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Linking the chemistry and physics of food with health and nutrition
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This article can be cited before page numbers have been issued, to do this please use: M. Huang, E. Su,
F. Zheng and C. Tan, Food Funct., 2017, DOI: 10.1039/C7FO00508C.
Volume 7 Number 1 January 2016 Pages 1–612 This is an Accepted Manuscript, which has been through the
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Linking the chemistry and physics of food with health and nutrition Accepted Manuscripts are published online shortly after
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Page 1 of 34 Food & Function
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DOI: 10.1039/C7FO00508C
4 1 Department of Food Science and Technology, College of Light Industry Science and
6 2 Beijing Laboratory for Food Quality and Safety, Beijing Technology and Business
10 ∗Corresponding author
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18 ABSTRACT
24 and luteolin exhibited relatively small size and homogeneous suspension compared to
26 to quercetin and luteolin than kaempferol during preparation, storage, heating and pH
30 techniques demonstrated that flavonoids could modulate the dynamic and packing
31 order of lipid chains, which were responsible for the stabilization of liposomes. Our
32 findings should guide the rationale designing for liposomal encapsulation technology
35 functional food.
36
37
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39
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40 1. INTRODUCTION
42 ubiquitous distributed in fruits and vegetables. They have gained great interests to act
47 unsaturated structure, thus decreasing their potential health benefits2. The low
48 aqueous solubility and poor bioactivity further limit their utilization as nutraceutical
49 ingredients3.
51 into water soluble carriers for chemical and biological protection. Liposomes are
52 artificial vesicles formed by one or more concentric lipid bilayers separated by water
53 compartments. They can entrap both hydrophilic and hydrophobic substances into
54 their aqueous compartment and lipid bilayer, respectively. In the last decades,
57 the nutraceutical compounds have been loaded into liposomes in food formulation,
3
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67 distinct affinities with lipid bilayer14. On the other hand, incorporation would also
68 modulate the physical properties of liposomes. Our previous studies revealed that
72 membrane may suffer from the lipid peroxidation once loading excessive
78 explored a new type of liposomes composed of mixed lipids including EYPC and
79 nonionic surfactant Tween 80. The presence of Tween 80 can confer the lipid bilayer
80 to high flexibility and steric stability. These liposomes were successfully applied to
81 increase the shelf life, oxidation stability and gastric stability of encapsulated
82 carotenoids10, 15
. In this study, our objective was to explore the potential of a
4
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84 flavonoid structure on the stability, three different kinds of flavonoids with different
90 and its stability were investigated. Furthermore, the comparative antioxidant capacity
96 2.1. Materials
97 Quercetin, kaempferol and luteolin (all 98% purity) were purchased from Shanghai
100 lysophosphatidylcholine and phosphatidic acid, and 15% fatty acid) was purchased
101 from Chemical Reagent Plant of East China Normal University (Shanghai, China).
102 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St.
103 Louis, MO, U.S.A.). L-ascorbic acid, FeCl3 and Tween 80 were purchased from
104 Thermo Fisher scientific Inc. All other reagents were of analytical grade.
5
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107 according to our recent report18. Briefly, a weighted amount of flavonoids was
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108 dissolved in 2 mL ethanol together with the lipids (1.72 g) composed of EYPC and
109 Tween 80 at a fixed mass ratio of 1:0.72 under magnetic stirring for 30min.The total
111 mass of lipids through the initial flavonoid concentration, mflavonoid/mlipids (% wt/wt).
