Studying the effect of Silver nanoparticles synthesized by Ulva fasciata aqueous extract against liver toxicity induced by CCl4 in rats Fawzia Alshubaily Biochemistry department

Studying the effect of Silver nanoparticles synthesized by Ulva fasciata aqueous extract against liver toxicity induced by CCl4 in rats
Fawzia Alshubaily
Biochemistry department, Faculty of science, King Abdulaziz University, Kingdom of Saudi Arabia
The development of green synthesis of metallic nanoparticles and their applications are considered to be among the most important areas of research. Ulva fasciata (sea lettuce) is one of the green seaweed that composed of highly active contents known for their antioxidant activity. The present study aimed to use U. fasciata as a reducing agent for the green synthesis of silver nanoparticles (AgNPs) and also to investigate the heaptoprotective effect of these nano particles against CCl4. In this study, aqueous extract of U. fasciata used for reduction of silver nitrate (AgNO3) to reduce Ag ions to Ag0. The bio-synthesized AgNPs were characterized by Ultra violet-visible spectroscopy (UV-Vis spectra) and transmission electron microscope (TEM) analysis. The results revealed a spherical shape of the AgNPs that are well distributed in solution with size range of 9-37 nm and an optical absorption at 430 nm. The rats (28 rats) used in this study randomly divided into 4 groups each of 7 animals; Control group, Ulva fasciata-AgNPs group (150 mg/kg b.wt/20 days), CCl4 group (2ml/kg/20 days of 1:1 v/v mixture of CCl4 and olive oil.) and CCl4& UF-AgNPs group. The results showed that CCl4 injection led to increase in liver function enzymes, level of urea and creatinine, hepatic oxidative stress (a significant increase in lipid peroxidation concomitant with a significant decrease in glutathione content and antioxidant enzyme activities) and histopathological disorders of liver tissues compared to control group. Rats received CCl4 along with U. fasciata–AgNPs showed significantly less severe damage and remarkable improvement in all of the measured parameters when compared to CCl4-rats. It could be concluded that the aqueous extract Ulva fasciata can be used as effective and eco-friendly reducing agent for biosynthesis of AgNPs. Also, AgNPs capped with U. fasciata can be used as potent antioxidant and heaptoprotective agent against the biochemical and histopathological alterations induced by CCl4-toxicity in liver tissues.

Key words: Green synthesis, Ulva fasciata, Silver nanoparticles, CCl4-toxicity
The green biosynthesis of nanoparticles employing either biological microorganism or plant extracts has emerged as a simple cost effective and environment friendly method. This process is scaled up for large scale synthesis alternative to more complex chemical synthetic procedures to obtain nanomaterials. Further, there is no need to use high pressure, energy, temperature and toxic chemicals during the green biosynthesis of nanoparticles (Seham et al., 2017). Different types of metals can be used for production of nanoparticles with dimension less than 100 nm including gold, silver, copper, zinc etc. Silver nanoparticles (Ag-NPs) have attracted great attention due to their specialized magnetic, electrical and optical properties. Ag-NPs can be considered as powerful antibacterial, antifungal and antioxidant agent (Seham et al., 2017) and have wide range of applications in different fields such as agriculture, medicine and industry (Leela and Anchana Devi, 2017).
Green biosynthesis of Ag-NPs by algal extract is more advantageous than other biological processes due to rapid growth rates of algae, high biomass production in a short time, cost effective, eco-safe and helpful for human therapeutic use (Abdel-Raouf et al., 2017). Ulva fasciata (sea lettuce) is one of the green seaweed that composed of highly active contents that have antitumor, antioxidant, hypocholesterimic and antimicrobial potentials (Mohapatra et al., 2016). The antioxidant activity of U. fasciata could be arising from bioactive compounds such as carotenoids, tocopherols and polyphenols that can directly or indirectly induced inhibition or suppression of free radical generation (Abdel-Raouf et al., 2017). Also, these green algae can be used as an effective and eco-friendly reducing agent for the synthesis of silver nanoparticles (AgNPs) (Abdel-Raouf et al., 2017).
Treatment of liver disorders by the usage of synthetic drugs could be associated with risk of relapses and danger of side effects. Therefore, natural products can be used as an effective, safe and alternative therapy for treatment of liver diseases without any side effects (Wang et al., 2016). CCl4 is one of toxic agent that can be used to induce liver damage and can be used for evaluation of heaptoprotective agents. Trichloromethyl free radicals formed during CCl4 metabolism can lead to oxidative damage by reacting with biological substances such as fatty acids, proteins, and nucleic acids (HYPERLINK “” l “!”Mahmoodzadeh et al., 2017). Thus, this article aimed to use U. fasciata as a reducing agent for the biosynthesis of silver nanoparticles (AgNPs) and investigation of their heaptoprotective effect against CCl4.

