Allium porrum and Bauhinia Variegata Mitigate Acute Liver Failure and Nephrotoxicity Induced by Thioacetamide in Male Rats

Abstract

The present work has been designed to investigate the hepatoprotective and renoprotective efficiency of alcoholic extract of Allium porrum and Bauhinia variegata leaves in thioacetamide-induced toxicity in adult Wistar rats. Allium porrum leaf extract, Bauhinia variegata leaf extract and their combinations were orally administered for 14 days then TAA (300 mg/kg) i.p. was injected once and the rats were sacrificed 2 days later. Plasma AST, ALT, GGT, total bilirubin, creatinine, urea, uric acid, triglyceride, cholesterol, HDL and LDL were measured. Liver MDA, GSH, CAT, SOD and TNF-α were evaluated. Histological examination was performed. The rats treated with TAA showed a significant increase in AST, ALT, GGT, total bilirubin, creatinine, urea, uric acid, total, triglyceride, cholesterol and HDL while it led to a significant decrease in protein and HDL. The treatment of rats with TAA resulted in a significant decrease of the hepatic GSH, SOD and CAT and a significant elevation of MDA and TNF-α. Allium porrum and Bauhinia variegata extracts alleviated the toxic effects of TAA on the liver and the kidney. In conclusion, treatment with Allium porrum and Bauhinia variegata extracts and their combination reduced deleterious effects of TAA on liver through antioxidant and anti-inflammatory properties.

Introduction

Acute liver failure (ALF) is a disease characterized by the rapid development of severe hepatocellular dysfunction, specifically coagulopathy and hepatic encephalopathy in patients with originally normal liver function. Hepatocellular necrosis and parenchymal inflammation usually accompany acute liver failure. ALF is accompanied by high mortality rate [1]. Thioacetamide (TAA) is a chemical agent commonly used for induction of ALF [2]. TAA has long been known as a hepatotoxicant; its biotransformation to thioacetamide sulfoxide (TAAS) occurs along the cytochrome P-450 (CYP)-dependent pathway (a CYP2E enzyme is mainly involved here). TAA is subsequently converted to thioacetamide disulfoxide, a toxic reactive metabolite [3]. These reactive metabolites covalently bind to liver macromolecules and dramatically increase the production of reactive oxygen species which then induces acute centrilobular liver necrosis [3]. Moreover, TAA evoked kidney injury [4].

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defense system, which stimulates tissue damage. In normal physiological state, the body’s antioxidant defense system remove ROS and help in repair the tissue damage [5]. Oxidative stress plays an important role in the etiology and pathogenesis of many diseases such as hepatotoxicity, nephrotoxicity, diabetes mellitus and cancers [6]. Dietary intake of antioxidants can inhibit or delay the oxidation of susceptible cellular substrates so prevent oxidative stress. Phenolic compounds such as flavonoids, phenolic acids, saponins and tannins have received much attention for their high antioxidative activity. Therefore, it is important to enrich our diet with antioxidants to protect our body against many diseases related to oxidative damage. The flavonoids are the principal active component found in Allium porrum (A porrum) and Bauhinia variegata (B variegata) leaves [7]. Both A porrum and B variegata leaves exhibit antioxidant properties [8, 9].

Allium is a genus belonging to the family Liliaceae. Among the extracts of Allium porrum leaves, methanolic extract showed maximum phytochemical constituents including sulphate [10], when compared with aqueous or acetone extracts. Allium porrum (Leek) exhibited hypolipidemic [11], hypoglycemic [12].

Bauhinia variegata belongs to the family Leguminosae. The phytochemical screening revealed that Bauhinia leaves contained terpenoids, flavonoids, tannins, saponins, reducing sugars, and steroids [13].

Different pharmacological activities have been reported for leaves of many Bauhinia species, including hepatoprotective, antioxidant The leaves of Bauhinia variegata is reported to have antidiabetic [14], antioxidant [15], anti-inflammatory [16], anticancer [17], and nephroprotective activities [8].

Hence, the present study was carried out to confirm the antioxidant and protective activities of the methanol extract of Allium porrum and Bauhinia variegata leaves in TAA-induced acute hepatotoxicity and nephrotoxicity in rats.