112 After dissolution, the liposomal system was brought to dryness by reduced pressure in
114 completely remove the solvent. The achieved thin film was then hydrated with 0.01 M
115 phosphate-buffered saline (150 mM NaCl, pH 7.4) under stirring at 300 r/min for 2 h
116 at 55 oC. To achieve homogenous liposomes, the liposomal suspensions were then
117 subjected to probe sonication processing in ice bath for 10 min at 300 W with a
118 sequence of sonication for 5 s and rest for 5 s using a sonicator (Thermo Fisher
119 Scientific™ Model 505 Sonic Dismembrator, USA). The final concentrations of
120 EYPC and Tween 80 were kept at 12.5 g/L and 9 g/L, respectively. The pure liposome
121 was prepared in the same way without addition of flavonoids. Finally, the samples
122 were sealed in headspace vials with nitrogen flushing and kept in refrigerator at 4 oC
125 The particle size and polydispersity index (PDI) of liposomes were analyzed by
127 Malvern, UK) with a He/Ne laser (λ=633 nm) at 25 °C. To eliminate multiple
6
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129 folds with the same phosphate buffer19. The average particle diameter and PDI were
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133 Fisher Scientific, Waltham, MA) equipped with a 780 nm excitation laser and a 10 ×
134 objective. The resulting laser spot diameter was about 3.0 µm with a spectral
135 resolution of 5 cm-1. The Raman measurements were performed with 24 mW and 50
136 µm slit aperture for 2 s integration time. The Raman samples (pure liposomes and
138 spectrum was baseline-corrected, and the Raman intensities were measured as peak
139 height. To give insight to the degree of longitudinal order of liposomes, changes in the
140 choline group and lateral packing of the chains and trans/guache population ratio were
141 recorded.
143 The encapsulation efficiency (EE) was indicated by the ratio of partitioning between
144 lipid membrane and water phase. Analysis on flavonoid partitioning between lipid
145 membrane and water phase was based on our previous report20. Briefly, a weighted
147 and then mixed with 1 mL of pure liposomes, the final concentration of flavonoid was
7
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150 liposomes. The flavonoids partitioning between liposomes and water phase was
152 liposomes (Ilipo) rationed to that in direct mixture of flavonoid and pure liposomes
153 (Imix).
155 To evaluate the shelf life of the liposomes, the samples were stored for 26 days under
156 N2 atmosphere at 4 oC and at 2 days intervals, two milliliter of the sample was taken
157 out to measure the EE and particle size. The measurement of leaked flavonoid during
158 storage was carried out in the same way as described above for the determination of
160 liposomes was incubated in 0.01M phosphate saline buffer (150 mM NaCl) at pH 2.5,
161 5.0, 7.4, and 8.5 at room temperature for 2 h. In the case of temperature assay,
162 liposomes were incubated at different temperatures (20, 37, 55 and 80 oC) at pH 7.4 in
163 a shaking water bath at 100 r/min for 2 h. Afterward, the liposomes were taken out for
164 measurements.
167 according to our previous method with a slight modification15. Two milliliter of
168 liposomes was mixed well with 0.5 mL of DPPH solution (1 mM in ethanol) followed
169 by incubation at 37 °C for 40 min in the dark. The control was prepared by replacing
170 the sample with mixture of distilled water and DPPH solution. A blank sample was
171 prepared by replacing the DPPH with ethanol. Then, the absorbance of solution after
8
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173 Molecular Devices, Sunnyvale, CA, USA). Lower absorbance of the solution
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174 indicates higher free radical scavenging ability. The ability of DPPH scavenging
177 Lipid peroxidation was evaluated by measuring the final product malonaldeyde
179 modification19. MDA is a colored complex formed by the reacting of fatty acid with
180 thiobarbituric acid (TBA). A high absorbance indicates a high lipid oxidation. Briefly,
181 one milliliter of liposome was mixed with FeCl3 (1 mL, 1 mM) and ascorbic acid (1
183 TBA-TCA-HCl solution containing TBA (15%, w/v), trichloroacetic acid (0.4%, w/v)
184 and hydrochloric acid (2.0%, w/v) was added and heated at 100 °C for 30 min. The
185 mixture was cooled immediately with ice bath to quench the lipid peroxidation
186 reaction, centrifuged for 5 min at 2000 r/min and filtered. Afterward, the absorbance
9
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192 The attenuated total reflectance (ATR)-FTIR spectra were collected by IR Affinity-1S
194 Kyoto, Japan). The liposomes were directly spread on the surface of the ZnSe-ATR
195 crystal. After air dried, the infrared absorption spectra were recorded at a resolution of
198 All measurements were repeated triplicate. All data are presented as means± standard
199 deviations. Data were analyzed using one-way analysis of variance with p<0.05 of
200 significance.
