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Physical, structural, thermal and morphological characteristics of zeinquercetagetin composite colloidal nanoparticles
Industrial Crops and Products 77 (2015) 476–483
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Physical, structural, thermal and morphological characteristics of zeinquercetagetin composite colloidal nanoparticles Cuixia Sun, Fuguo Liu, Jie Yang, Wei Yang, Fang Yuan, Yanxiang Gao ∗ Beijing Laboratory for Food Quality and Safety, College of Food Science & Nutritional Engineering, China Agricultural University, 100083, China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 31 August 2015 Accepted 12 September 2015 Keywords: Zein Quercetagetin Composite colloidal nanoparticles Thermal behaviors Structural properties
a b s t r a c t The anti-solvent precipitation method was applied for the preparation of zein–quercetagetin composite colloidal nanoparticles with zein to quercetagetin mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1. Nephelometry analysis indicated that the turbidity of zein–quercetagetin composite colloidal nanoparticles was decreased from 68.4 to 35.6 NTU at zein to quercetagetin mass ratio of 25:1 (Z–Q25:1 ). The result of fourier transform infrared spectroscopy revealed that the primary interactions between zein and quercetagetin were hydrogen bonds and hydrophobic effects. Fluorescence quenching of zein was ascribed to the binding of quercetagetin to zein and the presence of quercetagetin in zein alcoholic solution resulted in changes in the circular dichroism intensities. Furthermore, differential scanning calorimetry thermograms showed that the endothermic peaks of the zein–quercetagetin composite colloidal nanoparticles were higher than that of native zein nanoparticles (ZNP, 208.15 ◦ C), especially for Z–Q20:1 (266.82 ◦ C). Scanning electron microscopy images exhibited that native ZNP were of nanospheres with the diameter around 100 nm and smooth surfaces and zein–quercetagetin composite colloidal nanoparticles showed more compact structure with rough edges and agglomeration was observed resulting from the close packing of nanoparticles. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Zein is generally regarded as safe (GRAS) food ingredient by the US Food and Drug Administration and behaves as an amphiphilic protein with hydrophilic top and bottom with hydrophobic outer surface (Dong et al., 2004). Zein can be easily converted into spherical colloidal nanoparticles by the anti-solvent precipitation method which makes it to be an ideal delivery system for drugs and micronutrients in food, pharmaceutical and biotechnological industries (Zhong and Jin, 2009). However, the native zein nanoparticles are susceptible to environmental stresses like pH, temperature and ironic strength, which may impair the stability of delivered bioactive compounds during the process of machining and storage. Phenolic compounds are known to be able to interact with proteins via noncovalent interactions like hydrophobic effects and hydrogen bonds which may lead to changes in physicochemical and functional properties of proteins such as thermal stability, solubility and digestibility (Labuckas et al., 2008). Nowadays, most researches are mainly focused on the interactions between water
∗ Corresponding author. Fax: +86 10 62737986. E-mail address: [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.indcrop.2015.09.028 0926-6690/© 2015 Elsevier B.V. All rights reserved.
soluble proteins and polyphenols, including interactions between bovine serum albumin and grape seed procyanidin oligomers (de Freitas et al., 2003), -lactoglobulin and (−)-epigallocatechin-3gallate (Shpigelman et al., 2010), also ␣- and -caseins with tea polyphenols (+)-catechin, (+)-epicatechin, (+)-epigallocatechin and (+)-epigallocatechin gallate (Hasni et al., 2011). However, little information is available on the interaction between alcohol-soluble proteins and flavonoids. Quercetagetin, as a characteristic alcohol-soluble flavonol compound, is abundant in Tagetes and has a similar structure to quercetin but an additional 6-OH group based on the molecular structure of the flavones backbone (2-phenyl-1,4-benzopyrone) as shown in Fig. 1, which endows it with stronger affinity to proteins (Cotin et al., 2012). Limited studies revealed that quercetagetin exhibited a range of pharmacological activities. Gong et al. (2012) highlighted that quercetagetin possessed stronger antioxidant activity than that of quercetin, and Baek et al. (2013) suggested the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases since only quercetagetin strongly suppressed c-Jun NH2 -terminal kinases activity according to the examination about the activity of four representative flavonoids (quercetagetin, quercetin, myricetin, and kaempferol) using an in vitro kinase screening system. We hypothesized the complexation of zein and quercetagetin would be an attractive method to
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OH 3¨@ 2¨@
6 HO
1 O 2
7 8
HO
A 5 OH
C
5¨@
1¨@
B
OH 4¨@
6¨@ 3 OH
4 O
Fig. 1. Chemical structure of quercetagetin.
develop new food grade materials with better thermal behaviors and structural properties by drawing advantages of both components. To the best of our knowledge, there is no information on the interaction between zein and quercetagetin, which partly stimulated this work. The objective of the present study was to investigate the effect of different mass ratios of zein to quercetagetin on the physical, structural, thermal and morphological characteristics of zein–quercetagetin composite colloidal nanoparticles. Fourier transform infrared (FTIR) spectroscopy was applied to explore intermolecular forces, fluorescence measurements were performed to provide information about the interaction between zein and quercetagetin, far-UV CD spectroscopy was used to explain the secondary structure changes of zein, differential scanning calorimetry (DSC) was occupied to probe thermal behaviors and scanning electron microscopy (SEM) was used for morphological characterization. Results from present work might be useful for the development of a potential carrier for bioactive compounds. 2. Materials and methods 2.1. Materials Zein with a protein content of 95% (w/w) was purchased from Gaoyou Group Co. Ltd. (Jiangsu, China). Absolute ethanol (99.9%) was acquired from Eshowbokoo Biological Technology Co., Ltd. (Beijing, China). Water purified by a MilliQ system (Millipore, MA, USA) was used for all the experiments. Quercetagetin was prepared from marigold (Tagetes erecta L.) flower with the method described by Gong et al. (2012). The marigold (Tageteserecta L.) flower powder was firstly defatted by the traditional Soxhlet-extraction with n-hexane as the solvent, and then the defatted material (1.0 g) was extracted with 10 mL of 70% (v/v) of ethanol–water solution in the shaker incubator at 60 ◦ C for 6 h. The solid–liquid mixture was filtered and each filtered extract was concentrated with a vacuum rotary evaporator, then centrifuged at 4200 rpm to get the precipitation, the separated solid precipitate was dispersed with 10 times of deionized water and ultrasound vibrated for 30 min to remove soluble polysaccharides, the water washing procedure was repeated for another time, then centrifuged to make the precipitate and lyophilized for a purified quercetagetin (91%, w/w). The purified quercetagetin was dissolved in 70% ethanol and then stored in an amber colored air-tight container at 20 ◦ C until used.