Materials and Methods

Plant Material

Bauhinia variegata Linn (July–September) and Allium porrum leaves (December–January) were collected from the farm of Faculty of Agriculture, Cairo University, Giza, Egypt. The plants were kindly identified by Mrs. Tersea Labib, taxonomist at Orman Botanical garden, Giza, Egypt and Dr. Mona Marzok, a researcher at the Herbarium of National Research Centre, Giza, Egypt. A voucher specimen was deposited at the Herbarium of the National Research Centre (NRC), Dokki, Giza, Egypt.

The fresh leaves were dried at room temperature in shad and ground to fine powder. Air dried fine powdered were extracted with 70% methanol in the percolator at room temperature till exhausted and concentrated under reduced pressure at 40 °C by the rotary evaporator till dryness.

Chemicals

Thioacetamide (TAA) was purchased from Sigma (St. Louis, MO, USA). Other chemicals and reagents were of high analytical grade and were purchased from standard commercial suppliers. TAA was prepared freshly by dissolving in sterile distilled water and stirred well until all crystals were dissolved.

Animals

Adult male Wistar albino rats weighing 150–170 g were obtained from the animal house of the National Research Centre (Dokki, Giza, Egypt) and were accommodated in a standard polypropylene cages and kept under constant environmental conditions with equal light–dark cycles. The rats were allowed to adapt in the laboratory environment for two weeks. They were served normal pellet diet and water ad libitum.

Ethics Statement

This experiment was carried out according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication No. 85–23, revised 1996) and under regulations of Animal Care and Use of National Research Centre in Egypt. All efforts were made to minimize suffering.

Experimental Design and Preparation of Samples

Forty rats were divided into five equal groups:-

• Group 1 (control group): rats received same volume of vehicle.

• Group 2 [hepatotoxic (TAA) group]: rats received TAA (300 mg/kg) intraperitoneally (IP) once [18].

• Group 3: rats received 200 mg/kg of Allium porrum leaf extract orally for 14 days, then TAA (300 mg/kg) intraperitoneally (IP) once then administered the extract for 2 more days before sacrifice.

• Group 4: rats received 200 mg/kg of Bauhinia variegata leaf extract orally for 14 days, then TAA (300 mg/kg) intraperitoneally (IP) once then administered the extract for 2 more days before sacrifice.

• Group 5: rats received 200 mg/kg Allium porrum and Bauhinia variegata extract orally for 14 days, then TAA (300 mg/kg) intraperitoneally (IP) once then administered the mixture for 2 more days before sacrifice.

At the end of the experimental period, blood samples were collected from a retro orbital vein and separated plasma was stored in Eppendorf tubes at −80˚ for biochemical analysis.

Immediately after blood sampling, animals were victimized by cervical dislocation under ether anesthesia and livers and kidneys were collected for biochemical and histopathological examinations. Liver and kidney were rapidly removed and washed in ice-cooled saline.. A weighed part of each tissue was homogenized with ice-cooled saline (0.9% NaCl) to prepare 20% homogenate. The homogenate was then centrifuged at 3000 rpm for 10 min at 5 °C using a cooling centrifuge (Laborzentrifugen, Sigma, Germany). The supernatant was used for various analyses. The remaining portions of liver and kidney were fixed immediately in 10% neutral buffered formalin, processed for light microscopy to get (5 μm) paraffin sections and stained with Hematoxylin & Eosin (H&E) to verify histological details. Moreover, some liver and kidney sections were subjected to immunochemistry.

Biochemical Investigation

Blood plasma was used for colorimetric determination of AST, ALT, GGT, bilirubin, creatinine, urea, uric acid and protein by using kits from Salucea Company, Netherlands. Moreover, hepatic inflammatory marker, TNF-α (R&D Systems, USA) was determined by enzyme-linked Immunosorbent assay, ELISA. On the other hand tissue (Hepatic and renal) MDA, SOD, CAT, NO and GSH were evaluated using colorimetric assay kits according to the instructions of the manufacturer (Bio Diagnostic, Egypt).

Histological Study

The specimens of liver and kidney were fixed in 10% neutral-buffered formalin, dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin wax. Tissue samples were cut into 5 μm thick sections and stained with hematoxylin– eosin (H&E) and evaluated for any histopathological changes.

