Hepatoprotective and antioxidant activities of Tamarix nilotica flowers

Abstract

Context: Tamarix nilotica (Ehrenb.) Bunge (Tamaricaceae) is used in the Egyptian traditional medicine as an antiseptic agent. This plant has been known since pharaonic times and has been mentioned in medical papyri to expel fever, relieve headache, to draw out inflammation, and as an aphrodisiac. No scientific study is available about the biological effect of this plant.

Objective: This study aimed to evaluate the hydro-alcoholic extract (80%) of T. nilotica flowers for hepatoprotective and antioxidant activities.

Materials and methods: Hepatoprotective activity was assessed using carbon tetrachloride–induced hepatic injury in rats by monitoring biochemical parameters. Antioxidant activity was evaluated in alloxan-induced diabetic rats. Biochemical markers of hepatic damage such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), and tissue glutathione were determined in all groups.

Results and conclusion: Carbon tetrachloride (5 mL/kg body weight) enhanced the SGOT, SGPT, and ALP levels. There was a marked reduction in tissue glutathione level in diabetic rats. The hydro-alcoholic extract of T. nilotica (100 mg/kg body weight) ameliorated the adverse effects of carbon tetrachloride and returned the altered levels of biochemical markers near to the normal levels.

Keywords::

• Antioxidant
• hepatoprotective
• in vivo
• Tamaricaceae
• Tamarix nilotica

Introduction

Free radicals are involved in a number of pathological conditions such as inflammatory diseases, atherosclerosis, cerebral ischemia, AIDS, and cancer (Thomas & Kalyanaraman, 1997). These free radicals are induced in the human body due to environmental pollutants, chemicals, physical stress, radiation, etc. There are several biomolecules in the body which act as free radical scavengers. Catalase and hydroperoxidase enzymes are among the most important antioxidants produced by the immune system. Consumption of antioxidants or free radical scavengers is necessary to compensate depletion of antioxidants of the immune system. There is an increasing interest in the use of medicinal plants as dietary supplements to act as antioxidants. Silymarin from Silybum marianum (L.) Gaertn. (Asteraceae) fruits and wheat germ oil are among the most widely used antioxidant plant products in the pharmaceutical market.

Tamarix nilotica (Ehrenb.) Bunge (Tamaricaceae) is a tree of 2–5 m; widespread in Egypt, growing in saline sandy soils, on the edges of salt marshes, coastal and inland sandy plains, and Nile banks (Boulos, 2000). The plant is locally known as alaabal. T. nilotica has been used in Egyptian traditional medicine as an antiseptic agent. This plant has been known since pharaonic times and has been mentioned in medical papyri to expel fever, relieve headache, to draw out inflammation, and as an aphrodisiac (Kamal, 1967). The wood yields a locally made charcoal, also used as a fuel; the timber is sometimes used for inferior carpentry. In our previous work, T. nilotica hydro-alcoholic (80%) flower extract showed significant in vitro antioxidant activity using 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity, superoxide anion scavenging activity, and iron chelating activity (AbouZid et al., 2008). The present work aimed to investigate the potential in vivo antioxidant and hepatoprotective activities of T. nilotica hydro-alcoholic (80%) flower extract in rats.

Materials and methods

Chemicals, solvents, and reagents

Ethyl alcohol (95%) was obtained from El Nasr Pharmaceutical Chemicals, Cairo, Egypt. Carbon tetrachloride (Analar) and alloxan were obtained from Sigma (St. Louis, MO, USA). Silymarin (Legalon^®) was obtained from Chemical Industries Development (CID), Giza, Egypt; α-tocopherol acetate was obtained from Pharco Pharmaceutical, Alexandria, Egypt, and biodiagnostic kits for assessment of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), and alkaline phosphatase (ALP) were obtained from bioMerieux (Durham, MO, USA).

Plant material

T. nilotica flowers were collected from Beni-Sueif governorate, Egypt (Maydoum’s desert oasis) in March 2006. The plants were identified by M. Abdelhalim, Plant Taxonomy Department, Agricultural Research Center, Egypt. Voucher specimens were deposited in the Herbarium of the Faculty of Pharmacy, Beni-Sueif University, Beni-Sueif, Egypt.

Preparation of T. nilotica flower extract

The air-dried flowers of T. nilotica (2.7 kg) were ground to a powder and extracted by maceration in 80% aqueous ethanol. The resulting extract was filtered, concentrated (36 g), and kept in tightly sealed sample tubes until used for the biological study.