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vigorously. The zein–quercetagetin complex solutions were added in 2 min to this beaker in a controlled way using a syringe. To acquire aqueous dispersions, approximately three quarters of the ethanol were removed under reduced pressure (0.1 MPa) by rotary evaporation at 50 ◦ C for 30 min. Finally, the zein–quercetagetin composite dispersions with a pH around 4.0 were stored in the refrigerator at 5 ◦ C for further analysis. Zein nanoparticle dispersions without quercetagetin addition were obtained by the same process above and used as the control sample. In this work, samples of native zein nanoparticles, zein–quercetagetin composite colloidal nanoparticles at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1were termed as ZNP, Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 , respectively. 2.3. Particle size measurement Mean particle size of native zein nanoparticles and zein–quercetagetin composite colloidal nanoparticles were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments Ltd., Worcestershire, UK) according to the descriptions of Chen et al. (2014) with slight modifications. Freshly prepared samples were diluted 10 times with distilled water at room temperature before measurements to avoid multiple particle effects. Results were described as cumulative mean diameter (size, nm) for particle size. All measurements were carried out at room temperature (25 ◦ C) and each sample was analyzed in triplicate. 2.4. Turbidity measurement Nephelometry experiments were performed in a HACH 2100N laboratory turbidimeter (Loveland, USA), and the turbidity of zein–quercetagetin composite colloidal dispersions was evaluated as stated by Yang et al. (2014). The optical apparatus was equipped with a tungsten-filament lamp with three detectors: a 90◦ scattered-light detector, a forward-scatter light detector, and a transmitted light detector. The calibration was performed using a Gelex Secondary Turbidity Standard Kit (HACH, Loveland, USA), which consists of stable suspensions of a metal oxide in a gel. All experiments were performed in triplicate. 2.5. Fourier transform infrared (FTIR) spectroscopy FTIR was used to study chemical structure characteristics of the freeze-dried native zein nanoparticles and zein–quercetagetin composite colloidal nanoparticles as suggested by Chen and Zhong (2014) with some modifications. Briefly, 2.0 mg samples were mixed with 198 mg pure potassium bromide (KBr) powder. The mixture was ground into fine powder, pressed into pellet and measured by a Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, UK). FTIR spectra were acquired at 400–4000 cm−1 wavenumbers with a resolution of 4 cm−1 . Pure KBr powder was used as a baseline. The data were analyzed using Omnic v8.0 (Thermo Nicolet, USA). 2.6. Fluorescence measurements
2.2. Preparation of zein–quercetagetin composite colloidal nanoparticles Zein–quercetagetin composite colloidal nanoparticles were prepared by the anti-solvent precipitation method adapted from Zhong and Jin (2009). Briefly, zein and quercetagetin at different mass ratios (30:1, 25:1, 20:1, 15:1 and 10:1 w/w) were dissolved in 40 mL 70% (v/v) ethanol–water solution to form the stock solutions. About 120 mL deionized water was put into a beaker and stirred
Fluorescence measurements were performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) by the method of Zhai et al. (2012). The excitation wavelength was set at 280 nm, and the emission spectra were collected in the range of 290–450 nm with a scanning speed of 100 nm/min. Excitation and emission slit widths were set at 10 nm. Each individual emission spectrum was the average of three runs. All data were collected at room temperature.
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Fig. 2. Size (A) and turbidity (B) curves of native zein and zein–quercetagetin composite colloidal nanoparticles. ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein–quercetagetin composite colloidal nanoparticles with different mass ratios of zein to quercetagetin at 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
2.7. Circular dichroism spectroscopy
2.8. Differential scanning calorimetry (DSC) analysis
Far-UV CD spectra were recorded between 190 and 260 nm using a CD spectropolarimeter (Pistar -180, Applied Photophysics Ltd. UK) as described by Zhang and Zhong (2013) with some modifications. The protein concentration was 0.2 mg/mL, a quartz cell with a 0.1 cm path length was used and a constant nitrogen flush was applied during data acquisition. The secondary structure contents of the samples were estimated using Dichroweb: the online Circular Dichroism Website http://dichroweb.cryst.bbk.ac. uk (Lobley et al., 2002; Whitmore and Wallace, 2004).
The thermal behaviors of freeze-dried zein–quercetagetin composite colloidal nanoparticles were analyzed by differential scanning calorimeter (DSC-60, Shimadzu, Tokyo, Japan) according to the description of Neo et al. (2013) with some modifications. In our experiments, 3.0 mg sample was placed inside an aluminum pan and sealed tightly by a perforated aluminum lid, heated from 30 to 300 ◦ C at a constant rate of 10 ◦ C/min with a constant purging of dry nitrogen at a rate of 30 mL/min. An empty aluminum pan was used as a reference. The peak temperature of melting point was
1650.55
3310.97
F
1650.61 1539.94
3311.10
1650.17 1539.01
E 3311.04
1651.77
D
3310.94 1652.30
C
3315.94
B
1538.99
1538.97 1539.01
1653.10 1540.17
3293.10
A 3500
3000
2000
1500
-1
1000
500
Wavelength(cm ) Fig. 3. FTIR spectra of zein and quercetagetin composite colloidal nanoparticles. A: ZNP (zein nanoparticle); B: Z–Q30:1 ; C: Z–Q25:1 ; D: Z–Q20:1 ; E: Z–Q15:1 ; F: Z–Q10:1 ; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
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ZNP Z-Q30:1
6000
Z-Q25:1 Z-Q20:1
5000
Z-Q15:1 Z-Q10:1
Intensity
4000
3000
2000
1000
0 300
320
340
360
380
400
420
440
Wavelength (nm) Fig. 4. Fluorescence spectra of ZNP and zein–quercetagetin composite colloidal nanoparticles. ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein–quercetagetin composite colloidal particles with different mass ratios of zein to quercetagetin at 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
computed using the universal analysis software from each thermal curve.
3. Results and discussion 3.1. Particle size and turbidity
2.9. Scanning electron microscopy (SEM) analysis Morphology of freeze-dried samples was observed by SEM (JEOL, JSM-6701F, Japan) at an accelerating voltage of 10.0 kV. Prior to the observation, the surfaces of samples were sputter-coated with a gold layer to avoid charging under the electron beam.
2.10. Statistical analysis All the data obtained were average values of triplicate determinations and subjected to statistical analysis of variance (ANOVA) using SPSS 18.0 for Windows (SPSS Inc., Chicago, USA). Least significant differences (P < 0.05) were accepted among the treatments.
