Protective effect of partially purified 35 kDa protein from silk worm (Bombyx mori) fecal matter against carbon tetrachloride induced hepatotoxicity and in vitro anti-viral properties

Abstract

Context: It has been found that many proteins from silkworm (Bombyx mori L.) fecal matter have been active against human immunodeficiency virus, Sendai virus, herpes simplex virus type-1, and nuclear polyhedrosis virus.

Objective: A partially purified 35 kDa protein from silkworm was screened for its hepatoprotective activity, and in vitro antioxidant, and antiviral properties against camelpox and goatpox viruses.

Materials and methods: The study investigated the efficiency of the partially purified 35kDa protein from silk worm fecal matter against CCl₄-induced liver damage measured in terms of enzyme levels such as aspartate aminotransferase (AST), alanine amino transferase(ALT), alkaline phosphatase (ALP) and total bilirubin, which maintain liver integrity. In vitro antioxidant potential of this protein was determined based on its ability to scavenge 2, 2-diphenylpicrylhydrazyl (DPPH) and superoxide anions scavenging activity. Further, in vitro cytotoxic effect on Vero cells and antiviral activity against goatpox and camelpox viruses were also studied.

Results: The protein had significant hepatoprotection against CCl₄-induced liver damage and scavenging of DPPH radical and superoxide anion activity. However, the protein did not inhibit the multiplication of either virus tested at its maximum non-toxic concentration (MNTC) in vitro.

Discussion and conclusion: The partially purified 35 kDa protein from silk worm Bombyx mori L fecal matter possessed protective effect against CCl₄-induced oxidative stress in rat model. The protein was found to be ineffective against camelpox and goatpox viruses at its MNTC in vitro.

Keywords::

• Bombyx mori
• 35 kDa protein
• hepatotoxicity
• antioxidant
• cytotoxicity
• antiviral activity

Introduction

Silkworm Bombyx mori L. fecal matter is identified as having beneficial effects against different pathophysiological states. The protein purified from silk worm Bombyx mori fecal matter was reported to act as an antiviral against viruses such as human immunodeficiency virus (HIV), Sendai virus (HVJ), herpes simplex virus type-1 (HSV) (Hiraki et al., 1997) and nuclear polyhedrosis virus (NPV) (Uchida et al., 1983; Neelagund et al., 2007) and also antibacterial against infectious clinical strains such as Staphylococcus aureus, Bacillus subtilis, Streptococcus haemolyticus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (Raghavendra & Neelagund, 2009). In addition the silkworm fecal matter was also explored in the field of medicine against various traditional infections in China and this implies that the silkworm fecal matter has high medicinal value (Zhang et al., 2008).

The high medicinal value of the silkworm fecal matter can be exploited to examine its efficacy on various diseases including toxicity of chemicals on liver. The liver is a key organ for metabolic, excretory and detoxification activities; thus it will be constantly exposed to xenobiotics (Wolf, 1999). The toxins absorbed from the intestinal tract pass through the liver resulting in a variety of liver ailments, which is one of the most serious health problems. Hence, certain plant extracts are applied to treat liver disorders (Karan et al., 1999; Chaudhari et al., 2009). Despite tremendous strides in modern medicine, there are few drugs which stimulate liver functions or regenerate hepatic cells (Satyapal et al., 2008). Natural drugs are considered less toxic and free from side effects to liver than synthetic drugs (Satyapal et al., 2008). So far, no studies have been reported regarding the beneficial property of a natural protein obtained from silk worm fecal matter. Therefore, the present study was undertaken to investigate the protective efficacy of the partially purified 35 kDa protein from silk worm fecal matter against carbon tetrachloride (CCl₄)-induced liver damage, in vitro antioxidant potential and antiviral activity against two DNA viruses such as camelpox and goatpox viruses.

Materials and methods

Chemicals

2, 2-Diphenyl-1-picrylhydrazyl (DPPH), nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide reduced (NADH), phenazine methosulfate (PMS), disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Merck Chemicals, Mumbai, India. Eagle’s minimum essential medium (EMEM) from GIBCO Invitrogen, Grand Island, NewYork, USA. Fetal bovine serum (FBS) from Hyclone, Logan, UT, and crystal violet from Qualigens, GlaxoSmithKline, Mumbai, India.

