Protective effect of Amaranthus spinosus against d-galactosamine/lipopolysaccharide-induced hepatic failure

Abstract

The current study is an effort to identify the hepatoprotective activity of the 50% ethanol extract of the whole plant of Amaranthus spinosus Linn. (Amaranthaceae) against d-galactosamine/lipopolysaccharide (d-GalN/LPS)-induced liver injury in rats. d-GalN/LPS (300 mg/kg body weight/30 µg/kg body weight)-induced hepatic damage was manifested by a significant (p <0.05) increase in the activities of marker enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase and gamma glutamyl transferase) and bilirubin level in serum while phospholipids significantly decreased. All other parameters, i.e. cholesterol, triglycerides and free fatty acids were increased significantly in both serum and liver compared to the control group. Pretreatment of rats with A. spinosus extract (400 mg/kg) significantly (p <0.05) reversed these altered parameters to normal compared to the intoxicated group. The biochemical observations were supplemented by histopathological examination of liver sections. There were no significant changes in the activities of marker enzymes, bilirubin level and lipids in the rats treated with A. spinosus extract alone. Results of this study revealed that A. spinosus extract could afford a significant protection against d-GalN/LPS-induced hepatocellular injury.

Keywords::

• Amaranthus spinosus
• d-galactosamine
• hepatitis
• lipopolysaccharide
• marker enzymes

Introduction

Hepatitis is a common disease in the world especially in the developing countries. It has been estimated that over 350 million people worldwide are infected with hepatitis B virus (HBV), and globally around 1 million die due to consequences of this infection annually (Al-Mahtab et al., 2008), while hepatitis C virus infection is around 2%, with 170 million people chronically infected with the virus and 3 to 4 million newly infected each year (Mukhopadhya, 2008). Africa was reported to have the highest hepatitis C virus (HCV) prevalence rate (Elsheikh et al., 2007). The high prevalence rate of HCV in developing countries may be due to the lack of proper health facilities or poor economical status and less public awareness. Steroids, vaccines and antiviral drugs which have been employed to treat liver disease, have potential adverse effects especially when administered long term. Presently, the use of herbal medicines for prevention and control of chronic liver diseases is the focus of attention for the physicians, pharmaceutical manufacturers and patients; the reasons for such a shift toward the use of herbals include the expensive cost of conventional drugs, adverse drug reactions, and their inefficacy. Hence, hepatoprotective drugs from plant sources seem to be attractive alternative (Aghel et al. 2007; Sehrawat et al. 2006).

Amaranthus spinosus Linn. (Amaranthaceae) which is commonly known as “Kate Wali Chaulai” in Hindi, is used as a vegetable in India. In Indian traditional medicine (Ayurveda) the whole plant is used as an aid in upset stomachs, laxative, diuretic, stomachic, antipyretic, to improve the appetite, biliousness, blood diseases, burning sensation, leprosy, bronchitis, piles and leucorrhea (Kirtikar & Basu, 2001; Mishra, 1986). Juice of Amaranthus spinosus is used by tribes of Kerala, India to prevent swelling around the stomach, while the leaves are boiled without salt and consumed for 2-3 days to cure jaundice and are also employed to cure some types of rheumatic pain and stomachache (Hema et al., 2006). The leaves and roots are applied as a poultice to relieve bruises, abscesses, burns, inflammatory wounds, menorrhagia, gonorrhea, eczema and inflammatory swelling (Parrotta, 2001).

The aqueous extract of A. spinosus leaves possesses significant immunostimulating activity (Lin et al., 2005) and the stem extract has shown antimalarial properties (Hilou et al., 2006). The plant has high nutritive values due to the presence of high concentration of antioxidant components, fibers, proteins and essential amino acids, especially lysine (Odhav et al., 2007; Teutonico & Knorr, 1985). A. spinosus has been reported to possess a very good scavenging system to combat the effects of air pollution (Mandal & Mukherji, 2001).The betalains (180 mg/100 g) in the stem of A. spinosus were identified as amaranthine and isoamaranthine along with hydroxycinnamates, quercetin and kaempferol glycoside concentrations (305 mg/100 g) (Stintzing et al., 2004). Betalains are well known for hepatic disorders, jaundice, scanty urine, wounds, and for having antioxidant, anticancer, antiviral and antiparasitosis properties (Kapadia et al., 1995; Patkai et al., 1997; Berghofer & Schoenlechner, 2002). It also contains amaranthoside and amaricin, along with stigmasterol glycoside, spinoside (7-p-coumaroyl-apigenin-4-O-β-d-glucopyranoside), α-xylofuranosyl uracil, β-d-ribofuranosyl adenine, β-sitosterol glucoside, and betaines such as glycinebetaine and trigonelline (Azhar-ul-Haq et al., 2004, 2006; Blunden et al., 1999).