Animals

Albino mice (25–30 g body weight) were used for the toxicological study. Adult male albino rats of Sprague–Dawley strain (130–150 g body weight) were used for hepatoprotective and antioxidant screening. The animals were kept in standard environmental conditions, fed with standard laboratory diet consisting of vitamin mixture (1%), mineral mixture (4%), corn oil (10%), sucrose (20%), cellulose (0.2%), casein-95% pure (10.5%), starch (54.3%), and water ad libitum. All of the methods used in the present study were approved by the ethics committee of the National Research Centre, Giza, Egypt.

Toxicological study

Determination of the LD₅₀ of the hydro-alcoholic (80%) extract of T. nilotica was estimated according to the Karber procedure (1931). Experiments were done to determine the minimal dose that kills all animals (LD₁₀₀) and the maximal dose that fails to kill any animal. Several doses at equal logarithmic intervals were chosen in between these two doses; each dose was injected in a group of six animals by subcutaneous injection. The animals were then observed for 24 h and symptoms of toxicity and mortality rates in each group were recorded and the LD₅₀ were calculated.

Hepatoprotective activity

Liver damage in rats was induced by intraperitoneal injection of 5 mL/kg body weight of 25% carbon tetrachloride in liquid paraffin (Klaassen & Plaa, 1969). Animals were randomly divided into three groups of six animals each. The first group received a daily oral dose of saline 1 mL/kg body weight for 1 week (control). The second group received a daily oral dose of 100 mg/kg body weight of the hydro-alcoholic (80%) extract of T. nilotica flowers. Administration of the extract was continued for 7 days. The third group received a daily oral dose of 25 mg/kg body weight silymarin (positive control). Blood samples were collected on days 0, 1, 3, and 7 after the carbon tetrachloride injections. Whole blood was obtained from the retro-orbital venous plexus through the eye canthus of anesthetized rats. Serum was isolated by centrifugation. SGOT, SGPT (Thewfweld, 1974), and ALP (Kind & King, 1954) activities were determined.

In vivo antioxidant activity

In vivo antioxidant activity of hydro-alcoholic (80%) extract of T. nilotica flowers was determined by measuring the glutathione level in the blood of alloxan-induced diabetic rats according to Beutler et al. (1963), using α-tocopherol as a positive control. Animals were randomly divided into four groups, six rats each. The first group was kept as negative control, whereas for other groups diabetes mellitus was induced according to Eliasson and Samet (1969). A single dose of 150 mg alloxan/kg body weight was injected intraperitoneally into each animal followed by an overnight fast. The second group of diabetic rats was kept untreated; the third group received the reference drug α-tocopherol 7.5 mg/kg body weight; the fourth group was orally administered hydro-alcoholic (80%) extract of T. nilotica flowers using a dose of 100 mg/kg body weight. The rats were kept for 1 week before the determination of glutathione in the blood. A blood sample (0.1 mL) was hemolysed by the addition of 0.9 mL double distilled water. One and half milliliter of the precipitating solution was added to the hemolysate, mixed, left for 5 min, then centrifuged at 3000 rpm for 15 min. Four milliliters of phosphate buffer (0.3 M) were added to 1 mL of the resulting supernatant followed by 0.5 mL of Ellman’s reagent. The optical density was measured within 5 min at 412 nm against blank solution. Standard glutathione solution (1 mL) was added to 4 mL phosphate buffer (0.3 M) and 0.5 mL Ellman’s reagent then the optical density was measured at 412 nm against blank solution. Blood glutathione level is calculated according to the following equation:

Statistical analysis

The data are the mean of triplicate measurements. The results are expressed as mean ± SE (n = 6). Statistical significance was determined by Student’s t-test with p < 0.01 considered significant.

Results and discussion

Toxicological study

In acute toxicity, the LD₅₀ of the extract was found to be 6.4 g/kg body weight. This LD₅₀ is categorized as unclassified according to the ranking system of European Economic Community (EC Directive 83.467 EEC, 1983).

Hepatoprotective activity

Carbon tetrachloride was used to induce liver damage and hence enhancing the levels of SGOT, SGPT, and ALP. Carbon tetrachloride is biotransformed by liver enzymes to a highly reactive free radical. This free radical can lead to lipid peroxidation, disruption of Ca²⁺ homeostasis, elevation of hepatic enzymes, and finally results in cell death (Clawson, 1989). Carbon tetrachloride has been used in animal models to investigate chemical toxin-induced liver damage. The extent of hepatic damage is assessed by the increased level of cytoplasmic enzymes (SGOT, SGPT, and ALP). Treatment with silymarin or 100 mg/kg body weight of hydro-alcoholic (80%) extract of T. nilotica flowers has significantly brought down the elevated levels of these biochemical parameters. Results are reported in Table 1.

