In vitro antidiabetic and anti-inflammatory effects of Fe-doped CuO-rice husk silica (Fe-CuO-SiO₂) nanocomposites and their enhanced innate immunity in zebrafish

2.1 Preparation of silica nanoparticles (SiO₂ NPs or SNPs) from rice husk

In first (I) process, raw rice husk (15 g) was washed in HCl solution (10 wt%) for refluxed 2 h, rinsed with deionized water, and then dried at 80 °C for 24 h. Sample was further calcined at 600 °C for 4 h to get white needle like crystals. In second (II) process, 2 g of white needle crystals were washed in HCl solution (10 wt%) for refluxed 2 h, rinsed with deionized water, and then dried at 80 °C for 24 h. After calcination (600 °C for 4 h) white ash formed. In third (III) process, 0.5 g of white ash added 15 ml of NaOH (1 N) solution for refluxed 2 h and centrifuged at 8000 rpm for 2 min. In the supernatant clear solution of Na₂SiO₃, the HCl (1 N) was added until get the pH 7. Sample was converted into silica gel and pyrolysis was carried out in a muffle furnace (80 °C for 2 h) to obtained silica (SiO₂) nanoparticles (SNPs). Fig. 1 displays the preparation procedures of SNPs from rice husk.

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Fig. 1

Preparation of SiO₂ NPs (SNPs) from rice husk.

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2.2 Synthesis of CuO NPs, CuO-SiO₂ nanocomposite (CuONC), and Fe-doped CuO-SiO₂ nanocomposite (FeCuONC)

For CuO NPs synthesis, 2.9 g of copper nitrate and 1.2 g of polyvinylpyrrolidone (PVP) were dissolved in 100 ml of distilled water with continuous stirring and heated the solution up to 600 °C. The NaOH solution (1 M) was further added dropwise with continuous stirring for 1 h to get black precipitate. Then mixture was centrifuged and dried at 80 °C for 1 h to get CuO NPs.

For CuO- SiO₂ nanocomposite (CuONC) synthesis, 1 g CuO NPs and 1 g SiO₂ NPs were mixed in 100 ml of distilled water and kept in shaker for 72 h. After that mixture was centrifuged at 2000 rpm for 20 min and dried at 80 °C for 1 h. Finally, sample was calcined at 600 °C for 4 h get blackish brown powder of CuONC.

For Fe-doped CuO-SiO₂ nanocomposite (FeCuONC) preparation, FeCl₃·6H₂O and CuO-SiO₂ in a concentration ratio of 1:6 was mixed in a beaker stirred for 1 h. Then, suspension was transferred into autoclave and heated at 180 °C for 3 h. After that suspension was centrifuged at 2000 rpm for 20 min and pellets were dried at 80 °C for 1 h. Finally, pellets were calcine at 600 °C for 4 h to get FeCuONC. Schematic of CuO NPs, CuONC, and FeCuONC synthesis is presented in Fig. 2.

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Fig. 2

Schematic of CuO NPs, CuONC, and FeCuONC synthesis.

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2.3 Antidiabetic activity

Antidiabetic activity (α-amylase inhibition assay) of prepared samples was examined according to method described earlier (Sivashanmugam, 2017). Briefly, α-amylase (0.1 g in 100 ml of ice-cold distilled water) was mixed with different concentrations (10–50 μg/mL) SNPs, CuONC, and FeCuONC and incubated for 10 min at 30 °C. Thereafter, starch substrate (0.55% in phosphate buffer) was added to start the reaction for 3 min. The reaction was terminated by adding 2 ml of DNS reagent (2% 3,5-dinitrosaliycilic acid and 13% sodium potassium tartrate in 0.5 M NaOH) and was boiled for 10 min in a water bath at 90–100 °C. The mixture was cooled to room temperature and absorbance recorded at 540 nm using UV-visible spectrophotometer. The α–amylase inhibitory activity was expressed as percentage inhibition and was estimated using the following equation. $$I_{\propto -Amylase\left(\%\right)}=\frac{A_{540Control}-A_{540Sample}}{A_{540Control}}\times 100$$