The average size curves of native ZNP and zein–quercetagetin composite colloidal nanoparticles are shown in Fig. 2A. The average particle size of ZNP was 100.3 nm, which was obviously decreased to 88.9, 64.0 and 73.1 nm for Z–Q composite colloidal particles with mass ratios of zein to quercetagetin at 30:1, 25:1 and 20:1, respectively. However, continuing adding quercetagetin into zein, the average particle sizes were significantly (p < 0.05) increased from 100.3 to 130.7 and 164.1 nm for samples of Z–Q15:1 and Z–Q10:1 , respectively, which might be attributed to the particle aggregation resulting from the interaction between zein and excessive quercetagetin. Nephelometry was performed to obtain some information about the physical–chemical driving forces involved in the formation of
ZNP Z-Q30:1 -20
80
ZNP Z-Q30:1
Z-Q25:1 Z-Q20:1 Z-Q15:1
Z-Q25:1
Z-Q10:1
Z-Q20:1
C D (m e d g )
60
Z-Q15:1
CD (mdeg)
40
Z-Q10:1
20
0 200
210
220
230
240
250
260
Wavelength (nm) -20
-40
Fig. 5. CD spectra of ZNP and zein–quercetagetin composite colloidal nanoparticles. ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein–quercetagetin composite colloidal nanoparticles with different mass ratios of zein to quercetagetin at 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
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Fig. 6. DSC thermograms of zein and quercetagetin composite colloidal nanoparticles. A: ZNP: zein nanoparticle; B: quercetagetin; C: Z–Q30:1 ; D: Z–Q25:1 ; E: Z–Q20:1 ; F: Z–Q15:1 ; G: Z–Q10:1 ; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
zein–quercetagetin composite colloidal nanoparticles. As shown in Fig. 2B, the turbidity of native zein colloidal dispersion was 68.4 NTU, and the presence of quercetagetin significantly (p < 0.05) decreased the turbidity of zein colloidal dispersions to 35.6, 40.6 and 42.0 NTU for samples of Z–Q30:1, Z–Q25:1 and Z–Q20:1 , respectively. Oppositely, the turbidity was greatly (p < 0.05) increased to 128.7 and 400.3 NTU for samples of Z–Q15:1 and Z–Q10:1 , respectively. The changes of turbidity could be also observed according to the illustrations in Fig. 2. The decreased turbidity suggested that the interaction between quercetagetin and zein led to the formation of the binary zein–quercetagetin composites with more compact structure. The increased turbidity might be attributed to the fact that excessive quercetagetin was bound to the surface of zein, resulting in the formation of the largely binary composites by noncovalent interaction (Le Bourvellec and Renard, 2012). Yang et al. (2014) reported that bovine lactoferrin (LF) and (−)-epigallocatechin-3-gallate (EGCG) formed LF–EGCG complex by the molecular interaction and the turbidity of LF–EGCG complex was increased when EGCG/LF molar ratio was more than a critical value of 25:1. According to the aforementioned analysis of size and turbidity, it could be concluded that there existed a certain relationship between size and turbidity since the change trend of particle size was consistent with that of turbidity in this work. 3.2. FTIR spectra The intermolecular interactions of zein and quercetagetin composites were identified by FTIR. The representative spectra of quercetagetin, ZNP and zein–quercetagetin composite colloidal nanoparticles are shown in Fig. 3. In the original spectra of ZNP (Fig. 3A), the band of hydrogen bonds was at 3293.10 cm−1 . However, after the formation of zein–quercetagetin composite colloidal nanoparticles, a shift of hydrogen bonds occurred, and the peaks were at 3315.94, 3310.94, 3311.04, 3311.10 and 3310.97 cm−1 in the spectra of Z–Q30:1 (Fig. 3B), Z–Q25:1 (Fig. 3C), Z–Q20:1 (Fig. 3D), Z–Q15:1 (Fig. 3E) and Z–Q10:1 (Fig. 3F), respectively. Besides, the peak intensity in spectra of zein–quercetagetin colloidal nanoparticles was significantly increased compared with that in the spectrum
of ZNP. This result indicated the formation of hydrogen bonds between zein and quercetagetin, which may be due to the interaction between amide groups of glutamine in zein and hydroxyl groups in quercetagetin as reported by Luo et al. (2011) who found strong hydrogen bonds formed between zein and ␣-tocopherol. As shown in Fig. 3A, typical amide I and amide II peaks for ZNP were 1653.10 and 1540.17 cm−1 , respectively. The amide I band was the prominent C O stretching and amide II absorption was mainly referred to C N stretching and C N H in plane bending (Torres-Giner et al., 2008a; Sessa et al., 2008). Comparing with the spectra of ZNP, the amide I and amide II peaks were shifted to 1652.30 and 1539.01, 1651.77 and 1538.97, 1650.17 and 1539.01, 1650.61 and 1539.94, and 1650.55 and 1538.99 cm−1 in the spectra of Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 , respectively. These findings suggested that another intermolecular force, electrostatic or hydrophobic interactions, might exist between zein and quercetagetin, which were in accordance with previous observations that the interactions between the zein and polyphenols (cranberry procyanidins, curcumin) consisted of hydrogen bonds and hydrophobic interactions (Zou et al., 2012; Liang et al., 2015). 3.3. Fluorescence spectra Fluorescence quenching refers to the interacting process between a fluorophore like a protein and an external quencher molecule such as a polyphenol that decreases the fluorescence intensity of proteins (Keppler et al., 2014) and it is regarded as a useful technique to provide unique information about the interactions between small molecules and proteins due to the sensitivity of intrinsic fluorescence to the microenvironment changes around proteins (Zhang et al., 2013). The effect of quercetagetin on the fluorescence emission spectra of ZNP is shown in Fig. 4. It can be observed that ZNP possessed a strong fluorescence emission peak at 304 nm after being excited at 280 nm. The fluorescence intensity of ZNP was gradually decreased upon increasing the concentration of quercetagetin, which indicated that the fluorescence of ZNP was quenched by quercetagetin. Fluorescence quenching may be ascribed to a variety of molecular interactions including molecular rearrangements, energy transfer, ground state complex formation
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Fig. 7. SEM images of zein and quercetagetin composite colloidal nanoparticles. (A) ZNP: zein nanoparticle; (B) Z–Q30:1 ; (C) Z–Q25:1 ; (D) Z–Q20:1 ; (E) Z–Q15:1 ; (F) Z–Q10:1 ; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
and collisional quenching (Lakowicz and Masters, 2008). The result in this study may be due to the binding of quercetagetin to zein and complexes formation between zein and quercetagetin, which was consistent with Joye et al. (2015) who found that resveratrol binding to zein resulted in fluorescence quenching of zein. 3.4. CD spectroscopy The secondary structure of zein was measured using far-UV CD spectroscopy as shown in Fig. 5. The CD spectrum of ZNP shows two negative peaks at 208 nm and 220 nm and a positive peak at 192 nm, which was characteristic of ␣-helical-rich secondary structure (Selling et al., 2007; Zhai et al., 2011). The quantitative analysis of secondary structural content estimated by SELCON3 as shown in Table 1 yielded 51.1% ␣-helix, 12.3% -sheet,
