Formation of Highly Antioxidative Liposomes from Crude Acetone Extracts of Canna indica, Cucumis melo and Prunus armeniaca
Liposomes are often used as a carrier to improve the therapeutic activity and
safety of drugs. The lipid composition of liposomes plays an important role
in determining the behavior of liposomes in phases. In this study, the phospholipid
and alkylresorcinol content in crude acetone extracts of seeds of Canna indica
L., Cucumis melo L. and Prunus armeniaca L. was analyzed in
order to assess the applicability in liposome delivery vesicles. The crude extracts
were used to form liposomes and their stability and resistance to oxidative
degradation was evaluated by spectroscopy. The liposomes formed from these extracts
were stable compared to control Phosphatidylcholine (PC) liposomes and had a
lower percentage of aggregation as a function of time, as measured by the Optical
Density (OD) at 400 nm (6.1-26.6% for extract liposomes and 43% for PC liposomes).
Lipid peroxidation measured by the Thiobarbituric Acid (TBA) method after 28
days incubation at room temperature was much lower for extract liposomes compared
to PC liposomes. Interactions of the extract liposomes with human white blood
cells resulted in a reduction of the free radical (O2-)
formation by 38-65% compared to PC liposomes.
March 04, 2012; Accepted: June 27, 2012;
Published: October 11, 2012
Liposomes have been studied and characterized for over 50 years since first
being described in the 1960s (Bangham et al., 1965).
These vesicles are composed of a lipid bilayer that spontaneously forms in the
presence of water (Mozafari, 2005). The amphiphilic
characteristics of liposomes and ease of vesicle formation (Grabielle-Madelmont
et al., 2003) allows them to encapsulate molecules and serve as carriers
for various biologically active compounds (Felnerova et
al., 2004; Brgles et al., 2009), such
as vitamins, enzymes, drugs, proteins, nucleic acids (Brgles
et al., 2007) and some vaccines (Chen et al.,
Liposomes have been used as a drug carrier to improve the therapeutic activity
and safety of drugs by altering their pharmacokinetics (Lian
and Ho, 2001), solubility, bioavailability and stability as well as reducing
their toxicity and enhancing targeting to their site of action. Furthermore,
due to their biodegradability and low permeability to small hydrophilic molecules,
liposomes have been used in many areas such as cancer chemotherapy, ophthalmology,
antimicrobial therapy, vaccines, gene therapy and diagnostic applications (Allen
et al., 1995).
The lipid composition of liposomes plays a significant role in determining
their phase behavior as well as other properties such as permeability, fusion,
aggregation and stability, which can all affect their efficacy in delivery and
biological systems (Barenholz, 2001), interaction with
cells in vivo and in vitro and the encapsulation efficiency of
various substances, especially drugs (Volodkin et al.,
2007). Many studies have attempted to enhance liposomal stability and minimize
oxidative degradation, because stability is a prerequisite for the exploitation
of liposomes in the delivery of therapeutic molecules (Sivakumar
and Rao, 2003; Lau et al., 2005). Several
methods were done to enhance stability include preparation of more stable bilayers,
coating the liposome surface with protective polymers, addition of polyethylene
glycol, preparation of polymerized liposomes and charge modification and freeze
drying (EI-Samaligy et al., 2006).
