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Encapsulation of Flavonoids in Liposomal Delivery Systems: Case of

Quercetin, Kaempferol and Luteolin

Article  in  Food & Function · June 2017

DOI: 10.1039/C7FO00508C

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Encapsulation of flavonoids in liposomal delivery

systems: the case of quercetin, kaempferol and
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Cite this: DOI: 10.1039/c7fo00508c

luteolin
a,b
Meigui Huang, Erzheng Su,a Fuping Zheng*b and Chen Tan *c

Received 2nd April 2017,
Accepted 19th June 2017
DOI: 10.1039/c7fo00508c
rsc.li/food-function
The instability of dietary flavonoids is currently a challenge for their incorporation in functional foods.
This study investigated the protective effects of liposome encapsulation on a variety of flavonoids and
their interaction mechanisms. It was found that the incorporation of flavonoids into the liposomal mem-
brane was strongly dependent on their structure and loading concentration. Liposomes loading quercetin
and luteolin exhibited a relatively small size and homogeneous suspension compared to those loading
kaempferol. Additionally, liposomes displayed a stronger retaining ability to quercetin and luteolin than
kaempferol during preparation, storage, heating and pH shock. After encapsulation, quercetin displayed
the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and lipid peroxidation inhibition capacity,
followed by kaempferol and luteolin. Raman and IR spectroscopy techniques demonstrated that flavo-
noids could modulate the dynamic and packing order of lipid chains, which were responsible for the
stabilization of liposomes. Our findings should guide the rational design of liposomal encapsulation
technology to efficiently deliver flavonoids in nutraceuticals and functional foods.

1. Introduction have become excellent candidates for drug delivery, including

Flavonoids, a group of natural compounds with various pheno-
lic structures, are ubiquitously distributed in fruits and veg-
etables. They have gained great interest to act as potential sca-
vengers of reactive species, owing to their protective effects
against degenerative conditions such as inflammatory, cancer
or cardiovascular diseases.1 For these reasons, flavonoids have
become very popular nutritional supplements. Unfortunately,
flavonoids are prone to oxidation and degradation because of
a highly unsaturated structure, thus decreasing their potential
health benefits.2 The low aqueous solubility and poor bioactiv-
ity further limit their utilization as nutraceutical ingredients.3
A strategy to overcome the aforementioned limitations is to
encapsulate flavonoids into water soluble carriers for chemical
and biological protection. Liposomes are artificial vesicles
formed by one or more concentric lipid bilayers separated by
water compartments. They can entrap both hydrophilic and
hydrophobic substances into their aqueous compartments and
lipid bilayers, respectively. In the last few decades, liposomes
a
Department of Food Science and Technology, College of Light Industry Science and
Engineering, Nanjing Forestry University, Nanjing 210037, PR China
b
Beijing Laboratory for Food Quality and Safety, Beijing Technology and
Business University, Beijing 100048, PR China. E-mail: zhengfp@btbu.edu.cn
c
Department of Food Science, College of Agriculture & Life Science,
Cornell University, NY, USA. E-mail: ct632@cornell.edu
epigallocatechin gallate,4 quercetin,5 fisetin,6 resveratrol,7 and
curcumin.8,9 More recently, nutraceutical compounds have
been loaded into liposomes in food formulation, including
carotenoids,10 antimicrobial agents,11 and functional pep-
tides.12 To our knowledge, however, few studies have investi-
gated the feasibility of liposomes as a delivery system for a
variety of flavonoids in food application.
Encapsulating and stabilizing capacities of liposomes are
closely related to the structure of inserted compounds.
Different structures of flavonoids could exhibit different
loading abilities in liposomal membranes and modulate
their effects on liposomal properties.13 For example, because
of the different numbers of hydroxyl groups and positions,
quercetin (3,5,7,3′,4′-pentahydroxylflavone), kaempferol
(3,5,7,4′-tetrahydroxylflavone) and luteolin (5,7,3′,4′-tetrahy-
droxylflavone) displayed distinct affinities to lipid bilayers.14
On the other hand, incorporation would also modulate the
physical properties of liposomes. Our previous studies
revealed that loading a high concentration of carotenoids
resulted in the penetration of oxygen into the liposomal
membrane and subsequent membrane instability.15
Flavonoids could exert pro-oxidant effects,16 therefore, we can
speculate that the integrity of the liposomal membrane may
suffer from lipid peroxidation upon loading excessive concen-
tration of flavonoids. This process is always accompanied by
the leakage of encapsulated nutraceuticals.17 Thus, an

This journal is © The Royal Society of Chemistry 2017 Food Funct.