Experimental animals

Wistar albino male rats weighing 240–250 g were procured from the Animal Experimental Laboratory of the National College of Pharmacy, Shivamogga, Karnataka, India and used throughout the study. They were housed in microlon boxes in a controlled environment (temperature 25° ± 2°C and 12 h dark/light cycle) with standard laboratory diet (M/s Sai Durga Feeds and Foods, Bangalore, Karnataka, India) and water ad libitum. The study was conducted after obtaining Institutional Animal Ethics Committee’s clearance (144/1999/CPCSEA/SMG under the Certification Reference No. NCP/IAEC/CLEAR/06/2007-08). As per the standard practice, the rats were segregated based on their gender and quarantined for 15 days before the commencement of the experiment.

CCl₄ induced hepatoxicity

Four groups of rats, each having 6 animals, were used in the experiment as detailed below.

• Group I: Untreated control

• Group II: Negative control treated with CCl₄ (carbon tetrachloride)

• Group III: 50 mg/kg bw of lyophilized partially purified protein and on day 7 CCl₄ was administered orally.

• Group IV: 100 mg/kg bw of lyophilized partially purified protein and on day 7, CCl₄ was administered orally.

Group I and II rats were given a single dose of 0.5 mL double distilled water (vehicle) once daily for 7 days. Group III and IV were simultaneously treated with 0.5 mL of lyophilized protein dissolved in double distilled water (50 and 100 mg/kg bw), respectively, for 7 days. On day 7, 6 h after the last treatment, group II, III and IV rats were given a single dose of CCl₄ in olive oil (1:1) at a dose of 2 mL/kg bw. After 24 h of CCl₄ administration, the rats were killed by cardiac puncture after being anesthetized lightly with diethyl ether, blood samples were collected and serum was separated by centrifuging at 800 g for 15 min, and the serum samples were subjected to biochemical investigations. Liver from each rat was dissected out for studying histopathological changes.

Serum enzymes

The biochemical parameters of serum enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) (Reitman & Frankel, 1957), alkaline phosphatase (ALP) (King, 1965) and total bilirubin (Malloy & Evelyn, 1937) levels were investigated.

Preparation of partially purified 35 kDa protein from silk worm (Bombyx mori) fecal matter

Isolation of partially purified 35 kDa protein and the yield of protein were reported in our previous study (Raghavendra & Neelagund, 2009).

In vitro antioxidant activity

DPPH-scavenging activity

The method described by Chidambara Murthy et al. (2002) and Singh et al. (2002), and was used for determining the antioxidant activity of partially purified 35 kDa protein on scavenging DPPH radical. Various concentrations (20, 40, 60, 80 and 100 μg) of partially purified protein were taken in 100 μL of methyl alcohol in separate test tubes and to each test tube 5 mL of 0.1 mM methanol solution of DPPH was added and shaken vigorously and allowed to stand at 27°C for 20 min. The blank was prepared without the sample and methyl alcohol was used for baseline correction. Changes in the absorbance of the samples were measured at 517 nm. Radical scavenging activity was calculated and expressed in terms of percentage inhibition.

Superoxide anion scavenging activity

Superoxide anion scavenging capacity of the partially purified protein was assessed as per Nishimiki et al. (1972). Reaction mixture containing 156 μM nitroblue tetrazolium, 468 μM NADH in 100 mM phosphate buffer (pH 7.4), with 0.1 mL of partially purified protein (25–125 μg) in methanol were mixed and the reaction was initiated by adding 100 μL of 60 μM phenazine methosulfate in 100 mM phosphate buffer (pH 7.4). The reaction mixture was incubated at 25°C for 5 min and the absorbance was measured at 560 nm against blank.

Histopathological studies

The liver tissue of treated rats was dissected out and fixed in 10% formalin, dehydrated in graded ethanol (50–100%), cleared in xylene and finally embedded in paraffin. Sections of tissues were prepared and then stained with hematoxylin and eosin (H&E) dye for photomicroscopic observation.