203 Hydrophobic flavonoids, quercetin, kaempferol and luteolin could insert into the
204 lipid bilayer because liposome bilayer contains both hydrophobic and hydrophilic
205 parts17. Raman spectroscopy has been recently proved as a facilitate and powerful
208 determining the Raman scattering from their characteristic peaks20. In this study, the
209 represented Raman spectra of the three flavonoids-loaded liposomes were presented
210 in Fig. 2A. The characteristic peaks of liposomes were observed at 1654, 1301, and
211 1441 cm-1, which were derived from ν(C=O), ν(C-C)ring and δ(CH2), respectively. The
212 most intense characteristic peak of flavonoids was found at around 1600cm-1, which
213 was assigned to ν(C=O) from phenyl ring vibrations in the central heterocyclic ring.
10
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214 This peak was sensitive to the OH bends on related molecules22, 23. In our case, it was
215 shifted to 1615, 1611 and 1575 cm-1 for quercetin, kaempferol and luteolin,
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217 to that of mixture of pure liposome and flavonoids (Imix), the intensity ratios at these
220 Insert in Fig. 2A shows the flavonoids partitioning by plotting Ilip/Imix versus various
221 loading concentrations. It was found that the values of Ilip/Imix progressively decreased
222 with the increase of flavonoid concentration, suggesting the decrease of encapsulation
223 efficiency. In the case of quercetin and luteolin, their intensity ratios greatly decreased
224 when concentration was >1%. The Ilip/Imix of liposomes loading 5% quercetin and
225 luteolin decreased by 40.34% and 35.09% compared to those loading 1% flavonoid,
226 respectively. One proposed explanation was that the large amounts of inserted
227 flavonoids led to the saturation of the bilayer, thus decreasing the encapsulation
229 maintained at a high level within the tested concentration. The higher encapsulation
230 efficiency of kaempferol was probably due to its flexible location either in the
232 Particle size and PDI reflect the colloidal stability and particle distribution. Fig. 2B
233 shows the concentration dependence of liposomal particle size. As the loading
234 concentration was increased, the particle size was progressively increased. This
235 variation trend was more apparent for kaempferol. The particle size of liposomes was
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236 increased 2.12-, 0.84-, and 1.05-fold after incorporation of 5% kaempferol, quercetin
237 and luteolin, respectively. This phenomenon was closely related to the location and
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239 flavonoids showed a broad transverse distribution in lipid membrane according to the
241 liposome volume25. Additionally, it was possible that the mechanical property of
242 EYPC membrane was affected by flavonoid incorporation, so that the ultrasonic
243 treatment cannot effectively break into the large multilamer liposomes into small
244 unilamelar particles10. The PDI data further explained the particle size change. At 1%
245 and 3%, the PDI values of flavonoids-loaded liposomes were below or very close to
246 0.3 (Fig. 2C), indicating a relatively homogenous and acceptable system26. However,
247 at 5% the PDI values were greatly increased to 0.533 and 0.416 for kaempferol and
248 luteolin, respectively. It was believed that the incorporation of high concentration of
249 luteolin and kaempferol, especially the latter, broadened the size distribution of
252 As liposomes are thermodynamically unstable system, the particles may tend to
255 monitoring the change in EE, particle size and PDI during storage at 4oC in the dark.