16.9% -turn and 19.7% unordered structure. This result was consistent with the report of Cabra et al. (2006) who found that the average ␣-helical structure content was 50%, with the majority of the remaining structure in a random coil state. The presence of quercetagetin resulted in changes in the CD intensities which could be observed in the magnified illustration in Fig. 5, suggesting a great effect on the changes of the second structure of ZNP. With zein to quercetagetin mass ratios of 30:1 and 25:1, the deconvolution of the secondary structure content indicated a significant (p < 0.05) decrease in ␣-helical content of ZNP from 51.1% to 44.9% and 40.4%, respectively. At the same time, -sheet content was increased from 12.3% to 14.8% and 17.5%, respectively. These findings might be due to the interaction between zein and quercetagetin. Mizutani et al. (2003) found the ␣-helix content was decreased with an increase in the ratio of linolenic
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Table 1 Secondary structure analysis of native zein and zein–quercetagetin composite nanoparticles Samples
ZNP Z–Q30:1 Z–Q25:1 Z–Q20:1 Z–Q15:1 Z–Q10:1
Content (%) ␣-Helix
-Sheet
-Turn
Unordered coils
51.1 44.9 40.4 56.4 51.9 52.6
12.3 14.8 17.5 7.1 10.5 9.9
16.9 16.6 13.8 18.3 16.1 17.2
19.7 23.8 28.4 18.2 21.5 20.3
ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
acid ethyl (LAE), suggesting the interaction between LAE and zein. However, continuing the rise of quercetagetin induced obvious increase in the CD intensities, especially for the sample of zein to quercetagetin mass ratio of 20:1, this result suggested a significant (p < 0.05) increase in ␣-helical content of ZNP from 51.1% to 56.4%. At the same time, -sheet content was decreased from 12.3% to 7.1%. These results indicated the addition of quercetagetin led to the conformation change of ZNP. Kanakis et al. (2011) pointed out that the presence of EGCG increased the ␣-helix content of -lactoglobulin, suggesting stronger structural stabilization of the protein.
nanoparticles was not changed but the structure seemed more compact than that of native ZNP. Continuing to increase quercetagetin into the zein solution with mass ratios of 15:1 (Fig. 7E) and 10:1 (Fig. 7F), the morphology of nanoparticles was gradually deviated from the spherical shape with rough edges, and agglomeration was observed resulting from the close packing of particles with larger diameter. These results may be due to the interaction between zein and quercetagetin since the excessive quercetagetin disturbed the intermolecular entanglements among polypeptide chains and the chains were not able to aggregate into spheres after solvent evaporation (Gomez-Estaca et al., 2012). The more compact structure of zein–quercetagetin composite colloidal nanoparticles could be applied to further confirm that the thermal stability of zein–quercetagetin composite colloidal particles was better than that of native ZNP (Fig. 6). 4. Conclusions The formation of composites between zein and quercetagetin led to the fluorescence quenching and changes in the CD intensities of zein, which was mainly due to the hydrogen bonds and hydrophobic effects. The composite colloidal nanoparticles of Z–Q20:1 exhibited higher endothermic peak and more compact structure with reduced turbidity and particle size. The complexation of zein and quercetagetin might be an attractive method to develop new food grade biopolymers with better thermal behaviors and structural properties which could be useful in the development of the potential delivery system for bioactive compounds.
3.5. DSC The DSC thermograms of quercetagetin, ZNP and zein–quercetagetin composite colloidal nanoparticles are presented in Fig. 6. The DSC curve of native ZNP showed a melting peak at 208.15 ◦ C (Fig. 6A) and the native quercetagetin thermogram displayed an endothermic peak at 214.26 ◦ C (Fig. 6B). Compared with the melting peak of native ZNP or quercetagetin, the endothermic peaks of the zein–quercetagetin colloidal nanoparticles were shifted to higher temperatures at 255.50, 257.0, 266.820, 245.5 and 221.51 ◦ C in the DSC thermograms of Z–Q30:1 (Fig. 6C), Z–Q25:1 (Fig. 6D), Z–Q20:1 (Fig. 6E), Z–Q15:1 (Fig. 6F) and Z–Q10:1 (Fig. 6G), respectively. This may be due to the formation of more compact structure between zein and quercetagetin during the anti-solvent process to form the composite particles, and more energy was required to destroy the molecular structures, especially for Z–Q20:1 composite colloidal nanoparticles which could be confirmed by the result of the reduced particle size (Fig. 2A) and lower turbidity (Fig. 2B) of Z–Q20:1 composite colloidal nanoparticles. In addition, the reduction of height and sharpness of the endotherm peak of zein-Q composite colloidal nanoparticles may be attributed to the presence of quercetagetin in the composite nanoparticles (Lai and Guo, 2011). Similar observation was also reported by Hu et al. (2012) who found there was a strong melting peak for sow lutein between 180 and 190 ◦ C, and a thermodynamic compatibility between lutein and zein by processing in solution enhanced dispersion by supercritical fluids. 3.6. SEM images SEM was applied to characterize the morphology of ZNP and zein–quercetagetin composite colloidal nanoparticles as shown in Fig. 7. The ZNP exhibited nanospheres with the diameter around 100 nm and had smooth surface and good dispersion (Fig. 7A), which was consistent with the findings of Wu et al. (2012). When quercetagetin was added into the zein solution at different mass ratios of 30:1 (Fig. 7B), 25:1 (Fig. 7C) and 20:1 (Fig. 7D), the morphology of zein–quercetagetin composite colloidal