Other strategies involve the addition of various molecules such as Alkylresorcinols
(ARs), vitamins, chitosan and bovine serum albumin to minimize phospholipid
oxidation and modify liposomal behavior (Guo et al.,
2003; Takeuchi et al., 2001). ARs are polyketide-derived
compounds (Alastair et al., 2004) and are defined
as amphiphilic 1, 3-dihydroxybenzene derivatives with an odd-numbered alkyl
chain at position 5 of the benzene ring. ARs have been reported to have anticancer,
antimicrobial, antiparasitic, antitumour, antioxidant, antifungal, antileukemic,
enzyme-inhibiting and DNA-cleaving properties (Francisco
et al., 2005). In addition, ARs are amphiphilic, exhibit strong affinity
for lipid bilayers and biological membranes and can affect membrane structure
and properties (Stasiuk and Kozubek, 2008). ARs are
abundant in plant seeds, fruits, higher plants, algae, mosses, fungi and bacteria
(Zarnowski et al., 2001). The antioxidant properties
of ARs may play a role in protecting both free fatty acids and phospholipids
against peroxidation, auto-oxidation and oxidation of biological membranes (Deszcz
and Kozubek, 2000). Canna indica, Cucumis melo and Prunus armeniaca
are known to have antioxidant activity and the extract of these plants may
be used as a main component of liposomes to enhance liposome stability, therapeutic
activity and to be used as a delivery or carrier system for the treatment of
In this study, the phospholipids and ARs content in acetone extracts of seeds
of Canna indica, Cucumis melo and Prunus armeniaca was
determined. These crude acetone extracts are composed of mixtures of individual
phospholipids, ARs and other compounds and were used to form highly antioxidative
liposomes. In addition, the interaction of the liposomes with white blood cells
MATERIALS AND METHODS
Materials: Plant fruit of Canna indica, Cucumis melo and
Prunus armeniaca were collected in may 2009 from local markets. The research
project was conducted from May 2009 to August 2011.
Acetone extraction of Canna indica, Cucumis melo and Prunus
armeniaca: Fifty grams of each type of plant seed were ground in a coffee
grinder. The resulting powder was then soaked with acetone and extracted by
continuous shaking for 25 h at Room Temperature (RT). The extracts were filtered
and the residues soaked again with acetone for an additional 25 h at RT. The
extracts were then filtered and combined and the final extracts were evaporated
to dryness in a rotary evaporator (RV 05-ST Janke and Kunkel, IKA, Germany)
to form a dry film.
Qualitative and quantitative analysis: The dry films of the extracted
material were dissolved in chloroform (1 mL). The extracts were then qualitatively
analyzed by Thin-layer Chromatography (TLC). Two developmental systems for TLC
were used: chloroform: ethyl acetate (85:15, v/v) to separate ARs, which were
specifically detected by immersing the plate for 5-10 sec in Fast Blue B solution
(Sigma-Aldrich Chemie GmbH, Germany) to yield pink-red spots (Kulawinek
et al., 2008) and chloroform:methanol:distilled water (65:25:4, v/v/v)
to separate phospholipids, which were specifically detected by immersing the
plate for 1-2 min in ammonium molybdate solution to yield blue spots (Atrouse
and Qato, 2000).
Phospholipids content was measured using a phosphate assay according to the
method described by Rouser et al. (1970). ARs
content was determined according to the method described by Sampietro
et al. (2009).
Liposome preparation and optical densities: Phosphatidylcholine (Phospholipid
GmbH, Cologne, Germany) (5 mg mL-1) in chloroform solution was evaporated
using the rotary evaporator. The lipid film was hydrated with Phosphate-buffered
Saline buffer (PBS) (100 mM, pH 7.4). Liposomes were formed by immersion in
an ultrasonication bath (Clifton, England) for 90 min (Kagan
et al., 1990). The dry films prepared from the crude acetone extracts
of Canna indica, Cucumis melo and Prunus armeniaca were
hydrated with PBS and sonicated as described above. The optical densities of
the liposome suspensions were measured at wavelengths ranging from 350-750 nm
(Trofimove and Nisnevich, 1990) using a Vis-Spectrophotometer
(Biotech Engineering Management Co. LTD, UK).
Liposome peroxidation assay (thiobarbituric acid method): To 500 μL
liposome solution, 500 μL PBS (100 mM, pH 7.4) and 100 μL FeSO4.7H2O
(4 mM) were added, followed by addition of 100 μL ascorbic acid (2 mM).
After incubation for 30 min at 37°C, the reaction was terminated by the
addition of 5.5% trichloroacetic acid. Next, 250 μL of Thiobarbituric Acid
(TBA) solution (in 50 mM NaOH) was added to 1 mL of the above reaction mixture,
followed by heating for 10 min. The mixtures were centrifuged at 3000 rpm for
10 min and the supernatant absorbance was determined at 532 nm (Choi
et al., 2002). The inhibition ratio (%) was calculated using the
where, A was the absorption of the control and As was the absorption
of the sample.