Immunohistochemical Assessment of iNOS

Immunohistochemical staining of iNOS antibody was performed with sections of four μm thick were deparaffinized and incubated with fresh 0.3% hydrogen peroxide in methanol for 30 min at room temperature. iNOS positive cells were determined with streptavidin biotin–peroxidase staining method. For immunohistochemistry examination, iNOS antibody as the primer antibody at 1:100 dilution, and biotinylated secondary antibody (DAKO-Universal) were used. The binding sites of antibody were visualized with 3,3′-Diaminobenzidine (Sigma) The specimens were counterstained with hematoxylin and evaluated by high-power light microscopic.

Statistical Analysis

The values were stated as mean ± S.E. of 6–8 rats and the variances between groups were tested for significance using analysis of variance (ANOVA), followed by LSD comparison test estimated by SPSS software, version 21. The level of statistical significance was at P < 0.01.

Results

Liver and Kidney Functions

Injection of TAA (300 mg/kg, i.p.) resulted in a marked significant (P ≤ 0.01) increase of plasma liver enzymes (AST, ALT,GGT), bilirubin, creatinine, urea and uric acid levels (Tables 1, 2) as compared to control values.On the other hand, the levels of the previous parameters in the A porrum + TAA or in the B variegata + TAA groups were significantly (P ≤ 0.01) less than those of TAA group.On the other hand, total protein decreased significantly (P ≤ 0.01) in TAA group or B variegata + TAA, while it did not change significantly in the A porrum + TAA group or in A porrum + B variegata + TAA group as compared to control. Moreover,ALT,AST,GGT,urea and uric acid levels of A porrum + B variegata + TAA group were significantly (P ≤ 0.01) lower than those of A porrum + TAA group or B variegate + TAA group.

Table 1.

Protective effects of Allium porrum and Bauhinia variegate leaves on liver function tests in plasma

Parameter	Group
	Control	TAA	TAA + A porrum	TAA + B variegata	TAA + A porrum + B variegata
ALT(U/l)	45.67 ± 2.11	360.5 ± 18.30*	172.70 ± 14.14*@	139.7 ± 5.92*@	103.20 ± 4.18*@#
AST(U/l	60.83 ± 3.02	431.8 ± 26.37*	196.20 ± 3.87*@	248.7 ± 8.77*@	128.50 ± 5.13*@#
GGT(U/l)	6.24 ± 0.25	18.34 ± 0.84*	12.86 ± 0.44*@	11.06 ± 0.48*@	9.39 ± 0.40*@#
Total bilirubin (mg/dl)	0.82 ± 0.04	2.61 ± 0.20*	2.00 ± 0.08*@	2.05 ± 0.07*@	2.01 ± 0.02*@
Total protein (g/dl)	7.10 ± 0.17	5.57 ± 0.20*	6.48 ± 0.24@	5.74 ± 0.26*	6.83 ± 0.15@

Each value is the mean ± SE, n = 8

*Significantly different from control group at P < 0.01

@Significantly different from TAA group at P < 0.01

#Significantly different from TAA + A porrum group or TAA + B variegata group at P < 0.01

Table 2.

Protective effects of Allium porrum and Bauhinia variegate leaves on kidney function tests in plasma

Parameter	Group
	Control	TAA	TAA + A porrum	TAA + B variegata	TAA + A porrum + B variegata
Creatinine (mg/dl)	0.93 ± 0.05	1.53 ± 0.08*	1.12 ± 0.02*@	1.20 ± 0.06*@	1.15 ± 0.02*@
Urea (mg/dl)	32.17 ± 1.48	77.34 ± 3.16*	58.49 ± 2.34*@	53.02 ± 1.98*@	41.86 ± 1.07*@#
Uric acid (mg/dl)	1.74 ± 0.05	2.95 ± 0.03*	2.29 ± 0.06*@	2.17 ± 0.04*@	1.82 ± 0.05*@#

Each value is the mean ± SE, n = 8

*Significantly different from control group at P < 0.01

@Significantly different from TAA group at P < 0.01

#Significantly different from TAA + A porrum group or TAA + B variegata group at P < 0.01

Plasma Lipid Profile

As shown in Table 3, total cholesterol, tryiglycerides, and LDL concentrations increased significantly (P ≤ 0.01) in TAA group, while HDL level decreased significantly. The A porrum and B variegate extracts mitigate the changes in lipid profile when compared to TAA group.The most pronounced effect on lipid profile was recorded in A porrum + B variegata + TAA group.

Table 3.

