Riboflavin conjugated temperature variant ZnO nanoparticles with potential medicinal application in jaundice

Abstract

A single step rapid synthesis of zinc oxide nanoparticles (ZnO NPs) in a green approach has been demonstrated via conjugating the natural product riboflavin by varying temperature. The conjugation of riboflavin to ZnO NPs was confirmed by ultraviolet-visible (UV-VIS) spectroscopy and photoluminescence (PL) intensity. The crystallinity and presence of functional groups in the temperature variant riboflavin conjugated ZnO NPs was determined by the X-ray diffraction (XRD) technique and Fourier transformed infrared (FT-IR) spectroscopy respectively. The average diameter of the synthesized nanoparticles is about ∼40 nm in spherical shape which has been preliminarily revealed by field emission scanning electron microscopy (FESEM) analysis and further confirmed by high-resolution transmission electron microscopy (HRTEM). The synthesized temperature variant ZnO nanoparticles with the aid of riboflavin showed significant ameliorative efficiency against jaundice stress at molecular and cellular levels in mice models. Biochemical parameters, different cytokine profiling (Th1 & Th2 cells) and their corresponding m-RNA expressions have been studied by the enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) in the presence of riboflavin conjugated ZnO NPs synthesized at 60 °C and 30 °C temperature (Ribo-ZnO30/Ribo-ZnO60). Substantial Ribo-ZnO60 assisted improvement (p > 0.001) of the thymus-dependent functions and protein expressions of other genes associated with jaundice were observed by Western blotting. The supplementation of Ribo-ZnO60 treatment is more active in histologically recovering the liver and kidney tissues than Ribo-ZnO30 due to their particle nature and more phases with uniform size.

---

1. Introduction

Obstructive jaundice is caused by occlusion of the common bile duct or its tributaries that leads to complications such as biliary infection, septic shock, hepatic parenchymal injury, and multiple organ dysfunctions which carry a high risk for mortality.¹ It is associated with many other clinical conditions such as gallstones, tumours of the bile duct, pancreatitis, wound breakdown, sepsis, coagulopathy, gastrointestinal hemorrhage, cardiovascular problems, and immune depression.² A healthy liver regulates its activities in an exquisite homeostatic mechanism, maintaining constant tissue mass relative to levels of metabolic stress in the body.³ In mammalian system, hepatocytes of the individual are the main functional cell of the organ, retain a remarkable capacity to adjust to changes in metabolic demand by cell division in the event of a metabolic deficit, or alternatively, through apoptosis in the event of excess metabolic capacity.^4,5 The mechanisms and mediators responsible for the pathogenesis of liver damage from acute biliary obstruction remain unclear.

Usages of different medicines have become resistive and expensive for the treatment of any diseases. With the improvement of human lifestyle and quality with technology, it is receiving abundant demands to develop a healthy environment for human beings because without environmental awareness, people are naturally exposed to various harmful bacteria and virus too.^6,7 Recent advancement in nanoscience and nanotechnology has introduced the potential applications of inorganic nanomaterials in different fields. Nanomaterials like ZnO, copper oxide nanoparticles (CuO), gold nanoparticles (GNPs), silver nanoparticles (AgNPs) and titanium dioxide (TiO₂) have drawn the attention of many researchers for their unique optical and chemical behaviours which can be easily altered by changing the morphology in different aspects.^8–11 Within the large family of metal oxide nanoparticles, the role of ZnO NPs already has been well established in various cutting edge applications like electronics, communication, sensor, cosmetics, environmental protection, biology and medicinal industry along with its anti-bacterial, anti-fungal, acaricidal, larvicidal and anti-diabetic activities.^12–14 ZnO NPs were also used as a potential phototherapy tool for treating jaundice for their biocompatibility.¹⁵ In absence of light ZnO NPs may also degrade bilirubin but its productivity is approximately 45% of light induced degradation of bilirubin.¹⁶ Green (biological) synthesis of nanoparticles (NPs)/novel metals, such as Ag, Au, Pd, Pt using microorganism or plant extract enriched with bioactive phytochemicals shows considerable interest in the last couple of years.^17,18

Riboflavin, a yellow-orange solid substance with poor water solubility is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). It is required for a variety of flavoprotein enzyme reactions including activation of other vitamins also.¹⁹ It is well known that riboflavin supplements have been used as part of the phototherapy treatment of neonatal jaundice which breaks down bilirubin and others toxin caused by jaundice.²⁰

The aim of the study is to synthesize ZnO NPs via green way using riboflavin, giving special emphasize on the growth of NPs at different temperatures for the substantial treatment of jaundice on the molecular level. The synthesized NPs were characterized using UV-VIS, PL spectra, XRD, FTIR, FESEM and HRTEM respectively. Different biochemical parameters, effected cytokines and other regulatory genes associated with jaundice had been investigated in response to temperature variant ZnO NPs treatments utilizing ELISA, RT-PCR with the corresponding expression of the protein by Western blotting techniques.

2. Materials and methods

2.1. Experimental ingredients

Zinc acetate dihydrate, riboflavin, tri-sodium citrate and other chemicals used in this experiment were purchased from Merck (Germany) and all are of analytic grade. Healthy male Swiss albino mice (weight of 32 ± 5 g) were obtained from animal house approved by committee for the purpose of control and supervision of experiments on the animal (CPCSEA), Chennai, India (Registration No. 50/PO/99/CPCSEA).