Whole plants of A. spinosus are used for the treatment of jaundice in the traditional system of medicine (Stintzing et al., 2004; Hema et al., 2006) but there are no scientific studies on the protective potential of 50% ethanol extract of A. spinosus against experimentally induced hepatitis in rats. Thus, the aim of the present study is to further evaluate the hepatoprotective activity of A. spinosus extract correlating biochemical and histological changes against d-GalN/LPS-induced hepatitis in rats.

Materials and methods

Plant material and preparation of extracts

Whole plants of Amaranthus spinosus Linn. (Amaranthaceae) were freshly collected from the botanical garden of the National Botanical Research Institute, Lucknow, India in October 2006. The plant material was identified and authenticated taxonomically by R.L.S. Shikarwar at the National Botanical Research Institute. A voucher specimen (NAB 75006) of the collected sample was deposited in the institutional herbarium for future reference. The plants were washed with distilled water to remove dirt, and then shade dried. The dried materials were powdered and passed through a 10-mesh sieve. The coarsely powdered material (1 kg) was extracted with petroleum ether thrice to remove the fatty material and further marc was extracted thrice with ethanol (50%, v/v). The extracts were filtered, pooled and concentrated at reduced temperature (5°C) on a rotary evaporator (Buchi, USA) and then freeze dried (Freezone^® 4.5, Labconco, Kansas City MO) at high vacuum (133 × 10⁻³ mBar) and at temperature -40° ± 2°C (A. spinosus extract, yield 6.12%, w/w). For the pharmacological tests the A. spinosus extract was suspended in double distilled water containing carboxymethyl cellulose (1%, w/v, CMC).

Chemicals

d-GalN and LPS (Sero type 011.B4 extracted by phenol water method from Escherichia coli) were obtained from Sigma (St. Louis, MO). Diagnostic kits used for the estimation of biochemical assays were purchased from Qualigen (Mumbai). Anesthetic ether i.p. was procured from Sherad Laboratories (Andhra Pradesh). All other chemicals used were of analytical grade.

Animals

Sprague-Dawley rats weighing (140 ± 20 g) of either sex were procured from the National Laboratory Animal Centre (NLAC), Central Drug Research Institute, Lucknow. They were kept in a departmental animal house in a well cross ventilated room at 27° ± 2°C, and relative humidity 44-56%, light and dark cycles of 10 and 14 h, respectively, for one week before and during the experiments. Animals were provided with standard rodent pellet diet (Amrut, India) and the food was withdrawn 18-24 h before the experiment, though water was allowed ad libitum. All studies were performed in accordance with the guidelines for the care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India (Reg. No. 222/2000/CPCSEA). All the chemicals used were of analytical grade from standard companies and the water represents double distilled water. A standard orogastric cannula was used for oral drug administration.

Experimental design

The animals were divided into four groups. Each group had six animal. Group 1 served as solvent control which received CMC (1 ml/kg body weight orally) for 10 days. Group 2 rats were given A. spinosus extract alone (400 mg/kg body weight orally) for 10 days. Hepatitis was induced in group 3 rats through intraperitoneal injections of D-GalN and LPS (300 mg/kg body weight and 30 µg/kg body weight, respectively) 18 h before the experiment (Omar et al., 1996). Group 4 rats were pretreated with A. spinosus extract for 10 days prior to the induction of D-GalN/LPS as in group 3.

The rats were anesthetized with anesthetic ether i.p. (Sherad Laboratories, Andhra Pradesh) and sacrificed by cervical dislocation after the experiment. Blood was collected and the serum was isolated for various biochemical assays. The liver tissue excised was washed with ice cold saline. A portion of the liver was then homogenized in 0.1 M Tris buffer and the homogenate was used for biochemical estimations.

Biochemical assays

The activities of serum aspartate transaminase (AST), alanine transaminase (ALT) (Reitman & Frankel, 1957), alkaline phosphatase (ALP) (King & Armstrong, 1934), lactate dehydrogenase (LDH) (King, 1965), gamma glutamyl transferase (γ-GT) (Rosalki & Rau, 1972) and levels of bilirubin (Malloy & Evelyn, 1937) were assayed. The extraction of serum and liver tissue lipids (Folch et al., 1957) followed by the estimation of total cholesterol (Zlatkis et al., 1953), triglycerides (Foster & Dunn, 1973), free fatty acids (Falholt et al., 1973) and phospholipids (Zilversmit & Davis, 1950) were carried out.