Acknowledgement Financial support from the National Natural Science Foundation of China (No. 31371835) is gratefully acknowledged. References Baek, S., Kang, N.J., Popowicz, G.M., Arciniega, M., Jung, S.K., Byun, S., Lee, K.W., 2013. Structural and functional analysis of the natural JNK1 inhibitor quercetagetin. J. Mol. Biol. 425, 411–423. Cabra, V., Arreguin, R., Vazquez-Duhalt, R., Farres, A., 2006. Effect of temperature and pH on the secondary structure and processes of oligomerization of 19 kDa alpha-zein. BBA-Proteins Proteom. 1764, 1110–1118. Chen, J., Zheng, J., McClements, D.J., Xiao, H., 2014. Tangeretin-loaded protein nanoparticles fabricated from zein/-lactoglobulin: preparation, characterization, and functional performance. Food Chem. 158, 466–472. Chen, H., Zhong, Q., 2014. Processes improving the dispersibility of spray-dried zein nanoparticles using sodium caseinate. Food Hydrocoll. 35, 358–366. Cotin, S., Calliste, C.A., Mazeron, M.C., Hantz, S., Duroux, J.L., Rawlinson, W.D., Alain, S., 2012. Eight flavonoids and their potential as inhibitors of human cytomegalovirus replication. Antivir. Res. 96, 181–186. de Freitas, V., Carvalho, E., Mateus, M., 2003. Study of carbohydrate influence on protein-tannin aggregation by nephelometry. Food Chem. 81, 503–509. Dong, J., Sun, Q., Wang, J.Y., 2004. Basic study of corn protein zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility. Biomaterials 25, 4691–4697. Gomez-Estaca, J., Balaguer, M.P., Gavara, R., Hernandez-Munoz, P., 2012. Formation of zein nanoparticles by electrohydrodynamic atomization: effect of the main processing variables and suitability for encapsulating the food coloring and active ingredient curcumin. Food Hydrocoll. 28, 82–91. Gong, Y., Hou, Z., Gao, Y., Xue, Y., Liu, X., Liu, G., 2012. Optimization of extraction parameters of bioactive components from defatted marigold (Tagetes erecta L.) residue using response surface methodology. Food Bioprod. Process. 90, 9–16. Hasni, I., Bourassa, P., Hamdani, S., Samson, G., Carpentier, R., Tajmir-Riahi, H.A., 2011. Interaction of milkaandb-casein with tea polyphenols. Food Chem. 126, 630–639. Hu, D., Lin, C., Liu, L., Li, S., Zhao, Y., 2012. Preparation, characterization, and in vitro release investigation of lutein/zein nanoparticles via solution enhanced dispersion by supercritical fluids. J. Food Eng. 109, 545–552. Joye, I.J., Davidov-Pardo, G., Ludescher, R.D., McClements, D.J., 2015. study of resveratrol binding to zein and gliadin: towards a more rational approach to resveratrol encapsulation using water-insoluble proteins. Food Chem. 185, 261–267. Kanakis, C.D., Hasni, I., Bourassa, P., Tarantilis, P.A., Polissiou, M.G., Tajmir-Riahi, H.A., 2011. Milk -lactoglobulin complexes with tea polyphenols. Food Chem. 127, 1046–1055.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Physical, structural, thermal and morphological characteristics of zeinquercetagetin composite colloidal nanoparticles Cuixia Sun, Fuguo Liu, Jie Yang, Wei Yang, Fang Yuan, Yanxiang Gao ∗ Beijing Laboratory for Food Quality and Safety, College of Food Science & Nutritional Engineering, China Agricultural University, 100083, China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 31 August 2015 Accepted 12 September 2015 Keywords: Zein Quercetagetin Composite colloidal nanoparticles Thermal behaviors Structural properties
a b s t r a c t The anti-solvent precipitation method was applied for the preparation of zein–quercetagetin composite colloidal nanoparticles with zein to quercetagetin mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1. Nephelometry analysis indicated that the turbidity of zein–quercetagetin composite colloidal nanoparticles was decreased from 68.4 to 35.6 NTU at zein to quercetagetin mass ratio of 25:1 (Z–Q25:1 ). The result of fourier transform infrared spectroscopy revealed that the primary interactions between zein and quercetagetin were hydrogen bonds and hydrophobic effects. Fluorescence quenching of zein was ascribed to the binding of quercetagetin to zein and the presence of quercetagetin in zein alcoholic solution resulted in changes in the circular dichroism intensities. Furthermore, differential scanning calorimetry thermograms showed that the endothermic peaks of the zein–quercetagetin composite colloidal nanoparticles were higher than that of native zein nanoparticles (ZNP, 208.15 ◦ C), especially for Z–Q20:1 (266.82 ◦ C). Scanning electron microscopy images exhibited that native ZNP were of nanospheres with the diameter around 100 nm and smooth surfaces and zein–quercetagetin composite colloidal nanoparticles showed more compact structure with rough edges and agglomeration was observed resulting from the close packing of nanoparticles. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Zein is generally regarded as safe (GRAS) food ingredient by the US Food and Drug Administration and behaves as an amphiphilic protein with hydrophilic top and bottom with hydrophobic outer surface (Dong et al., 2004). Zein can be easily converted into spherical colloidal nanoparticles by the anti-solvent precipitation method which makes it to be an ideal delivery system for drugs and micronutrients in food, pharmaceutical and biotechnological industries (Zhong and Jin, 2009). However, the native zein nanoparticles are susceptible to environmental stresses like pH, temperature and ironic strength, which may impair the stability of delivered bioactive compounds during the process of machining and storage. Phenolic compounds are known to be able to interact with proteins via noncovalent interactions like hydrophobic effects and hydrogen bonds which may lead to changes in physicochemical and functional properties of proteins such as thermal stability, solubility and digestibility (Labuckas et al., 2008). Nowadays, most researches are mainly focused on the interactions between water
∗ Corresponding author. Fax: +86 10 62737986. E-mail address: [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.indcrop.2015.09.028 0926-6690/© 2015 Elsevier B.V. All rights reserved.
soluble proteins and polyphenols, including interactions between bovine serum albumin and grape seed procyanidin oligomers (de Freitas et al., 2003), -lactoglobulin and (−)-epigallocatechin-3gallate (Shpigelman et al., 2010), also ␣- and -caseins with tea polyphenols (+)-catechin, (+)-epicatechin, (+)-epigallocatechin and (+)-epigallocatechin gallate (Hasni et al., 2011). However, little information is available on the interaction between alcohol-soluble proteins and flavonoids. Quercetagetin, as a characteristic alcohol-soluble flavonol compound, is abundant in Tagetes and has a similar structure to quercetin but an additional 6-OH group based on the molecular structure of the flavones backbone (2-phenyl-1,4-benzopyrone) as shown in Fig. 1, which endows it with stronger affinity to proteins (Cotin et al., 2012). Limited studies revealed that quercetagetin exhibited a range of pharmacological activities. Gong et al. (2012) highlighted that quercetagetin possessed stronger antioxidant activity than that of quercetin, and Baek et al. (2013) suggested the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases since only quercetagetin strongly suppressed c-Jun NH2 -terminal kinases activity according to the examination about the activity of four representative flavonoids (quercetagetin, quercetin, myricetin, and kaempferol) using an in vitro kinase screening system. We hypothesized the complexation of zein and quercetagetin would be an attractive method to
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OH 3¨@ 2¨@
6 HO
1 O 2
7 8
HO
A 5 OH
C
5¨@
1¨@
B
OH 4¨@
6¨@ 3 OH
4 O
Fig. 1. Chemical structure of quercetagetin.