Material and Methods:
Chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Collection and preparation of Ulva fasciata:
The algal sample was manually collected from shallow water beside the shore of Abu-qur coast Alexandria Egypt and was identified according to Aleem (1978) and Coppejans et al. (2009). Samples were immediately brought to the laboratory in new plastic bags containing sea water, washed thoroughly with tap water and filtered seawater to remove extraneous materials. Algal material was shade-dried for 5 days and oven dried at 60oC until constant weight was obtained, then was grind into fine powder using electric mixer and stored at 0oC (Abdel-Raouf et al., 2017) for future use.

Fig. (1): Ulva fasciataPreparations of U. fasciata aqueous extracts: 
One gram of dry powder U. fasciata was added to 100 mL Distilled Deionized Water (DD H2O), boiled for 1 h then filtrated.

Biosynthesis of silver nanoparticles (AgNPs):
Silver nanoparticles were prepared by reduction of Ag+ ions to Ag0 according of methods of Devi et al. (2012). 10 mL of U. fasciata aqueous extract was added slowly to 90 mL of freshly prepared 0.1 mM of silver nitrate (AgNO3) with stirring and heating at 40°C until reduction of Ag+ ions and changing the color were observed.

Characterization of AgNPs
UV–Visible spectroscopy analysis:
The bio-reduction of silver ions (Ag +) in aqueous extracts of marine algae and the formation of AgNPs was monitored by UV-V in spectroscopy analysis. The reduction was confirmed by sampling the reaction mixture at regular intervals and the absorption maximum was scanned by UV–vis spectra, at the wavelength of 300–700 nm (Abdel-Raouf et al., 2017).

Transmission Electron Microscopic analysis:
The morphological analysis of the size, shape and the silver nanoparticles state was monitored by using Transmission Electron Microscopic (TEM) analysis. A drop of aqueous silver nanoparticle sample was loaded on carbon-coated copper grid and it was allowed to dry completely for an hour at room temperature. The clear microscopic views were observed and documented in different ranges of magnifications.

Biochemical study:
The experiment was conducted on 28 male rats (170 to 200g body weight (B.WT)). Rats were acclimated to controlled laboratory conditions for two weeks. Rats were maintained on stock rodent diet and tap water that were allowed ad libitum.
Experimental Design:
Animals (28 rats) were randomly divided into 4 groups each of 7 animals as follows:
Control group: rats fed on balanced diet containing 2% olive oil for 20 days.

Ulva fasciata -AgNPs group (UF- AgNPs): rats were administrated daily by nanoparticles Ulva fasciata aqueous extract by gastric intubation at dose level of 150 mg/kg b.wt (Abdel-Raouf et al., 2017).

CCl4 group: rats were treated daily with hepatotoxic agent CCl4 (2ml/kg of 1:1 v/v mixture of CCl4 and olive oil.) (Bhuvaneswari et al., 2014) for 20 days.

CCl4& UF-AgNPs: rats received UF-AgNPs (150 mg/kg b.wt/day) along with CCl4 (2ml/kg of 1:1 v/v mixture of CCl4 and olive oil.) for 20 days.

Animals from each group were sacrificed 24 hrs post the last dose of treatment. Blood samples were collected though heart puncture after light anesthesia and allowed to coagulate and centrifuged to obtain serum for biochemical analysis. Also, liver tissue was removed for biochemical investigation and histological examination.

Biochemical Analysis:
The activity of serum aspartate transaminase (AST) and alanine transaminase (ALT) was estimated according to Reitman and Frankel (1957), serum gamma glutamyl transferase (GGT) was assessed according to Rosalki (1975). Serum urea was measured by enzymatic colourimetric method as described by Coulomb and Farreau (1963) and serum creatinine was measured by the method of Husdan and Rapoport (1968).

