Vol 3, Issue 3, 2021 (149-158)
http://journal.unpad.ac.id/idjp
*Corresponding author,
e-mail : amalia@unpad.ac.id (Eri Amalia)
https://doi.org/10.24198/idjp.v3i3.36140
©2021 E. Amalia et al
Preparation of organic-solvent free liposome of Piper albi Linn extract in solution and
powder form
Eri Amalia1, Iyan Sopyan1, Norisca Aliza Putriana 1, Sriwidodo1, Anas Subarnas2
1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,
Padjadjaran University, 45363, Indonesia.
2Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Padjadjaran
University, 45363, Indonesia.
e-mail : amalia@unpad.ac.id
Submitted :1/02/2022, Revised :3/02/2022, Accepted 10/02/2022, Published : 11/02/2022
Abstract
White pepper (Piper albi Linn) is widely used as a spicy enhancer commodity in
many countries. Moreover, it is also known to contain piperine as a potential bioactive
substance responsible for numerous beneficial pharmacological activities. Nevertheless,
utilization of these bioresources as a product remains less, due to its poorly water-soluble
characteristic in nature which affects its low bioavailability. Therefore, our research
focused on the preparation of piperine standardized white pepper extract and its
modification in the liposome delivery system in both solution and liposome powder form
to improve its bioavailability.
The liposome solution was prepared without organic solvent to dissolve the
mixture of lipid phase of soy lecithin and cholesterol, thus requiring suitable wet-
hydrating the lipid film and overnight self-hydration method for liposome formation.
Results showed that the liposome is successfully formed with an average size of 398.7
nm observed by microscopic and particle size analyzer evaluation, and giving
encapsulation efficiency of 98.92%±1.17%. Furthermore, the liposome solution was
dried by using the spray dry method by employing sucrose as the carrier. This method
successfully reveals that sucrose gives maximum protection to the stability of liposome
structure, where the rehydration of powder showed a similar spheric shape to the initial
liposome solution.
This research gives interesting finding that white pepper is feasible to be
processed as a beneficial standardized raw material in liposome delivery system in both
solution and powder form, without of employment of toxic organic solvent. Therefore,
can be further processed as a commercial solvent-free product in the future.
E.Amalia et al / Indo J Pharm 3 (2021) 149-158
151
Keywords: Piperine, Piper albi Linn, liposome, extract, organic solvent-free, drug
delivery system, spray dry
1. Introduction
White pepper (Piper albi Linn) is one
of the national commodities mostly as a
potential spicy taste enhancer. White
pepper contains many substituents such as
alkaloids, flavonoids, lignan, aromatic
compounds, amides, and essential oil.[1]–
[3]. Among them, alkaloid piperine is
believed as the most potential bioactive
compound with several important
pharmacological activities such as
antimicrobial, antioxidant,
immunomodulatory anti-inflammatory,
and anticancer activity [3]. White pepper
from kepulauan Bangka Belitung
Province- Indonesia is known to have
good quality with a higher piperine
substituent. The application of white
pepper extract and piperine as a product is
still limited due to its poor solubility in
water (0.004 mg/ml at 18oC) character,
thus affect to its low bioavailability in the
body and limiting its biological activity
[4]. Besides its solubility matter, it is also
less in the peppercorn as 3.84–5.35 % [1].
Therefore, modification of piperine is
required to improve its bioavailability.
One of the most promising techniques
applied is by preparation of a liposome
delivery system. A liposome is a unique
artificial vesicle composed of a
combination of phospholipid which has
hydrophilic and lipophilic regions to form
a bilayer membrane similar to a natural
cell membrane. The liposome structure is
special which can be engineered to
entrapped lipophilic or hydrophilic active
substance on its suitable region of the
liposome. [5][6]. Piperin known as a
lipophilic substance with a log P value of
2.25 expected to be entrapped in the inner
bilayer membrane, and with the
hydrophilic head of the structure may
improve its release profile when applied
as several pharmaceutical dosage forms
[4]. Nevertheless, most of the current
liposome preparation is employ organic
solvents such as chloroform, methanol, or
ethanol during the film hydration process,
glass rotary evaporation vessel, and
sonication or extruder to reduce the size of
liposome, thus not applicable in most
industrial facilities. Therefore, this
research tries to prepare liposomes of
extracted white pepper, in solution and
powder form, which is organic solvent-
free and in simple technique. This method
is promising to be upscaled on a larger
industrial scale thus possible to be
manufactured as a product that can give
benefit for human health.
2. MATERIALS AND METHODS
2.1 Materials
White pepper (Piper albi Linn)
was obtained from kepulauan Bangka
Belitung Province, Indonesia. Piperine
with >97% purity was purchased from
Sigma-Aldrich (Saint Louis, USA),
Cholesterol Food grade was purchased
from Dyets, Inc (Bethlehem), Soy lecithin
was purchased from Cargill Food
E.Amalia et al / Indo J Pharm 3 (2021) 149-158
151
(Tianjin, China). Tween 80 (Polysorbate
80) was purchased from PT Brataco (Indonesia), and sucrose was purchased
from Merck (Germany).