Histopathology

The liver tissues were fixed with 10% phosphate buffered neutral formalin, dehydrated in graded (50-100%) alcohol and embedded in paraffin. Thin sections (5 μM) were cut and stained with routine hematoxylin and eosin (H & E) stain for photo microscopic assessment. The initial examination was qualitative, with the purpose of determining histopathological lesions in liver tissue (Amresh et al., 2007a).

Statistical analysis

All the data were presented as mean ± SEM and analyzed by SPSS for Windows, version 9 (SPSS, Chicago, IL) for the possible significant interrelation between the various groups, the independent samples t-test. A value of p < 0.05 was considered statistically significant.

Results

Group 3 rats intoxicated with d-GalN/LPS caused significant elevation (p < 0.05) in serum activities with higher levels of AST (3.77-fold), ALT (3.91-fold), ALP (2.60-fold), LDH (1.80-fold), γ-GT (2.62-fold) and bilirubin (1.85-fold) compared to control group 1. While the pretreatment of A. spinosus extract (400 mg/kg) significantly reduced the elevated serum parameters by 96.36, 75.07, 55.92, 13.34, 22.87, and 33.33% of the AST, ALT, ALP, LDH, γ-GT, and bilirubin, respectively compared to the intoxicated group 3 (Figures1 and 2).

Figure 1.  Levels of serum AST, ALT, ALP and LDH in control and experimental groups of rats. Values expressed as mean ± SD, n = 6. ^ap < 0.05 compared with group 1; ^bp < 0.05 compared with group 3. Group 1, control (only vehicle); group 2, ASE (400 mg/kg alone); group 3, D-GalN and LPS 300 mg/kg and 30 µg/kg intoxicated; group 4, pretreatment of ASE 400 mg/kg and D-GalN/LPS.

Figure 2.  Levels of serum γ-GT and bilirubin in control and experimental groups of rats. Values expressed as mean ± SD, n = 6. ^ap < 0.05 compared with group 1; ^bp < 0.05 compared with group 3. Group1, control (only vehicle); group 2, ASE (400 mg/kg alone); group 3, d-GalN and LPS 300 mg/kg and 30 µg/kg intoxicated; group 4, pretreatment of ASE 400 mg/kg and d-GalN/LPS.

The rats treated with d-GalN/LPS in group 3 showed significant (p < 0.05) increase in the levels of cholesterol (1.34 and 1.77-fold), triglycerides (2.54 and 1.96-fold) and free fatty acid (1.47 and 3.35-fold) while the levels of phospholipids were found to be reduced (0.64 and 0.712-fold) significantly in serum and liver, respectively compared to control group 1. Pretreatment of A. spinosus extract (400 mg/kg) showed significant (p < 0.05) protection in the levels of cholesterol (0.78 and 0.83-fold), triglycerides (0.54 and 0.69-fold), free fatty acids (0.83 and 0.63-fold) and phospholipids (1.35 and 1.29-fold) in serum and liver, respectively compared to intoxicated group 3 (Tables 1 and 2). Pretreatment of A. spinosus extract afforded a significant protection against d-GalN/LPS-induced liver injury.

Table 1.  Levels of serum cholesterol, triglycerides, free fatty acids and phospholipids in control and experimental groups of rats.

Parameters	Cholesterol (mg/dL)	Triglycerides (mg/dL)	Free fatty acids (mg/dL)	Phospholipids (mg/dL)
Group 1	85.26 ± 5.25	40.78 ± 3.41	34.68 ± 1.34	91.84 ± 4.23
Group 2	85.31 ± 6.06	41.02 ± 2.03	34.82 ± 2.33	93.76 ± 3.44
Group 3	114.64 ± 6.54^a	103.86 ± 4.86^a	51.06 ± 3.76^a	58.76 ± 2.92^a
Group 4	90.46 ± 4.86^b	56.77 ± 2.04^b	42.89 ± 2.32^b	79.64 ± 3.74^b

Values expressed as mean ± SD, n = 6. ^ap <0.05 compared with group 1. ^bp <0.05 compared with group 3. Group1, control (only vehicle); group 2, ASE (400 mg/kg alone); group 3, d-GalN and LPS 300 mg/kg and 30 µg/kg intoxicated; group 4, pretreatment of ASE 400 mg/kg and d-GalN/LPS.