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optimal flavonoid loading concentration should be found in
the design of the liposome delivery system.
Egg yolk phosphatidylcholine (EYPC), a natural phospholi-
pid, is generally recognized as a safe food ingredient. Previous
experiments in our laboratory have explored a new type of lipo-
some composed of mixed lipids including EYPC and nonionic
surfactant Tween 80. The presence of Tween 80 can confer
high flexibility and steric stability to the lipid bilayer. These
liposomes were successfully applied to increase the shelf life,
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oxidation stability and gastric stability of encapsulated caroten-
oids.10,15 In this study, our objective was to explore the poten-
tial of a liposomal delivery system for flavonoids. To systemi-
cally study the effects of the flavonoid structure on the stabi-
lity, three different kinds of flavonoids with different positions
and numbers of hydroxyl groups were selected, including quer-
cetin (3,5,7,3′,4′-hydroxyl groups), kaempferol (3,5,7,4′-hydroxyl
groups) and luteolin (5,7,3′,4′-hydroxyl groups), as shown in
Fig. 1. Liposomal vehicles composed of EYPC and Tween 80
were prepared by thin-film evaporation integrated with the
sonication dispersion method. The flavonoid encapsulation
efficiency of liposomes and their stability were investigated.
Furthermore, the comparative antioxidant capacity of querce-
tin-, kaempferol- and luteolin-loaded liposomes was deter-
Fig. 1 Molecular structure of quercetin (3,5,7,3’,4’-pentahydroxylfl-
avone), kaempferol (3,5,7,4’-tetrahydroxylflavone) and luteolin (5,7,3’,4’-
tetrahydroxylflavone). The red circle emphasizes the difference in the
critical position of the hydroxyl group for target compounds.
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mined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and
lipid peroxidation assays. The relationship between the lipo-
some stability and structural change of the liposomal mem-
brane resulting from the insertion of flavonoids was discussed.
2. Materials and methods
2.1. Materials
Quercetin, kaempferol and luteolin (all 98% purity) were pur-
chased from Shanghai yuanye Bio-Technology Co., Ltd
(Shanghai, China). Egg yolk phosphatidylcholine (EYPC, com-
posed of approximately 80% phosphatidylcholine, 5% lysopho-
sphatidylcholine and phosphatidic acid, and 15% fatty acid)
was purchased from the Chemical Reagent Plant of East China
Normal University (Shanghai, China). 2,2-Diphenyl-1-picryl-
hydrazyl (DPPH) was purchased from Sigma Chemical Co.
(St Louis, MO, USA). L-Ascorbic acid, FeCl3 and Tween 80 were
purchased from Thermo Fisher scientific Inc. All other
reagents were of analytical grade.
2.2. Preparation of flavonoid-loaded liposomes
Flavonoid-loaded liposomes were prepared by the thin-film
evaporation method according to our recent report.18 Briefly, a
weighed amount of flavonoids was dissolved in 2 mL ethanol
together with the lipids (1.72 g) composed of EYPC and Tween
80 at a fixed mass ratio of 1 : 0.72 under magnetic stirring for
30 min. The total amount of flavonoids was adjusted to 1, 3,
and 5%, which was expressed relative to the mass of lipids
through the initial flavonoid concentration, mflavonoid/mlipids
(% wt/wt). After dissolution, the liposomal system was brought
to dryness by reduced pressure using a rotary evaporator, fol-
lowed by further vacuum-drying in an oven at 30 °C overnight
to completely remove the solvent. The achieved thin film was
then hydrated with 0.01 M phosphate-buffered saline (150 mM
NaCl, pH 7.4) under stirring at 300 rpm for 2 h at 55 °C. To
achieve homogeneous liposomes, the liposomal suspensions
were then subjected to probe sonication processing in an ice
bath for 10 min at 300 W with a sequence of sonication for 5 s
and rest for 5 s using a sonicator (Thermo Fisher Scientific™
Model 505 Sonic Dismembrator, USA). The final concen-
trations of EYPC and Tween 80 were kept at 12.5 g L−1 and
9 g L−1, respectively. The pure liposome was prepared in the
same way without the addition of flavonoids. Finally, the
samples were sealed in headspace vials with nitrogen flushing
and kept in a refrigerator at 4 °C in the dark until use.
2.3. Particle size distribution analysis
The particle size and polydispersity index (PDI) of liposomes
were analyzed by dynamic light scattering (DLS) using a
Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) with
a He/Ne laser (λ = 633 nm) at 25 °C. To eliminate multiple
scattering phenomena by particle interaction, the liposome
dispersion was diluted 10-fold with the same phosphate
buffer.19 The average particle diameter and PDI were calcu-
lated from 14 runs for each recording.
This journal is © The Royal Society of Chemistry 2017

