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Table of Contents
ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 5  |  Page : 193-197

Protective effects of Aristolochia longa and Aquilaria malaccensis against lead induced acute liver injury in rats


1 Department of Cellular and Molecular Biology, Faculty of natural science and life, University of Echahid Hamma Lakhdar-Eloued, El-oued 39000, El-oued, Algeria
2 Laboratory of Animal Ecophysiology, Faculty of Science of Sfax, 3018 Sfax, Tunisia

Date of Submission27-Jun-2017
Date of Decision01-Jul-2017
Date of Acceptance08-Jul-2017
Date of Web Publication7-Dec-2017

Correspondence Address:
Derouiche Samir
Department of Cellular and Molecular Biology, Faculty of natural science and life, University of Echahid Hamma Lakhdar-Eloued, El-oued 39000, El-oued
Algeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2221-6189.219611

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  Abstract 


Objective: To investigate the protective effects of Aristolochia longa (A. longa) and Aquilaria malaccensis (A. malaccensis) on acute hepatotoxicity induced by lead in female albino rats. Methods: Twenty five (25) apparently healthy female Wistar rats were randomly divided into five groups of five rats in each: control, Pb, Pb + A. longa (Ar), Pb+ A. malaccensis (Aq), and Pb+ A. longa (Ar) + A. malaccensis (Aq) lead (100 mg/kg b.w.) as Pb (C2H3O2)2 added in their drinking water for 75 days. A. longa (rhizome powder at a dose of 10 g/kg of diet) and A. malaccensis (heartwood powder at a dose 10 g/kg of diet) were added to the feed during the last 15 days of lead exposed in the animals. Results: Obtained results revealed that lead treatment caused a significant increase in serum GOT, GPT and ALP activities and in liver of MDA level and CAT activity. In contrast, it led to an decrease in the liver GOT, GPT and GST activities and in GSH level in rats. Also, the results clearly showed that lead causes alterations of hepatic tissue in comparison with controls. Our results showed that treatment with A. malaccensis and A. longa a partial correction of the previous parameters. The histological observations confirmed the hepatoprotection results by the biochemical parameters. Conclusions: Results demonstrated beneficial effects of A. longa and A. malaccensis treatment in Pb-induced oxidative stress and tissue damage in liver.

Keywords: Aristolochia longa, Aquilaria malaccensis, Acute hepatotoxicity, Acetate lead, Stress oxidative


How to cite this article:
Samir D, Khaoula Z, Safa G, Yahia K, Anouar F. Protective effects of Aristolochia longa and Aquilaria malaccensis against lead induced acute liver injury in rats. J Acute Dis 2017;6:193-7

How to cite this URL:
Samir D, Khaoula Z, Safa G, Yahia K, Anouar F. Protective effects of Aristolochia longa and Aquilaria malaccensis against lead induced acute liver injury in rats. J Acute Dis [serial online] 2017 [cited 2017 Dec 17];6:193-7. Available from: http://www.jadweb.org/text.asp?2017/6/5/193/219611




  1. Introduction Top


Lead is among the most known and abundant heavy metals. it is present in the different biological systems and in the environment. It is recognized by its toxicity and its harmful effect, even in small amounts[1]. Lead is present in soil and aquatic environments including drinking water also present in several products manufactured in industry[2]. This metal causes a disturbance in biochemical and physiological aspects in many living species, including humans. such as disturbances in respiratory, neurological, hepatic and reproductive systems[3]. Lead causes an imbalance between the release of free radicals and the antioxidant system in the cell which generates a state of oxidative stress which is the origin of lead toxicity in the body. Macromolecules are the main targets of ROS. The latter cause lipid peroxidation, protein oxidation and DNA alterations, leading to the development of cancer, diabetes, neurodegenerative diseases and cardiovascular diseases[4]. Aquilaria malaccensis (A. malaccensis) and Aristolochia longa (A. longa) are very well known plants by their use in the popular medicine against cancer in several regions in Algeria. A. malaccensis has numerous biological activities, including activity, anti-microbial, anti-tumor, anti-allergic, anti-oxidant[5]. A. longa also has several therapeutic uses as against ovarian insufficiencies, healing, diuretic[6], analgesic, anti-inflammatory, anti-hyperglycemic[7]. The purpose of this study is to evaluate the effect of A. malaccensis and A. longa on hepatotoxicity, oxidative stress and liver tissue alteration induced by lead in rats.


  2. Materials and Methods Top


2.1. Chemicals

Lead nitrate Pb(C2H3O2)2 and all other chemicals used in the study were obtained from Sigma-Aldarich Corporation (St. Louis, Missouri, USA).