Table 1.  Effect of hydro-alcoholic extract of Tamarix nilotica flowers and silymarin on biochemical parameters in liver damaged rats (n = 6).

Group	SGOT (U/L)				SGPT (U/L)				ALP (KAU)
	0 day	1 day	3 days	7 days	0 day	1 day	3 days	7 days	0 day	1 day	3 days	7 days
Control	29.4 ± 0.9	28.6 ± 0.4	136.9 ± 5.1	151.7 ± 5.9•*	31.6 ± 1.1	30.9 ± 0.7	143.9 ± 6.1	149.2 ± 5.7*	6.8 ± 0.1	7.1 ± 0.1	57.9 ± 1.8	63.4 ± 2.3•*
T. nilotica	31.6 ± 1.2	31.2 ± 0.9	62.8 ± 2.7	43.2 ± 1.5•*	28.9 ± 0.0	28.2 ± 0.7	73.1 ± 2.6	39.8 ± 1.3•*	7.5 ± 0.1	7.2 ± 0.1	26.3 ± 0.9	18.9 ± 0.4•*
Silymarin	32.3 ± 1.1	30.6 ± 0.9	49.2 ± 1.3	29.7 ± 0.6•	27.8 ± 0.5	26.8 ± 0.4	56.2 ± 1.8	29.2 ± 0.8•	7.3 ± 0.1	6.9 ± 0.1	18.3 ± 0.6	6.9 ± 0.1•

*Statistically significant from zero time at p <0.01. •Statistically significant from 3 d after CCL₄ at p <0.01.

Antioxidant activity

Oxidative stress plays a significant role in many diseases including liver damage (Kiso et al., 1984). Oxidative stress is the state of imbalance between the level of antioxidant defense system and production of oxygen-derived species. It has been hypothesized that one of the principal causes of carbon tetrachloride–induced liver injury is formation of lipid peroxides by its free radical derivatives. Thus, the antioxidant activity or the inhibition of the generation of free radicals is important in the protection against carbon tetrachloride–induced hepatopathy (Castro et al., 1974). In vivo antioxidant activity was determined by measuring tissue glutathione level in alloxan-induced diabetic rats. Tissue glutathione level in the diabetic rats is decreased to the extent of 60%. Treatments with α-tocopherol or T. nilotica flower extract increased tissue glutathione levels to the near healthy levels (Table 2). It can be argued that the hepatoprotective effect of the hydro-alcoholic extract of T. nilotica flowers may be at least partly due to the prevention of the depletion in the tissue glutathione levels.

Table 2.   In vivo antioxidant activity of Tamarix nilotica extract and α-tocopherol.

Group	Blood glutathione (mg%)	% Change from control
Control (1 mL saline)	36.1 ± 1.1	—
Diabetic	21.7 ± 0.4*	39.9
Diabetic, α-tocopherol (7.5 mg/kg)	35.3 ± 0.8	2.2
Diabetic, Tamarix nilotica (100 mg/kg)	34.6 ± 0.9	4.2

*Statistically significant difference from control group at p <0.01.

Conclusions

A Literature review indicated that T. nilotica contains many phenolic and flavonoid constituents. Ethyl ester of kampferol 3-O-β-D-glucuronide, the methyl and ethyl esters of quercetin 3-O-β-D-glucuronide are the main flavonoids isolated from the flowers of the plant. The Methyl ester of ferulic acid 3-O-sulfate, coniferyl alcohol 4-O-sulfate, kampferol 4’-methyl ether, kampferol 3-O-β-D-glucupyranuronide, tamarixetin, tamarixetin 3-O-β-D-glucupyranuronide, and quercetin 3- O-β-D-glucupyranuronide were isolated from the leaves of the plant (El-Sisi et al., 1973; Nawwar et al., 1982, 1984a, 1984b; Barakat et al., 1987; AbouZid et al., 2009; Orabi et al., 2009, 2010). In this study the in vivo antioxidant activity of T. nilotica flowers was demonstrated. In vitro antioxidant activity of the flowers of this plant was previously reported (AbouZid et al., 2008). There is a possibility that the proven in vitro and in vivo antioxidant activity of T. nilotica flowers which may be due to its phenolic constituents is involved in the hepatoprotective property.

Acknowledgement

Thanks to Dr. Abdelhalim M. Mohamed, Flora Research and Plant Taxonomy Department, Agricultural Research Institute, Egypt for botanical identification of the plant used in this study.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.