Food & Function
2.4. Raman spectra analysis
Raman spectra of liposomes were obtained using a DXR
Raman microscope (Thermo Fisher Scientific, Waltham, MA)
equipped with a 780 nm excitation laser and a 10× objective.
The resulting laser spot diameter was about 3.0 μm with a
spectral resolution of 5 cm−1. The Raman measurements were
performed with 24 mW, 50 μm slit aperture and 2 s integration
time. The Raman samples ( pure liposomes and flavonoid-
loaded liposomes) were prepared as described above. An ordin-
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ary Raman spectrum was baseline-corrected, and the Raman
intensities were measured as peak heights. To give insight into
the degree of longitudinal order of liposomes, changes in the
choline group and lateral packing of the chains and
trans/guache population ratio were recorded.
2.5. Encapsulation efficiency (EE)
The encapsulation efficiency (EE) was indicated by the ratio of
partitioning between the lipid membrane and water phase.
Analysis on flavonoid partitioning between the lipid mem-
brane and water phase was based on our previous report.20
Briefly, a weighted amount of flavonoid was dissolved in 10 μL
of dimethylsulphoxide (DMSO) solution, and then mixed with
1 mL of pure liposomes and the final concentration of flavo-
noid was equal to that in flavonoid-loaded liposomes. Taking
1% quercetin-loaded liposome for example, 0.01 mL of flavo-
noid-DMSO solution was mixed with 1 mL of pure liposomes.
The flavonoid partitioning between liposomes and the water
phase was calculated by plotting the intensity of the Raman
characteristic peak of flavonoids in liposomes (Ilipo) divided to
that in the direct mixture of flavonoids and pure liposomes
(Imix).
2.6. Stability assays of flavonoid-loaded liposomes
To evaluate the shelf life of the liposomes, the samples were
stored for 26 days under a N2 atmosphere at 4 °C and at 2 days
intervals, two milliliters of the sample were taken out to
measure the EE and particle size. The measurement of the
leaked flavonoid during storage was carried out in the same
way as described above for the determination of flavonoid par-
titioning. To evaluate the stability of liposomes against pH,
1 mL of liposomes was incubated in 0.01 M phosphate saline
buffer (150 mM NaCl) at pH 2.5, 5.0, 7.4, and 8.5 at room
temperature for 2 h. In the case of temperature assay, lipo-
somes were incubated at different temperatures (20, 37, 55 and
80 °C) at pH 7.4 in a shaking water bath at 100 rpm for 2 h.
Afterwards, the liposomes were taken out for measurements.
2.7. DPPH radical scavenging assay
The DPPH radical-scavenging activity of flavonoid-loaded lipo-
somes was measured according to our previous method with a
slight modification.15 Two milliliters of liposomes were mixed
well with 0.5 mL of DPPH solution (1 mM in ethanol) followed
by incubation at 37 °C for 40 min in the dark. The control was
prepared by replacing the sample with a mixture of distilled
water and DPPH solution. A blank sample was prepared by
This journal is © The Royal Society of Chemistry 2017
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replacing the DPPH solution with ethanol. Then, the absor-
bance of the solution after incubation was determined at
525 nm using a UV spectrometer (SpectraMax M2, Molecular
Devices, Sunnyvale, CA, USA). A lower absorbance of the solu-
tion indicates higher free radical scavenging ability. The ability
of DPPH scavenging (DPPHscav) was calculated using the fol-
lowing equation:
 