2.4 Anti -inflammatory activity

The in vitro anti-inflammatory activity (inhibition of protein denaturation) was assessed as reported earlier (Nguemnang et al., 2019). In brief, 1 ml of aqueous solution of different concentrations (100, 200, 300, 400 and 500 μg/mL) of SNPs, CuONC, and FeCuONC along with standard solution (diclofenac) was mixed with 1 ml of aqueous solution of bovine serum albumin (5%) and incubated at 37 °C for 15 min. Protein denaturation was carried out by placing the reaction mixture in a water bath at 80 °C for 10 min. Afterwards, mixture was cool down at room temperature and absorbance was measured at 660 nm by UV-visible spectrophotometer. The inhibition of protein denaturation was expressed as percentage inhibition and calculated using the following equation. $$\text{\%}\text{of inhibition}=\frac{\mathit{Absorbanc}{e}_{\mathit{Control}}-Absorbanc{e}_{\mathit{Sample}}}{\mathit{Absorbanc}{e}_{\mathit{Control}}}\times 100$$

2.5 Zebrafish maintenance

All the investigations were accomplished by standard animal practice of OECD guidelines for testing chemicals. Zebrafish (Dania rerio) were obtained from a native aquarium fish supplier (zebrafish wild-type AB strain). Zebrafish were maintained in wealth section loaded up with the adapted water (75 g NaHCO₃, 18 g ocean salt, 8.4 g CaSO₄, per 1000 L). Breeding of fishes was stimulated by sustaining female and male ratio of 3:1. Photo-periodic cycle was sustained by preserving them for 10 h dark and 14 h light. Eggs are collected in early morning and reared up in E3 medium (5 mM/L sodium chloride, 0.18 mM/L, potassium chloride, 0.33 mM/L, calcium chloride, and 0.33 mM/L magnesium sulfate) with 2 μg/mL gentamycin. The fish embryos were instigated from natural depositing and incubated at 28.5 °C.

2.6 Toxicity study of zebrafish embryos and larvae

In vivo toxicity of CuONC and FeCuONC was examined in zebrafish embryos and larvae. In brief, thirty zebrafish embryos of 24-hour post-fertilization (hpf) were exposed to different concentrations (25–200 μl) of CuONC and FeCuONC in E3 medium for 24, 48, 72, and 96 h. Microscopic observation was carried out at different time interval (24–96 h) to visualize the morphological abnormalities. The hatching rate, malformation, and embryos death were also determined. All the experiments were performed in triplicate.

3.1 X-ray diffraction study

XRD spectra of prepared samples are presented in Fig. 3. The CuONC and FeCuONC exhibited the peaks at 32.6°, 35.6°, 38.8°, 48.7°, 53.5°, 58.5°, 61.6°, 66.3°, 68.3⁰, 72.5°, and 75.2° for the respective planes. Two highly intense diffraction peaks are observed at values 35.6° and 38.8° with a preferred orientation the (−111) and (111) axis and which can be indexed as monoclinic structure of CuO. Due to amorphous nature peak of SNPs was not appear in nanocomposite. Absence of Fe peak in FeCuONC suggested that Fe ions were homogeneously mixed with the nanocomposite. The average crystallite size was calculated from the peak width of most intense peak using Debye-Scherrer equation (Shahid et al. 2018). The average crystallite size of CuONC, and FeCuONC was around 36 nm, and 62 nm, respectively. Due to magnetic property Fe-doping might increase the agglomeration crystallites. This could be the possible reason of higher crystallite size of FeCuONC as compared to CuONC. Fe-doping might also increase the oxygen vacancies at the surface of FeCuONC that enhance its biological activities (Arfan et al., 2019).

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Fig. 3

XRD pattern of SNPs, CuO NC, and FeCuONC.

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3.2 FTIR analysis

FTIR spectra was recorded in the range of 500–4000 cm⁻¹. The CuONC and FeCuONC show characteristic vibrations of monoclinic CuO (Fig. 4). Presence of new band at 1738 cm⁻¹ in FeCuONC in comparison to CuONC suggests new bond formation after Fe-doping. The 3011 cm⁻¹ band of SNPs is due to the stretching vibration of O–H bonds in Si-OH and the HO-H of water molecules adsorbed on the surface (Adam et al. 2011). The Si-O-Si bond asymmetric stretching, symmetric and bending vibration were observed as reported in other studies (Rahman et al. 2009). In FTIR spectra of CuO-SiO₂ nanocomposite (CuONC), characteristic bands of SiO₂ were observed with marginal shifting of bands and slight changes in intensities of absorption bands at 1038 and 795 cm⁻¹ (Farrukh et al. 2019). The absence of absorption modes related to secondary phases or other impurities confirms the successful Fe-doping in CuO-SiO₂ NC.