Protective effects of Allium porrum and Bauhinia variegate leaves on triglycerides, cholesterol and HDL

Parameter	Group
	Control	TAA	TAA + A porum	TAA + B variegata	TAA + A porrum + B variegata
Cholesterol (mg/dl)	82.09 ± 2.475	127.5 ± 4.636*	92.25 ± 3.016@	94.22 ± 4.24@	75.89 ± 1.96@#
Triglycerides (mg/dl)	59.38 ± 2.8611	99.69 ± 3.040*	78.57 ± 1.867*@	73.97 ± 1.65*@	74.10 ± 2.81*@
HDL (mg/dl)	38.03 ± 1.966	21.83 ± 0.74*	28.29 ± 1.94*@	26.70 ± 1.47*@	36.02 ± 1.92@#
LDL (mg/dl)	28.43 ± 0.78	90.28 ± 3.25*	48.00 ± 1.32*@	51.79 ± 2.01*@	29.11 ± 0.56*@#

Each value is the mean ± SE, n = 8

*Significantly different from control group at P < 0.01

@Significantly different from TAA group at P < 0.01

#Significantly different from TAA + A porrum group or TAA + B variegata group at P < 0.01

Hepatic and Renal Oxidative Stress Parameters

The results indicated that administration of the A porrum and B variegate extracts to rats given TAA decreased hepatic and renal MDA concentration and increasesd GSH, CAT, SOD levels in the two tissues significantly(P ≤ 0.01) as compared to TAA group (Table 4). The treatment of the rats with the two extracts in combination + TAA led to a significant increase (P ≤ 0.01) in hepatic or renal GSH and SOD levels as compared to A porrum + TAA group or B variegate + TAA group. Moreover, the tested extracts reduced the elevation in hepatic TNF-α due to TAA treatment (Fig. 1). Also,the pretreated with A porrum and B variegate extracts lessen the elevation of hepatic nitric oxide in rats given TAA.The renal NO level did not change significantly in all treatment groups (Table 5).

Table 4.

Protective effect of Allium porrum and Bauhinia variegate leaves extract on renal oxidative stress parameters

Parameter	Group
	Control	TAA	TAA + A porrum	TAA + B variegata	TAA + A porrum + B variegata
MDA (nmol/mg)	6.83 ± 0.25	7.43 ± 0.34*	7.00 ± 0.21@	7.10 ± 0.17@	6.79 ± 0.19@
GSH (mg/g tissue)	2.50 ± 0.06	1.48 ± 0.03*	1.85 ± 0.01*@	2.00 ± 0.03*@	2.37 ± 0.02@#
CAT (U/g tissue)	7.49 ± 0.27	5.16 ± 0.13*	6.34 ± 0.22*@	6.75 ± 0.21*@	6.59 ± 0.40*@
SOD (U/g tissue)	32.96 ± 1.65	18.04 ± 0.07*	24.78 ± 1.04*@	22.53 ± 0.87*@	34.28 ± 1.26*@#

Each value is the mean ± SE, n = 8

*Significantly different from control group at P < 0.01

@ Significantly different from TAA group at P < 0.01

# Significantly different from TAA + A porrum group or TAA + B variegata group at P < 0.01

Fig. 1.

Hepatoprotective effect of A porrum and B variegata leaves extract on hepatic GSH and antioxidant enzymes of rats treated with TAA. *: Significant difference from control (P ≤ 0.01). a: Significant difference from TAA group (P ≤ 0.01). @: Significant difference from A porrum group (P ≤ 0.01). #: Significant difference from A porrum or B variegata group (P ≤ 0.01)

Table 5.

Protective effect of Allium porrum and Bauhinia variegate leaves extract on hepatic and renal nitric oxide (μmol.g. tissue)

Parameter	Group
	Control	TAA	TAA + A porrum	TAA + B variegata	TAA + A porrum + B variegata
Hepatic NO	15.43 ± 1.51	27.00 ± 2.18*	20.56 ± 1.15*	22.55 ± 0.89*	18.67 ± 1.85@
Renal NO	29.50 ± 1.50	24.48 ± 2.23	30.41 ± 2.11	32.78 ± 2.62	30.57 ± 3.17

Each value is the mean ± SE, n = 8, NO: Nitric oxide

*Significantly different from control group at P < 0.01

@Significantly different from TAA group at P < 0.01

Histopathological Results

Liver

The control liver showed normal lobular architecture with central veins, radiating hepatic cords, blood sinusoid and prominent nuclei (Fig. 2-a).