Asample  Acontrol
DPPHscav ð%Þ ¼ 1   100:
Ablank
2.8. Assay of lipid peroxidation
Lipid peroxidation was evaluated by measuring the final
product malonaldehyde (MDA) induced by Fe3+/ascorbate
according to an earlier method with a slight modification.19
MDA is a colored complex formed by the reaction of fatty acid
with thiobarbituric acid (TBA). A high absorbance indicates a
high lipid oxidation. Briefly, one milliliter of liposome was
mixed with FeCl3 (1 mL, 1 mM) and ascorbic acid (1 mL,
1 mM) followed by incubation at 37 °C for 60 min. Then, 5 mL
of TBA–TCA–HCl solution containing TBA (15%, w/v), trichlor-
oacetic acid (0.4%, w/v) and hydrochloric acid (2.0%, w/v) was
added and heated at 100 °C for 30 min. The mixture was
cooled immediately in an ice bath to quench the lipid peroxi-
dation reaction, centrifuged for 5 min at 2000 rpm and fil-
tered. Afterwards, the absorbance of the supernatant was
recorded by using a spectrometer at 535 nm (A535nm). The
change in thiobarbituric acid-reactive substances (TBARS) was
calculated as follows:
Ac  As
Change in TBARS ð%Þ ¼  100
Ac
where As and Ac are the absorbance of flavonoid-loaded lipo-
somes and flavonoid-free liposomes, respectively.
2.9. FTIR measurements
The attenuated total reflectance (ATR)-FTIR spectra were col-
lected by using an IR Affinity-1S spectrometer equipped with a
single-reflection ATR accessory (Shimadzu Corp., Kyoto,
Japan). The liposomes were directly spread on the surface of
the ZnSe-ATR crystal. After air drying, the infrared absorption
spectra were recorded at a resolution of one data point every
8 cm−1 in the region of 800–4500 cm−1.
2.10. Statistical analysis
All measurements were repeated in triplicate. All data are pre-
sented as means ± standard deviations. Data were analyzed
using one-way analysis of variance with p < 0.05 of
significance.
3. Results and discussion
3.1. Flavonoids loading
Hydrophobic flavonoids, quercetin, kaempferol and luteolin
could be inserted into the lipid bilayer because the liposome
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bilayer contains both hydrophobic and hydrophilic parts.17
Raman spectroscopy has been recently proved as a facile and
powerful technique for on-line quantitative analysis of multi-
component samples.21 Previously, we successfully evaluated
the carotenoid encapsulation efficiency of lipid vesicles by
determining the Raman scattering from their characteristic
peaks.20 In this study, the represented Raman spectra of the
three flavonoid-loaded liposomes are presented in Fig. 2A. The
characteristic peaks of liposomes were observed at 1654, 1301,
and 1441 cm−1, which were derived from ν(CvO), ν(C–C)ring
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respectively. One proposed explanation was that large amounts
of inserted flavonoids led to the saturation of the bilayer, thus
decreasing the encapsulation efficiency.20 Interestingly, the
intensity ratios of kaempferol-loaded liposomes were main-
tained at a high level within the tested concentration. The
higher encapsulation efficiency of kaempferol was probably
due to its flexible location either in the hydrophilic head or in
the hydrophobic core.14
Particle size and PDI reflect the colloidal stability and par-

ticle distribution. Fig. 2B shows the concentration dependence

and δ(CH2), respectively. The most intense characteristic peak
of flavonoids was found at around 1600 cm−1, which was
assigned to ν(CvO) from phenyl ring vibrations in the central
heterocyclic ring. This peak was sensitive to the OH bends on
related molecules.22,23 In our case, it was shifted to 1615, 1611
and 1575 cm−1 for quercetin, kaempferol and luteolin, respect-
ively. By comparing the intensity of flavonoids encapsulated in
liposomes (Ilip) to that of the mixture of pure liposomes and
flavonoids (Imix), the intensity ratios at these characteristic
peaks (Ilip/Imix) can reflect the encapsulation efficiency (EE) of
liposomes to flavonoids. The decrease in Ilip/Imix was an indi-
cation of EE reduction. The insert in Fig. 2A shows the flavo-
noid partitioning by plotting Ilip/Imix versus various loading
concentrations. It was found that the values of Ilip/Imix pro-
gressively decreased with the increase of flavonoid concen-
tration, suggesting the decrease of encapsulation efficiency. In
the case of quercetin and luteolin, their intensity ratios greatly
decreased when the concentration was >1%. The Ilip/Imix of
liposomes loading 5% quercetin and luteolin decreased by
40.34% and 35.09% compared to those loading 1% flavonoid,
of liposomal particle size. As the loading concentration was
increased, the particle size progressively increased. This vari-
ation trend was more apparent for kaempferol. The particle
size of liposomes was increased 2.12-, 0.84-, and 1.05-fold after
the incorporation of 5% kaempferol, quercetin and luteolin,
respectively. This phenomenon was closely related to the
location and orientation of flavonoids in the liposomal mem-
brane. It has been demonstrated that flavonoids showed a
broad transverse distribution in the lipid membrane according
to the polarity of respective flavonoids.24 The spanning orien-
tation could expand the liposome volume.25 Additionally, it
was possible that the mechanical properties of the EYPC mem-
brane were affected by flavonoid incorporation, so that the
ultrasonic treatment cannot effectively break the large multila-
meller liposomes into small unilamellar particles.10 The PDI
data further explained the particle size change. At 1% and 3%,
the PDI values of flavonoid-loaded liposomes were below or
very close to 0.3 (Fig. 2C), indicating a relatively homogeneous
and acceptable system.26 However, at 5% the PDI values were
greatly increased to 0.533 and 0.416 for kaempferol and luteo-

