we could see that
phospholipids did not exit on the appearance
of drugs but drugs uniformly dispersed in phospholipids and formed the
structure of spherical shape.
Fig. 5 Scanning electron micrographs of phospholipids complex
Spectrophotometric Characterization
Construction of standard graph of Methanolic extract
The absorbance was measured by
spectrophotometer at 204 nm using 6.8 pH Buffer
Table 6. Absorbance of drug at 204 nm
|
Sr. No.
|
Concentration ( µg/ml)
|
Absorbance
|
|
1
|
0
|
0
|
|
2
|
10
|
0.194
|
|
3
|
20
|
0.428
|
|
4
|
30
|
0.621
|
|
5
|
40
|
0.812
|
|
6
|
50
|
0.984
|
Fig. 6 Standard Calibration Curve of phosphate buffer pH 6.8.
Ex-Vivo
Absorption Studies
Table 7. Absorbance of
extract Phospholipids complex and plain extract
|
Sr. No.
|
ME-Complex (cumulative abs µm)
|
Extract (cumulative abs µm)
|
|
1
|
0
|
0
|
|
2
|
165
|
108
|
|
3
|
280
|
238
|
|
4
|
521
|
420
|
|
5
|
734
|
650
|
|
6
|
780
|
678
|
Fig. 7 Absorption of extract
Phospholipid complex and plain extract
The absorption of the Methanolic extract-Phospholipid
complex was found to be greater than plain Methanolic extract at different time
intervals. The curve between absorption and time for Methanolic extract
Phospholipid complex is shown in Fig. 7 which clearly indicates the enhanced
absorption of the Methanolic extract-Phospholipid complex.
CONCLUSION
In the present study pomegranate peel extracts phospholipid
complex were prepared by solvent evaporation
method and evaluated by
using various physiochemical parameters. The physiochemical parameters investigation showed that
pomegranate peels formed complex with phosphotidylcholine with
better bioavailability. The
IR, DSC, SEM studies confirmed theformation of complex. The dissolution
profile of the complex was found to be improved. Thus, it can be concluded that
phospholipid complex of pomegranate peels may be of potential use for improving
its bioavailability.
ACKNOWLEDGEMENT: Authors are thankful to the Principal of Dr. Vedprakash Patil Pharmacy College,
Georai Tanda, Aurangabad for providing necessary facilities
and support. Authors are also thankful to Lipoi; Ludwigshafen, Germany for supplying
phosphotidylcholine as gift sample.
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