2.2. Synthesis of riboflavin conjugated ZnO NPs

Riboflavin conjugated ZnO NPs were synthesized in a colloidal solution by the co-precipitation technique. Initially, 10 ml riboflavin solution (2 mM) was added drop-wise in 50 ml zinc acetate solution (10 mM) under continuous stirring condition at 50 °C for 20 min and the pH of the reaction mixture was adjusted to 9.0. The yellow coloured reaction mixture was left for 1 h for complete reaction and the precipitate was collected by centrifugation at 16 000 rpm for 10 min at 4 °C. The precipitate was divided into two parts and calcinated at 30 °C and 60 °C for 24 h distinctly and the samples were designated as Ribo-ZnO30 and Ribo-ZnO60 respectively. Yield of riboflavin conjugated ZnO NPs synthesized through this process were calculated by using the following formula:

2.3. Characterization of Ribo-ZnONPs

Various physicochemical procedures were used to characterize the synthesized ZnO NPs of different temperature. UV-VIS light spectra of the synthesized nanoparticles were recorded (λ25 spectrophotometer, Perkin Elmer Germany) and PL spectra were noted for excitation at (λ) 272 nm with an excitation slit of 5 mm (PMT voltage 500) in the range of 400–700 nm were measured in Agilent. Similar weights of different nanoparticles (Ribo-ZnO30 & Ribo-ZnO60) were considered for UV-VIS and PL data analysis. The crystallinity of the nanoparticles was analysed by the XRD patterns in the range of 2θ from 25° to 80° using powder diffractometer, Model D8, BRUKER AXS, by Cu Kα radiation (α = 0.15425 nm). About 1% KBr plate of the riboflavin conjugated to ZnO NPs was studied for analysing associated chemical groups in FTIR (JASCO FTIR instrument-410, USA). The morphology and the size distribution of Ribo-ZnO30 and Ribo-ZnO60 samples were analysed by the electron microscopes (FESEM; INSPECT F50, Netherland & HRTEM; JEM–2100 HRTEM, JEOL, Japan).

2.4. Experimental design for anti-hyperbilirubinemia test

For anti-hyperbilirubinemia studies, induction of Jaundice was carried out by following standard protocol.²¹ Briefly, 36 mice were divided into 4 groups (n = 9) and maintained under standard laboratory conditions (temperature 25 °C ± 2 °C with day/night circle of 12 h/12 h). For the day period all groups provided blue light by Philips TL 20W/52 lamps (λ = 450; 20 μW cm⁻² nm; Philips, Amsterdam, Netherlands).²² Hyperbilirubinemia was induced by treating mice with 1 ml kg⁻¹ body weight CCl₄ solution (50% CCl₄ in olive oil) daily for a period of 3 weeks. The experimental procedure was tabulated as follows:

Groups	Treatment	Drug administration	Remarks
I	N.A.	N.A.	Control
II	Olive oil (1 ml kg^−1 body weight)	N.A.	Hyperbilirubinemia
III	CCl4 in olive oil (1 ml kg^−1 body weight)	Ribo-ZnO30 (5 mg kg^−1 body weight)	Ribo-ZnO30 treated
IV	CCl4 in olive oil (1 ml kg^−1 body weight)	Ribo-ZnO60 (5 mg kg^−1 body weight)	Ribo-ZnO60 treated

Free access to dry plate diet (Hindustan Liver, Kolkata) and water adlimitom were provided in this study. The experiments were carried out according to the guideline of CPCSEA (control and supervision of experiments on animal), approved by the institutional animal ethics committee (IAEC; Approval no. TOX/DEY'S/IAEC/09/14). Animals belonging to group I and II received a subcutaneous injection (SC) of 0.5 ml physiological saline only. Hyperbilirubinemic mice belongs to group III and IV received subcutaneous injection of 5 mg kg⁻¹ body weight Ribo-ZnO30 and Ribo-ZnO60 dissolved in physiological saline water respectively. The total duration of the experiments was continued for 4 weeks. After the treatment, the animals were fasted overnight, anesthetized with anaesthetic ether and killed by cervical dislocation. Blood samples were collected from the heart immediately after sacrifice and stored in both with or without anticoagulant (heparin) containing containers. Serum was collected by centrifugation and stored at −20 °C. For further study, Liver and kidney tissues were dissected out and stored in vacuum desiccators at −20 °C to prevent exposure from auto-oxidation environment by removing adhered blood.

2.4.1. Animal ethical permission. All the animals used for our study were procured from a Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) approved animal house (registration number: 50/PO/99/CPCSEA) and all experiments performed were under the guidelines of both CPCSEA and IAEC. The approval number – Tox/DEY'S/IAEC/09/14.

2.5. Biochemical assay

Total bilirubin was measured by using the kit provided by Accurex Biomedical Pvt. Ltd., Mumbai, India. Liver function enzymes (ALP; alkaline phosphatase & ACP; aspartate aminotransferase, SGOT; serum glutamic oxaloacetic transaminase, SGPT; serum glutamate-pyruvate transaminase) and kidney function test (urea & creatinine) were measured by using individual kits (Merckotest®, Merck, India). Cholesterol level and Triglyceride was assayed using kit provided by Merckotest®, Merck, India. Lipid peroxidation (LPO) was measured by the determination of the thiobarbituric acid-reactive substances (TBARS) according to a standard protocol where the amount of malondialdehyde (MDA) was calculated from the extinction coefficient of MDA (1.56 × 105 M⁻¹ cm⁻¹).²³ Antioxidant activities like superoxide dismutase (SOD) level was assayed by the method based on the reduction of nitroblue tetrazolium (NBT) to blue formazan by superoxides, produced phytochemically in the reaction system.²⁴ Reduced glutathione (GSH) and glutathione peroxidase (GPx) activities were determined by using the standard protocol respectively.^25,26

2.6. Cytokine profiling by ELISA test

Cytokine profiles of treated and untreated mice were studied by using commercial ELISA kits (R&D System, Minneapolis, MN, USA). The levels of interleukin 1β (IL-1β), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-10 (IL-10) from the serum of treated and control samples were determined. The assays were performed by the respective Quantikine Immunoassay Kit (Minneapolis, U.S.A.).