2.2 Methods
2.2.1 Preparation of Piper albi Linn
extract
Piper albi Linn extract was prepared by
grinding the peppercorn and passthrough
80 mesh. One hundred grams of powder
was extracted by using 250 ml of 95%
ethanol and blended for 15 in lower speed
and maceration process for 24 hours. Filter
the ethanol and evaporate ¾ of the ethanol
to remain ¼ portion. Add 1g/10 ml of KOH,
to the remaining filtrate, gently shake and
let it for 30 minutes. Filter the solution, and
add purified water the let precipitation of
the piperine. Allow the crystallization
process for 24 hours at room temperature
protected from light. Separate the
supernatant from piperine precipitate. Two
types of precipitation will be identified as
yellowish-brown mass and glisten yellow
crystal, and carefully separated. Weigh
each precipitation and performed assay by
using HPLC at 342 nm.
2.2.2 Preparation of white pepper
extract liposome
Accurately weighed 18 g of soy lecithin and
1.5 g of cholesterol (ratio 12:1) and mixed
at 60°C±5°C until homogenous liquid
formed. The glisten yellow crystal obtained
from the preparation of Piper albi Linn
extract was accurately weighed in 300 mg
and mixed to the lipid phase. The water
phase containing 2% of tween 80 was
prepared and mixed homogenously at 25֯C
±5°C. Half portion of the water phase was
poured into a clean glass container. The
lipid phase was poured to form a thin layer
above the water. Slowly add the remaining
water phase on top of the lipid layer to help
the hydration. Allow the overnight
hydration or until it is completely hydrated.
After sufficient hydration, stir the liposome
solution by using a magnetic stirrer at a low
speed to homogenize and reduce the
particle of the liposomes.
2.2.3 Preparation of liposome powder
Accurately weigh sucrose and dissolved in
150 ml of water to make lipid to sucrose
ratio 1:6. Transfer the sucrose solution into
300 ml of liposome solution, mixed
homogenously by using a magnetic stirrer
at low speed. Dry the 300 ml of the mixture
by using BÜCHI Mini Spray Dryer B-290
at inlet 123oC, outlet 61oC, aspirator 90%
and pump 25%. Keep the powder of
liposome at room temperature for further
analysis.
2.2.4 Analysis of liposome formation by a
microscope
The morphology of white pepper extract
liposome solution and rehydration after
drying was analyzed by using inverted
microscopy phase contrast (Olympus,
Japan). The liposome obtained by the above
method preparation was observed directly
without the addition of immersion oil at
400x magnification.
150
E.Amalia et al / Indo J Pharm 3 (2021) 149-158
152
Figure 1. White pepper from Kepulauan Bangka
Belitung Province Indonesia used in this experiment
(A) has been extracted and yielded in 2 types of
material as yellowish-brown mass (B, left picture)
and glisten yellow crystal (B, right picture). Piperine
content of these 2 types of material was analyzed by
HPLC compared to the chromatogram of piperine
with purity > 97.0% k (C)
2.2.5 Analysis of liposome powder by a
Scanning Electron Microscopy
(SEM)
The morphology of white pepper extract
liposome powder was examined by a
Scanning electron microscope (SEM) using
TM3000 Tabletop microscope HITACHI
(Japan).
The powder liposome was placed onto
carbon tape on top of the stub. Insert the
sample stub into the chamber and operate
the at a low vacuum 15.0 kV, with an
emission current 47800 nA, a filament
current 1750 mA magnification. Observed
at a magnific of 300x and 2500x by using
version 02-03 software.
2.2.6 Characterizations of particle size
and zeta potential of the liposome
solution
The white pepper extract liposome solution
was diluted into 3000 ppm of lipid
concentration by using purified water. The
size distribution and zeta potential of the
liposomal formulations were characterized
by dynamic light scattering (DLS) using a
Nano Particle analyzer (SZ-100 Nano
Partica, Horiba Scientific). The
measurement was determined through a
helium-neon (He-Ne) laser beam with a
scattering angle of 90° and temperature of
the holder 25°C. The particle size and zeta
potential were analyzed in triplicate and
data were analyzed by HORIBA SZ-100 for
Windows [Z Type] Ver2.40 software.