Fig. 2 (A) Raman spectra and peak assignments of pure liposomes and flavonoid-loaded liposomes. All spectra were averaged and presented in the
baseline scale. The loading concentration of flavonoids was 5%. The inset graph was the loading concentration dependence of encapsulating efficacy
(EE, %) as deduced from the Raman intensity ratio (Ilip/Imix). Each point is expressed as the mean value (n ≥ 8). (B) Average particle size and (C) poly-
dispersity index (PDI) of liposomes loading different concentrations of flavonoids. Each point is expressed as the mean value ± standard deviation (n = 3).

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lin, respectively. It was believed that the incorporation of high
concentration of luteolin and kaempferol, especially the latter,
broadened the size distribution of liposomes and led to the
heterogeneous suspension.
3.2. Storage stability of flavonoid-loaded liposomes
As liposomes are thermodynamically unstable systems, the
particles may tend to degrade or aggregate, resulting in the
leakage of entrapped compounds during storage.19 Here, we
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evaluated the shelf life of flavonoid-loaded liposomes by moni-
toring the change in EE, particle size and PDI during storage
at 4 °C in the dark. As presented in Fig. 3, liposomes loading
1% and 3% flavonoids displayed a slow release within 12 days
of storage, indicating the strong retaining ability of the liposo-
mal membrane to the flavonoids. In contrast, a considerable
leakage was observed for liposomes loading 5% flavonoids
throughout the storage period. Approximately 60.4% of the
incorporated kaempferol leaked after 26 days of storage, much

Fig. 3 Change in EE (%), average particle size (nm) and polydispersity index (PDI) of liposomes loading different concentrations of quercetin (A, D
and G), luteolin (B, E and H) and kaempferol (C, F and I) during storage at 4 °C in the dark. The data are acquired by dynamic light scattering and
expressed as the mean ± standard deviation (n = 3).

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higher than 15.5% of quercetin and 29.4% of luteolin. The
stability of liposomes was affected by the orientation of flavo-
noids in the liposomal membrane, involving the lipophilicity
and planar structure. Owing to the great planarity and hydro-
philicity, luteolin and quercetin could decrease the per-
meability of the liposomal membrane.27 Moreover, it was
proved that quercetin and luteolin exhibited high affinity to
liposomes due to the good match between their planar con-
figuration and liposomes.28,29 Therefore, quercetin and luteo-
lin played rigidifying roles on the membrane.30 Because of the
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flexible position, some kaempferol molecules at high concen-
tration may adopt a horizontal orientation to the liposomal
membrane. Such a position probably caused the disorganizing
effects and accelerated the leakage of kaempferol.
The change of average particle size and PDI confirmed the
EE data. The stability of flavonoid-loaded liposomes displayed
strong flavonoid structure- and concentration-dependency. At
1%, only a slight particle aggregation was observed during
storage, while further increasing loading concentration to 3%
and 5%, the particles would aggregate seriously from 15 days.
The aggregation was more apparent in the case of kaempferol
and luteolin. The average particle size increased by 124% and
268% for liposome loading 5% kaempferol and luteolin after
26 days, respectively. On the other hand, the PDI values of lipo-
Fig. 4 Average particle size (nm) of liposome loading different concentrations of quercetin (A, D), luteolin (B, E), and kaempferol (C, F) as a function
of temperature and pH. Each point represents the mean value ± standard deviation (n = 3).
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some loading 1% and 3% quercetin and luteolin were below or
very close to 0.3 during the whole storage period, indicating
a homogeneous suspension. However, the PDI values of
kaempferol-loaded liposomes progressively increased during
storage regardless of the loading concentration. The values
even reached 0.558, 0.822 and 0.805 after 26 days for 1%, 3%
and 5%, respectively. Based on these data, we concluded that
flavonoids exhibited different storage stability at 4 °C in the
dark, ranging from the strongest to the weakest: quercetin >
luteolin > kaempferol.
3.3. Resistant ability against temperature and pH
To investigate the resistance of liposomes against external
environmental conditions, the change of particle size was
monitored as a function of temperature and pH. Fig. 4A shows
that pure liposomes underwent a slight particle size change
from 4 to 55 °C, while beyond this range a sudden increase in
particle size was observed. As DLVO theory described, once the
velocity or kinetic energy of the particles is high enough to
overcome the energy barrier of electrostatic repulsion, they will
collide and aggregate. There was no doubt that the energy
input by temperature was the main reason for the liposomal
particle aggregation. Note that after the incorporation of quer-
cetin and luteolin, liposomes exhibited high resistance to
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temperature with a slight particle size change even at 80 °C.