2.2. Plant material

A. longa roots and A. malaccensis heartwood were collected in herbalists shops from a local market of El-Oued. The plant material was washed using distilled water and then drying in room temperature for 48 to 92 h and then grounded into powder and stored at room temperature until use.

2.3. Animals and Experiments

Female albino rats aged 8 weeks old weighting approximately 220 g, were obtained at the Animal Service of the Pasteur Institute, Algeria. Animals have free access to water and food. After the adaptation period, rats were divided into five groups (n=5) and kept in animal's house of biology Department, El Oued University, Algeria. Standard diet and water were available ad libitum for the duration of the experiments unless otherwise noted. The animals were carried under the same conditions, photoperiod (12h of light/12h of black) with a relative humidity of 65.3% and an ambient temperature of (23 ± 2) °C for two weeks. The experiments on rats were performed according to the National Institute of Health's standards for animal care and authorized by the ethics committee of our institution. The experiment was conducted over a period of 70 days. After a period of adaptation, the animals were divided into five experimental groups of 5 animals each:

Group I (control group): animals served as normal control; Group II (Pb-treated group): animals received acetate lead (100 mg/kg b.w.) as Pb(C2H3O2)2 added in drinking water for 70 days in animals; Group III (Pb+ Aq-treated group): animals received powder of heartwood of A. malaccensis alone at a dose of 10 g/kg diet for 15 days; Group IV (Pb+Ar-treated group): animals received powder of rhizome of A. longua alone at a dose of 10 g/kg diet for 15 days. Group V (Pb+Aq+Ar-treated group): animals received both powder of heartwood of A. malaccensis and rhizome of A. longua for 15 days.

Blood was collected, some of which was centrifuged at 3 000 rpm for 10 min to obtain the plasma which was kept frozen at -20 °C until used for biochemical analysis of GOT, GPT and ALP activities.

2.4. Tissue preparation

For each lot, 1 g of liver was taken from the rats. the tissues are bryoed and homogenized in a TBS buffer solution (50 Mm Tris, 150 mM NaCl, pH 7.4), then centrifuged for 15 min at 10 000 g and 4 °C the supernatant obtained is stored at – 20 °C while waiting for the determination of liver GOT and GPT activities and the oxidative stress parameters.

2.5. Plasma Biochemical parameters

Enzymes marker were measured according to classic methods. Alkaline phosphatase (ALP) and transaminases (GOT and GPT) activities were estimated using commercial kits from Spinreact laboratories, Spain (ALP, ref. 1001130; GOT, ref. 1001160 and GPT, ref. 1001170).

2.6. Tissue stress oxidative parameters

2.6.1. Estimation of lipid peroxidation levels

The malondialdehyde dosage is based on the condensation of MDA in acidic and hot medium with thiobarbituric acid according to Yagi et al.[8]. The reaction results in the formation of a pink complex between two molecules of thiobarbituric acid which can therefore be measured by Absorption spectrophotometry at 532 nm and the level of MDA in liver was expressed as nmol/mg protein.

2.6.2. Determination of reduced glutathione (GSH) level

The GSH level was measured by Ellman method[9]. The complex formed between GSH and 5,5'dithiodis-2-nitrobenzoic acid (DTNB) releases thionitrobenzoic acid (TNB) quantify by reading the absorbance at 412 nm. The GSH rate results were expressed as nmol GSH/mg prot.

2.6.3. Determination of Glutathione-S-transferase (GST) activity

The activity of Glutathione S Transferase was measured by the method of Habig et al.[10] using the spectrophotometric technique, which is based on the kinetics of formation of a complex between CDNB (1-chloro-2-4-dinitrobenzene) and GSH (glutathione). The formed complex can be detected by increasing the optical density at 340 nm. The enzymatic activity of GST was expressed as nmol CDNB conjugate/min/mg protein.

2.6.4. Determination of the enzymatic activity of catalase The assay of the catalase enzyme activity consists in measuring the catalase-induced loss of H2O2 contained in the sample by the measurement of H2O2 absorbance at 560 nm using a spectrophotometer according to the Aebi, 1984 method[11].

2.7. Histopathology analysis

Part of the liver of each group was removed and stored immediately in a 10% formalin solution, treated by the technique of the paraffin and then cut (5 μm thickness), and stained with hematoxylin and eosin for histological examination.

2.8. Statistics analysis

The values are presented by the means with their standard deviations (mean ± SD). Statistical comparison between means was done by Student's t-test, and statistical significance was defined as P <0.05.