Liver was dissected, thoroughly washed with ice-cold 0.9% NaCl, weighed, minced and homogenized (10% w/v) using 66 mmol/L chilled phosphate buffer (pH 7.0). The tissue homogenates were centrifuged at 6000 rpm for 15 min and the supernatants were used to estimate the level of malondialdehyde (MDA) (Yoshioka et al., 1979), the activity of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) (Kaminski and Jewezska, 1979), glutathione content (GSH) (Gross et al., 1967) and the activity of superoxide dismutase (SOD) (Minami and Yoshikawa, 1979) and catalase (CAT) (Aebi, 1984).

Histopathological Examination
For histopathological study the tissue samples were taken rapidly from each rat, and fixed in 10% formalin. All the samples were dehydrated in ascending grades of ethanol, cleared in butanol and embedded in parablast. Sections of 5-6 µm thick sections were obtained and stained with the following stains:
1- Haematoxylin and Eosin (H&E) staining for general histological studies.

2- Masson’s Trichrome stain for collagen fibers.

Statistical analysis
Results were presented as mean ± SE (n = 7). Experimental data were analyzed using one way analysis of variance (ANOVA). Duncan’s multiple range test was used to determine significant differences between means. Statistical analyses were performed using computer program Statistical Packages for Social Science (SPSS, 1998). Differences between means were considered significant at P < 0.05.

The bio-reduction of silver ions (Ag +) was visually confirmed by changing the color of reaction mixture from colorless to brownish-yellow after 3 min of reaction. The brown color increased by increasing the incubation period (Fig. 2).

Fig. (2): Changing the color of reaction mixture after adding U. fasciata aqueous extracts to AgNO3 (colorless)

UV-visible spectroscopy analysis:
The bio-synthesis of AgNPs was confirmed by UV–Visible spectrophotometer analysis. The results obtained that the absorption spectrum of reaction mixture at different wavelengths ranging between 400-500 nm revealed a peak at 430 nm (Fig. 3).

Fig. (3): UV-Visible spectra showing absorbance for silver nanoparticle synthesized using Ulva fasciataTransmission electron microscope (TEM): 
The result of TEM analysis of AgNPs showed that U. fasciata aqueous extract strongly affected the size and shape of the AgNPs. Also, the results revealed that AgNPs nano particles bio-synthesized by U. fasciata have spherical shape, well distribution in solution and the range of particles size is 9-37 nm.

Fig. (4): Transmission electron microscopic image of AgNPs biosynthesized (9-37 nm) by the reduction of AgNO-3 ions using Ulva fasciata aqueous extract
Results of biological study:
The results revealed that administration of CCl4 to rats showed significant elevation of the activity of liver markers enzymes AST, ALT and GGT as compared to the control, whereas treatment of rats with U. fasciata–AgNPs and CCl4 resulted in significant reduction in the activity of liver markers compared to CCl4-rats (Table 1).

Table (1): Effect of Ulva fasciata silver nanoparticles on liver enzymes of rats intoxicated with CCl4
Parameters Control UF-AgNPsCCl4 CCl4&UF-AgNPs
AST (U/ml) 29.40±0.36c 29.14±0.38c 67.34±1.12a 38.12±0.89b
ALT (U/ml) 21.77±0.56c 21.45±0.49c 53.45±0.98a 30.18±0.77b
?GT (U/ml) 3.76±0.37c 3.62±0.42c 7.27±0.41a 4.88±0.51b
Means in the same row with different superscripts are significantly different at (P<0.05), Values are expressed as mean ± S.E. (n=7)
UF-AgNPs: Ulva fasciata –Silver nanoparticles
CCl4: Carbone tetrachloride
The data in table 2 indicated that the serum levels of urea and creatinine were significantly increased in the CCl4-treated rats as compared to the control. Treatment of CCl4-administrated rats with Ulva fasciata –AgNPs showed significantly decreased levels of serum urea and creatinine as compared to the CCl4 injected rats (Table 2).

