JOURNAL OF MEDICINAL FOOD

J Med Food 16 (6) 2013, 551–557

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2012.0162

Biological Activity of Liposomal Vanillin

Leniher Castan,1 Grisel del Toro,2 Adolfo A. Fernández,3 Manuel González,4
Emilia Ortı́z,1 and Daliana Lobo1
Departments of 1Biomedical Engineering, 3Computer Science, and 4Biology, Faculties of 1Electrical Engineering,
3
Mathematics & Computer Science, and 4Natural Sciences, University of Oriente, Santiago de Cuba, Cuba.
2
Food and Pharmacy Institute, Havana University, Havana, Cuba.

ABSTRACT This article presents a study of vanillin encapsulation inside multilamellar liposomes, with emphasis on the
evaluation of antioxidant activity, the hemolytic effect, and the antisickling properties of these products. Egg phosphati-
dylcholine-cholesterol and egg phosphatidylcholine-cholesterol-1-O-decylglycerol liposomes were prepared by mechanical
dispersion, all with vanillin included. Vesicles were characterized by determination of encapsulation efficiency and vanillin
retention capacity. Antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The hemolytic
effect of liposomes was also evaluated by spectrophotometry, as well as the antisickling activity by the Huck test using optical
microscopy. Results showed that the lipid composition of liposomes did not significantly affect the encapsulation efficiency.
Stable vesicles were obtained with a high retention percentage of vanillin. Liposomes exhibited a high capture of the DPPH
radical compared to free vanillin and 1-O-decylglycerol (C10) in solution. Vesicles caused no significant hemolisys in normal
erythrocytes, nor in those coming from patients with sickle cell anemia. Vanillin encapsulated in liposomes retained its
antisickling activity, with a greater effect for C10-containing vesicles. Our results show that vanillin encapsulation in
liposomes is a way to enhance the pharmacologic properties of this molecule using a suitable vehicle.

KEY WORDS:  alkylglyerols  aromatic aldehyde  artificial vesicles  drug delivery  sickle cell disease

INTRODUCTION Recently developed drug delivery systems such as lipo-

somes serve as efficient vehicles, and there are several FDA-
V anillin (4-hydroxy-3-methoxybenzaldehyde) is
an aromatic derivative aldehyde of Vanilla planifolia,
which has been traditionally used in the food and cosmetic
approved formulations.13 These vesicles can be modified to
influence the process of transporting the encapsulated drug.
Among the changes that have been made in lipid membranes
industries.1 This flavoring agent has some pharmacological
is the inclusion of 1-O-alkylglycerols.14 These lipid ethers,
properties. Reports had shown that vanillin can display
derivatives of shark liver oil, have antitumor, antimicrobial,
chemopreventive effects in chemical carcinogenesis mod-
and anti-inflammatory activities.15–17 They are also absorp-
els,2 antimutagenic activity,3 inhibition of metastasis,4 an-
tion promoters and enhance the passage of several molecules
tioxidant properties,5 and antibacterial and antinflammatory
through the blood–brain barrier, the gut, and cell mem-
action.6,7 Vanillin also has antisickling properties, as has
branes.18,19 The lipidic ether 1-O-decylglyerol is an example
been demonstrated in multiple studies.8,9
of this family of compounds.15–19 The 1-O-alkylglycerol
Although vanillin has these medicinal effects, its bio-
ethers may be used to modify liposomes and improve the
logical activity in animal models has been limited because it
capacity of these systems to transport molecules through the
has low oral bioavailability, probably due to its degradation
barriers mentioned above. The 1-O-alkylglycerols, and spe-
in the gastrointestinal tract.10 The half-life of this molecule
cifically 1-O-decylglycerol (C10), can also synergize the ef-
is very short, fast disappearing from peripheral blood.11 Its
fects of drugs encapsulated in the liposomes, due to its
low solubility in water raises the problem of administering it
pharmacological properties.
by nonaqueous vehicles, which are often toxic.12 All of these
In this work, we performed the encapsulation of vanillin
factors make it necessary to have a suitable vehicle for the
in liposomal vesicles, some of them modified with the
efficient administration of vanillin.
synthetic ether C10, and then studied the influence of en-
capsulation on some pharmacological properties of vanillin.