257 release within 12 days storage, indicating the strong retaining ability of liposomal
12
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258 membrane to the flavonoids. By contrast, a considerable leakage was observed for
259 liposomes loading 5% flavonoids throughout the storage period. Appropriately 60.4%
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260 of the incorporated kaempferol leaked after 26 days storage, much higher than 15.5%
261 of quercetin and 29.4% of luteolin. The stability of liposome was affected by the
263 planar structure. Owing to the great planarity and hydrophilicity, luteolin and
264 quercetin could decrease the permeability of liposomal membrane27. Moreover, it was
265 proved that quercetin and luteolin exhibited a high affinity for liposome due to the
266 good match between their planar configuration and liposome28,29. Therefore, quercetin
267 and luteolin played rigidifying roles on the membrane30. Because of the flexible
268 position, some kaempferol molecules at high concentration may adopt a horizontal
269 orientation to liposomal membrane. Such position probably caused the disorganizing
271 The change of average particle size and PDI confirmed the EE data. The stability of
274 during storage, while further increasing loading concentration to 3% and 5%, the
275 particles would aggregate seriously from 15 days. The aggregation was more apparent
276 in the case of kaempferol and luteolin. The average particle size increased by 124%
277 and 268% for liposome loading 5% kaempferol and luteolin after 26 days,
278 respectively. On the other hand, the PDI values of liposome loading 1% and 3%
279 quercetin and luteolin were below or very close to 0.3 during the whole storage period,
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281 liposomes were progressively increased during storage regardless of the loading
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282 concentration. The values even reached 0.558, 0.822 and 0.805 after 26 days for 1%,
283 3% and 5%, respectively. Based on these data, we concluded that flavonoids exhibited
288 the change of particle size was monitored as a function of temperature and pH. Fig.4A
289 shows that pure liposomes underwent slight particle size change from 4 to 55oC, while
290 beyond this range a sudden increase in particle size was observed. As DLVO theory
291 described, once the velocity or kinetic energy of the particles is high enough to
292 overcome the energy barrier of electrostatic repulsion, they will collide and aggregate.
293 There was no doubt that the energy input by temperature was the main reason for the
294 liposomal particle aggregation. Note that after incorporation of quercetin and luteolin,
295 liposomes exhibited high resistance to temperature with a slight particle size change
296 even at 80 oC. The resistance ability was stronger when loading low concentration of
297 flavonoids (1 and 3%). By contrast, serious aggregation of liposomes was found at
298 high loading concentration of kaempferol (3 and 5%). At 5%, the particles size was
299 increased by 21.57%, 12.14%, and 39.78% after 2 h heat treatment at 80oC for
300 quercetin, luteolin and kaempferol, respectively. The different temperature resistant
14
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303 Fig. 4 also shows that the particle size was increased after incubation at both acid
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304 and alkaline pH. Compared to liposomes loading kaempferol, those loading quercetin
305 and luteolin exhibited slighter size change with pH (Fig. 4D, E and F). It was reported
307 That is, flavonoids tend to distribute in hydrophobic region due to protonation of
308 phenolic hydroxyl group in more acidic pH, while in alkaline pH it would interact
309 with polar head groups due to the deprotonation effect31. Quercetin and luteolin were
310 able to deeply embed in planar lipid bilayer at acidic pH, but preferentially interacted
311 with polar head group at the lipid-water interface at physiological pH14, 31. That is why
312 quercetin and luteolin incorporation led to the small particle size change against pH.
313 Kaempferol has higher pKa values (7.05) than quercetin (6.30) and luteolin (6.50)32. It
314 suggested a higher deprotonation of polar groups in kaempferol and thus a shallow
315 penetration into the lipid bilayer. The kaempferol molecules that reside near the
319 hydrogen-donating molecules, has been widely used to evaluate the free radical
320 scavenging effectiveness in foods systems34. In the DPPH assay, the absorbance
321 decreases when color changes from purple to yellow. Fig. 5A shows the DPPH•
323 increase of flavonoid concentration, the DPPH scavenging activity was significantly
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325 molecules in preventing penetration of acyl chain regions by radical species14, 35. The
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326 trend of antioxidant activity ranging from the strongest to weakest was quercetin >
327 kaempferol > luteolin. The higher the loading concentration of flavonoid, the stronger
329 73.97%, 67.07% and 53.73% for quercetin, kaempferol, and luteolin, respectively.