Table (2): Effect of Ulva fasciata silver nanoparticles on kidney function of rats intoxicated with CCl4
Parameters Control UF-AgNPsCCl4 CCl4&UF-AgNPs
Urea (mg/dl) 27.22±0.62c 25.93±0.56c 53.65±0.72a 32.72±0.83b
Creatinine (mg/dl) 0.92 ± 0.08c 0.89±0.06c 2.25±0.11a 1.22±0.07b
Means in the same row with different superscripts are significantly different at (P<0.05), Values are expressed as mean ± S.E. (n=7)
UF-AgNPs : Ulva fasciata –Silver nanoparticles
CCl4: Carbone tetrachloride
CCl4 intoxication resulted in significant increases in malondialdehyde (MDA) and xanthine oxidase (XO) and decreases in glutathione content (GSH) level and the activity of xanthine dehydrogenase (XDH), superoxide dismutase (SOD) and catalase (CAT) of hepatic tissues compared to control rats. Treatment of CCl4 – intoxicated rats with U. fasciata–AgNPs resulted in significant reduction of MDA and XO activity with remarkable elevation in GSH level and antioxidant enzymes relative to CCl4 group (Table 3).

Table (3): Effect of Ulva fasciata silver nanoparticles on antioxidant status of rats intoxicated with CCl4
Parameters Control UF-AgNPsCCl4 CCl4&UF-AgNPs
MDA(n mol/ml) 198.21±4.37c 182.25±4.76c 392.21±5.17a 261.42±4.16b
XO(mU/mgprotein) 2.71±0.15c 2.64±0.14c 4.12±0.19a 3.25±0.22b
XDH(mU/mg protein) 3.51±0.18a 3.57±0.17a 1.52±0.15c 3.12±0.16b
GSH(mg/g tissue) 28.22±1.12a 28.88±1.14a 16.11±0.78c 23.66±1.55b
SOD(U/mg protein) 45.27±1.91a 45.90±1.86a 25.47±1.32c 38.54±1.43b
CAT(U/g protein) 3.22±0.14a 3.37±0.12a 1.63±0.09c 2.82±0.11b
Means in the same row with different superscripts are significantly different at (P<0.05), Values are expressed as mean ± S.E. (n=7)
UF-AgNPs : Ulva fasciata –Silver nanoparticles
CCl4: Carbone tetrachloride
Histopathological Examination
Histopathological examination of liver tissues revealed that control rats have normal liver architecture with central vein, and cytoplasm and prominent nucleus and nucleolus were preserved (Figure 4A). The same observation was observed when the experimental animals were treated by Ulva fasciata–AgNPs (Figure 4B). Whereas, the liver tissues of CCl4 intoxicated rats characterized by inflammatory cell collection, scattered inflammation across liver parenchyma, focal necrosis and swelling up of vascular endothelial cells (Figure 4C). Treatment of CCl4-rats with Ulva fasciata –AgNPs appeared to significantly prevent the CCl4 toxicity as revealed by the hepatic cells which were preserved cytoplasms. This also caused a marked decrease in inflammatory cells (Figure 4D)

Fig (4): Histological structure of a rat liver. (A): control rats with normal liver lobular architecture, well brought out central vein and prominent nucleus and nucleolus; (B): rat treated with Ulva fasciata–AgNPs showing the normal appearance of liver tissue; (C): CCl4-rats showing liver section with severe toxicity with congested blood vessels with inflammatory cell collection, spaces of sinusoids, and hepatocytes degeneration. (D): Liver section of rats treated with E.O and CCl4 showing only a few inflammatory cells around portal tract.

The green algae Ulva fasciata composed of important nutritional components with high therapeutic value and can be used for biosynthesis of silver nanoparticles (Hamouda et al., 2017). The results indicated that the color change of the reaction mixture from colorless to brownish yellow was noticed obviously after 3 min of reaction and the intensity of brown color increased by the time. The formation of brown color can be considered as a sign of reduction of Ag+ to AgNPs by U. fasciata aqueous extract (Prakash et al., 2015). Sajidha Parveen and Lakshmi (2016) concluded that the reduction time of AgNO3 by red algae (Amphiroa fragilissima) was visually evident from the colour change (brownish-yellow) of reaction mixtures within 20 min. Prakash et al. (2015) reported that the appearances of brown color in the reaction may be due to excitation of surface plasmon resonance (SPR) and reduction of AgNO3 .