2.7. m-RNA expression by RT-PCR

Molecular biological parameters were estimated by following the protocol of Alkaladi et al.²⁷ For real-time polymerase chain reaction (RT-PCR), total RNA from the liver tissues of treated and control mice were extracted by using MagJET RNA Kit (Thermo scientific, USA) and cDNA was synthesized by using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo scientific, USA). Every RT reaction contained 1 μg RNA template, 1 μl random hexamer primer, 4 μl 5× reaction buffer (250 mM Tris–HCl (pH 8.3), 250 mM KCl, 20 mM MgCl₂, 50 mM DTT), 1 μl Ribolock RNase inhibitor (20 U/μl; Thermo scientific, USA), 2 μl dNTP mix (10 mM; Thermo scientific, USA) and 1 μl RevertAid H Minus M-MuLV Reverse Transcriptase (200 U/μl; Thermo scientific, USA). 2× Dream Taq Green PCR Master Mix (Thermo scientific, USA) were used to start the PCR reaction and performed by using Light Cycler® 480 Real-Time PCR (Roche Applied Science, Penzberg, Germany). Agarose gel electrophoresis of amplified PCR products of selected genes was visualized in UV-transilluminator. GAPDH was used as reference control. Primers (Operon Biotechnologis, Gmbh, Germany) for selected genes were as follows:

IL-1β: forward 5′-TTGACGGACCCCAAAAGAT-3′; reverse 5′-GAAGCTGGATGCTCTCATCTG-3′.

TNF-α: forward 5′-GGCAGGTCTACTTTGGAGTCATTGC-3′, reverse 5′-ACATTCGAGGCTCCAGTGAATTCGG-3′.

IL-6: forward 5′-TGGAGTCACAGAAGGAGTGGCTAAG-3′, reverse 5′-TCTGACCACAGTGAGGAATGTCCAC-3′.

IL-8: forward 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′, reverse 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′.

IL-10: forward 5′-CGGGAAGACAATAACTG-3′, reverse 5′-CATTTCCGATAAGGCTTGG-3′.

GAPDH: forward 5′-CCC GTA GAC AAA ATG GTG AAGGTC-3′, reverse 5′-GCC AAA GTT GTC ATG GAT GAC C-3′.

2.8. Western blotting analysis

Protein expression profiles of IL-1β, TNF-α, IL-6, IL-8 and IL-10 from liver tissues were studied by the Western blot analysis. Initially, the liver tissues (30 mg) were homogenized in phosphate buffer saline (PBS; pH 7.4) containing 1× protease inhibitor cocktail. The supernatants were collected as cytosolic proteins by centrifuging homogenates at 2900 × g for 10 min at 4 °C. The remaining pellets were further dissolved in lysis buffer A (20 mM HEPES, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.05% Triton X-100, 1× protease inhibitor cocktail, pH 7.9) and centrifuged at 3500 × g for 10 min at 4 °C. The supernatants were also collected as cytosolic proteins and both the fractions were precipitated by adding 8 volume of ice-cold acetone after incubating overnight at −20 °C. The pellets were dissolved in rehydration buffer (8 M urea, 5% β-mercaptoethanol, 10% SDS, 2% CHAPS and 50 mM DTT) and the protein content was determined by the Bradford method.²⁸ The proteins (∼20 μg) were resolved on a 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to the nitrocellulose membranes and blocked with 2% bovine serum albumin (BSA) in TBST (137 mM NaCl, 25 mM Tris, 1 mM Na₂-EDTA and 0.1% Tween 20). Thereafter, the membranes were incubated with the rabbit anti-mouse IL-1β, TNF-α, IL-6, IL-8 and IL-10 antibodies (Santa Cruz Biotech, California, USA) at 4 °C overnight at a dilution of 1 : 200–1 : 1000. After washing several times with TBST, the membranes were incubated with the HRP-conjugated goat anti-rabbit IgG antibody (1 : 2000 dilution) at room temperature for 3 h, washed with TBST and incubated with a chemo-luminescent peroxides substrate (Sigma, St. Louis, USA). Reactions were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotec, Uppsala, Sweden).

2.9. Histological studies

The liver and kidney tissues of treated and control mice were fixed by using Bouin's fluid and then washed several times with different graded alcohol (50%, 70%, 80%, & 95%) to remove the excess fluid for embedding in paraffin. The embedded tissues were sliced by using the rotary microtome and the paraffin sections were washed in xylol before staining with haematoxylin and eosin. The histological study of liver and kidney tissues were analysed by the compound microscope at 100× magnification.

2.10. Statistical analysis

The whole experimental setup was repeated twice and data were averaged and tabulated as the mean ± standard deviation (S.D.). The statistical analyses of the data estimated from each group of all conditions were done by One-way analysis of variance (ANOVA) and all pair wise multiple comparison procedures (Holm–Sidak method) by using the Sigma Stat (version 3.2).

3. Results and discussions

3.1. Optimizing the riboflavin concentration on ZnO NPs synthesis

The yellow coloured ZnO–riboflavin complexes precipitated which upon heating produces riboflavin conjugated ZnO NPs as the zinc ions (Zn²⁺) of zinc acetate in aqueous solution reduced by the free electrons of amine and carbonyl groups of riboflavin. Optimizing the yield of ZnO NPs, concentration of riboflavin plays a vital role. It has been observed that 20 (mM mM⁻¹) molar ratio of zinc acetate to riboflavin (i.e. riboflavin (2 mM): zinc acetate (10 mM) = 1 : 5) was optimal concentration for the synthesis of ZnO NPs.

Insufficient amount of bioactive compound present in 15–20 molar ratio of zinc acetate to riboflavin lowers the yield of ZnO, whereas 30–35 molar ratio of zinc acetate to riboflavin yield nearly same amounts of ZnO (Fig. 1). The findings suggest that the amount of bioactive compounds present in the case of 20 molar ratios of zinc acetate to riboflavin was sufficient to reduce all Zn²⁺ ions present in the reaction mixture.