Fig. 2.

Hepatoprotective effect of A porrum and B variegata leaves extract on hepatic TNF-α of rats treated with TAA *: Significant difference from control (P ≤ 0.01). a: Significant difference from TAA group (P ≤ 0.01). #: Significant difference from A porrum or B variegata group (P ≤ 0.01)

Light microscopic examination of in the liver of rats administered TAA caused degenerative changes, gross necrosis, and cellular infiltration of the centrilobular hepatocytes with marked congestion of central veins. The hepatocytes appeared to be suffering from certain degree of cytoplasmic vacuolations, fatty infiltration and nuclei of most cells revealed clear signs of pyknosis. The sinusoids between the hepatic cells were markedly dilated, congested with increase in Kupffer cells (Fig. 2-b).

The liver of Allium porrum + TAA group showed reduction in degree of hepatocyte necrosis and degeneration and there were a mild inflammatory cell infiltrates and hemorrhage around central lobular veins with signs of pyknotic and apoptotic cells. The sinusoids between the hepatic cells were still dilated, congested with decreased in Kupffer cells (Fig. 2-c).

In rats treated with Bauhinia variegata + TAA, although hepatic lesions were minimal, some of the cells still showed hemorrhage around central lobular veins with signs of pyknotic and apoptotic cells. The sinusoids between the hepatic cells were still dilated, with reduced in Kupffer cells (Fig. 2-d).

The pre-treatment of rats with combination of the two plants showed more improvement in histological structure of liver tissues pronounced in normalized appearance of liver lobules with strains of hepatocytes. Hemorrhage and dilated, sinusoids with few Kupffer cells were also observed (Fig. 2-e). From this result we observed that the improvement was more pronounced in combination of the two extracts treated rats.

Kidney

Histological sections from control rats showed normal structures of glomerulus, urinary space, proximal, distal and collecting tubules appeared normal (Fig. 3-a).

Fig. 3.

a Section of liver from control rat showing normal lobular architecture with central veins (CV), radiating hepatic cords, blood sinusoid (S) and prominent nucleus (N). b Section of liver from a rat treated with TAA showing degenerative changes, gross necrosis (arrowhead), marked congestion of central veins (CV cytoplasmic vacuolations, fatty infiltration (arrow) of hepatocytes, pyknotic nuclei (P), haemorrahge of blood sinusoids with increase in Kupffer cells (K). c Section of liver from rat treated with A.porrum leaf extract and TAA showing moderated improvement with necrosis (arrowhead), degeneration, mild inflammatory cells infiltrates and hemorrhage around central lobular veins (H) and pyknotic nuclei (P). The sinusoids still dilated, congested with decreased in Kupffer cells (K). d Section of liver from rat treated with B.variegata leaf extract and TAA showing hepatic lesions were minimal; some of the cells still necrosis (arrowhead), pyknotic nuclei (P). The sinusoids between the hepatic cells were still dilated and hemorrhage (H), with reduced in Kupffer cells (K). e Section of liver from rat treated with the two plant extracts in combination and TAA showing remarkable improvement in histological structure of liver tissues, pronounced in normalized appearance of liver lobules with strains of hepatocytes. Hemorrhage (H) and dilated, sinusoids (S) with few Kupffer cells (K) (H & E X400)

Examination of experimental rats after administration TAA revealed glomerular changes in the form of shrinkage and widening of urinary space. In addition, some glomeruli showed mesengial cell proliferation in apart or segment.

The tubules (both proximal and distal convoluted ones) showed many changes. The lining epithelium showed injury in the form of cytoplasmic vacuolation, (vacuolar degeneration) loss of nuclei, necrosis and pyknotic nuclei. The interstitium was hemorrhage and infiltrated by mononuclear cells (Fig. 3-b).

Pretreatment of rats treated with Allium porrum revealed moderate improvement, degeneration of some glomeruli, vacuolation of convoluted tubules, mild hemorrhage of interstitium and pyknotic nuclei (Fig. 3-c).

The rats treated with Bauhinia variegata showed widening of urinary space still, also degeneration of some tubules, few pyknotic cells and mild hemorrhage of interstitium (Fig. 3-d) were observed.

However, the group treated with combination of the two plants extract + TAA showed remarkable improvement and almost normal appearance of the kidney tissue with interstitial hemorrhage and few pyknotic cells (Fig. 3-e).