In vitro antiviral activity

Vero cells (CCL-81) between passages 75 and 86 were propagated in Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS. Maintenance medium containing 2% FBS was used for maintenance of cells. The camelpox virus (CMLV) (Genus: Orthopoxvirus, family: Poxviridae) (CMLV/96, P60) and goatpox virus (Uttarkashi, GTPV/78) (genus: Capripoxvirus, family: Poxviridae) adapted to Vero cells were used as source of the viruses. The Vero cell monolayer was infected with CMLV at 0.01 multiplicity of infection and allowed to adsorb the virus on to cells at 37°C for 1 h. Then the monolayer was fed with maintenance medium and incubated for 2–3 days until >80%. Cytopathic effect (CPE) was observed with a change of medium on every alternate day. The virus was harvested when infected Vero cells showing virus specific CPE (syncytia, ballooning, and rounding of cells). After harvesting, the cells were freeze-thawed thrice and the virus was titrated in Vero cell monolayer. The titer was calculated as per Reed and Muench (1938) and expressed as log tissue culture infective dose 50% (TCID₅₀). The titrated virus was stored in aliquots at −80°C until further use.

Cytotoxicity assay of the protein

Determination of the maximum nontoxic concentration/Dose

The cytotoxicity of partially purified 35 kDa protein to Vero cells was determined (Bhanuprakesh et al., 2007, 2008). Various concentrations of partially purified protein (25, 12.5, 6.25, 3.125, 1.56, 0.39 mg/mL with two-fold dilutions) were prepared in maintenance medium and added to the 24 h confluent Vero cell monolayer in 96-well tissue culture plates and incubated in an atmosphere of 5% CO₂ at 37°C for 4 days. Each concentration was tested in triplicate along with controls. Cells were examined daily for morphological changes (rounding, degeneration), if any. After 96 h, cell morphology was compared between treated and untreated cultures (control). The highest concentration of the protein that showed no cellular morphological changes was considered as the maximum non-toxic concentration/dose (MNTC/MNTD). Further, the medium was decanted from the wells and the cells were trypsinized and counted by the Trypan blue dye exclusion staining method to assess the viability of the cells. The concentration of the protein at which 50% cytotoxicity was observed (i.e., 50% viability of cells) was recorded as the CyC₅₀ value. The concentration of protein at which cell viability was above 80% was further used in the study for assessment of antiviral activity of the protein against camelpox/goatpox viruses in a dose-dependent manner.

Antiviral effect (cytopathic effect inhibitory assay)

Assessment of in vitro antiviral activity of the partially purified protein was initially performed in 96-well microtiter plates in triplicate against camelpox and goatpox virus. Vero cells were seeded at 5 × 10⁴ cells/well. After 24 h the growth medium was removed and replaced with serial dilutions of partially purified protein in maintenance medium. The pretitrated camelpox vaccine virus (CMLV) (10^5.5 TCID₅₀/mL) and goatpox virus (10^6.5 TCID₅₀/mL) at different dilutions (10⁻¹ to 10⁻¹²) in 100 μL was used along with various non-toxic concentrations (1.56, 0.78, 0.39, 0.1953 mg/mL) of protein. To ensure the cytotoxic effect of the protein, a set of controls such as virus, protein (at different dilutions) and appropriate cell controls were included in the test. The plates were incubated at 37°C in a humidified CO₂ incubator. The media was changed at every 48 h interval. The cells were observed for cytopathic effect (CPE) regularly under an inverted microscope.

Statistical analysis

The data of antioxidant activities were expressed as mean ± standard error mean (SEM). The difference among the means was analyzed by one-way ANOVA test. Statistical significance was calculated at P <0.05.