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Fig. 4

FTIR analysis of SNPs, CuO NC, and FeCuONC.

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3.3 Optical characterization

Optical behavior of CuO-SiO₂ nanocomposite (CuONC) and Fe/CuO-SiO₂ nanocomposite (FeCuONC) was examined by UV-visible spectrophotometer. Fig. 5 represents the absorption spectra of CuONC and FeCuONC. The absorption peaks for CuONC were at 192 nm, 200 nm, 361 nm, and 376 nm while for FeCuONC were at 361 nm, 396 nm, and 576 nm. Two prominent peaks at 200 nm and 376 nm were assigned to the absorption of CuONC and two most prominent peaks at 361 nm and 576 nm were assigned to the absorption of FeCuONC (Das et al. 2013). Tauc plot shows that the bandgap energy of CuONC was decreased from 5.5 eV to 5.13 eV after Fe-doping.

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Fig. 5

Optical study CuO NC and FeCuONC.

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3.4 Scanning electron microscopy characterization

Structural morphology of prepared nanostructures was visualized by scanning electron microscope (SEM) (Fig. 6). Both SNPs and CuO-SiO₂ nanocomposite (CuONC) exhibited almost needle like morphology with smooth surfaces (Fig. 6A and B). However, Fe-doped CuO-SiO₂ nanocomposites (FeCuONC) displayed the rectangular morphology with smooth surfaces (Fig. 6C). These SEM images also suggested the slight agglomeration of nanostructures. The average size of prepared nanostructures was approximately 30–70 nm which was according to XRD results. EDX was carried out to examine the elemental composition of Fe/CuO-SiO₂ nanocomposite (FeCuONC). EDX spectra indicated that Fe, Cu, Si, and O were main elements in FeCuONC without impurity (Fig. 6D). These results were in agreement with previous studies (Subhalakshmi et al. 2019).

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Fig. 6

SEM-EDX characterization. SEM image of SNPs (A), CuONC (B), FeCuONC (C), and SEM-EDX spectra of FeCuONC.

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3.5 Particle size analyzer

Particle size analyzer (PSA) was applied for examine the hydrodynamic size distribution of prepared samples (Fig. 7). The hydrodynamic diameter of prepared samples was higher than size calculated from XRD and SEM. This could be due to agglomeration of nanostructures in aqueous state (Baalousha and Lead, 2012). Fig. 7A and B indicate the optimized hydrothermal method effectively produces a well-dispersed and narrow distribution of particles diameter. Aqueous suspension of FeCuONC was fairly stable.

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Fig. 7

Hydrodynamic size distribution of FeCuONC.

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3.6 Thermogravimetric analysis

Thermal property of FeCuONC was analysed by TGA (Fig. 8). TGA graph predicts the 30% mass decomposition of FeCuONC 30–900 °C. The decomposition rate of FeCuONC slow as 5% weight loss per 100 °C temperature increment up to 900 °C. It expressions that 80 % thermal immovability of the complex at 900 °C. Although several studies have been carried out on the catalytic improvement of the thermal decomposition properties of FeCuONC by different nano-additives. However, that may be explained by two proposed approaches. Firstly, the higher surface area results in less sputtering of the particles during decomposition. This causes less mechanical loss and more efficient heat transfer within the nanocomposite, causing high heat release. Another reason for the higher heat release may be attributed to the efficient combustion of NH₃ with chlorine oxides in the presence of Fe/CuO-SiO₂ nanocomposite (FeCuONC) (Joshi et al. 2008).

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Fig. 8

TGA analysis of FeCuONC.

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3.7 Antidiabetic activity

The α-amylase assay is a rapid and inexpensive technique to assess the antidiabetic activity (Laoufi et al. 2017). Antidiabetic activity of prepared samples was examined in the concentrations range of 10–50 µg/ml. Fig. 9 shows that SNPs, CuONC, and FeCuONC exhibited a concentration-dependent antidiabetic activity. Moreover, FeCuONC has highest antidiabetic activity as compared to other studied nanostructures in all tested concentrations. Hence, FeCuONC has great potential to reduce the rate of digestion and absorption of carbohydrate and thereby contribute for effective management of diabetes. Our results are aligned with several recent reports that observed the antidiabetic activity of metal oxide based nanocomposites (Faisal et al., 2022; Selvan et al., 2022).

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Fig. 9

Antidiabetic activity of SNPs, CuONC, and FeCuONC.