Table 2.  Levels of cholesterol, triglycerides, free fatty acids and phospholipids in the liver of control and experimental groups of rats.

Parameters	Cholesterol (mg/dL)	Triglycerides (mg/dL)	Free fatty acids (mg/dL)	Phospholipids (mg/dL)
Group 1	11.57 ± 1.46	23.52 ± 1.22	0.68 ± 0.0.16	25.78 ± 1.12
Group 2	12.18 ± 2.54	24.38 ± 1.32	0.71 ± 0.0.14	26.06 ± 0.84
Group 3	20.48 ± 0.77^a	46.33 ± 2.45^a	2.28 ± 0.152^a	18.36 ± 1.34^a
Group 4	17.02 ± 1.13^b	32.17 ± 0.72^b	1.44 ± 0.097^b	23.67 ± 0.37^b

Values expressed as mean ± SD; n = 6. ^ap < 0.05 compared with group 1; ^bp <0.05 compared with group 3. Group1, control (only vehicle); group 2, ASE (400 mg/kg alone); group 3, d-GalN and LPS 300 mg/kg and 30 µg/kg intoxicated; group 4, pretreatment of ASE 400 mg/kg and d-GalN/LPS.

There were no significant changes in the activities of marker enzymes, bilirubin level and lipids in the rats treated with A. spinosus extract (400 mg/kg) alone as compared to the control, thereby showing the absence of any adverse toxic effects of A. spinosus extract.

Diffused areas of hepatitis, especially in the perivenular region which extends to the central zone with inflammatory collections, were observed in d-GalN/LPS-induced rats (Figure 3A) when compared to control (Figure 3B). Pretreatment with A. spinosus extract reversed to a large extent the hepatic lesions produced by d-GalN/LPS as it was evident from the absence of cellular necrosis and inflammatory infiltrate around the central zone (Figure 3C) which further confirms the hepatoprotective potential of the A. spinosus extract. The rats, given A. spinosus extract alone, did not show any abnormal change in the architecture of the liver (Figure 3D) as compared to the control rats.

Figure 3.  (A) Photomicrograph of d-GalN/LPS intoxicated rat liver section which shows loss of architecture and cell necrosis (perivenular) extending to the central zone. The cell necrosis with inflammatory collections is more prominent in the central zone than around central vein. Hematoxylin-eosin stain (100×). (B) Photomicrograph of control rat liver section showing normal architecture. Hematoxylin-eosin stain (100×). (C) Photomicrograph of liver section of rat pre-treated with ASE prior to d-GalN/LPS challenge showing central vein surrounded by hepatocytes with sinusoidal dilatation with occasional inflammatory cells. No hepatic necrosis was seen around central vein or in the central zone. Hematoxylin-eosin stain (100×). (D) Photomicrograph of liver section of rat treated with ASE alone for 10 days showing normal liver parenchyma with central vein and cords of hepatocytes. Hematoxylin-eosin stain (100×).

Discussion

d-Galactosamine is a well established hepatotoxicant which induces a diffuse type of liver injury closely resembling human viral hepatitis (Decker & Keppler, 1972). The toxicity of d-GalN results from inhibition of RNA and protein synthesis in the liver (Konishi et al., 1974). The metabolism of d-GalN may deplete several uracil nucleotides including UDP-glucose, UDP-galactose and UTP (Decker & Keppler, 1974) which are trapped in the formation of uridine-diphosphogalactosamine. Accumulation of UDP-sugar nucleotides (Endo et al., 1992; Manabe et al., 1996) may contribute to the changes in the rough endoplasmic reticulum and to the disturbance of protein metabolism. Further, intense galactosamination of membrane structures is thought to be responsible for loss in the activity of ionic pumps. The impairment in the calcium pump, with consequent increase in the intracellular calcium is considered to be responsible for cell death (Tsai et al., 1997). d-Galactosamine/lipopolysaccharide (d-GalN/LPS) (300 mg/kg body weight/30 µg/kg body weight) induced highly elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin in the untreated toxic control group. Elevated serum enzymes are indicative of cellular leakage and loss of functional integrity of the cell membrane in liver (Amresh et al., 2007b; Drotman & Lawhorn, 1978). Hence, significant rise in the serum enzyme levels could be taken as an index of liver damage (Lim et al., 2000). Pretreatment with 50% ethanol extract of whole plant of Amaranthus spinosus attenuated the increased activities of these enzymes in serum caused by d-GalN/LPS recovery towards normalization, and suggests that A. spinosus extract causes parenchymal cell regeneration in liver, thus protecting membrane fragility, thereby, decreasing enzyme leakage. Determination of serum bilirubin represents an index for the assessment of hepatic function, and any abnormal increase in the levels of bilirubin in the serum indicate hepatobiliary disease and severe disturbance of hepatocellular function (Martin & Friedman, 1992). Increased levels of bilirubin in this study is in agreement with previous reports showing that d-GalN-induced hepatitis is characterized by increased levels of bilirubin in serum (Sree Rama Murthy & Srinivasan, 1993). The A. spinosus extract-mediated suppression of the increased bilirubin level suggests the possibility of the A. spinosus extract being able to stabilize biliary dysfunction.