The resistance ability was stronger when loading low concen-
tration of flavonoids (1 and 3%). In contrast, a serious aggrega-
tion of liposomes was found at a high loading concentration
of kaempferol (3 and 5%). At 5%, the particle size was
increased by 21.57%, 12.14%, and 39.78% after 2 h heat treat-
ment at 80 °C for quercetin, luteolin and kaempferol, respect-
ively. The different temperature resistant abilities of liposomes
could be explained by the different rigidifying effects of flavo-
noids on the liposome membrane.10
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Fig. 4 also shows that the particle size was increased after
incubation at both acid and alkaline pH. Compared to lipo-
somes loading kaempferol, those loading quercetin and luteo-
lin exhibited a slight size change with pH (Fig. 4D, E and F). It
was reported that pH can significantly affect the position of
flavonoid molecules in the lipid bilayer. That is, flavonoids
tend to distribute in the hydrophobic region due to the proto-
nation of the phenolic hydroxyl group in more acidic pH,
while in alkaline pH it would interact with polar head groups
due to the deprotonation effect.31 Quercetin and luteolin were
able to deeply embed in planar lipid bilayers at acidic pH, but
preferentially interacted with polar head groups at the lipid–
water interface at physiological pH.14,31 That is why quercetin
and luteolin incorporation led to a small particle size change
against pH. Kaempferol has higher pKa values (7.05) than
quercetin (6.30) and luteolin (6.50).32 It suggested a higher
deprotonation of polar groups in kaempferol and thus a
shallow penetration into the lipid bilayer. The kaempferol Fig. 5 DPPH scavenging (A) and TBARS change (B) of liposomes at
molecules that reside near the water-accessible surface would different concentrations of flavonoids. Results are expressed as the
induce serious liposome aggregation.33 mean ± standard deviation (n = 3). Different letters represent a signifi-
cant difference ( p < 0.05).
3.4. Antioxidant activity
A DPPH model based on the reduction of DPPH• in the pres-
ence of hydrogen-donating molecules has been widely used to give rise to the weakest DPPH scavenging activity of luteolin.
evaluate the free radical scavenging effectiveness in food Additionally, the antioxidant activity of flavonoids was
systems.34 In the DPPH assay, the absorbance decreases when correlated with their modification of membrane fluidity.36–38
the color changes from purple to yellow. Fig. 5A shows the Quercetin was demonstrated to rigidify the hydrophobic
DPPH• scavenging ability of liposomes at different concen- regions of membrane lipid bilayers.39 The localization of luteo-
trations of flavonoids. With the increase of flavonoid concen- lin into the membrane interior also favored the restrictions on
tration, the DPPH scavenging activity was significantly the fluidity of membrane components, which could sterically
increased ( p < 0.05). It is understandable because the flavo- hinder the diffusion of radical species and decrease the radical
noids served as “guarding” molecules in preventing the pene- reaction.40
tration of acyl chain regions by radical species.14,35 The trend The liposomal membrane may suffer from peroxidation due
of antioxidant activity ranging from the strongest to the to the polyunsaturated acyl chain structure in the processing
weakest was quercetin > kaempferol > luteolin. The higher the and storage period of liposomes. Antioxidant efficiency against
loading concentration of flavonoid, the stronger the scaven- lipid peroxidation in an iron dependent system initiated by
ging capacities were. At 5%, the scavenging values were appro- Fe3+/ascorbate was widely evaluated by TBARS assay.16 The
priately 73.97%, 67.07% and 53.73% for quercetin, kaemp- effect of flavonoid incorporation on the TBARS production of
ferol, and luteolin, respectively. The DPPH scavenging activity liposomes was determined, as shown in Fig. 5B. It was clearly
is mainly affected by the structural features in flavonoids, i.e. observed that the change in TBARS for pure liposome (0%)
the number and position of the hydroxyl group and its capa- was considerably increased to 116.3%, indicating the serious
bility to donate hydrogen. The number of hydrogen donors is lipid peroxidation by iron. The incorporation of flavonoids can
5 (quercetin), 4 (kaempferol) and 4 (luteolin). Note that 3-OH significantly inhibit the lipid peroxidation, depending on the
is highly susceptible to radicals,36 which could explain the type and concentration of flavonoids. Higher inhibition ability
high scavenging activity of quercetin and kaempferol. In con- was found at 3% ( p < 0.05). Additionally, quercetin exerted the
trast, the absence of 3-OH and fewer hydrogen donors might strongest inhibition ability of lipid peroxidation, followed by