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3.8 Anti-inflammatory activity

Anti-inflammatory activity (inhibition of protein denaturation) of SNPs, CuONC, and FeCuONC was assessed in the concentrations range of 100–500 µg/ml. Diclofenac sodium was used as standard materials for comparison with the inhibitory effects of prepared samples. Results showed that all three samples (SNPs, CuONC, and FeCuONC) inhibits the denaturation of egg albumins in a dose-dependent manner (Fig. 10). Moreover, FeCuONC has highest anti-inflammatory activity in comparison to studied nanostructures in all tested concentrations. Earlier studies also reported that inhibition on denaturation of egg albumin was increases with increasing in concentration different nanostructures (Anoop and Bindu, 2015; Govindappa et al. 2011).Figs. 11a-12b

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Fig. 10

Anti-inflammatory activity of SNPs, CuO NC, and FeCuONC.

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Fig. 11a

Hatching rate, malformation, and mortality rate in zebrafish embryos exposed to CuONC for different hours of post-fertilization (hpf).

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Fig. 11b

Representative images of zebrafish embryos and larvae exposed to CuONC. Control group shows the normal appearance for 24–96 hpf. Tail bent (TB), Yolk-sac edema (YSE) and head malformation (HM), and spinal card bend (SB) were markers of malformation caused by CuONC 24–96 hpf.

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Fig. 12a

Hatching rate, malformation, and mortality rate in zebrafish embryos exposed to FeCuONC for different hours of post-fertilization (hpf).

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Fig. 12b

Representative images of zebrafish embryos and larvae exposed to FeCuONC. Control group shows the normal appearance for 24–96 hpf. Tail bent (TB), Yolk-sac edema (YSE) and head malformation (HM), and spinal card bend (SB) were markers of malformation caused by FeCuONC 24–96 hpf.

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3.9 Toxicity study in zebrafish embryos and larvae

The present results represented the biological consequences and teratogenic effects of CuO-SiO₂ nanocomposite (CuONC) and Fe-doped CuO-SiO₂ nanocomposites (FeCuONC) in zebrafish. Our results are in agreement with foregoing reports on metal oxide nanocomposite toxicity in zebrafish (AshaRani et al. 2008). Aforementioned studies have employed nanocomposite of a comparable size range, but then again large surface area and the capability to produce sensitive oxygen species show a role in fashionable the toxicity of nanocomposites (Lee et al. 2007). It is also known that the toxicity varies with different capping agents (Chen et al. 2008). In this study, in vivo toxicity of CuONC and FeCuONC to zebrafish embryos and larvae is shown in Figs. 11 and 12. Results showed that prepared CuONC and FeCuONC deposition of on the egg surfaces increases with increasing the concentration of CuONC and FeCuONC. The mortality of zebrafish embryos and larvae exposed to different concentrations (25, 50, 100, 200 µl/ml) of CuONC and FeCuONC was resolute at specific time points. The CuONC and FeCuONC at the concentration of 200 µl/ml exhibited noticeable malformations. As shown in Fig. 11 experience in the direction of the CuONC produced embryos characteristically the malformations like spinal card bent (SB) yolk-sac edema (YSE), tail bent (TB), head malformation (HM) and delayed hatching going on the embryos of zebrafish by the side of the higher concentration (100, 200 µl/ml) during the 24–96 hpf. The FeCuONC exhibited higher characteristic malformations comparable to the spinal cord bent (SB), yolk sac edema (YSE), tail bent (TB), head malformation (HM) and delayed hatching continuously the embryos of zebrafish at higher concentration (100, 200 µl/ml) during the 24–96 hpf. Such type of malformations was also caused by CuO NPs, ZnO NPs, ZrO-NPs, and silica NPs as reported in earlier studies (Karthiga et al. 2019; Ganesan et al. 2016). Comparable wrinkles in backbone of zebrafish embryos were beforehand perceived upon treatment through cadmium, where cadmium twisted somite formation consequently important to kinks and undulations (Chow and Cheng 2003). Zebrafish embryos exposed to quantum dots also demonstrated these defects (King-Heiden et al. 2009). As compared to control, CuONC and FeCuONC exposure showed the substantial effects on mortality of zebrafish embryos. Composed 25, 50 µl/ml of CuONC and FeCuONC established toxicity, killing of 80% of zebrafish embryos at 96 hpf. Conversely 100, 200 µl/ml CuONC and FeCuONC preserved groups exhibited 100% mortality at 96 hpf. Overall, innate immunity response of FeCuONC in zebrafish was higher than CuONC