develop new food grade materials with better thermal behaviors and structural properties by drawing advantages of both components. To the best of our knowledge, there is no information on the interaction between zein and quercetagetin, which partly stimulated this work. The objective of the present study was to investigate the effect of different mass ratios of zein to quercetagetin on the physical, structural, thermal and morphological characteristics of zein–quercetagetin composite colloidal nanoparticles. Fourier transform infrared (FTIR) spectroscopy was applied to explore intermolecular forces, fluorescence measurements were performed to provide information about the interaction between zein and quercetagetin, far-UV CD spectroscopy was used to explain the secondary structure changes of zein, differential scanning calorimetry (DSC) was occupied to probe thermal behaviors and scanning electron microscopy (SEM) was used for morphological characterization. Results from present work might be useful for the development of a potential carrier for bioactive compounds. 2. Materials and methods 2.1. Materials Zein with a protein content of 95% (w/w) was purchased from Gaoyou Group Co. Ltd. (Jiangsu, China). Absolute ethanol (99.9%) was acquired from Eshowbokoo Biological Technology Co., Ltd. (Beijing, China). Water purified by a MilliQ system (Millipore, MA, USA) was used for all the experiments. Quercetagetin was prepared from marigold (Tagetes erecta L.) flower with the method described by Gong et al. (2012). The marigold (Tageteserecta L.) flower powder was firstly defatted by the traditional Soxhlet-extraction with n-hexane as the solvent, and then the defatted material (1.0 g) was extracted with 10 mL of 70% (v/v) of ethanol–water solution in the shaker incubator at 60 ◦ C for 6 h. The solid–liquid mixture was filtered and each filtered extract was concentrated with a vacuum rotary evaporator, then centrifuged at 4200 rpm to get the precipitation, the separated solid precipitate was dispersed with 10 times of deionized water and ultrasound vibrated for 30 min to remove soluble polysaccharides, the water washing procedure was repeated for another time, then centrifuged to make the precipitate and lyophilized for a purified quercetagetin (91%, w/w). The purified quercetagetin was dissolved in 70% ethanol and then stored in an amber colored air-tight container at 20 ◦ C until used.
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vigorously. The zein–quercetagetin complex solutions were added in 2 min to this beaker in a controlled way using a syringe. To acquire aqueous dispersions, approximately three quarters of the ethanol were removed under reduced pressure (0.1 MPa) by rotary evaporation at 50 ◦ C for 30 min. Finally, the zein–quercetagetin composite dispersions with a pH around 4.0 were stored in the refrigerator at 5 ◦ C for further analysis. Zein nanoparticle dispersions without quercetagetin addition were obtained by the same process above and used as the control sample. In this work, samples of native zein nanoparticles, zein–quercetagetin composite colloidal nanoparticles at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1were termed as ZNP, Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 , respectively. 2.3. Particle size measurement Mean particle size of native zein nanoparticles and zein–quercetagetin composite colloidal nanoparticles were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments Ltd., Worcestershire, UK) according to the descriptions of Chen et al. (2014) with slight modifications. Freshly prepared samples were diluted 10 times with distilled water at room temperature before measurements to avoid multiple particle effects. Results were described as cumulative mean diameter (size, nm) for particle size. All measurements were carried out at room temperature (25 ◦ C) and each sample was analyzed in triplicate. 2.4. Turbidity measurement Nephelometry experiments were performed in a HACH 2100N laboratory turbidimeter (Loveland, USA), and the turbidity of zein–quercetagetin composite colloidal dispersions was evaluated as stated by Yang et al. (2014). The optical apparatus was equipped with a tungsten-filament lamp with three detectors: a 90◦ scattered-light detector, a forward-scatter light detector, and a transmitted light detector. The calibration was performed using a Gelex Secondary Turbidity Standard Kit (HACH, Loveland, USA), which consists of stable suspensions of a metal oxide in a gel. All experiments were performed in triplicate. 2.5. Fourier transform infrared (FTIR) spectroscopy FTIR was used to study chemical structure characteristics of the freeze-dried native zein nanoparticles and zein–quercetagetin composite colloidal nanoparticles as suggested by Chen and Zhong (2014) with some modifications. Briefly, 2.0 mg samples were mixed with 198 mg pure potassium bromide (KBr) powder. The mixture was ground into fine powder, pressed into pellet and measured by a Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, UK). FTIR spectra were acquired at 400–4000 cm−1 wavenumbers with a resolution of 4 cm−1 . Pure KBr powder was used as a baseline. The data were analyzed using Omnic v8.0 (Thermo Nicolet, USA). 2.6. Fluorescence measurements
2.2. Preparation of zein–quercetagetin composite colloidal nanoparticles Zein–quercetagetin composite colloidal nanoparticles were prepared by the anti-solvent precipitation method adapted from Zhong and Jin (2009). Briefly, zein and quercetagetin at different mass ratios (30:1, 25:1, 20:1, 15:1 and 10:1 w/w) were dissolved in 40 mL 70% (v/v) ethanol–water solution to form the stock solutions. About 120 mL deionized water was put into a beaker and stirred
Fluorescence measurements were performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) by the method of Zhai et al. (2012). The excitation wavelength was set at 280 nm, and the emission spectra were collected in the range of 290–450 nm with a scanning speed of 100 nm/min. Excitation and emission slit widths were set at 10 nm. Each individual emission spectrum was the average of three runs. All data were collected at room temperature.
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Fig. 2. Size (A) and turbidity (B) curves of native zein and zein–quercetagetin composite colloidal nanoparticles. ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein–quercetagetin composite colloidal nanoparticles with different mass ratios of zein to quercetagetin at 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
2.7. Circular dichroism spectroscopy
2.8. Differential scanning calorimetry (DSC) analysis
Far-UV CD spectra were recorded between 190 and 260 nm using a CD spectropolarimeter (Pistar -180, Applied Photophysics Ltd. UK) as described by Zhang and Zhong (2013) with some modifications. The protein concentration was 0.2 mg/mL, a quartz cell with a 0.1 cm path length was used and a constant nitrogen flush was applied during data acquisition. The secondary structure contents of the samples were estimated using Dichroweb: the online Circular Dichroism Website http://dichroweb.cryst.bbk.ac. uk (Lobley et al., 2002; Whitmore and Wallace, 2004).
The thermal behaviors of freeze-dried zein–quercetagetin composite colloidal nanoparticles were analyzed by differential scanning calorimeter (DSC-60, Shimadzu, Tokyo, Japan) according to the description of Neo et al. (2013) with some modifications. In our experiments, 3.0 mg sample was placed inside an aluminum pan and sealed tightly by a perforated aluminum lid, heated from 30 to 300 ◦ C at a constant rate of 10 ◦ C/min with a constant purging of dry nitrogen at a rate of 30 mL/min. An empty aluminum pan was used as a reference. The peak temperature of melting point was
1650.55
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E 3311.04
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Wavelength(cm ) Fig. 3. FTIR spectra of zein and quercetagetin composite colloidal nanoparticles. A: ZNP (zein nanoparticle); B: Z–Q30:1 ; C: Z–Q25:1 ; D: Z–Q20:1 ; E: Z–Q15:1 ; F: Z–Q10:1 ; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
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ZNP Z-Q30:1
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Wavelength (nm) Fig. 4. Fluorescence spectra of ZNP and zein–quercetagetin composite colloidal nanoparticles. ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein–quercetagetin composite colloidal particles with different mass ratios of zein to quercetagetin at 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
computed using the universal analysis software from each thermal curve.