330 The DPPH scavenging activity is mainly affected by the structural features in
331 flavonoids, i.e. the number and position of hydroxyl group and its donate hydrogen
333 (luteolin). Note that 3-OHis highly susceptible to radical36, which could explain the
334 high scavenging activity of quercetin and kaempferol. To the contrary, the absence of
335 3-OH as well as lower hydrogen donate might give rise to the weakest DPPH
337 was correlated with their modification on membrane fluidity36-38. Quercetin was
338 demonstrated to rigidify the hydrophobic regions of membrane lipid bilayers39. The
339 localization of luteolin into the membrane interior also favored the restrictions on
340 fluidity of membrane components, which could sterically hinder the diffusion of
342 Liposomal membrane may suffer from peroxidation due to the polyunsaturated acyl
343 chain structure in the processing and storage period of liposome. Antioxidant
345 Fe3+/ascorbate, was widely evaluated by TBARS assay16. The effect of flavonoids
16
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347 Fig. 5B. It was clearly observed that the change in TBARS for pure liposome (0 %)
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348 was considerably increased to 116.3%, indicating the serious lipid peroxidation by
349 iron. Incorporation of flavonoids can significantly inhibit the lipid peroxidation,
351 ability was found at 3% (p<0.05). Additionally, quercetin exerted the strongest
352 inhibition ability of lipid peroxidation, followed by kaempferol and luteolin. Similar
353 to the DPPH scavenging activity, the lipid peroxidation inhibition capacity is closely
354 related to the orientation of flavonoids in lipid bilayer. Due to the broad dynamic
355 distribution in the membrane, flavonoids can access all segments of the lipid
356 molecules, thus preventing peroxidation efficiently24. It should be also pointed out
358 loading of flavonoids may fragment the liposomes, resulting in the destabilization and
359 disruption of the phospholipid arrangement26. That may be the reason that the
362 IR and Raman spectroscopy techniques are recognized as quick and efficient tools
364 containing fatty acid and phosphide group has characteristic absorption bands. These
367 To investigate the structural change of lipid bilayer by flavonoids incorporation, the
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368 choline group conformation of polar head, acyl chain conformation and the intra- and
369 inter- molecular membrane order were monitored by the change of Raman
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370 intensities20. The gauche conformation of C-C bond in the choline group (O-C-C-N+)
371 appeared in the range of 710-720 cm-1, which would transfer to trans conformation
373 at around 770 cm-1 was detected in pure and flavonoids-loaded liposome, indicating
374 that the choline group mainly took on the gauche conformation. Raman band of C-C
375 from the angle vibration at 1444 cm-1 is relatively insensitive to the polar headgroup.
376 Therefore, we applied the peak intensity ratio (I718/I1444) to semi-quantify the variation
377 of gauche conformation20. It was found that this ratio decreased sharply after
378 incorporation of kaempferol of 5%, but slightly by quercetin and luteolin (Fig. 6). One
379 probable reason was that the multiple hydroxyl groups of flavonoids tended to render
380 lipid bilayer more planar, thus inducing some degree of gauche conformation44.