Cell isolation: Blood samples (with EDTA) obtained from volunteers were
centrifuged and the buffy coat layer was removed and washed three times with
PBS. Then, 1 mL 0.155 M ammonium chloride was added to lyse the residual erythrocytes.
The White Blood Cell (WBC) viability was determined to be 95-98% by stained
blood films (Wright stain). The WBCs were then suspended in PBS (Nilsson
and Palmblad, 1988).
Superoxide ion production: Free radical production was determined by
measuring the reduction of Nitro Blue Tetrazolium (NBT) to Formazan (Tonetti
et al., 1991). In brief, cells (1x106 mL-1)
were incubated with liposomes for 10 min at 37°C, followed by the addition
of 50 μL NBT solutions. After incubation for 20 min at 37°C, the reaction
mixture was centrifuged at 1000 rpm for 10 min. The absorbance was determined
at 520 nm.
Statistical analysis: All analyses were performed on triplicate samples.
The results were expressed as Mean±SD. The Students
t-test was used for the evaluation of statistical significance (p<0.05).
RESULTS AND DISCUSSION
Qualitative TLC analysis of Canna indica, Cucumis melo and
Prunus armeniaca acetone extracts showed that they contained PLs, ARs and
||Determination of PLs and ARs content obtained from acetone
extracts of Canna indica, Cucumis melo and Prunus armeniaca
using the phosphate assay method and micro method, respectively
|Data represented as Mean±SD, Values are statistically
significant at p<0.05
The extracted materials were then separated by TLC and developed with different
methods to yield spots that were specifically stained with ammonium molybdate
or Fast Blue B to detect the presence of PLs and ARs, respectively. These qualitative
experiments were followed by quantitative determinations of the PLs content
using a phosphate assay and the ARs content using the micro method (Table
1). Acetone extract from Canna indica, Cucumis melo and Prunus
armeniaca contained 650, 1600 and 230 μg g-1 PLs and 2800,
2000 and 2380 μg g-1 ARs, respectively. These results illustrate
that there were large differences in the quantities of PLs in each extract and
that Cucumis melo contained the largest amount of PLs followed by Canna
indica and Prunus armeniaca. Smaller differences were observed in
the quantities of ARs with Canna indica having the largest amount followed
by Prunus armeniaca then Cucumis melo. These crude lipid mixtures
were used to prepare liposomes from each extract: Canna indica (LCI),
Cucumis melo (LCM) and Prunus armeniaca (LPA). The liposome optical
density, susceptibility to oxidation degradation and interaction with human
WBCs was then evaluated.
Figure 1 shows the optical densities of the liposomes incubated
for up to 28 days at RT. The differences in optical densities seen for LCI (Fig.
1b), LCM (Fig. 1c) and LPA (Fig. 1d)
compared to PC liposomes (Fig. 1a) may be due to the different
compounds within each sample. Liposome aggregation as a function of time was
followed by monitoring liposome suspension turbidity at 400 nm (Fig.
1), which showed changes in aggregation from 0 to 28 days of 26.6, 11.8
and 6.1% for LCI, LCM and LPA, respectively. These values were lower than those
seen for PC liposomes (43%). The aggregation tendency depends on the surface
properties of liposomes, which is may be changed by the peroxidation of constituting
LCI, LCM and LPA were less susceptible to oxidation during the incubation period
(28 days) than PC liposomes (Table 2). It was observed that
all the formed liposomes have different activity against lipid peroxidation.