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kaempferol and luteolin. Similar to the DPPH scavenging
activity, the lipid peroxidation inhibition capacity is closely
related to the orientation of flavonoids in the lipid bilayer. Due
to the broad dynamic distribution in the membrane, flavo-
noids can access all segments of the lipid molecules, thus pre-
venting peroxidation efficiently.24 It should be also pointed out
that flavonoids could act as both antioxidants and pro-
oxidants.15,16 High loading of flavonoids may fragment the
liposomes, resulting in the destabilization and disruption of
the phospholipid arrangement.26 That may be the reason that
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the antioxidant ability was decreased at 5% of flavonoids.
3.5. Structure properties of liposomes
IR and Raman spectroscopy techniques are recognized as
quick and efficient tools by providing vibrational spectroscopy
on a “submolecular” level. Liposomes containing fatty acids
and phosphide groups have characteristic absorption bands.
These absorption-active groups in phospholipids are sensitive
to foreign biological compounds like flavonoids.41,42
To investigate the structural change of the lipid bilayer by
flavonoid incorporation, the choline group conformation of
polar head, acyl chain conformation, and the intra- and inter-
molecular membrane order were monitored by the change of
Raman intensities.20 The gauche conformation of the C–C
bond in the choline group (O–C–C–N+) appeared in the range
of 710–720 cm−1, which would transfer to trans conformation
located at about 770 cm−1 in the doped lipid bilayer.43 In our
case, no observable band at around 770 cm−1 was detected in
pure and flavonoid-loaded liposomes, indicating that the
choline group mainly took on the gauche conformation. The
Raman band of C–C from the angle vibration at 1444 cm−1 is
relatively insensitive to the polar head group. Therefore, we
applied the peak intensity ratio (I718/I1444) to semi-quantify the
variation of the gauche conformation.20 It was found that this
ratio decreased sharply after the incorporation of 5% kaemp-
ferol, but slightly with quercetin and luteolin (Fig. 6). One prob-
able reason was that the multiple hydroxyl groups of flavo-
Fig. 6 Order parameters of liposomes loading different concentrations of flavonoids as deduced from Raman spectra. Each point is expressed as
the mean value ± standard deviation (n = 3).
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Food & Function
noids tended to render the lipid bilayer more planar, thus
inducing some degree of gauche conformation.44 Additionally,
the decrease of the number of gauche conformers resulting
from flavonoid incorporation suggested a new spatial arrange-
ment of these lipid parts and enhancement of the molecule
packing.45
A distinct feature in lipid bilayers is the bands located at
2885 and 2854 cm−1 derived from the methylene C–H antisym-
metric stretching and symmetric stretching, respectively. The
changes in both the lateral packing of acyl chains and the
trans–gauche population ratio are expressed as the ratio of
intensity of 2885 to 2854 cm−1.20 Fig. 6 shows that the modu-
lating effects of flavonoids on the lipid chain lateral packing
were concentration dependent. The ratio I2885/I2854 increased
with increasing loading concentration of flavonoids from 1%
to 3%. However, further increase of the loading content would
lead to the decrease of this ratio. It is reasonable because the
insertion of hydrophobic flavonoids tends to fluidify the lipo-
somal membrane due to the presence of stiffer cyclic carbon
rings, and thereby disrupted the compact packing lipids.30
IR spectroscopy can provide a possibility to monitor the
alternations of vibration frequency features for different parts
of lipid molecules. Fig. 7 presents the infrared absorption
spectra of pure liposomes and flavonoid-loaded liposomes
ranging from 600 to 2000 cm−1. Some characteristic peaks of
liposomes were presented at 1236 cm−1, 1096 cm−1 and
970 cm−1, which were derived from νas (PO2−), νs (PO2−), and
