Modified TiO2 Characterization
X-ray diffraction (XRD)
XRD pattern of the three types of TiO2 E171 samples were shown in figure 1. All the characteristics peaks for anatase TiO2 appeared in the XRD pattern of anatase TiO2 E171 and modified with thyme and honey ; see figure (1-A). All the assigned peaks were in a good agreement with the JCPDS no (73-1764) for anatase TiO2 [24]. The three samples of anatase TiO2 E171 showed the anatase phase in the tetragonal crystal structure with lattice parameter a=b=3.776 nm, 3.774 nm , 3.777 nm for anatase TiO2 E171 and modified with thyme and honey respectively. While the lattice parameter c is 9.47 nm, 9.479 nm, 9.481 nm for anatase TiO2 E171 and modified with thyme and honey respectively. A small increase in the lattice parameter c was observed in modified anatase TiO2 E171 samples, which suggest the enlargement of the crystal dimension in this direction and increase of the particle size.
Figure (1-b) represents XRD pattern of rutile TiO2 E171, all the assigned peaks were in a good agreement with the JCPDS no (87-0920) for rutile TiO2 [25]. The rutile phase with tetragonal crystal structure were detected in the three samples of rutile TiO2 E171 and modified with thyme and honey. The calculated lattice parameters were a=b= 4.553 nm, 4.588 nm, 4.584 nm for rutile TiO2 E171 and modified with thyme and honey respectively and lattice parameter c=2.95 nm, 2.956 nm, 2.956 nm for rutile TiO2 E171 modified with thyme and honey respectively. A small increase in the lattice parameter a=b was observed in modified rutile TiO2 E171 samples, which suggest the enlargement of the crystal dimension in this direction and increase of the particle size.
Figure (1-c) represents XRD pattern of food grade TiO2 E171, all the assigned peaks were in a good agreement with the JCPDS no (87-0920) for rutile TiO2 [25]. The rutile phase with tetragonal crystal structure were detected in the three samples of food grade TiO2 E171 and modified with thyme and honey. The calculated lattice parameters were a=b= 4.594 nm, 4.5879 nm, 4.5369 nm for food grade TiO2 E171 and modified with thyme and honey respectively and lattice parameter c=2.9558 nm, 2.9578 nm, 2.9519 nm for rutile food grade E171 modified with thyme and honey respectively. A small decrease in both lattice parameter a=b and c was observed in modified food grade TiO2 E171 samples, which suggest the quenching of the crystal dimension of modified food grade TiO2 E171with thyme and honey and decrease of the particle size.
The effect of modification with thyme and honey on the structure of TiO2 E171 was observed as increase in the peak intensity and decrease in peak broadening with peak shift to lower 2θ. This result may be due to the increase of the particle size and improvement the crystallinity of TiO2 E171 after modification with thyme and honey [26] [27].
The average crystalline size of TiO2 E171 was calculated by Scherer's equation[28] (see table 1).
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where d is the average mean diameter of NPs, λ is the wavelength of X-ray radiation source, 0.89 is a constant crystalline shape factor, θ is the Bragg’s diffraction angle, and β is the angular full width at half maximum (FWHM) of XRD peaks recorded at diffraction angle 2θ.
The modification of anatase and rutile TiO2 E171 with thyme and honey causes an observed increase in the calculated crystal size, while it causes decrease in the crystal size of food grade TiO2 E171. This change in the particle size after biomodification may attributed to the interaction kinetics of biomolecules with nanoparticles [29] [30].
Table 1
Particle size of TiO2 E171 samples as calculated from Scherer's equation
Sample |
Particle size (nm) |
Anatase TiO2 E171 |
53.36 ± 9.52 |
Anatase TiO2 E171 modified with thyme |
81.48 ± 24.3 |
Anatase TiO2 E171 modified with honey |
92.43 ± 15.85 |
Rutile TiO2 E171 |
90.5 ± 16.22 |
Rutile TiO2 E171 modified with thyme |
97.5 ± 12.27 |
Rutile TiO2 E171 modified with honey |
106.5 ± 12.61 |
Food grade TiO2 E171 |
102.76 ± 27.68 |
Food grade TiO2 E171 modified with thyme |
70.97 ± 10.72 |
Food grade TiO2 E171 modified with honey |
92.08 ± 15.78 |
High resolution transmission microscope (HRTEM)
The HRTEM images and their corresponding particle size distributions of all TiO2 E171 samples are shown in figure 2. It's observed in HRTEM images that all TiO2 E171 samples have a non-uniform spherical shape. While the modification of anatase TiO2 E171 with thyme and honey causes decrease in primary particle size (120.23 ± 34.5 nm, 188.9 ± 62.9 nm, 128.79 ± 37.62 nm for anatase TiO2 E171 and modified with thyme and honey respectively), the modification of rutile and food grade TiO2 E171 with thyme and honey causes increase in primary particle size (189.147 ± 42 nm, 168.44 ± 59.61 nm, 158.66 ± 34.62 nm for rutile TiO2 E171 and modified with thyme and honey respectively and 136 ± 44.5 nm, 111.07 ± 38.55 nm, 110.77 ±17.78 nm for food grade TiO2 E171 and modified with thyme and honey respectively).