Immunohistochemical Reaction of iNOS

Liver

The liver section of control rat demonstrated the negative immune-reaction of iNOS(Fig. 4-a). Sections prepared from the liver tissue of TAA group demonstrate the strong positive immune-reaction of iNOS in the form of fine brown granules within the cytoplasm of hepatocytes (Fig. 4-b).

Fig. 4.

a Section of kidney from control showing normal structures of glomeruli (G) and tubules (T). b Section of kidney from a rat treated with TAA showing shrinkage glomerular (G) and widening of urinary space (US). In addition cytoplasmic vacuolation of the lining epithelium (V), loss of nuclei, necrosis (star) and pyknotic nuclei (arrowhead) i. The interstitium was hemorrhage and infiltrated by mononuclear cells. (H). c Section of kidney from rat treated with A porrum leaf extract and TAA showing moderate improvement, degeneration of some glomeruli, cytoplasmic vacuolation of the lining epithelium (V), necrosis (star) mild hemorrhage of interstitium (H) and pyknotic nuclei (arrowhead). d Section of kidney from rat treated withB variegata leaf extract and TAA showing improvement, urinary space still widening (US), also necrosis (star) of some tubules, few pyknotic nuclei (arrowhead) and mild hemorrhage of interstitium (H). e Section of kidney from rat treated the two plant extracts in combination and TAA showing almost normal appearance of the kidney tissue with interstitial hemorrhage (H) and few pyknotic nuclei (arrowhead) and inflammatory cells (arrow) (H & E X400)

iNOS immunopositivity decreased in the livers of TAA-intoxicated rats and pretreated with A.porrum (Fig. 4-c) or B. variegata extracts (Fig. 4-d) but still not exact as control group.

In the group treated with TAA and combination of two plants, iNOS expression was nearly similar to controls (Fig. 4-e).

Kidney

The kidney of the control rat showed a negative immune reaction to iNOS (Fig. 5-a).

Fig. 5.

a Section of liver from control showing negative immune-reaction of iNOS. b Section of liver from a rat treated with TAA showing strong iNOS immunopositivity in the form of fine brown granules within the cytoplasm of hepatocytes. c Section of liver from rat treated with A porrum leaf extract and TAA showing moderate iNOS immunopositivity reaction. d Section of liver from rat treated with B variegata leaf extract and TAA showing remarkable reduced iNOS immunopositivity. e Section of liver from rat treated the two plant extracts in combination and TAA showing iNOS immunopositivity nearly similarly to control (iNOS, immunostaining with hematoxylin counterstain × 400)

In the group received TAA showed highly expressed iNOS reaction in the cytoplasm of glomeruli and tubular cells. Positive immunohistochemical staining of iNOS demonstrated brown cytoplasmic staining (Fig. 5-b).

The administration of TAA and A.porrum showed decreased expression of iNOS (Fig. 5-c). In the group received TAA and B. variegata showed minimally expressed of iNOS reaction in the cytoplasm of glomeruli and tubular cells (weak brownish staining in the cytoplasm) (Fig. 5-d).

In addition, TAA and combination of two plants group showed the recovery to nearly normal expression of iNOS (Fig. 5-e).

Discussion

Thioacetamide confirms to be a highly valuable agent as an experimental inducer of hepatic damage. The pathological roles of TAA at high dose is mainly restricted to acute liver inju-ry and they are known to encourage oxidative stress, lipid peroxidation [19], as well as a decrease in the antioxidant status [20]. TAA induces the formation of reactive metabolites (free radicals) derived from thioacetamide-S-oxide and by ROS generated as intermediates, which leads to the cell death. The metabolites produced are covalently attached to the cellular macromolecules and motivate the oxidative stress [3]. The present study indicated a rise in the liver and kidney oxidative stress in rats treated with TAA as manifested by an increase of MDA, and a decrease of GSH, CAT and SOD levels. The MDA is a product of lipoper-oxidative process and known to be an impor-tant indicator of oxidative stress[21]. Here, the pretreatment of A porrum and B variegata extracts to rats given TAA reduced hepatic or renal MDA level and elevated GSH,SOD and catalase depleted concentrations which may indicate a decrease of oxidative stress. Both A porrum and B variegata leaf extracts exhibited antioxidant properties and they can scavenge free radicals since they contain polyphenols and flavonoids [22–24]. Flavonoids have several biological effects including antioxidant and hepatoprotection [25–27].