Results

Hepatoprotective activity

The result of hepatoprotective activity of protein on CCl₄-intoxicated rats is shown in Table 1. In the CCl₄ intoxicated group (II), serum AST, ALT, ALP and total bilirubin were increased to 478.6, 660.8, 111.2 and 456.8% respectively, when compared to the control group. The elevated levels of serum AST, ALT, ALP, and total bilirubin were significantly reduced to 110.2, 97, 63.4, 53.1% and 312.4, 349.2, 78.1, and 108.3%, respectively in the animal groups treated with 50 and 100 mg dose of partially purified protein. Rats treated with partially purified protein 100 mg/kg bw showed highly significant hepatoprotective activity (P <0.001).

Table 1.  Effect of protein on CCl₄ induced hepatotoxicity in rats.

Group	Treatment	AST	ALT	ALP	Total bilirubin
Group I	Control	242.02 ± 18.39	139.38 ± 14.6	456.08 ± 25.12	0.44 ± 0.03
Group II	CCl4	1400.05 ± 78.82***	1060.47 ± 65.67***	963.48 ± 55.22***	2.45 ± 0.16***
Group III	Protein (50 mg) + CCl4	666.9 ± 43.04**	538.27 ± 67.88**	589.47 ± 34.85**	1.59 ± 0.09**
Group IV	Protein (100 mg) +CCl4	339.5 ± 24.3**	236.6 ± 31.7**	541.4 ± 32.68**	1.19 ± 0.05**

Values are the mean ± SEM of six rats; ***P <0.001 significant compared to control; **P <0.01 significant compared to CCl₄-intoxicated control.

DPPH-scavenging activity

Figure 1 illustrates the dose-dependent decrease in the concentration of DPPH radical due to scavenging activity of the protein. The results indicate that the protein has better scavenging activity that was enhanced with increased concentration. The calculated inhibition concentration (IC₅₀) value for the protein was found to be 52 µg/mL.

Figure 1.  Radical scavenging activity of partially purified protein by DPPH method.

Superoxide anion scavenging activity

The superoxide anion derived from dissolved oxygen by phenazine methosulfate/nicotinamide adenine dinuleotide coupling reaction reduces to nitroblue tetrazolium. The decreased absorbance at 560 nm indicates the consumption of superoxide anion in the reaction mixture. The partially purified protein showed the scavenging activity with an IC₅₀ value of 111 μg/mL (Figure 2).

Figure 2.  Superoxide anion scavenging activity of partially purified protein.

Histopathological observations

Histologically, the liver from control animals (Group I) had normal hepatic cells with well-preserved cytoplasm, prominent nucleus, nucleolus and visible central veins, whereas the liver collected from CCl₄-intoxicated rats showed massive fatty degenerations, necrosis, ballooning and infiltration of lymphocytes with the loss of cellular boundaries. However, the histological architecture of liver sections of the rats treated with different doses of protein showed more or less normal lobular pattern with mild degree of fatty tissue changes and necrosis, which were similar to the control group (Figure 3).

Figure 3.  Effect of partially purified protein against CCl₄-induced histological liver injury: (A) normal control rats showing hepatic cells with well-preserved cytoplasm (H), well brought out central vein (CV) and prominent nucleus and nucleolus; (B) liver section of CCl₄-treated rats showing massive fatty changes (F), necrosis (NC), and the loss of cellular boundaries; (C) liver section of rats treated with CCl₄ and protein 100 mg/kg bw, retaining normal hepatic architecture with fewer areas of fatty change, and necrosis.

Cytotoxicity and antiviral activity

Cytotoxicity of partially purified 35 kDa protein on host cells (Vero cells from an African green monkey kidney cell line) was studied. The maximum non-toxic concentration/dose (MNTC/MNTD) of protein was found at a concentration of 1.56 mg/mL at which there was no visible toxicity to the cells. The cells tolerated MNTCs of protein and hence below MNTCs were utilized for antiviral efficiency testing. When the protein was tested for its antiviral activity below its MNTC against camelpox virus and goatpox virus it failed to inhibit the replication of these viruses in vitro. The titers of both the viruses remained 10^6.5 and 10^5.1/mL, respectively, both in control and treated wells. This indicates that the protein is non-inhibitory/avirucidal (data not shown) against these viruses; rather, infected and treated cells showed virus-specific cytopathic effects.