UV- visible spectroscopy
The UV–visible absorption spectra of all E171 samples are shown in Fig 3 (a, b and c). The absorption peaks were (282.35, 283.17 and 283.17 nm) for anatase, rutile, and food grade TiO2 (E171) respectively. The absorption peak of honey extract was observed at 280.53 nm, while the absorption peak of thyme detected at 483.12 nm. After modification with honey the three types of TiO2 showed a blue shift with the absorption peaks at 222.4, 269.63 and 223.91 nm for anatase, rutile, and food grade TiO2 (E171) respectively. Similar after modification with thyme all the three types of TiO2 showed a blue shift with the absorption peaks at 261.76, 268.12 and 268.12 nm for anatase, rutile, and food grade TiO2 (E171) respectively. This blue shift is the result of changing the particle size after modification, which confirm the modification of the three types of TiO2 with honey and thyme [31].
The presence of broad peaks in all the spectra of honey and thyme modified TiO2 samples can attributed to two reasons; the first reason is the presence of bioactive molecules of honey and thyme. It has been concluded that honey has several important peaks in the region 250 – 450 nm due to the absorption of benzoic, salicylic and aryl-aliphatic acids [32]. Also, the reported result for thyme revealed that it has various absorption peaks in the UV-vis region due to the presence of carvacrol, thymol. While, the second reason is the excitation of plasmonic resonances as absorption spectra of larger metal colloidal dispersions can exhibit broad peaks or additional bands with the lower absorbance in the UV-visible range due to the excitation of plasmonic resonances or higher multiple Plasmon excitation [33].
FTIR spectroscopy
Different functional groups can be observed in the FTIR spectrum of thyme and honey due to the presence of bioactive groups such as carvacrol, thymol, carbohydrates, organic acids and sugar, see fig. 4 (a ,b and c). The broad band observed approximately between 3000 to 3500 cm-1 corresponding to OH vibration of hydroxyl group. Band observed approximately at 2900 cm−1 due to C–H stretching of methyl and isopropyl groups on the phenolic ring. Band at approximately 1730 cm−1, 1600 cm−1 due to C=O and C=C functional groups respectively. The vibrational band due to C–C in ring of aromatics group were observed at 1496 cm−1, 1319 cm−1 and 1010 cm−1, while C-O were observed at 1171 cm−1 [34] [35].
The functional groups belonging to TiO2 were observed approximately at 458 cm−1 corresponds to the Ti - O – Ti vibrational band and around 693 cm-1 correspond to TiO2 Modes [36] [37].
The presence of functional groups belonging to honey and thyme in the FTIR spectrum of the three types of TiO2 particles confirm the success of modification. New bands where observed in FTIR spectrum of anatase modified with honey due to the presence of honey such as the bands at 3279,1647, and 1074cm-1 which corresponding the stretching vibration of the –OH group, vibrations of the –OH groups and the stretching vibrations of C–O in C–OH group and stretching of C–C in the carbohydrate structure respectively. Bands at 3244,1651cm-1 corresponding OH vibration of hydroxyl group, the C=C bonds respectively. All these bands are shifted from their original position in honey, this confirming honey and thyme interaction with the surface of anatase TiO2.
Cytotoxicity
Cell viability against Caco2 cells for all types of TiO2 E171 was evaluated using MTT assay , fig. 5. At low concentration (0.125, 0.0625 and 0.03125 mM) a small reduction in cell viability approximately from 1 to 6% were observed. While at high concentration (0.25 and 0.5 mM) the reduction of cell viability increased approximately from 10 to 20%. Similar result was obtained by Ji-Soo Hwang et.al.[38]. An enhancement in the cell viability was observed in the three types of TiO2 E171 after modification with honey and thyme, which is in a good agreement with the reported result for honey [39] and thyme [40]. However, samples modified with honey showed lower cytotoxicity than that modified with thyme.
Antioxidant activity
Free radical scavenging activity of TiO2 E171 after modification of thyme was measured with DPPH assay and plotted in fig. 7. An increase approximately from 10 to 15% was observed in all samples at low concentrations and approximately from 5 to 10% at high concentrations. Between the three types of TiO2 E171 food grade has the largest radical scavenging activity at all concentrations, while the other two types have a similar radical scavenging activity. The modification of TiO2 E171 with thyme appears to increase the radical scavenging activity of the three types of TiO2 E171. The calculated IC50 is 19.6 mM, 81.6 mM and 6.8 mM for anatase, rutile and food grade modified TiO2 E171 respectively. This agreed with the previous studies, which report that thyme extract can be used as a natural antioxidant to prolong stability of oils [41].
In the body, honey can take up free radicals and contribute to better health. The radical scavenging activity after modification with honey increased by approximately 2 to 6% with lower concentration and increased by approximately 1 to 4% with higher concentration. The calculated IC50 is 62.6 mM, 93.56 mM, and 60.16 mM for anatase, rutile and food grade modified TiO2 E171 respectively [42].
In comparison of the effect of modification with honey and thyme on the radical scavenging activity of the three types of TiO2 E171, thyme was found to have the higher radical scavenging activity. Moreover, food grade TiO2 E171 modified with thyme have the highest radical scavenging activity among the three types of TiO2 E171, fig. 8. These results demonstrate that there no correlation between cytotoxicity and radical scavenging activity [43].