Manuscript received 23 June 2012. Revision accepted 25 February 2013 Specifically, we studied the antioxidant activity by evalua-
tion of 2,2-diphenyl-1-picrylhydrazyl (DPPH) capture.
Address correspondence to: Leniher Castan, MSc, Department of Biomedical En-
gineering, Faculty of Electrical Engineering, University of Oriente, Avenida de las
Since vanillin can be used in sickle cell anemia and hemo-
Américas s/n, Santiago de Cuba 90 900, Cuba, E-mail: castan@fie.uo.edu.cu lysis is frequent in this pathology,8 we also assayed the

551
552 CASTAN ET AL.
hemolytic activity of liposomes to determine if those vesi-
cles may aggravate this problem. Finally, we assayed the
capacity of vesicles to inhibit the sickle deformation.
MATERIALS AND METHODS
Reagents
To obtain the liposomal vesicles, egg yolk phosphati-
dylcholine (ePC) obtained by the method of Pangborn
et al.20 was used. The C10 was provided by the Food and
Pharmacy Institute, Havana University. Cholesterol (Cho)
was purchased from Sigma, vanillin was provided by
Panreac, and organic solvents (chloroform and methanol)
by UNI-chem.
Preparation of liposomes
Organic solutions were prepared containing ePC, Cho,
vanillin, and C10 in a chloroform–methanol 2:1 mixture.
Two preparations were developed, one containing ePC and
Cho at the molar ratio 1:0.5 and another containing ePC–
Cho–C10 at the molar ratio 1:0.5:0.1. In all solutions, 3 mg
of vanillin were added to ensure a molar 1:1 ratio with ePC.
Each preparation was performed in triplicate. The organic
solutions were evaporated in a rotary evaporator (IKA),
until a thin lipid film formed on the walls of the vessel
containing the mixture. One milliliter of saline was then
added to the reaction vessel and subjected to vortexing
(IKA), thereby obtaining a suspension containing lipo-
somes. Subsequently, the vesicles were purified by centri-
fugation 60 min at 10,000 g in a refrigerated centrifuge
(Heal Force) and washed three times to remove the un-
encapsulated solute, then resuspended in 1 mL of saline and
stored at 4C. Encapsulation efficiency (EE%) of vanillin in
the liposomal vesicles was determined indirectly as follows:
Cvi  Cvs
EE% ¼ · 100%
Cvi
where Cvi is the concentration of vanillin added to the initial
mixture and Cvs is the concentration of vanillin in the su-
pernatants after purification.
The vanillin concentration was determined spectropho-
tometrically at 610 nm using the method reported for the
determination of this flavoring agent in foods,21 by reaction
with Folin Ciocalteu’s reagent. The retention percent was
determined by quantification of vanillin released from the
liposomes after 7 days of storage and subsequent centrifu-
gation.
Free radical scavenging capacity
To determine the scavenging activity of the compounds, a
0.5 mL ethanol solution of DPPH at 0.1 mM and 1 mL of
different preparations were mixed in a vial. These reaction
mixtures were prepared in triplicate for each liposomal
preparation and incubated at 25C for 1 h. Then, they were
subjected to centrifugation at 10,000 g for 60 min, and the
supernatants were collected. DPPH was also mixed with
ethanolic solutions of vanillin to the concentration of
1.5 mg/mL. Reactivity was also evaluated using solutions of
C10 (10 lmol/mL) and ePC (20 lmol/mL) with the same
concentration into liposomes, proceeding in a similar man-
ner. In the supernatants of the preparations, as well as in the
solutions of vanillin, C10, and ePC, the absorbance was
determined at 517 nm. A solution of DPPH itself was used as
the negative control, while the positive control was an
ascorbic acid solution.
The equation to determine the percentage of DPPH rad-
ical capture is
Ap  Am
Ip DPPH (%) ¼ · 100%
Ap
where Ip is capture percent, A is the absorbance, and the
subscripts m and p represent the sample and the DPPH or
white, respectively.