Fig. 1 Effect of riboflavin/zinc acetate ratio on yield (%) of ZnO NPs synthesis.

3.2. Characterization of temperature dependent synthesized nanoparticles

Optical analysis of synthesized sample dried at 30 °C (Ribo-ZnO30) by UV-VIS spectra (Fig. 2A) showed that the characteristics absorption band near (λ) 365 nm for ZnO, two other associated absorption bands at (λ) 270 nm and (λ) 446 nm were due to the conjugated riboflavin compounds. Riboflavin conjugated ZnO calcinated at 60 °C (Ribo-ZnO60) showed sharp surface plasmon resonance (SPR) bands at (λ) 377 nm along with bands at (λ) 270 nm and (λ) 446 nm. Ribo-ZnO60 samples showed higher absorption intensity than the Ribo-ZnO30 samples due to the formation of more intense phases of ZnO NPs.

Fig. 2 (A) UV-VIS spectra of Ribo-ZnO30 & Ribo-ZnO60 (B) PL spectra of Ribo-ZnO30 & Ribo-ZnO60. In both UV-VIS and PL measurements aqueous solution of 0.5 mg ml⁻¹ Ribo-ZnO were used.

It has been observed that the PL intensity of Ribo-ZnO30 represents the low emission intensity, while Ribo-ZnO60 showed a sharp narrow emission band at (λ) 330 nm and a broadband at (λ) 532 nm for excitation at (λ) 272 nm. More emission intensity of Ribo-ZnO60 was shown due to the better crystallinity and more phase of ZnO than Ribo-ZnO30 (Fig. 2B).

The XRD patterns of the synthesized samples were shown in Fig. 3A. Several characterization peaks for ZnO NPs in XRD were showed 2θ values at 31.75, 34.41, 36.22, 47.59, 56.60, 62.85, 66.40, 67.95, 69.02, 72.54 and 76.87 corresponds to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) respectively. All the peaks were duly assigned by using JCPDS file no. 36-1451.²⁹ The less intensity of Ribo-ZnO30 confirmed that the small particle size, as well as less phase formation at room temperature whereas crystallinity and phase formation increases with a higher formation.

Fig. 3 (A) XRD pattern of synthesized Ribo-ZnO30 & Ribo-ZnO60 respectively (B) FTIR spectra of Ribo-ZnO30 & Ribo-ZnO60 respectively showing appearance of transmittance bands (with arrow) for different functional groups.

The presence of characteristic bands for several functional groups of synthesized samples was shown in the FT-IR spectra (Fig. 3B). The IR peaks at (λ) 3418 cm⁻¹ for vibrational –OH and stretching of coordinated water was observed at around (λ) 3500 cm⁻¹.³⁰ Aromatic groups of riboflavin were present and confirmed by –C=N conjugated system and –C–N were observed at (λ) 1549 cm⁻¹ and 1407 cm⁻¹ respectively. At (λ) 1071–1065 cm⁻¹ and 1220–1210 cm⁻¹, the peaks stand for the C–O stretching of primary alcohol, benzene derivatives, and the –C–N symmetry stretching were represented by peak at (λ) 1222 cm⁻¹. The symmetric and asymmetric vibration of –C–N, –C=O and –C–O stretching vibration observed at (λ) 1641 cm⁻¹, 1371 cm⁻¹ and 1065 cm⁻¹ respectively. Aromatic ring deformation was confirmed by the peak at (λ) 700 cm⁻¹. The presence of ZnO was confirmed by the peak at (λ) 482 cm⁻¹ for both Ribo-ZnO30 and Ribo-ZnO60.³¹ The IR bands of different groups confirm the presence of riboflavin molecules with close association to ZnO NPs. A less significant difference in the IR spectra of Ribo-ZnO30 and Ribo-ZnO60 was observed. FT-IR spectra of synthesized NPs confirm the presence of intense stretching and vibrational bands for several compounds of riboflavin. This result supports that the bioactive riboflavin compounds were absorbed on the surface of ZnO nanoparticles. Some functional groups show the less intensity in IR bands for Ribo-ZnO60 as they were calcinated at the higher temperature.

The morphology of the synthesized nanoparticles was shown in Fig. 4. The FESEM image of the Ribo-ZnO30 and Ribo-ZnO60 reveal the size of the synthesized materials are in nano range (30–40 nm) and the Ribo-ZnO60 particles are distinctly observed in regular spherical shape.

Fig. 4 FESEM view of synthesized Ribo-ZnO30 & Ribo-ZnO60.

The HRTEM micrograph shows that the Ribo-ZnO30 is attained merely amorphous with less crystallinity in nature whereas the Ribo-ZnO60 are equal sized spherical shaped particles. The HRTEM micrographs support the size and nature of synthesized particles as evident from FESEM and XRD respectively (Fig. 5).

Fig. 5 HRTEM view of synthesized Ribo-ZnO30 & Ribo-ZnO60.

3.3. Mechanism of riboflavin conjugated ZnO NPs synthesis

Riboflavin conjugated ZnO NPs synthesis is a green route for nanoparticles synthesis. The presence of riboflavin molecules in a close association with ZnO molecules was previously confirmed by UV-VIS and FT-IR spectra. The detailed scheme of ZnO formation was represented in Fig. 6.

Fig. 6 Mechanism of riboflavin conjugated ZnO NPs synthesis.