3. Results and discussion 3.1. Particle size and turbidity
2.9. Scanning electron microscopy (SEM) analysis Morphology of freeze-dried samples was observed by SEM (JEOL, JSM-6701F, Japan) at an accelerating voltage of 10.0 kV. Prior to the observation, the surfaces of samples were sputter-coated with a gold layer to avoid charging under the electron beam.
2.10. Statistical analysis All the data obtained were average values of triplicate determinations and subjected to statistical analysis of variance (ANOVA) using SPSS 18.0 for Windows (SPSS Inc., Chicago, USA). Least significant differences (P < 0.05) were accepted among the treatments.
The average size curves of native ZNP and zein–quercetagetin composite colloidal nanoparticles are shown in Fig. 2A. The average particle size of ZNP was 100.3 nm, which was obviously decreased to 88.9, 64.0 and 73.1 nm for Z–Q composite colloidal particles with mass ratios of zein to quercetagetin at 30:1, 25:1 and 20:1, respectively. However, continuing adding quercetagetin into zein, the average particle sizes were significantly (p < 0.05) increased from 100.3 to 130.7 and 164.1 nm for samples of Z–Q15:1 and Z–Q10:1 , respectively, which might be attributed to the particle aggregation resulting from the interaction between zein and excessive quercetagetin. Nephelometry was performed to obtain some information about the physical–chemical driving forces involved in the formation of
ZNP Z-Q30:1 -20
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Fig. 5. CD spectra of ZNP and zein–quercetagetin composite colloidal nanoparticles. ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein–quercetagetin composite colloidal nanoparticles with different mass ratios of zein to quercetagetin at 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
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Fig. 6. DSC thermograms of zein and quercetagetin composite colloidal nanoparticles. A: ZNP: zein nanoparticle; B: quercetagetin; C: Z–Q30:1 ; D: Z–Q25:1 ; E: Z–Q20:1 ; F: Z–Q15:1 ; G: Z–Q10:1 ; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
zein–quercetagetin composite colloidal nanoparticles. As shown in Fig. 2B, the turbidity of native zein colloidal dispersion was 68.4 NTU, and the presence of quercetagetin significantly (p < 0.05) decreased the turbidity of zein colloidal dispersions to 35.6, 40.6 and 42.0 NTU for samples of Z–Q30:1, Z–Q25:1 and Z–Q20:1 , respectively. Oppositely, the turbidity was greatly (p < 0.05) increased to 128.7 and 400.3 NTU for samples of Z–Q15:1 and Z–Q10:1 , respectively. The changes of turbidity could be also observed according to the illustrations in Fig. 2. The decreased turbidity suggested that the interaction between quercetagetin and zein led to the formation of the binary zein–quercetagetin composites with more compact structure. The increased turbidity might be attributed to the fact that excessive quercetagetin was bound to the surface of zein, resulting in the formation of the largely binary composites by noncovalent interaction (Le Bourvellec and Renard, 2012). Yang et al. (2014) reported that bovine lactoferrin (LF) and (−)-epigallocatechin-3-gallate (EGCG) formed LF–EGCG complex by the molecular interaction and the turbidity of LF–EGCG complex was increased when EGCG/LF molar ratio was more than a critical value of 25:1. According to the aforementioned analysis of size and turbidity, it could be concluded that there existed a certain relationship between size and turbidity since the change trend of particle size was consistent with that of turbidity in this work. 3.2. FTIR spectra The intermolecular interactions of zein and quercetagetin composites were identified by FTIR. The representative spectra of quercetagetin, ZNP and zein–quercetagetin composite colloidal nanoparticles are shown in Fig. 3. In the original spectra of ZNP (Fig. 3A), the band of hydrogen bonds was at 3293.10 cm−1 . However, after the formation of zein–quercetagetin composite colloidal nanoparticles, a shift of hydrogen bonds occurred, and the peaks were at 3315.94, 3310.94, 3311.04, 3311.10 and 3310.97 cm−1 in the spectra of Z–Q30:1 (Fig. 3B), Z–Q25:1 (Fig. 3C), Z–Q20:1 (Fig. 3D), Z–Q15:1 (Fig. 3E) and Z–Q10:1 (Fig. 3F), respectively. Besides, the peak intensity in spectra of zein–quercetagetin colloidal nanoparticles was significantly increased compared with that in the spectrum
of ZNP. This result indicated the formation of hydrogen bonds between zein and quercetagetin, which may be due to the interaction between amide groups of glutamine in zein and hydroxyl groups in quercetagetin as reported by Luo et al. (2011) who found strong hydrogen bonds formed between zein and ␣-tocopherol. As shown in Fig. 3A, typical amide I and amide II peaks for ZNP were 1653.10 and 1540.17 cm−1 , respectively. The amide I band was the prominent C O stretching and amide II absorption was mainly referred to C N stretching and C N H in plane bending (Torres-Giner et al., 2008a; Sessa et al., 2008). Comparing with the spectra of ZNP, the amide I and amide II peaks were shifted to 1652.30 and 1539.01, 1651.77 and 1538.97, 1650.17 and 1539.01, 1650.61 and 1539.94, and 1650.55 and 1538.99 cm−1 in the spectra of Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 , respectively. These findings suggested that another intermolecular force, electrostatic or hydrophobic interactions, might exist between zein and quercetagetin, which were in accordance with previous observations that the interactions between the zein and polyphenols (cranberry procyanidins, curcumin) consisted of hydrogen bonds and hydrophobic interactions (Zou et al., 2012; Liang et al., 2015). 3.3. Fluorescence spectra Fluorescence quenching refers to the interacting process between a fluorophore like a protein and an external quencher molecule such as a polyphenol that decreases the fluorescence intensity of proteins (Keppler et al., 2014) and it is regarded as a useful technique to provide unique information about the interactions between small molecules and proteins due to the sensitivity of intrinsic fluorescence to the microenvironment changes around proteins (Zhang et al., 2013). The effect of quercetagetin on the fluorescence emission spectra of ZNP is shown in Fig. 4. It can be observed that ZNP possessed a strong fluorescence emission peak at 304 nm after being excited at 280 nm. The fluorescence intensity of ZNP was gradually decreased upon increasing the concentration of quercetagetin, which indicated that the fluorescence of ZNP was quenched by quercetagetin. Fluorescence quenching may be ascribed to a variety of molecular interactions including molecular rearrangements, energy transfer, ground state complex formation