Hemolytic effect
The study of the hemolytic activity was performed using
the spectrophotometric method of Stanley.22 Five tests were
performed in whole blood from patients with sickle cell
disease (SCD) from the special hematology consultation at
Children’s South Hospital of Santiago de Cuba, with previous
informed consent and in accordance with the ethical stan-
dards of the institution. Five tests were also conducted with
blood from volunteer donors, provided by the Provincial
Blood Bank of Santiago de Cuba. The exclusion criteria were
infants born prematurely, those who had received transfu-
sions within two months before the taking of samples, and
pregnant women. Patients with low hemoglobin values and
those who were receiving any type of treatment were also
excluded.
Heparinized whole blood was centrifuged at 1400 g for
10 min and the cell pellet was subjected to three successive
washes with phosphate buffer solution (PBS), pH = 7.4.
Thereafter, the pellet was diluted with PBS to a final volume
of 10 mL. The hemolytic effect was studied in triplicate for
each preparation. Positive control was a solution of eryth-
rocytes treated with Na2CO3 at 0.1%, and negative control
was erythrocytes with PBS (Table 1). After adding, com-
pounds were stirred and mixtures were allowed to stand
30 min and subsequently centrifuged. Supernatants were
collected, and the absorbance was determined at 545 nm to
detect the release of hemoglobin. Using the absorbance
Table 1. Preparation of Reaction Mixtures
for the Study of Hemolytic Effect
0.1% PBS Erythrocytes Liposomes
Na2CO3 pH = 7.4 solution solution
(mL) (mL) (mL) (mL)
Positive control 9.80 — 0.20 —
Negative control — 9.80 0.20 —
Reaction mix — 9.79 0.20 0.01
PBS, phosphate-buffered saline.
 BIOLOGICAL ACTIVITY OF LIPOSOMAL VANILLIN
values, the percentage of hemolysis caused was calculated,
according to the formula:
Am  Acn
%H ¼ · 100%
Acp  Acn
where the subscripts cn and cp represent the negative and
positive controls, respectively.
Effects on SS erythrocytes
To study the effect of liposomal preparations on SS
erythrocytes from patient with SCD, five tests were per-
formed with whole blood from patients with SCD. Reaction
mixtures were obtained by mixing 200 lL of blood with 200
lL of liposomal preparations. This ensures sufficient
amount of vanillin to be able for reacting with S hemoglobin
(HbS) as has been reported previously by our group.23 Each
experiment was performed with six replicates. Liposomal
preparations and free vanillin were evaluated. The mixtures
were incubated at 36C, and then a 10-lL aliquot of each
one was deposited in a slide for running the Huck test.24 This
involves placing a cover slip over deposited blood, and then
sealing it with paraffin to subject the samples to anoxygenic
conditions. The mixtures were prepared in triplicate. Slides
were observed under an optic microscope (Novel) with a
digital camera (Canon) at the initial time (t0) and at 24 and
48 h. In each case, a sickle cell counting was performed. The
observation field was fixed to 200 cells.
Statistical analysis
Analysis of variance was performed using simple classi-
fication, coupled with the Duncan’s test with a significance
level of P < .05. All data processing was performed using
Statistica for Windows version 8.1.
RESULTS AND DISCUSSION
Characterization of liposomes
Characteristics of vesicles are shown in Table 2. The
encapsulation efficiency showed a mean value of
47.5% – 2.6% in the preparation containing C10 and
50.3% – 1.3% in the one composed by ePC–Cho alone,
without statistically significant differences between the two
results (P > .05). We obtained high retention values of
Table 2. Characterization of Liposome Vesicles
Liposome Encapsulation Vanillin retained
composition yield (%) after 7 days (%)
ePC–Cho 50.3 – 1.3 98.48 – 0.37
ePC–Cho–C10 47.5 – 2.6 94.63 – 0.64
Values are given as the arithmetic mean – standard deviation of three
replicates. No significant differences were found with analysis of variance
(P > .05).
ePC, egg yolk phosphatidylcholine; Cho, cholesterol; C10, 1-O-decylgly-
cerol.