In aqueous solution, zinc acetate dihydrate disassociated as Zn²⁺ ions and acetate ions. Free amine (–NH) and carbonyl groups (C=O) of riboflavin interact with Zn²⁺ ions. In the presence of H₂O and NaOH, they donate an electron and reduced to Zn⁰.³² The Zn⁰ residues accumulate together to form zinc hydroxide particles with the help of water molecules which on heating produces ZnO NPs. The size of the synthesized particle was another vital parameter for its applicability in biomedical fields.³³ During aging Zn⁰ atoms accumulated in such a way that each particle of ZnO surrounded by many riboflavin molecules. Electrostatic repulsion of riboflavin molecules plays an important role in size controlling.³⁴

3.4. Anti-jaundice activity of riboflavin conjugated ZnO NPs

3.4.1. Bio-chemical activities. Jaundice exposure significantly increased the bilirubin and direct bilirubin levels whereas supplementation of Ribo-ZnO30/Ribo-ZnO60 treatment decreased the bilirubin and direct bilirubin levels as shown in Fig. 7A. More significant remediation was observed under riboflavin conjugated ZnO60 treatment.

Fig. 7 (A) Levels of direct bilirubin & indirect bilirubin (B) levels of ACP, ALP, SGOT, & SGPT. The data represented here are statistically significant, where a is p < 0.001 vs. control; b is p < 0.01 vs. control; c is p < 0.001 vs. disease and d is p < 0.01 vs. disease.

Among the most sensitive and widely used liver enzymes are ACP, ALP, AST/SGOT and ALT/SGPT. These enzymes were increased significantly in jaundice induced serum of mice. The levels of ACP, ALP, AST/SGOT and ALT/SGPT were significantly lowered by riboflavin conjugated ZnO30/ZnO60 treatments (Fig. 7B). These findings suggest that the presence of hepatocyte necrosis or membrane damage, as a result enzymes are released into the circulation, indicated by elevated serum enzyme levels.³⁵

Kidney function test (urea, creatinine) were also elevated due to jaundice exposure which are depressed to some extent by riboflavin conjugated ZnO30/ZnO60 treatment (Table 2). Both the levels of cholesterol and triglyceride of serum were significantly (p < 0.001) increased due to the induction of jaundice treatment (Table 1). The ameliorations of Ribo-ZnO30/ZnO60 were observed in both cases.

Table 1 Effect of Ribo-ZnO30/Ribo-ZnO60 on kidney function and total lipid content^a

Groups	Urea (mg dl^−1)	Creatinine (mg dl^−1)	Cholesterol (mg dl^−1)			Triglycerides (mg dl^−1)
			Total	HDL	LDH
a n = 9 for each group. All values have been expressed as the mean ± S.D. where * stands for significant (p < 0.01); and ** stands for more significant (p < 0.001).
Control	30.1 ± 2.8	0.63 ± 0.22	87.54 ± 3.1	32.92 ± 1.84	54.62 ± 8.1	49.1 ± 5.7
Disease	49.7 ± 3.5*	0.88 ± 0.21	116.2 ± 4.3	21.17 ± 2.4	73.8 ± 6.4	65.2 ± 4.1
Ribo-ZnO30	38.4 ± 2.6	0.74 ± 0.15	109.4 ± 3.5	24.52 ± 1.33**	66.52 ± 4.3*	58.3 ± 2.9
Ribo-ZnO60	33.5 ± 1.9	0.67 ± 0.26	95.37 ± 2.9	27 ± 1.24	59.3 ± 10.2	53.4 ± 6.2

---

Table 2 shows considerable (p < 0.001) decreased antioxidant (SOD, GSH and GPX) activity of serum due to jaundice induction with the increment of lipid peroxidation activity in the form of MDA (malondialdehyde). The supplementation of Ribo-ZnO60 treatment was found to be more active against the jaundice-induced effect on antioxidant enzymes than Ribo-ZnO30.

Table 2 Effect of Ribo-ZnO30/Ribo-ZnO60 on the serum lipid peroxidation & antioxidants levels^a

Groups	Lipid peroxidation (n mol ml^−1)	SOD (U/min 100 mg protein)	GSH (μM in plasma)	GPx (U/ml)
a n = 9 for each group. All values have been expressed as the mean ± S.D. where * stands for significant (p < 0.01); and ** stands for more significant (p < 0.001).
Control	5.3 ± 0.9	6.2 ± 0.734	34.8 ± 2.1	126.3 ± 2.4
Disease	12 ± 2.5**	4.7 ± 1.1*	25.6 ± 1.9	117.7 ± 1.8*
Ribo-ZnO30	8.6 ± 1.4*	5.1 ± 0.6*	29.4 ± 2.3*	120.2 ± 2.3
Ribo-ZnO60	6.5 ± 2.3	5.8 ± 1.2	31.5 ± 1.7	122.4 ± 3.2

---

Presence of CCl₄ induces the generation of Reactive Oxygen Species (ROS), by reducing antioxidant defenses; enzymes and substrates, results in oxidative stress in different tissues. Administration of CCl₄ in mice activated by liver enzyme cytochrome P450 damages hepatic cells by forming trichloromethyl free radical (CCl₃). These free radicals bind covalently to sulfhydryl groups of glutathione and protein thiols groups in cells to initiate a chain of reactions leading to membrane-lipid peroxidation and cell necrosis.^36–39 Significant changes of the biochemical parameter in serum due to jaundice exposure is an evidence of above-mentioned findings.

3.4.2. Riboflavin conjugated ZnO NPs mediated upregulation and downregulation of different factors as determined by ELISA & RT-PCR. Serum cytokine profiles revealed that the Th1 cytokines (IL-1β, TFN-α, IL-6) were elevated rapidly after the exposure of jaundice diseases (Fig. 8A). It caused a 1.75 fold secretion of IL-1β and 1.96 fold secretion of TFN-α, 1.84 fold secretion of IL-6 in comparison to the control mice. Additionally, Th2 cytokine such as IL-8 cytokines are also increasing (1.67 fold more) whereas IL-10 secretion was decreasing (1.91 fold lower) than their corresponding control values (Fig. 8A). Supplementation of riboflavin conjugated ZnO30/ZnO60 treatment lowered the serum Th1 cytokines and also IL-8. IL-10 Together, these results suggested that jaundice exposure altered the levels of Th1/Th2 cytokines, and Ribo-ZnO60 treatment ameliorated the action of jaundice exposure significantly.⁴⁰

Fig. 8 (A) Levels of cytokine released (B) fold change of m-RNA expression by RT-PCR.