νas (N+–CH3), respectively. The major characteristic peaks of
–OH phenolic bending from flavonoids are located at around
1300–1400 cm−1. The relatively strong band located at
1735 cm−1 represents the stretching vibrations of ester carbo-
nyl groups, i.e. ν(CvO) in the polar–apolar interface region of
the lipid bilayer. The position of the ν(CvO) band maximum
is sensitive to the conformation and/or orientation of ester
groups and to the hydration level of the carbonyl region in the
lipid bilayer. In our case, the position of ν(CvO) is close to the
lower wavelength number compared to previous reports.46 This
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Food & Function
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Fig. 7 IR spectra of liposomes loading different concentrations of quercetin (A), luteolin (B) and kaempferol (C) in the range of 600–2000 cm−1.
was probably due to the presence of the hydrogen bond
between the proton-donor OH group of flavonoids and the
proton-acceptor CvO group of lipids.47 The relatively weak
band at 1651 cm−1 is the result of CvO vibrations from the
central heterocyclic ring. It is noteworthy that the ν(CvC)
band appeared at 1600 cm−1, which can be interpreted as the
insertion of flavonoids into the lipid resulting in the dramatic
changes of the infrared absorption spectra.48 Note that an
additional band was found at 1565 cm−1 in kaempferol, but
was not observable in quercetin- and luteolin-loaded lipo-
somes. This phenomenon implied the involvement of inter-
actions between the functional group of flavonoids and the
lipid membrane through the hydrogen bond.48 Additionally,
the band representing the PO2− antisymmetric stretching
slightly shifted from 1236 to 1244, 1244 and 1241 cm−1 after
the incorporation of quercetin, kaempferol and luteolin,
respectively. The observation supports the concept of the invol-
vement of polar groups of the flavonoids but can also be an
indication of the flavonoid localization with respect to the type
of interaction with the lipid.49 The presence of the band at
1565 cm−1 only in kaempferol provides a proof of different
interactions with the lipid among flavonoids. As discussed pre-
viously, quercetin and luteolin well matched with liposomes,
which could rigidify the molecules. Nevertheless, kaempferol
orients flexibly to the lipid membrane and loosens the packing
of lipid molecules.
4. Conclusions
We performed a systematic study on the potential of liposomal
encapsulation for three different kinds of flavonoids.
Furthermore, the relationship between the organization of
incorporated flavonoids in a lipid bilayer and the chemical
structure was revealed. It has been demonstrated that lipo-
somes displayed different encapsulating capacities to flavo-
noids because of the various structures of flavonoids and
loading concentration. The stability and antioxidant assays
revealed a mutually protective relationship between incorpor-
ated flavonoids and liposomes. Liposomes loading quercetin
exhibited higher stability and stronger antioxidant capacity
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than those loading luteolin and kaempferol. Raman and IR
spectroscopy techniques demonstrated that flavonoids could
modulate the dynamics and structures of liposomal mem-
branes, highly depending on their molecular structures and
incorporation concentration. These modulations were closely
related to the stabilization of liposomes. Our findings
suggested that liposomes could be used as an ideal carrier to
deliver flavonoids in functional foods.
Acknowledgements
This research was financially supported by projects of the
National Natural Science Foundation of China for Young
Scholars (31401679 and 31401559); the priority academic
program development of Jiangsu higher education institutions
(PAPD), a grant from Nanjing Forestry University (Grant
Number GXL2014047) and the fund of the Beijing Laboratory
for Food Quality and Safety, Beijing Technology and Business
University.
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