Discussion

In the present study, a partially purified 35 kDa protein from silk worm Bombyx mori fecal matter was investigated for its antioxidant and hepatoprotective activities in CCl₄-treated rats. This study proved the usefulness of partially purified 35 kDa protein from silk worm fecal matter in controlling the hepatic disorders induced by CCl₄. Further, the protein was found to have a major defense mechanism involving free radical scavenging potential as measured by DPPH and superoxide anion scavenging activities (Figures 1 and 2). It is clear from the experimental observation that the dose-dependent decrease in the concentration of DPPH radical was due to scavenging ability of the protein. The protein showed good scavenging activity that was enhanced by increasing concentration. The calculated IC₅₀ value for the protein was found to be 52 μg/mL for DPPH and 111 μg/mL for superoxide anion scavenging activity. The antioxidant reacts with DPPH, which is a stable free radical, and converts DPPH into 1,1-diphenyl-2 picrylhydrazine (Chidambara Murthy et al., 2002). DPPH is known to abstract labile hydrogen (Constantin et al., 1990; Matsubara et al., 1991; Jain et al., 2008) simultaneously. The hepatoprotective effects of partially purified protein were assessed by biochemical studies in terms of measuring the levels of AST, ALT, ALP and total bilirubin in the serum under experimental conditions. The protein also protected the liver against CCl₄-induced histopathological changes such as necrosis, fatty changes, ballooning and degeneration. In addition, CCl₄ damages the liver, and the changes induced resemble histologically that of the viral hepatitis (James & Pickering, 1976). The aforementioned enzymes normally remain in the cytosol of hepatocytes. But, when the hepatocyte membrane is damaged, these enzymes are released into the blood stream. Estimation of these enzymes in the serum is a quantitative marker for the extent of liver damage (Ansari et al., 1991). Toxicity begins with the change in endoplasmic reticulum, which results in the loss of metabolic enzymes located in the intracellular structures (Recnagal, 1983). The toxic metabolite CC1₃ radical is produced, which further reacts with oxygen to give trichloromethyl peroxy radical. Cytochrome P450 2E1 is the enzyme responsible for this conversion. This radical binds covalently to the macromolecule and causes peroxidative degradation of the lipid membrane of the adipose tissue. In this view, the reduction in levels of AST and ALT by the protein is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by CC1₄. These findings are in agreement with the fact that the serum levels of AST and ALT return to normal with the healing of hepatic parenchyma and regeneration of hepatocytes (Thabrew et al., 1987). A number of recent reports (Tsoi & Fong, 2005; Oh et al., 2006; Lee et al., 2006) revealed that protein molecules from various plant sources possess antioxidant, hepatoprotective and antiviral activities. Some of the proteins from silk worm fecal matter have been found to have marked antiviral activity on enveloped viruses, but not on non-enveloped viruses (Hiraki et al., 1997). An enveloped animal virus, vesicular stomatitis virus (VSV, an RNA virus), was inhibited by chlorophyll derivatives (CPD) from silk worm excreta (Lim et al., 2002) and some of the virus deactivating protein was isolated from digestive juice of the silkworm Bombyx mori (Uchida et al., 1983). However, these reports have not described the mechanism of action of these proteins. In this investigation, the purified protein did not inhibit the two pox viruses (DNA viruses), camelpox virus and goatpox virus, in vitro.

In conclusion, it is to be inferred that the partially purified 35 kDa protein obtained from silk worm Bombyx mori fecal matter possess preventive as well as curative roles against CCl₄-induced oxidative stress in liver due to its antioxidant property. Moreover, the protein was non-inhibitory to two pox viruses studied. Further, complete purification and characterization of the hepatoprotective protein is in progress and the intention is to screen different groups of viruses for antiviral activity in the future.

Acknowledgements

We acknowledge the Poxvirus Disease Laboratory, IVRI, Mukteswar, Uttarakhand for in vitro antiviral studies, and the National College of Pharmacy, Shivamogga, Karnataka, India, for their cooperation in conducting the animal experiments.

Declaration of interest

We are grateful to the University Grants Commission, New Delhi, for financial support for this research work.