The RT-PCR analysis revealed that the m-RNA expression of IL-1β, TFN-α, IL-6 and IL-8 of liver tissue were up-regulated due to jaundice exposure which are further under expressed by Ribo-ZnO30-treatment in comparison to their respective controls (Fig. 8B). The remeditative activity was much more effective under the riboflavin conjugated Zn60-treated mice. Additionally expression of IL-10 was down-regulated by jaundice induction whereas riboflavin conjugated ZnO30/ZnO60 treatment significantly up-regulated this factor which also corroborates earlier findings.⁴¹

The Western blot analysis revealed that the protein profiles of IL-1β, TFN-α, IL-6 and IL-8 were similar to the expression level of mRNA in the treated mice (Fig. 9). The expression level of IL-10 was much lower in jaundice exposed groups (Fig. 9). These results suggested the immune-modulatory effect of riboflavin conjugated ZnO NPs against jaundice affected physiological changes on the mice.

Fig. 9 Protein expression profiles by Western blotting.

Endotoxemia induces the release of inflammatory cytokines like tumour necrosis factor, IL-1, IL-6, and IL-8 which have multiple biological activities of cells by causing large changes in the biological, physiological, and immunological status of the subject. Any changes in cytokine concentrations in acute cholangitis suggested that IL-l, IL-6, and IL-8 were all associated in the pathophysiology of liver disease.⁴²

3.4.3. Riboflavin conjugated ZnO NPs induced histological changes of liver and kidney. The histological study of liver tissues by the compound microscope at 100× magnification revealed the regular arrangement of hepatic cells and normal shape of central vein of control animals (Fig. 10A). The hepatic cell arrangement was distorted and the central vein was dilated due to jaundice induction (Fig. 10B). The riboflavin conjugated ZnO NPs at both conditions (temperature of 30 °C, 60 °C) treatment helped the hepatic cells and the central vein to regain their regular arrangement and normal shape, respectively (Fig. 10C) whereas more remeditative activity was seen in the case of Ribo-ZnO60 against jaundice treatment (Fig. 10D). This study supports the earlier findings.⁴³

Fig. 10 Histological study of liver tissues, (A) control; (B) hyperbilirubinemia; (C) mice treated with Ribo-ZnO30 and (D) treated with Ribo-ZnO60.

In the histological study of the kidney tissues, normal arrangement of the glomerulus, bowman space and proximal/distal tubules of kidney tissues were seen in Fig. 11A at 100× magnification, whereas jaundice induction distorted these arrangements significantly (Fig. 11B). Riboflavin conjugated ZnO30/ZnO60 supplementation recovered the normal arrangement glomerulus, bowman space and proximal/distal tubules of kidney tissue effectively in comparison to jaundice exposed mice respectively (Fig. 11C and D) which also is an agreement with other studies. These studies suggest that level of nitrite in serum was increased and renal lesions was observed after CCl₄ treatment. Highly reactive trichloromethyl (CCl₃) radical initiates the process of lipid peroxidation which is considered to be the most important mechanism in the pathogenesis of renal damage induced by CCl₄ Alteration of antioxidant status with CCl₄ may potentially cause nephropathies in rat.^44,45

Fig. 11 Histological study of kidney tissues, (A) control; (B) hyperbilirubinemia; (C) mice treated with Ribo-ZnO30 and (D) treated with Ribo-ZnO60.

The concrete mechanism of Ribo-ZnO mediated anti-hyperbilirubinemia was not fully understood. There are some previous work which may validate probable mechanism of the anti-hyperbilirubinemia activity. ZnO nanoparticles used as a phototherapy vehicle to degrade bilirubin in jaundice.^15,46 Though ZnO in absence of light also can degrade bilirubin nearly 45% of photoinduced degradation.¹⁶ Briefly, ZnO NPs were exited with blue light and they donate the energy to unconjugated bilirubin (4Z,15Z-bilirubin or ZZ-bilirubin). Upon absorption of photons some conformational changes occurred in ZZ-bilirubin resulting the formation of some photoisomers like 4Z,15E (ZE-bilirubin), 4E,15Z (EZ-bilirubin) and 4E,15E (EE-bilirubin). These photoisomers are more water soluble and excreted through renal filtration.^47,48 Some other more water soluble isomer like lumirubin was formed from ZZ-bilirubin through EZ-bilirubin.⁴⁹ Being slow conversion of lumirubin to ZZ-bilirubin, these were rapidly excreted through bile and renal filtration.^48,50 The riboflavin also degrades riboflavin by forming photoisomers and the process is identical to those discussed earlier in this section.⁵¹ Another view supporting that, riboflavin become excited to higher energy state (triplet form) in presence of oxygen. Excited riboflavin releases energy in the form of singlet oxygen and then transferred to bilirubin. This singlet oxygen triggers photo degradation of bilirubin.^52,53 On the basis of such knowledge we can conclude that, Ribo-ZnO NPs exhibit synergistic effects on hyperbilirubinemic mice by degrading bilirubin.

4. Conclusion

This study demonstrated that jaundice exposure induced the immune system at the cellular levels as demonstrated by the biochemical parameters, Th1/Th2 cytokine imbalance, protein expressions, hepatocytes morphology and other physiological consequences in mice model. Interestingly, temperature dependent riboflavin conjugated ZnO NPs appeared to be a very active agent as a potential blocker against the jaundice-induced stress. ZnO NPs supplementation cures hyperbilirubinemia by degrading bilirubin. Furthermore, riboflavin conjugated ZnO NPs works as an anti-hyperbilirubinemic agent more efficiently along with nutrition supplements. From the current experimental results, we envision that supplementation of riboflavin conjugated ZnO NPs to the day-to-day regime may defend the human beings against jaundice triggered stress and toxicity.