381 Additionally, the decrease of the number of gauche conformers resulting from
382 flavonoids incorporation suggested a new spatial arrangement of these lipid parts and
384 A distinct feature in lipid bilayers is the bands located at 2885 and 2854 cm-1
385 derived from the methylene C-H antisymmetric stretching and symmetric stretching,
386 respectively. The changes in both the lateral packing of acyl chains and the
387 trans-gauche population ratio are expressed as the ratio of intensity of 2885 to 2854
388 cm-1 20. Fig. 6 shows that the modulating effects of flavonoids on the lipid chain
389 lateral packing were concentration dependent. The ratio I2885/I2854 increased with
18
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391 increase of loading content would lead to the decrease of this ratio. It is reasonable
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392 because the insertion of hydrophobic flavonoids tends to fluidify liposomal membrane
393 due to the presence of stiffer cyclic carbon rings, and thereby disrupted the compact
396 frequencies features for different parts of lipid molecules. Fig. 7 presents the infrared
397 absorption spectra of pure liposome and flavonoids-loaded liposomes ranging from
398 600 to 2000 cm-1. Some characteristic peaks of liposome were presented at1236 cm-1,
399 1096 cm-1 and 970 cm-1, which were derived from νas (PO2-), νs (PO2-), and νas
400 (N+-CH3), respectively. The major characteristic peaks of -OH phenolic bending from
401 flavonoids are located at around 1300-1400 cm-1. The relatively strong band located at
402 1735 cm-1 represents the stretching vibrations of ester carbonyl groups, i.e. ν(C=O) in
403 the polar-apolar interface region of lipid bilayer. The position of the ν(C=O) band
404 maximum is sensitive to the conformation and/or orientation of ester groups and to
405 the hydration level of the carbonyl region in lipid bilayer. In our case, the position of
406 ν(C=O) is close to the lower wavelength number compared to previous reports46. This
407 was probably due to the presence of hydrogen bond between proton-donor OH group
408 of flavonoids and proton-acceptor C=O group of lipid47. The relatively weak band at
409 1651 cm-1 is the result of C=O vibrations from the central heterocyclic ring. It is
410 noteworthy that the ν(C=C) band appeared at 1600 cm-1, which can be interpreted as
411 the insertion of flavonoids into lipid resulted in the dramatic changes of the infrared
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412 absorption spectra48. Note that an additional band was found at 1565 cm-1 in
413 kaempferol, but was not observable in quercetin- and luteolin-loaded liposome. This
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415 flavonoids and lipid membrane through hydrogen bond48. Additionally, the band
417 1244 and 1241 cm-1 after incorporation of quercetin, kaempferol and luteolin,
418 respectively. The observation supports the concept on the involvement of polar groups
419 of the flavonoids but can also be an indication of the flavonoid localization with
420 respect to the type of interaction with lipid49. The presence of the band at 1565 cm-1
421 only in kaempferol provides a proof of different interaction with lipid among
422 flavonoids. As discussed previously, quercetin and luteolin well matched with
423 liposome, which could rigidify the molecules. Nevertheless, kaempferol orientate
424 flexibly to lipid membrane and loosen the packing of lipid molecules.
425 4. CONCLUSIONS
426 We performed a systematic study on the potential of liposomal encapsulation for three
427 different kinds of flavonoids. Furthermore, the relationship between the organization
428 of incorporated flavonoids in lipid bilayer and the chemical structure was revealed. It
429 has been demonstrated that liposomes displayed different encapsulating capacities to
430 flavonoids because of the various structures of flavonoids and loading concentration.
431 The stability and antioxidant assays revealed a mutually protective relationship
433 exhibited higher stability and stronger antioxidant capacity than those loading luteolin
20
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434 and kaempferol. Raman and IR spectroscopic techniques demonstrated that flavonoids
435 could modulate dynamics and structures of liposomal membrane, highly depending on
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436 their molecular structures and incorporation concentration. These modulations were
437 closely related to the stabilization of liposomes. Our findings suggested that liposome
439 ACKNOWLEDGEMENTS
440 This research was financially supported by projects of the National Natural Science
441 Foundation of China for Young Scholars (31401679 and 31401559); the priority
443 grant from Nanjing Forestry University (Grant Number GXL2014047) and the fund
444 of the Beijing Laboratory for Food Quality and Safety, Beijing Technology and
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560 probe for the study of the hydration of lipid self‐ assemblies. I. Methodology and general
584
585
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DOI: 10.1039/C7FO00508C
589 flavone). The red circle emphasizes the difference in critical position of hydroxyl
591 Fig. 2. (A) Raman spectra and peak assignments of pure liposome and
592 flavonoids-loaded liposomes. All spectra were averaged and presented in baseline
593 scale. The loading concentration of flavonoids was 5%. Insert graph was the loading
595 intensity ratio (Ilip/ Imix). Each data was expressed as mean value (n≥8). (B) Average
596 particle size and (C) polydispersity index (PDI) of liposomes loading different
597 concentrations of flavonoids. Each data was expressed as the mean value± standard
599 Fig. 3. Change in EE (%), average particle size (nm) and polydispersity index (PDI)
600 of liposome loading different concentrations of quercetin (A, D and G), luteolin (B, E
601 and H) and kaempferol (C, F and I) during storage at 4 oC in the dark. Each data was
602 acquired by dynamic light scattering and expressed as the mean ± standard deviation
603 (n=3).