UV–Visible spectrophotometer analysis recorded that the SPR of silver nanoparticles bio-synthesized by U. fasciata aqueous extract produced a maximum peak at 430 nm. A study of Sushmita (2014) has reported that the UV-Visible spectroscopy of silver nanoparticles from Murrayakoenigii and Zea mays produced absorption peak at 420 – 440nm and thus confirmed the formation of nanoparticles. Kasthuri et al. (2009) concluded that the frequency and width of the SPR depends on the size and shape of the metal nanoparticles as well as on the dielectric constant of the metal itself and the surrounding medium.

The results of TEM showed that the micrograph of AgNPs by U. fasciata aqueous extract has spherical shaped, well distribution in solution with particles size ranged from 9-37 nm. It has been suggested that the formation, shape, size, and distribution of nanoparticles is depending on physiochemical properties such as temperature, time, pH, optical and concentration of the substrate (Prasad, 2014). In a study done by Abirami and KowsalyaHYPERLINK “” l “1788264_ja” (2015) found that the AgNPs synthesis by U. fasciata have spherical shape and average size ranging from 28-41 nm. Sangeetha and Saravanan (2014) reported that the average size of silver nanoparticles synthesized by U. lactuca was 20 nm and spherical in shape. The bioactive constituents of U. fasciata can act as both reducing and capping agents that form stable and shape-controlled AgNPs in the solution (Abdel-Raouf et al., 2017 and Chahardolia et al., 2017).

This study revealed that injection of rats with CCl4 resulted in severe liver damage associated with elevation of the serum activity of ALT, AST, GGT, levels serum urea and creatinine when compared to normal group. Toxicity of CCL4 induces change of transition function of liver cells and increase membrane permeability which leads to the leakage of liver enzymes into extracellular space (Fu et al., 2008). The study done by Khan and Siddique (2012) showed that elevation in the plasma levels of urea and creatinine induced by CCl4 can be attributed to the damage of nephron structural integrity. The present investigation demonstrated that CCl4 toxicity induced significant elevation in the level of MDA and XO and significant reduction in GSH level and the activity of XDH, SOD and CAT of hepatic tissues compared to control rats. CCl4 affects the cytochrome P450 in liver tissues and produces trichloromethyl radicals that can react with polysaturated fatty acids and leads to formation of lipid peroxides (Bhuvaneswari et al., 2014). In addition, over formation of these radicals disrupts the balance between ROS production and antioxidant defense system associated with disruption of cellular functions through some events and causes liver damage and necrosis (Bhuvaneswari et al., 2014).

The results obtained indicated that the AgNPs capped with U. fasciata may protect against CCl4 induced toxicity in rats by decreasing the activity of liver enzymes (ALT, AST, and GGT), level of urea and creatinine, reducing level of hepatic MDA and XO activity and by increasing the GSH level and the activity of XDH, SOD and CAT compared to CCl4 injected rats. The effect of AgNPs on liver enzymes could be related to the fact that AgNPs have affinity for thiol (-SH) group within the protein molecule causing change in the functional state of proteins and inactivate amino transaminases Adeyemi and Whiteley (2013). The heaptoprotective activity of U. fasciata- AgNPs could be attributed to active components of U. fasciata aqueous extract such as carotenoids, tocopherols and polyphenols that have free radical scavenging activity (Abdel-Raouf et al., 2017) and can protect the liver cells from damage induced by CCl4. Rizk et al. (2016) concluded that sulphated polysaccharides of U. fasciata can be regarded as potential anti-peroxidative, atheroprotective, hypolipidemic, and antiatherogenic agents, and may be used in the protection of ROS -induced oxidative damage, hyperlipidemia and atherosclerotic complications. Also, the authors reported that Ulva fasciata polysaccharides alleviate the oxidative stress by its inhibitory effect of lipid peroxidation by reducing the formation of MDA and enhance the antioxidant defense via increasing GSH retention (Rizk et al., 2016).

Through the results of the study, it have been confirmed that the aqueous extract of Ulva fasciata can be used as effective and eco-friendly reducing agent for AgNO3 and producing AgNPs with high stability, spherical shape, well distribution in solution, size range between 9-37 nm and absorption peak at 400-500 nm in UV-Visible spectrum. Also, the results concluded that AgNPs capped with U. fasciata can be used as potent antioxidant and heaptoprotective agent that can significantly attenuated the histopathological alterations induced by CCl4-toxicityin liver tissues.

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