Authors contributions

S. Das, N. Bala, P. Nandi and R. Basu designed the work structure. S. Saha maintained and collected the animal's organs, tissue, blood and serum and prepared the samples for some experiments. N. Bala and M. Maiti prepared the nanoparticles. M. Sarkar, N. Bala and M. Maiti performed all of the biochemical and molecular/cellular level experiments and prepared a report of some analysis in written form. S. Das provided valuable comments and suggestions and planning of the experimental facilities.

Conflict of interest statements

The authors declare no competing financial interest.

Acknowledgements

The support for experimental facilities of Biophysics Laboratory, Jadavpur University is gratefully acknowledged.

References

1. S. Sherlock and J. Dooley, Diseases of the liver and biliary system, John Wiley & Sons, 2008.
2. T. P. Kingham, C. Correa-Gallego, M. I. D'Angelica, M. Gönen, R. P. DeMatteo, Y. Fong, P. J. Allen, L. H. Blumgart and W. R. Jarnagin, Hepatic parenchymal preservation surgery: decreasing morbidity and mortality rates in 4152 resections for malignancy, J. Am. Coll. Surg., 2015, 220(4), 471–479.
3. E. Fabbrini, S. Sullivan and S. Klein, Obesity and nonalcoholic fatty liver disease: biochemical, metabolic, and clinical implications, Hepatology, 2010, 51(2), 679–689.
4. I. Aurich, L. P. Mueller, H. Aurich, J. Luetzkendorf, K. Tisljar, M. M. Dollinger, W. Schormann, J. Walldorf, J. G. Hengstler, W. E. Fleig and B. Christ, Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers, Gut, 2007, 56(3), 405–415.
5. S. Sekiya and A. Suzuki, Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors, Nature, 2011, 475(7356), 390–393.
6. D. Schwartz and F. Collins, Medicine. Environmental biology and human disease, Science, 2007, 316(5825), 695–696.
7. W. N. Rom, Environmental and occupational medicine, ed. S. B. Markowitz, Lippincott Williams & Wilkins., 2007.
8. K. G. Stamplecoskie and J. C. Scaiano, Light emitting diode irradiation can control the morphology and optical properties of silver nanoparticles, J. Am. Chem. Soc., 2010, 132(6), 1825–1827.
9. C. Burda, X. Chen, R. Narayanan and M. A. El-Sayed, Chemistry and properties of nanocrystals of different shapes, Chem. Rev., 2005, 105(4), 1025–1102.
10. P. N. Njoki, I. I. S. Lim, D. Mott, H. Y. Park, B. Khan, S. Mishra, R. Sujakumar, J. Luo and C. J. Zhong, Size correlation of optical and spectroscopic properties for gold nanoparticles, J. Phys. Chem. C, 2007, 111(40), 14664–14669.
11. P. C. Ray, Size and shape dependent second order nonlinear optical properties of nanomaterials and their application in biological and chemical sensing, Chem. Rev., 2010, 110(9), 5332–5365.
12. C. Hanley, J. Layne, A. Punnoose, K. M. Reddy, I. Coombs, A. Coombs, K. Feris and D. Wingett, Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles, Nanotechnology, 2008, 19(29), 295103.
13. R. D. Umrani and K. M. Paknikar, Zinc oxide nanoparticles show antidiabetic activity in streptozotocin-induced Type 1 and 2 diabetic rats, Nanomedicine, 2014, 9(1), 89–104.
14. K. R. Raghupathi, R. T. Koodali and A. C. Manna, Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles, Langmuir, 2011, 27(7), 4020–4028.
15. T. Bora, K. L. Lakshman, S. Sarkar, A. Makhal, S. sardar, S. K. Pal and J. Dutta, Modulation of defect-mediated energy transfer from ZnO nanoparticles for the photocatalytic degradation of bilirubin, Beilstein J. Nanotechnol., 2013, 4, 714–725.
16. N. Polley, Development of low cot biomedical pectrocopic tool, Masters of Engineering thesis, Jadavpur University, 2013.
17. S. Iravani, Green synthesis of metal nanoparticles using plants, Green Chem., 2011, 13(10), 2638–2650.
18. P. Raveendran, J. Fu and S. L. Wallen, Completely “green” synthesis and stabilization of metal nanoparticles, J. Am. Chem. Soc., 2003, 125(46), 13940–13941.
19. S. Hustad, P. M. Ueland and J. Schneede, Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma by capillary electrophoresis and laser-induced fluorescence detection, Clin. Chem., 1999, 45(6), 862–868.
20. L. Pataki, B. Matkovich, Z. Navak, E. Martonyi, A. Molnar, I. Varga and F. Roman, Riboflavin (vitamin B2) treatment of neonatal pathological jaundice, Acta Paediatr. Hung., 1985, 26(4), 341–345.
21. N. Polley, S. Saha, A. Adhikari, S. Banerjee, S. Darbar, S. Das and S. K. Pal, Safe and symptomatic medicinal use of surface-functionalized Mn₃O₄ nanoparticles for hyperbilirubinemia treatment in mice, Nanomedicine, 2015, 10(15), 2349–2363.
22. G. Bortolussi, L. Zentilin, G. Baj, P. Giraudi, C. Bellarosa, M. Giacca, C. Tiribelli and A. F. Muro, Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler–Najjar syndrome type I by AAV9-mediated gene transfer, FASEB J., 2012, 26(3), 1052–1063.
23. S. N. Chatterjee and S. Agarwal, Liposomes as membrane model for study of lipid peroxidation, Free Radical Biol. Med., 1988, 4, 51–72.