553
vanillin, 98.48% – 0.37% for ePC–Cho and 94.63% – 0.64%
for ePC–Cho–C10, with no significant differences between
the two preparations (P > .05). The absence of statistically
significant differences between the encapsulation efficien-
cies and the retention capacity of the tested liposomes
suggests that the presence of C10 does not affect these pa-
rameters at the molar ratio used in the study. The high re-
tention percentage of vanillin in storage conditions affirms
the stability of the obtained liposomal vesicles.
Free radical scavenging capacity
Differences were statistically significant (P < .05) in the
capture of DPPH for tested liposomes of different compo-
sition. As shown in Figure 1, C10 vesicles showed a capture
of 98.56%, a percentage that is significantly (P < .05) higher
than the rest of the preparations. The preparation ePC–Cho
showed a scavenging activity value of 76.44%, which is
significantly higher than C10 (23.81%), ePC (45.01%), and
the unencapsulated vanillin (43.90%). These results dem-
onstrate that the incorporation of C10 into the liposomal
structure increases the ability to scavenge the DPPH radical.
Vesicles appeared to be stable after centrifugation in the
retention percent determination step, with very low libera-
tion of entrapped material to the medium. Therefore, this is
evidence that the antioxidant potential was not due to van-
illin being released from the liposomes as it was being
centrifuged in this assay.
The fact that the liposomal preparation without C10
showed a greater reactivity with DPPH than C10, ePC, and
vanillin can be explained by assuming a synergism between
the antioxidant action of vanillin and the ePC vesicle
structure. It has been shown that vanillin has antioxidant
capacity, and the antioxidant properties of ePC have also
been confirmed, through studies of its interaction with sin-
glet oxygen.25 It can be assumed that in the vesicles under
study, the capacity of immobilized vanillin to capture free
radicals is added to the free radical scavenging effect of
ePC. ePC contains double bonds and is able to react with
FIG. 1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging percent
of different tested preparations. All values are given as the arithmetic
mean – standard deviation of three replicates. Different letters indi-
cate statistically significant differences in ANOVA coupled with
Duncan’s test (P < .05). Control (positive), ascorbic acid; Cho, cho-
lesterol; ePC–Cho–C10, liposomes with 1-O-decylglycerol; ePC–
Cho, liposomes without 1-O-decylglycerol; C10, 1-O-decylglycerol;
ePC, egg yolk phosphatidylcholine.
554 CASTAN ET AL.
oxygen- and nitrogen-reactive species. It is noteworthy that
the preparation with C10 is the one that has the best scav-
enging activity. This result cannot be explained by merely
taking into account the direct capture of DPPH by C10,
since its structure has no double bonds or other groups ca-
pable of interacting significantly with free radicals. C10 is
likely to form curvature regions in the lipid bilayers, which
modify lipid packaging. These regions can be considered
areas where the membrane lipid density decreases, favoring
the inward diffusion of substances such as DPPH. In pre-
vious works, it has been demonstrated that lipids which do
not form bilayers, such as stearylamine (SA) and phospha-
tidylglycerol (PG), are inducers of curvature regions in li-
posomes and this allows proteins like epidermal growth
factor (EGF) to pass through them.26,27 C10, like SA and
PG, does not form bilayers,28 so it is very likely that cur-
vatures formed by this ether are of sufficient size to let large
molecules pass through. For many substances, phospholip-
ids with their electric charges and external groups may
constitute barriers to movement through the membranes. In
the areas of lower density and packing of the polar heads of
phospholipids, molecules like DPPH and other reactive
species may diffuse into the bilayers and have a greater
probability of interaction with the immobilized vanillin
within. This would increase the capacity of DPPH scav-
enging, as observed in the experiment under analysis. A fact
that supports this hypothesis is the property of alkylglycer-
ols to act as absorption promoters.29
Hemolysis
The polymerization of HbS causes the sickle deformation
in SS erythrocytes and hemolysis;24 therefore, any com-
pound with the potential to treat SCD needs to have a low
hemolytic effect to prevent increase of hemolysis due to the
illness. This fact makes it necessary to test the compounds
for hemolytic effect.