2.2.7 Encapsulation efficiency
Ten ml of the solution was let to stand for
48 hours at room temperature in a closed lid
tube. Transfer 1 ml of liposome solution
taken from the middle part of the solution
to 10 ml of volumetric flask. Add 1 ml of
water, shake vigorously, and add methanol
to volume. The piperine standard was
prepared in methanol at a concentration of
5 ppm. Samples and standards are analyzed
by using the Waters Alliance HPLC system
(5 μm particle size, 250 × 10 mm i.d.) and
20 µl volume injection. The mobile phase
consisted of acetonitrile-methanol-water
(65:5:30), at a flow rate of 1.0 ml/min refers
to a previous study [7]. The results were
processed by Empower® 3, software.
Chromatographic separations were done on
a Jupiter®, C-18 chromatogram was
recorded at 342 nm.
Encapsulation efficiency (EE%) was
calculated by below formula :
𝐸𝐸% = 𝑊𝑡
𝑊𝑖 x 100%
Where :
Wt = amount of piperine in liposome
Wi = amount of theoretical piperine in
white pepper extract liposome
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2.2.8 Assay of piperine in white pepper
extract liposome powder
Accurately weighed 100 mg of powder
liposome piperine and transfer to 10 ml of
volumetric flask. Add 3 ml of water, shake
vigorously, and add methanol to volume.
The standards curve preparation and further
analysis were conducted by HPLC refer to
as mentioned in the encapsulation
efficiency analysis.
3. RESULTS
3.1 Preparation of Piper albi Linn extract
Extraction of 100 g white pepper (figure
1A) by using 95% ethanol with 24 hours of
maceration process resulting in 1.98 g of
piperine or 1.98% (w/w). The purification
process gave the result of 2.85 g of
yellowish-brown mass containing 43.75%
of piperine (figure 1B, left) and 1.21 g of
glistening yellow crystal containing
60.97% of piperine (figure 1B, right) as
seen in the chromatogram (figure 1C). The
yellowish-brown mass from sticky resin-
like and has strong pepper characteristic
odor, glistening crystals still appear in the
mass due to difficulties of complete
separation. Meanwhile the glisten yellow
crystal has a crystalline, weak pepper
characteristic odor, and pungent taste. The
further optimization process of extraction is
possible to increase the yield of the
percentage of piperine.
3.2 Preparation of white pepper extract
liposome
Preparation of white pepper extract
liposome solution by wet-hydrating the
lipid film and overnight self-hydration
resulting in a yellow milky solution with
pH 5.8 (figure 2A).
Figure 2. Liposome piperin solution (A) and
liposome piperin powder (B)
3.3 Preparation of Liposomes Powder
The liposome solution is dried by spray-
dried technique resulting in yellow dried
white pepper extract liposome powder. The
powder has a sweet taste at the beginning
and a pungent taste after (figure 2B),
indicating the fruitful of the encapsulation
process. The dried powder has hygroscopic
characteristics and is stored at room
temperature for further analysis.
3.4 Analysis of liposome formation by a
microscope
The white pepper extract liposome was
successfully prepared in a spherical shape
and micrometer size. No needle-like
piperine crystals were observed in the
liposome solution, indicating the piperin
was successfully entrapped in the
hydrophobic region of the liposome (figure
3A). The liposome was observed visually
as a large Multilamelar vesicle (MUV) at
400x magnification. The dry powder of the
liposome was rehydrated in a concentration
of 1 mg/ μl by using purified water and
successfully form the spheric liposome
(figure 3B). A giant liposome was taken
during initial hydration and it was observed
that the MUV was formed (figure 3C).
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Further analysis results in a smaller size of
particles are given by using a particle size
analyzer instrument.
3.5 Analysis of liposome powder by a
Scanning Electron Microscopy (SEM)
The morphology of white pepper extract
liposome powder examined by a Scanning
electron microscope (SEM) shows a
surface of liposome protected by sucrose
(figure 4 A and 4B). The threshold of small
individual liposomes was identified, and
they are agglomerated into clump powder
during the drying process.
3.6 Characterizations of particle size and
zeta potential of the liposome solution
The size distribution of liposomes analyzed
by using particle size analysis shows that
the mean size is 398.7 nm with a
Polydispersion index (PI)= 0,655 (Figure
5A). Histogram shows that 10.0% of
particle in 159.3 nm size; 50.0% of particle
in 331.0 nm; 70.0% of particle in 464.3 nm;
90.0% of particle in 739.2 nm. Distribution
of particles in 3000 ppm lipid concentration
shows potential zeta -16.8 mV indicating a
threshold of delicate dispersion (figure 5B)
3.7 Encapsulation efficiency
The formula of white pepper extract is
designed to contain 300 mg of glisten
yellow crystal containing only 60.97% of
piperine in 300 ml of final liposome
solution. This equals 0.6097 mg of
piperine/ml of theoretical concentration of
white pepper extract in liposome formula.
Assay of piperine liposome solution results
in 0.6031 mg of piperine/ml, which reveals
the encapsulation efficiency of
98.92±1.17%.