  3. Results Top


3.1. Initial body weight, body weight gain and relative liver weight

The results obtained [Table 1] show that treatment with 100 mg/ kg bw of lead acetate results in a significant reduction (P<0.001) in body weight and a significant increase (P<0.05) in the liver relative weight compared to controls. But after the treatment time, the animals that received the powder of rhizome A. longa and heartwood A. malaccensis alone or combined showed partial reversion of this this change.
Table 1: Changes in body weight and biochemical parameters of control and experimental groups (n=5).

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3.2. Biochemical parameters

The results shown in [Table 1] showed that the GOT and ALP activities are significantly increased (P<0.05 and P<0.01) in the serum and significantly decreased (P<0.05 and P<0.01) in the liver respectively. But no significant difference for serum GPT activity in the group exposed to lead acetate compared to the control group. Treatment with powder of heartwood A. malaccensis or of rhizome A. longua partially restored the activity of serum GOT GPT and ALP activities. However, our results clearly show a significant increase (P<0.01) in the activity of liver transaminases (GOT and GPT) in A. malaccensis, A. longua and A. longua + A. malaccensis treated groups compared to Pb group.

3.3. Stress oxidative parameters

Our results illustrated in [Table 2] showed a significantly elevated (P<0.05 and P<0.01) in lipid peroxidation level and catalase activity respectively and a significant decrease (P<0.001) in GSH concentration and GST activity in the Pb group compared to the control. In contrast our results also show that the level of MDA and the catalase activity are significantly decreased (P<0.01) in the liver and that the hepatic GSH concentration and GST activity are significantly increased in A. malaccensis, A. longua and A. longua + A. malaccensis groups compared to Pb group.
Table 2: Liver tissue MDA, GSH levels and antioxidant enzyme activities of control and experimental groups (n=5).

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3.4. Histopathological studies

Observation of histological sections of the liver of the control rat by the light microscope showed a normal structure with clear liver cells [Figure 1]A. However, histological sections of the Pb- exposed rat liver revealed hepatic damage and lysis as haemorrhage, necrosis, inflammatory dilation, and sinusoidal dilatation [Figure 1]B. Treatment with A. malacenssis and / or A. longa in diets in lead- contaminated rats for 15 days provided protection against liver histological damage induced by this metal. These plants induce a remarkable regeneration of liver tissue. However, A. malacenssis partially decreased severe damage to liver tissue [Figure 1]C, [Figure 1]D, [Figure 1]E.
Figure 1: The histological examination of livers.
Histological examination from a control rat showing normal architecture (Figure 1A,×400); Acetate lead treated rats liver showing necrosis and hemorrhage (blue arrow), inflammatory (black arrow) and sinusoidal dilatation (green bolt) (Figure 1B, ×400). Powder rhizome of A. longa -treated rat liver showing normal appearance of hepatocytes (Figure 1D, ×400). Liver sections of the animals administered powder of heartwood A. malaccensis alone (Figure 1C,×400) or combined with rhizome of A. longa (Figure 1E,×400) of showed moderate degree of liver damage, hemorrhage and inflammatory cell, protection from hepatocyte degradation.