604 Fig. 4. Average particle size (nm) of liposome loading different concentrations of
605 quercetin (A, D), luteolin (B, E), and kaempferol (C, F) as a function of temperature
606 and pH. Each point represents the mean value ± standard deviation (n=3).
607 Fig. 5. DPPH scavenging (A) and TBARS change (B) of liposomes at different
25
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608 concentration of flavonoids. Results are expressed as the mean ± standard deviation
611 as deduced from Raman spectra. Each data was expressed as the mean value ±
614 luteolin (B) and kaempferol (C) in the range of 600-2000 cm-1.
615
616
617
618
619
26
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Page 27 of 34
622
621
620
Fig. 1.
27
Luteolin
Quercetin
Kaempferol
Food & Function
DOI: 10.1039/C7FO00508C
View Article Online
623 Fig. 2.
EE (%)
Kaempferol
150
Raman intensity (a.u.)
60
100
300 1611
100 0.4
PDI
0.3
0 1301 0.2
0.1
1800 1700 1600 1500
1400 1300 1200 0 1 2 3 4 5
624 Raman shift (cm-1) Loading concentration (%)
625
28
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626 Fig. 3.
100
A 1% B C
3%
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5%
80
EE (%)
60
20
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
Storage time (days)
700
D 0%
E F
600 1%
3%
Average particle size (nm)
500 5%
400
300
200
100
0
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 30
Storage time (days)
1.0
G 0% H I
1%
0.8 3%
5%
0.6
PDI
0.4
0.2
0.0
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 30
627 Storage time (days)
628
629
630
631
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Food & Function Page 30 of 34
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632
633 Fig. 4.
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400
A B C
0%
350 1%
3%
5%
Average particle size (nm)
300
200
150
100
50
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Temperature (oC)
300
D 0%
E F
1%
250 3%
5%
Average particle size (nm)
200
150
100
50
2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9
634 pH
635
30
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DOI: 10.1039/C7FO00508C
636 Fig. 5.
A 100
0%
1%
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3% d
80
DPPH scavenging (%) 5% d
c
60
d
a a
20 a
0
Quercetin Luteolin Kaempferol
B 140
0%
1% a a
a
3%
120 5%
Change in TBARS (%)
100
b
40
b bc d
b
20 c c c
c
0
Quercetin Luteolin Kaempferol
637
638
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Food & Function Page 32 of 34
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DOI: 10.1039/C7FO00508C
639 Fig. 6.
0.28 1.4
Quercetin Quercetin
Luteolin Luteolin
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1.2
0.20
I2885/I2854
I718/I1444
0.16
0.08 0.8
0 1 2 3 4 5 0 1 2 3 4 5
Loading concentration (%) Loading concentration (%)
640
641
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642 Fig. 7.
A B 0% 0% 1236
0% 1236 C
1236
1% 1% 1%
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3% 3% 3%
5%
5% 5%
2000 1800 1600 1400 1200 1000 800 600 2000 1800 1600 1400 1200 1000 800 600 2000 1800 1600 1400 1200 1000 800 600
644
645
646
647
648
649
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Food & Function Page 34 of 34
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DOI: 10.1039/C7FO00508C
500
Liposome
Quercetin
Luteolin
400 Kaempferol
OH
OH 300
R
OH O
OH
OH
R
OH O
R
OH
O
100
OH
OH
0
R
Fatty acid chain OH 1800 1700 1600 1500 1400 1300 1200
OH O
Raman shift (cm-1)
Lipid polar head Flavonoids
R
652 OH O
34