Figure 3. microscopic observation of white pepper extract liposome solution (A) and rehydration of the
liposome powder (B) at 400x magnification shows spheric shape. Giant liposome was also taken for
observation and shown multilamellar vesicle was successfully formed by wet layer-lipid film hydration
technique (C).
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3.8 Assay of piperine in liposome powder
The dried powder of white pepper extract
liposome powder which was analyzed
using HPLC (Figure 5C) shows that the
assay of piperine was 0.1704 mg/100 mg
powder or 0.104%(w/w).
4. DISCUSSION
Piperine is a bioactive substance of
white pepper (Piper albi Linn) with many
beneficial pharmacological effects by in
vitro and in vivo laboratory evaluations.
Nevertheless, its poor water-soluble
characteristic results in low of its
bioavailability in the body [8]. Preparation
of piperine in a liposome delivery system
will encapsulate the compound inside the
lipid region of the membrane-like liposome
structure. The structure will mask its
hydrophobic character inside and give good
water dispersion due to the hydrophilic
polar head of liposome present in the outer
part of the structure for better release and
bioavailability profile.
The 24 hours of maceration
extraction process yield 1.98% (w/w) of
piperine. The result is lower than the
previous report which is 3.84–5.35 % [1].
The purest extract result was a glisten
yellow crystal containing only 60.97% of
piperine (figure 1B, right). The further
optimization process of extraction and
purification is important to be studied to
obtain a higher yield of piperine with better
purity.
Figure 5. The size distribution of white pepper
extract liposome at lipid concentration 3000 ppm
analyzed by using particle size analyzer shows that
the mean size 398.7 nm with Polydispersion index
(PI)= 0.665 (Figure A) and zeta potential value of
the liposomal is -16.8 mV (Figure B).
Figure 4. The morphology of white pepper extract liposome powder a Scanning electron microscope (SEM)
at 300x magnification (A) and 2500x magnification (B) shows a threshold of small liposome protected by
sucrose agglomeration during the drying process.
E.Amalia et al / Indo J Pharm 3 (2021) 149-158
156
Omitting the organic solvent in the
process of liposome formation was
intended to prepare organic solvent-free
products which may cause problems due to
safety issues. Among several types of
phospholipid sources, soy lecithin was used
due to its abundance availability with less
price in the market and especially due to
less risk of microbial and viruses
contamination [6]. a combination of soy
lecithin and cholesterol in the ratio of 12:1
(w/w) is effective to form a stable vesicle
liposome with encapsulating efficiency of
98.92±1.17% of the piperine loaded.
The white pepper extract liposome is
spherical in the range and mostly observed
as LUV and MLV (Figure 2A).
Interestingly, smaller particles analyzed by
particle size analyzer result in the most
particle size in 398.7 nm and zeta potential
value -16.8 mV. This indicates the formula
is possible to be improved further by the
addition of phosphate buffer in suitable pH
Among several methods for tor
solvent removal, the spray drying method
was employed due to its advantages in a
time-efficient and suitable in our formula
which is consisted of heat-stable material.
Employing sucrose with lipid: sucrose ratio
1:6 successfully protect the structure of
liposome during the drying process.
Nevertheless, 49.21% of the powder
adheres to the inner wall of the drying
chamber and thus cannot be collected, thus
yielding powder as 50.79%. Improvement
of yield can be optimized by using a
combination or single carrier such as
maltodextrin since its glass transition
temperature (Tg) is 162°C higher than
sucrose which is 60°C [9]–[11]. This
procedure could avoid the sticky
characteristic of powder during the drying
process which is set at 123°C. Nevertheless,
experiment and evaluation need to be
performed since it was reported that it often
leads to liposomes breaking down and
resulting in nonfunctional liposomes in
rehydration [12].
5. CONCLUSION
The solution and powder liposome were
successfully prepared without applying
organic solvent for dispersion of lipid phase
to form lipid bilayer liposome structure.
Selection of type and concentration of each
concentration of material used is important
for maximum encapsulation of the piperine.
Self-hydration of the wet layer-lipid film is
suitable for the formation of liposomes
without organic solvent. Sucrose is known
to be suitable as a carrier to protect
liposomes during the drying process. Our
experiment result is important as the initial
finding for liposome preparation without
organic solvent in simple hydration
technique thus can be applied larger
industrial scale.
Conflict of interest
The authors declare that there is no conflict
of interest regarding the publication of this
paper.
Acknowledgment
This work was supported by the
Directorate for Research, Community
Service, and Innovation (DRPMI)
Universitas Padjadjaran. The present study
was supported by the Head lecture
acceleration grant (Hibah Percepatan
Lektor Kepala) Universitas Padjadjaran, no
48/UN6.3.1/PT.00/2021. We would also
thank Rizqa Nurul A. and Ferris Dzaky RN.
for technical support during the experiment.
E.Amalia et al / Indo J Pharm 3 (2021) 149-158
157
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