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  4. Discussion Top


This study evaluated the effects of treatment with powder of rhizome A. longa and heartworm A. malaccensis on enzymes markers, oxidative stress and liver histology outcome of acetate lead induced hepatotoxicity in rats. In the present study, The reduction in body weight is a sign of loss of the overall physiology of the rat. Exposure of lead acetate to doses (100 mg/kg) in rats resulted in a decrease in body weight and an increase in the relative weight of the liver, which is in agreement with several studies[12],[13] which suggest that lead induces a reduction in body weight and food consumption by its effect on nerve centers Responsible for the regulation of satiety and hunger. The reduction in body weight can also be explained by a reduction in muscle mass and cachexia due to lead-induced oxidative stress[14]. On the other hand the origin of the increase in the liver relative weight probably due to lead-induced necrosis and apoptosis on this organ[15]. After the treatment time, the animals that received A. longa and A. malaccensis showed reversion partial of this less body weight, due probably the improvement of behavior and biochemical parameters. improvement of relative liver weight is probably due to the inhibitory effect of these plants against the accumulation of lead in liver which reduces their injurious effect on liver and therefore decreases their relative weight. Also, the protective effect of these plants may be because of the existence of anti-inflammatory compounds such as phenolic acids and flavonoids[16]. In the present study, lead exposure results in a significant decrease of transaminases (GOT and GPT) activities in liver and a significant increase in GPT, GOT and ALP activities in serum. Our results in agreement with several studies[17],[18],[19]. Disruption of the activity of these enzymes may be the result of many factors, including hepatic lysis by cell necrosis, cell leakage, and loss of functional integrity of the tissue membrane of liver cells[20]. Also, these effects are due to lipid peroxidation induced by the generation of ROS under the effect of lead toxicity and alterations of the cell membrane causing the leakage of enzymes in the blood[21]. Then, our results indicates that the use of rats by A. longa or A. malaccensis or both significantly restored the enzymatic activity in serum or tissues compared to exposed lead acetate rats, which means that these plants inhibit hepatic damage caused by Pb. This is due to the reduction of accumulation of free radicals and protection against oxidative stress by Aristolochia. The anti-inflammatory property of Aristolochia species is probably the result of a direct interaction between aristolochic acid and phospholipase A2 derivatives. Aristolochic acid is the main constituent of A. longa. In addition, phospholipase A2 is an enzyme that catalyses the hydrolysis of the membrane arachidonic acid, which leads to the formation of local inflammatory mediators such as prostaglandins (PG), leukotrienes, and thromboxanes. Arachidonic acid is a key biological intermediate that is converted into a large number of eicosanoids with potent biological activity[22]. In our study, the results show in our results lead induces an increase in MDA levels and CAT activity and a decrease in GSH level and GST activity in the liver tissue. These results are consistent with several studies[23],[24],[25]. There is a complex interaction between antioxidants and pro-oxidant like ROS, which modulates oxidative stress generation[26]. These changes are explained by the increase in the of free radicals, which can be due to the increase of 5-aminolevulinic acid (ALA) under the effect of lead[27]. These also shows that lead is able to promote the generation of ROS, which results in lipid peroxidation in hepatic tissues, suggesting their deleterious effects in this tissue. Thus, another mechanism, lead can have a direct peroxidation activity that has a high binding affinity on the cell membrane[28]. Since oxidative stress is the first response to the environmental pollutants, Hepatic cells stimulate the antioxidant and detoxification system to limit the damage caused by heavy metals. The involvement of enzymatic antioxidants such as GST play an important role in protecting cells against the release of free radicals and therefore limit oxidative stress[29]. Increased CAT activity can be explained as a tissue defense mechanism against increased H2O2 fluxes during Pb-induced oxidative stress[30]. Our results show that treatment with A. malaccensis and/or A. longa could prevent lead-induced alteration, increasing the antioxidant activity of GSH and GST and decreasing lipid peroxidation. Suggest that both plants possess antioxidant activity based on the elimination of free radicals and the restoration of the oxidizing / antioxidant balance during toxicity induced by this metal. The presence of polyphenols and flavonoids in A. malaccensis and A. longa may be responsible for antioxidant activity and are considered good chelating agents for metal ions. In addition, the existence of (-OH) and (-OCH3) groups (number and position) in the structure of phenolic acids explains their powerful antioxidant power of these molecules[31], and the flavonoids inactivate and stabilize the free radicals by their group Hydroxyl (C3-OH). They are also capable of chelating metal ions (released from their binding or transport proteins)[32]. According to the results of the present study, exposure to lead causes tissue toxicity of the liver by causing histological lesions, including necrosis, inflammation and hemorrhage. These results are in agreement with the study of Shalan et al.[33]. These alterations may be due to lipid peroxidation induced by the excessive production of free radicals under the influence of exposure to lead[34]. Treatment with A. malaccensis and/or A. longa in intoxicated rats has been able to regenerate the structure of the liver. Our results suggest that these plants, especially A. longa, could reduce hepatic lesions induced by (Pb (C2H3O2) 2). The biochemical and oxidative stress parameters are also correlated with the histological study. This can be attributed to the anti-radical effect of A. malaccensis and A. longa. These plants reduced the oxidative stress caused by lead, allowing limit of histological lesions and the normalization of the physiological state of the body.

Lead is a strong hepatotoxic agent by inducing oxidative stress. A. longa and A. malaccensis were able to moderate this toxicity by decreasing oxidative stress and decreasing the alteration of the liver tissue.


  Acknowledgements Top


The first author would like to thank the Department of Cellular and Molecular Biology, Faculty of Sciences of Nature and Life, University of El Oued, Algeria and the University of Biskra, Biskra, Algeria, for the permission to utilize the facilities to make this work. This work was supported by the research project F03220140012 funded by the ministry of higher education, Algeria.

Conflict of interest statement

We declare that we have no conflict of interest.



 
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