Figure 2 shows the hemolysis percent of the compounds
on erythrocytes from apparently healthy individuals (AA)
and patients with SCD (SS erythrocytes). For the ePC–Cho–
C10 preparation, higher values of hemolysis were detected:
2.04% on AA erythrocytes and 2.56% on SS erythrocytes.
Hemolysis caused by ePC–Cho liposomes showed a lower
average, 1.43% for AA and 1.78% for SS erythrocytes.
Comparing the effect of both types of liposomes on SS
erythrocytes, the differences were not statistically signifi-
cant (P > .05). Similar results were observed in experiments
with blood from voluntary donors. The action of each kind
of liposome preparation was also compared for both types of
erythrocytes. In this case, there were no significant increases
of hemolysis with any of the two preparations (P > .05).
Although not significant, there was a trend toward in-
creased hemolysis in preparations containing C10. This is
consistent with previous studies where hemolytic events in
SS erythrocytes were increased by the presence of this li-
pidic ether.30 These results may be caused by the ability of
C10 to interact with cell membranes. Since polymerization
of HbS causes intracellular membrane damage and hemo-
FIG. 2. Hemolytic effect calculated in vitro by the spectrophoto-
metric method for C10-modified and unmodified liposomes on eryth-
rocytes of sickle cell disease patients (SS) and volunteer donors (AA).
No statistically significant differences were observed between treat-
ments when analysis of variance was applied (P > .05). All values are
given as the arithmetic mean – standard deviation of three replicates.
SS, erythrocytes from patient with SCD; AA, erythrocytes from healthy
individuals.
globin release with values of up to 40% of hemolysis in
patients with SCD,24 it can be considered that C10 acts to
increase the hemolytic effect in the case of SS erythrocytes.
In general, the hemolytic effect of the tested preparations is
very low compared with the maximum hemolysis reported
as permissible in the literature, which is 10%. This is the
maximal hemolysis that a compound may cause without
toxicity in the in vitro test developed by Stanley et al.22
Effect on erythrocytes SS
All tested preparations showed a marked decrease in the
number of sickle cells at 24 h of incubation, compared to
positive control (Table 3). For the positive control (Fig. 3a, b),
a significant increase (63% – 4.2%) in the number of sickle
cells at 48 h was observed, compared with the number of cells
at 24 h of incubation, 25% – 3.2% (P < .05).
The liposomal preparation containing C10 showed the
lowest values in sickle cell formation, with values of
6.0% – 1.1% at 24 h and 5.0% – 2.8% at 48 h, with erythro-
cytes appearing similar to echinocytes (Fig. 3c, d), which are
red cells with prolongations regularly distributed in the
Table 3. Counts of Sickle Cells at Different Times
Sickle cells (%)
24 h 48 h
Positive control 25.3 – 3.2a 63.0 – 4.2b
ePC–Cho–C10 6.0 – 1.1c 5.0 – 2.8c
ePC–Cho 15.0 – 1.7d 13.5 – 2.1d
Vanillin 13.3 – 0.5d 12.5 – 3.6d
Sickle cells were counted at different times after subjecting the erythrocytes
with each compound to anoxygenic conditions by the Huck test. Values are
given as the arithmetic mean – standard deviation of six replicates.
abcd
Different letters indicate significant differences using analysis of
variance coupled with Duncan’s test (P < .05).
 BIOLOGICAL ACTIVITY OF LIPOSOMAL VANILLIN
FIG. 3. Effects of liposomes and unencapsulated vanillin on SS
erythrocytes (magnification, · 40). Pictures were taken at 24 and 48 h
after treatment in oxygen-lacking conditions: (a, b) positive control
(untreated cells); (c, d) cells treated with vanillin encapsulated in
ePC–Cho–C10 liposomes; (e, f) cells treated with vanillin encapsu-
lated in ePC–Cho liposomes; (g, h) cells treated with free vanillin.