Any liver disease will show an increased blood cholesterol level (McIntyre & Rosalki, 1992). d-GalN/LPS significant increase of cholesterol noted in this study might have been due to the inability of the diseased liver to remove cholesterol from circulation. Pretreatment of A. spinosus extract (400 mg/kg) also showed protection against serum and liver lipid changes caused by d-GalN/LPS evidencing a broad spectrum of hepatoprotective property. This finding could be correlated with the results of the previous studies (Black et al., 1983).

Hepatocellular damage due to alcohol, virus and drug-induced hepatitis causes modest hypertriglyceridemia (Glickman & Sebesin, 1982), which is due to the biochemical changes interfering with the transport of triglycerides out of liver. Our study also showed an increased accumulation of triglycerides in d-GalN/LPS-induced rats which are in agreement with previous reports (Cartwright et al., 1982; Black et al., 1983). The administration of endotoxic LPS resulted in an increase in the free fatty acid content in rats (Dhuley & Naik, 1998). Accumulation of free fatty acids is a consequence of changes in hepatic lipid metabolism. This is well correlated in our study with the increased levels of free fatty acids due to the administration of d-GalN/LPS, which is responsible for the increment of phospholipase A₂ activity as a consequence of the high level of intracellular calcium causing the hydrolysis of liver membrane phospholipids and release of arachidonic acid (Kramer, 1993). This might account for the decreased levels of phospholipids observed in the serum and liver of d-GalN/LPS challenged rats.

Rats pretreated with A. spinosus extract (400 mg/kg) prior to the induction of hepatic damage showed a restoration of the altered lipid levels induced by d-GalN/LPS towards near normalcy thereby showing the modulating effect of A. spinosus extract against d-GalN/LPS-induced changes in the lipid levels in rats. Histopathological observations supported the protective effect of Amaranthus spinosus in d-GalN/LPS administered rats. There were no significant changes in the activities of marker enzymes, bilirubin level and lipids in the rats treated with A. spinosus extract alone (400 mg/kg) thereby showing the absence of any adverse toxic effects of A. spinosus extract. The hepatic lesion-produced central zone with inflammatory collections induced by d-GalN/LPS was prevented by treatment with A. spinosus extract. This can be well correlated with the protective actions of picroliv, which showed lipid changes against hepatotoxicity induced by paracetamol and d-GalN (Ansari et al., 1991).

Thus, the present study confirms the hepatoprotective action of A. spinosus extract against d-GalN/LPS-induced hepatitis in rats. The curative efficacy of A. spinosus extract was very promising as evidenced by the reversal of the altered values following the administration probably by promoting regeneration of hepatocytes. The hepatoprotective action may be mediated through the inhibition of UDP sugar derivatives, enhancement of glycoprotein biosynthesis, stabilization of cell membrane and inhibition of lipid accumulation by its hypolipidemic property. Preliminary qualitative phytochemical screening of 50% ethanol extract of whole plant of Amaranthus spinosus has given the positive tests for alkaloids, glycosides, flavonoids, saponins, carbohydrates, protein, amino acids, lipids, steroids, phenolic acid and tannins (Trease & Evans, 1983). Previous phytochemical investigations of Amaranthus spinosus showed that the presence of polyphenolic compounds such as betalains, quercetin, rutin, kaempferol glycosides, β-sitosterol and stigmasterol which may account for the hepatoprotective action of A. spinosus extract against d-GalN/LPS-induced hepatitis in rats (Berghofer & Schoenlechner, 2002; Suryavanshi et al., 2007).