24. C. Beauchamp and I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Anal. Biochem., 1971, 44, 276–287.
25. O. W. Griffith, Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinyl pyridine, Anal. Biochem., 1980, 106, 207–212.
26. D. E. Paglia and W. N. Valentine, Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase, J. Lab. Clin. Med., 1967, 70, 158–169.
27. A. Alkaladi, A. M. Abdelazim and M. Afifi, Antidiabetic Activity of Zinc Oxide and Silver Nanoparticles on Streptozotocin-Induced Diabetic Rats, Int. J. Mol. Sci., 2014, 15, 2015–2023.
28. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, 72(1–2), 248–254.
29. N. Bala, S. Saha, M. Chakraborty, M. Maiti, S. Das, R. Basu and P. Nandy, Green synthesis of zinc oxide nanoparticles using Hibiscus subdariffa leaf extract: effect of temperature on synthesis, anti-bacterial activity and anti-diabetic activity, RSC Adv., 2015, 5, 4993–5003.
30. S. S. Xantheas and T. H. Dunning Jr, Ab initio studies of cyclic water clusters (H₂O)_n, n = 1–6. I. Optimal structures and vibrational spectra, J. Chem. Phys., 1993, 99(11), 8774–8792.
31. G. Xiong, U. Pal, J. G. Serrano, K. B. Ucer and R. T. Williams, Photoluminescence and FTIR study of ZnO nanoparticles: the impurity and defect perspective, Phys. Status Solidi C, 2006, 3(10), 3577–3581.
32. S. A. Kumari, B. K. Babu, N. Muralikrishna, K. Mohana Raoa, V. L. Sinduri, K. Praveen, C. V. P. Rao, B. S. Latha, K. Ramarao, V. Veeraiah and K. Praveen, Riboflavin metal complexes, Pharma Chem., 2015, 7(2), 307–315.
33. J. Huang, L. Bu, J. Xie, K. Chen, Z. Cheng, X. Li and X. Chen, Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidone-coated iron oxide nanoparticles, ACS Nano, 2010, 4(12), 7151–7160.
34. H. Zhang and D. Wang, Controlling the Growth of Charged-Nanoparticle Chains through Interparticle Electrostatic Repulsion, Angew. Chem., 2008, 120(21), 4048–4051.
35. R. B. Drotman and G. T. Lawhorn, Serum enzymes as indicators of chemically induced liver damage, Drug Chem. Toxicol., 1978, 1(2), 163–171.
36. D. E. Johnston and C. Kroening, Mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes, Pharmacol. Toxicol., 1998, 83(6), 231–239.
37. R. O. Rechnagel Jr and E. A. Glende, Carbon tetrachloride hepatotoxicity: an example of lethal cleavage, Crit. Rev. Toxicol., 1973, 2(3), 263–297.
38. P. Muriel, Regulation of nitric oxide synthesis in the liver, J. Appl. Toxicol., 2000, 20(3), 189–195.
39. H. Jaeschke, G. J. Gores, A. I. Cederbaum, J. A. Hinson, D. Pessayre and J. J. Lemasters, Mechanisms of Hepatotoxicity, Toxicol. Sci., 2002, 65(2), 166–176.
40. L. Nehéz and R. Andersson, Compromise of immune function in obstructive jaundice, Eur. J. Surg., 2002, 168(6), 315–328.
41. M. G. Elferink, P. Olinga, A. L. Draaisma, M. T. Merema, K. N. Faber, M. J. Slooff, D. K. Meijer and G. M. Groothuis, LPS-induced downregulation of MRP2 and BSEP in human liver is due to a posttranscriptional process, Am. J. Physiol., 2004, 287(5), G1008–G1016.
42. R. B. Sartor, Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases, Am. J. Gastroenterol., 1997, 92, 5–11.
43. T. J. Davern, N. Chalasani, R. J. Fontana, P. H. Hayashi, P. Protiva, D. E. Kleiner, R. E. Engle, H. Nguyen, S. U. Emerson, R. H. Purcell and H. L. Tillmann, Acute hepatitis E infection accounts for some cases of suspected drug-induced liver injury, Gastroenterology, 2011, 141(5), 1665–1672.
44. S. F. Assimakopoulos and C. E. Vagianos, Bile duct ligation in rats: a reliable model of hepatorenal syndrome, World J. Gastroenterol., 2009, 15(1), 121–123.
45. R. A. Khan, M. R. Khan, S. Sahreen and J. Bokhari, Prevention of CCl₄-induced nephrotoxicity with Sonchus asper in rat, Food Chem. Toxicol., 2010, 48(8), 2469–2476.
46. S. Sarkar, A. Makhal, S. Baruah, M. A. Mohmood, J. Dutta and S. K. Pal, Nanoparticle-sensitized photodegradation of bilirubin and potential therapeutic application, J. Phys. Chem. C, 2012, 116, 9608–9615.
47. E. M. Bruzell, Phototherapy of newborns suffering from hyperbilirubinemia. An experimental study, PhD thesis, Norwegian University of Science and Technology, 2003.
48. A. F. McDonagh and D. A. Lightner, Like a shrivelled blood orange-Bilirubin, jaundice, and phototherapy, Pediatrics, 1985, 75, 443–455.
49. R. Bonnett, Chemical aspects of photodynamic therapy, Gordon and Breach Science Publishers, Amsterdam, 2000, pp. 103–113.
50. J. F. Ennever, A. T. Costarino, R. A. Polin and W. T. Speck, Rapid clearance of a structural isomer of bilirubin during phototherapy, J. Clin. Invest., 1987, 79, 1674–1678.
51. I. Knox, J. F. Ennever and W. T. Speck, Urinary excretion of an isomer of bilirubin during phototherapy, Pediatr. Res., 1985, 19, 198–201.
52. H. B. Kostenbauder and D. R. Sanvordeker, Riboflavin enhancement of bilirubin photocatabolism in vivo, Experientia, 1973, 29, 282.
53. D. R. Sanvordeker and H. B. Kostenbauder, Mechanism for riboflavin enhancement of bilirubin photodecomposition in vitro, J. Pharmacol. Sci., 1974, 63, 404.

---