This peroxidation inhibition of the LPA liposomes (71.8%) was higher than that
of LCI (52.7%) and LCM (34.5%), while PC liposomes (17.6%) was the least. This
difference may be due to the presence of different compounds in the liposomes
formed from the acetone extracts. Since phospholipids in liposomes are susceptible
to oxidation during incubation, the presence of antioxidant molecules (polyphenolic
compounds) such as ARs, xanthones, flavonoids and others (Yen
and Chuang, 2000) could minimize the oxidation degradation of liposomes
during the incubation period at room temperature and could be potentially responsible
for the powerful activity of the extracts and the main factor for the protection
of the formed liposomes from peroxidation compared to the PC liposomes. The
difference in the inhibition of peroxidation may be due to the type and concentration
of the antioxidant compounds in the acetone extract.
To assess the antioxidant properties of the molecules present in the extracts,
LCI, LCM and LPA were incubated with human WBCs. The scavenging activity of
the extract liposomes was measured in terms of their capacity to inhibit free
radical generation. Free radicals are molecules or molecule fragments with unpaired
electrons in the outer orbital (Sanchez-Moreno, 2002),
which renders them very reactive and capable of initiating chain reactions that
result in oxidation degradation of lipids and tissue damage.
|| Time course (days) of different liposome optical densities,
(a) Phosphatidylcholine liposomes (PC), Liposomes formed from crude acetone
extract of, (b) Canna indica (LCI), (c) Cucumis melo (LCM)
and (d) Prunus armeniaca (LPA)
||Percent inhibition of lipid peroxidation in LCI, LCM and LPA
liposomes compared to PC liposomes (control) as a function of time
|Data represented as Mean±SD, Values are statistically
significant at p<0.05
Free radicals are produced via metabolic pathways and phagocytosis of some
WBCs. One free radical is O2-, which can form oxidants
that are toxic towards phagocytes. O2- reduces NBT into
Formazan, which can be spectrophotometrically measured at 570 nm (Sanchez-Moreno,
||Effect of incubating different liposomes with human white
blood cells on O2- radical production compared to
control (without liposomes). Mean of triplicate±SD, p<0.05
Liposomes formed from the acetone extracts were found to have the ability to
scavenge free radical species (O2-), which indicates that
they have antioxidant power to inhibit O2- production
(Fig. 2). In general, control cells themselves produced free
radicals (0.15) but the presence of liposomes enhanced the production of O2-
in these cells to 0.7 for PC liposomes and 0.34, 0.45 and 0.25 for LCI, LCM
and LPA, respectively. These results indicate that the antioxidant activity
of the additional molecules present in the extract liposomes enhanced their
scavenging activity towards free radicals by reducing O2-
production and subsequently inhibited Formazan production by 38% (LCM), 52%
(LCI) and 65% (LPA) as compared to PC liposomes.
This experiment demonstrated that liposomes can influence certain functional
responses of human neutrophils in vitro to produce superoxide ions. The
increase was composition dependent, indicating that the extract liposomes (LCI,
LCM and LPA) may contain different compounds (especially antioxidant molecules)
that resulted in reduction of superoxide ions formation compared to PC liposomes.
Our qualitative experiments (TLC) on these extracts revealed the presence of
different molecules, including ARs, which exhibit antioxidant activity and are
powerful free radical scavengers.
In general, present results indicated that the molecular mixture obtained from
these extracts can be used to form liposomes that have high stability as determined
by the decrease in liposome aggregation (as measured by changes in turbidity),
minimal oxidative degradation of lipids and a capacity to reduce free radical
production. These features would be especially useful in systems using liposomes
as vehicles for drug encapsulation or as a delivery system for several types
Liposomes prepared from Canna indica, Cucumis melo and Prunus
armeniaca extracts exhibited high antioxidative property and could minimize
lipid oxidation. Their interaction with human WBCs resulted in reduced amounts
of free radical formation, indicating that these liposomes are both stable and
exhibit antioxidant effects that reduce the production of free radicals by virtue
of their lipid composition and in particular the presence of alkylresorcinols.
Further studies are needed to analyze all the compounds present in these extracts
and to develop these liposomes as a model for biological and drug systems for
use in vitro and in vivo.
This study was supported by the Deanship of the Academic Research (Grant No.
792/14/120) at Mutah University. The technical assistance of Abdulla Allawneh
is greatly acknowledged.
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