The increase in the activities of AST, ALT, and GGT in plasma of rats treated with TAA is likely due to the liver injury that resulted in increased permeability of plasma membrane leading to leakage of the enzymes to the blood stream [28]. An increase in bilirubin level mediates the pathological alteration in biliary flow. It was suggested that the increase in serum total bilirubin concentration after TAA administration might be referred to the insufficiency of normal uptake, conjugation and excretion of bilirubin by the damaged hepatic parenchyma [29].

Moreover, the pathological changes induced in liver and kidney by TAA is likely due to the elevation of MDA level (An indicator of lipid peroxidation). Lipid peroxidation of hepatic cell membrane is one of the principle causes of hepatic injury. MDA is a dialdehyde able to react with primary amines on proteins or DNA to form crosslinks [30]. The aldehyde degradation products of lipid peroxides are toxic to cells and may induce necrotic cell death [30].

Treatment of rats with A.porrum or B. variegata leaf extracts mitigated the liver and kidney histopathological changes in rats given TAA which may be attributed to their antioxidant properties, reduction in MDA level, maintaing antioxidant enzymes (SOD,CAT) and enhancement of GSH. The antioxidant enzymes, SOD and CAT eliminate superoxide radicals [31]. GSH is one of the most numerous, non-enzymatic tripeptide antioxidants in the hepatocytes, which protects the membrane protein thiols of hepatocytes from deleterious effects of reactive oxygen species metabolites [32].

The protective effects of A porrum or B Variegated leaf extracts may be attributed to their active principles, saponin and flavonoids that are effective in alleviating oxidative stress and maintain antioxidant enzymes in tissue injury induced by carbon tetrachloride [33]. Moreover, another suggested mechanism of protection by A porrum or B variegata leaf extracts is the alleviation in the increase of TNF- α, NO and iNOS levels induced by TAA.

The cytokine TNF-α has been implicated in numerous studies as having a critical role in the pathogenesis of acute liver failure [34]. TNF-α has a role in regulating many physiological events as apoptosis and inflammatory processes [35]. It has been previously reported that TNF-α activity is increased in many forms of experimental and clinical forms of liver injury [36]. It was suggested that the oxidative stress and elevated level of TNF-α play an essential role in hepatic cell injury [37]. The another study, demonstrated that TNF-α directly induced mitochondrial ROS production and caused DNA damage and dysfunction [38].

Hepatitis induces iNOS expression in hepatocytes, suggesting that NO overproduction might have an important role in progression of hepatitis to cirrhosis. NO reacts with reactive oxygen species to form reactive nitrogen species, which generates peroxynitrite, a strong biological oxidant generally implicated in toxic effects on cells and tissues.

It was reported that Bauhinia purpurea leaf extract significantly suppressed the cholesterol high fat diet induced hyperlipidemia in rats, suggesting the antihyperlipidemic and antiatherogenic potential of the extract [24]. Allium ampeloprasum can reduce liver TG accumulation induced by high fat diets. Our study showed that administration of A porrum or B variegata leaf extracts was able to reduce hyperlipidemia evoked by TAA. The two extracts lightened the increase of plasma cholesterol,triglyceides and low density lipoprotein in TAA treated rats. On the other hand, they maintained HDL level. The active ingredients present in the two extracts may recover the disorders in the lipid metabolism noted in TAA treated rats. Both flavonoids and saponin possess antihyperlipidemic property [39, 40].

The present investigation showed that the combined effect of A porrum and B Variegata extracts were pronounced in reduction of the toxic effects of TAA on liver and kidney. The mixture of some phenolic and flavonoid compounds exhibited the synergistic effects and can increase the antioxidant effects [41]. A polyherbal formulation offered a synergistic hepatoprotective effects due to the various phytochemicals present in it in TAA-induced hepatotoxicity.

The present study elucidated the protective effects of A porrum and B variegata extracts against TAA-evoked hepatic and renal injuries. Different mechanisms may explain the protective effects of the two plants. Both A porrum and B variegata extracts lowered the oxidative stress as evidenced by a decrease of MDA and enhancement of GSH, SOD and CAT. Moreover, they have anti-inflammatory property by decreasing TNF-α, NO and iNOS beside hypolipidemic effect.