Each experiment was performed with six replicates.
surface and are more flexible than sickle cells. These aver-
ages differ significantly (P < .05) for the positive control and
the other preparations for each incubation time. No signifi-
cant differences between the two incubation times (P > .05)
were observed. The change of the red cells into echinocytes
is reversible, and the erythrocytes may recover their normal
shape.31
Compared with the positive control, the liposomal prep-
aration without C10 (Fig. 3e, f) showed a significant dif-
ference (P < .05) in the number of sickle cells, with the
average value of 15% – 1.7% at 24 h and 13.5% – 2.1% at
48 h; no significant differences between stages (P > .05)
were observed.
Unencapsulated vanillin (Fig. 3g, h) showed significant
differences (P < .05) with the positive control and did not
differ significantly (P > .05) from the preparation without
C10. The average number of sickle cells for vanillin was
13.3% – 0.5% at 24 h and 12.5% – 3.6% at 48 h and no sig-
nificant differences were found for these values (P > .05).
555
Previous works8,30 have shown the potential of these
compounds to inhibit sickle deformation. Investigations
showed that vanillin and C10 exert their effects by different
routes. Vanillin is covalently attached to HbS, forming
Schiff bases with the free terminal amino groups and in-
hibiting the polymerization process of this molecule.8,9 C10
appears to exert its actions by modifying the critical micelle
concentration of the cell membrane.30 In SCD, there is a
reduction in the mechanical resistance of the erythrocyte
membranes due to dehydration and the polymerization of
HbS.24 Membrane becomes more rigid and less flexible due
to tight junctions between the phospholipids, leading to ir-
reversible sickle deformation and hemolysis.24,32 There are
also alterations in the cytoskeleton. Incorporation of C10
makes the membrane more flexible by intercalation between
tight lipids, and thus modifies the lipid composition and
concentration of cellular bilayers.33
Considering these routes, the synergistic effect observed
in C10-containing preparations can be attributed to an in-
hibition of HbS polymerization by vanillin and an influence
of C10 in the micellar state of the cell membrane. Further-
more, C10 may promote the entry of vanillin into erythro-
cytes. In vivo it is likely that the previously postulated
mechanisms act together. An important consideration is the
fact that experiments were performed with whole blood
from patients with SCD. In the blood, there are multiple
components besides erythrocytes, including plasma pro-
teins, lipids, and electrolytes. Encapsulation of vanillin
could prevent the interaction of this molecule with such
components, which could potentially decrease its interaction
with erythrocytes.
Regarding the administration route of liposomal vanillin,
some considerations must be made. During oral adminis-
tration, the liposome stability may be affected. Mechanical
agitation due to peristaltism in the gastrointestinal tract may
cause breakdown of membranes and liberation of the en-
trapped material.34 The changes in pH through the medium
lead to changes in the superficial charge of vesicles, which
may promote aggregation and changes in membrane per-
meability.35 The bile salts can destroy the membrane
structure and promote the active principal releasing before it
reaches the blood.36 The enzymes present in the gut and the
liver may also affect membrane integrity.36,37 For that rea-
son, oral administration of liposomes is not always suc-
cessful and some alternatives, such as the polymer coats,38
have been developed to improve the oral stability of vesi-
cles. Parenteral administration is preferred for liposomes,
and this may be an alternative to oral administration of
vanillin. Nevertheless, both administration methods should
be tested.
The fact that modification of multilamellar liposomes
with C10 enhances the pharmacological properties of a
solute such as vanillin without increasing hemolytic events
is evidence that this type of vehicle may improve the
pharmacokinetics and pharmacodynamics of an active in-
gredient. This suggests the possibility of using these com-
pounds for the future treatment of diseases such as sickle
cell anemia. Considering that the synergism observed in the
556 CASTAN ET AL.
present study was prompted by a structural component of the
liposomal vesicles (C10), the result can be considered as an
attempt to incorporate the liposomal vesicles to the phar-
macological activity.
In summary, liposomal vesicles are valuable tools as
vehicles for vanillin administration. The fact that the
encapsulation of this molecule does not affect their phar-
macological properties supports the use of liposomes in fu-
ture pharmacokinetic and pharmacodynamic studies, both
in vivo and in vitro.
ACKNOWLEDGMENT
The authors thank the Biochemistry Group, Department
of Biology, Faculty of Natural Sciences, University of
Oriente.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist
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