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Fig. 7. SEM images of zein and quercetagetin composite colloidal nanoparticles. (A) ZNP: zein nanoparticle; (B) Z–Q30:1 ; (C) Z–Q25:1 ; (D) Z–Q20:1 ; (E) Z–Q15:1 ; (F) Z–Q10:1 ; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
and collisional quenching (Lakowicz and Masters, 2008). The result in this study may be due to the binding of quercetagetin to zein and complexes formation between zein and quercetagetin, which was consistent with Joye et al. (2015) who found that resveratrol binding to zein resulted in fluorescence quenching of zein. 3.4. CD spectroscopy The secondary structure of zein was measured using far-UV CD spectroscopy as shown in Fig. 5. The CD spectrum of ZNP shows two negative peaks at 208 nm and 220 nm and a positive peak at 192 nm, which was characteristic of ␣-helical-rich secondary structure (Selling et al., 2007; Zhai et al., 2011). The quantitative analysis of secondary structural content estimated by SELCON3 as shown in Table 1 yielded 51.1% ␣-helix, 12.3% -sheet,
16.9% -turn and 19.7% unordered structure. This result was consistent with the report of Cabra et al. (2006) who found that the average ␣-helical structure content was 50%, with the majority of the remaining structure in a random coil state. The presence of quercetagetin resulted in changes in the CD intensities which could be observed in the magnified illustration in Fig. 5, suggesting a great effect on the changes of the second structure of ZNP. With zein to quercetagetin mass ratios of 30:1 and 25:1, the deconvolution of the secondary structure content indicated a significant (p < 0.05) decrease in ␣-helical content of ZNP from 51.1% to 44.9% and 40.4%, respectively. At the same time, -sheet content was increased from 12.3% to 14.8% and 17.5%, respectively. These findings might be due to the interaction between zein and quercetagetin. Mizutani et al. (2003) found the ␣-helix content was decreased with an increase in the ratio of linolenic
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Table 1 Secondary structure analysis of native zein and zein–quercetagetin composite nanoparticles Samples
ZNP Z–Q30:1 Z–Q25:1 Z–Q20:1 Z–Q15:1 Z–Q10:1
Content (%) ␣-Helix
-Sheet
-Turn
Unordered coils
51.1 44.9 40.4 56.4 51.9 52.6
12.3 14.8 17.5 7.1 10.5 9.9
16.9 16.6 13.8 18.3 16.1 17.2
19.7 23.8 28.4 18.2 21.5 20.3
ZNP: zein nanoparticle; Z–Q30:1 , Z–Q25:1 , Z–Q20:1 , Z–Q15:1 and Z–Q10:1 represent zein and quercetagetin composites at different mass ratios of 30:1, 25:1, 20:1, 15:1 and 10:1, respectively.
acid ethyl (LAE), suggesting the interaction between LAE and zein. However, continuing the rise of quercetagetin induced obvious increase in the CD intensities, especially for the sample of zein to quercetagetin mass ratio of 20:1, this result suggested a significant (p < 0.05) increase in ␣-helical content of ZNP from 51.1% to 56.4%. At the same time, -sheet content was decreased from 12.3% to 7.1%. These results indicated the addition of quercetagetin led to the conformation change of ZNP. Kanakis et al. (2011) pointed out that the presence of EGCG increased the ␣-helix content of -lactoglobulin, suggesting stronger structural stabilization of the protein.
nanoparticles was not changed but the structure seemed more compact than that of native ZNP. Continuing to increase quercetagetin into the zein solution with mass ratios of 15:1 (Fig. 7E) and 10:1 (Fig. 7F), the morphology of nanoparticles was gradually deviated from the spherical shape with rough edges, and agglomeration was observed resulting from the close packing of particles with larger diameter. These results may be due to the interaction between zein and quercetagetin since the excessive quercetagetin disturbed the intermolecular entanglements among polypeptide chains and the chains were not able to aggregate into spheres after solvent evaporation (Gomez-Estaca et al., 2012). The more compact structure of zein–quercetagetin composite colloidal nanoparticles could be applied to further confirm that the thermal stability of zein–quercetagetin composite colloidal particles was better than that of native ZNP (Fig. 6). 4. Conclusions The formation of composites between zein and quercetagetin led to the fluorescence quenching and changes in the CD intensities of zein, which was mainly due to the hydrogen bonds and hydrophobic effects. The composite colloidal nanoparticles of Z–Q20:1 exhibited higher endothermic peak and more compact structure with reduced turbidity and particle size. The complexation of zein and quercetagetin might be an attractive method to develop new food grade biopolymers with better thermal behaviors and structural properties which could be useful in the development of the potential delivery system for bioactive compounds.
3.5. DSC The DSC thermograms of quercetagetin, ZNP and zein–quercetagetin composite colloidal nanoparticles are presented in Fig. 6. The DSC curve of native ZNP showed a melting peak at 208.15 ◦ C (Fig. 6A) and the native quercetagetin thermogram displayed an endothermic peak at 214.26 ◦ C (Fig. 6B). Compared with the melting peak of native ZNP or quercetagetin, the endothermic peaks of the zein–quercetagetin colloidal nanoparticles were shifted to higher temperatures at 255.50, 257.0, 266.820, 245.5 and 221.51 ◦ C in the DSC thermograms of Z–Q30:1 (Fig. 6C), Z–Q25:1 (Fig. 6D), Z–Q20:1 (Fig. 6E), Z–Q15:1 (Fig. 6F) and Z–Q10:1 (Fig. 6G), respectively. This may be due to the formation of more compact structure between zein and quercetagetin during the anti-solvent process to form the composite particles, and more energy was required to destroy the molecular structures, especially for Z–Q20:1 composite colloidal nanoparticles which could be confirmed by the result of the reduced particle size (Fig. 2A) and lower turbidity (Fig. 2B) of Z–Q20:1 composite colloidal nanoparticles. In addition, the reduction of height and sharpness of the endotherm peak of zein-Q composite colloidal nanoparticles may be attributed to the presence of quercetagetin in the composite nanoparticles (Lai and Guo, 2011). Similar observation was also reported by Hu et al. (2012) who found there was a strong melting peak for sow lutein between 180 and 190 ◦ C, and a thermodynamic compatibility between lutein and zein by processing in solution enhanced dispersion by supercritical fluids. 3.6. SEM images SEM was applied to characterize the morphology of ZNP and zein–quercetagetin composite colloidal nanoparticles as shown in Fig. 7. The ZNP exhibited nanospheres with the diameter around 100 nm and had smooth surface and good dispersion (Fig. 7A), which was consistent with the findings of Wu et al. (2012). When quercetagetin was added into the zein solution at different mass ratios of 30:1 (Fig. 7B), 25:1 (Fig. 7C) and 20:1 (Fig. 7D), the morphology of zein–quercetagetin composite colloidal
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