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On the Anti Oxidative Stress Potential of Zataria multiflora Boiss (Avishan shirazi) in Rats



Mona Babaie , Narguess Yasa , Azadeh Mohammadirad , Reza Khorasani and Mohammad Abdollahi
 
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ABSTRACT

The present study was undertaken to explore the antioxidants effects of Zataria multiflora Boiss in rats. Antioxidant activity was measured by inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, Total Antioxidant Power (TAP) and Thiobarbituric Acid Reactive Substances (TBARS) in serum of treated rats. Rats received methanolic extract of Z. multiflora by intragastric intubation at doses of 50, 100 and 200 mg kg-1 daily for 14 consecutive days. The acute toxicity test (LD50) demonstrated that Z. multiflora is not lethal up to a dose of 2000 mg kg-1 after oral administration. Treatment of rats with Z. multiflora extract showed significant antioxidant activity in the DPPH test as compared to the control. Z. multiflora at doses of 50 and 100 mg kg-1 significantly increased TAP and decreased TBARS as compared to the control. Administration of Z. multiflora at a dose of 200 mg kg-1 per day did not significantly alter serum DPPH, TAP and TBARS. Antioxidant activities of Z. multiflora at doses of 50 and 100 mg kg-1 were in all experiments comparable to that of α-tocopherol. Further studies are needed to elucidate whether Z. multiflora as herbal medicine could be useful in the management of human diseases resulting from oxidative stress.

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Mona Babaie , Narguess Yasa , Azadeh Mohammadirad , Reza Khorasani and Mohammad Abdollahi , 2007. On the Anti Oxidative Stress Potential of Zataria multiflora Boiss (Avishan shirazi) in Rats . International Journal of Pharmacology, 3: 510-514.

DOI: 10.3923/ijp.2007.510.514

URL: https://scialert.net/abstract/?doi=ijp.2007.510.514

INTRODUCTION

Oxidative stress results from an imbalance between the generation of oxygen derived radicals and the organism’s antioxidant potential and thus plays important role in pathogenesis of many chronic diseases. Antioxidants are generally believed to protect body against oxidative stress by several mechanisms. These mechanisms include enzymatic degradation of free radicals, binding metals which stimulate the production of free radicals and scavenging free radicals (Abdollahi et al., 2004). Antioxidants may prevent the development of many chronic diseases associated with oxidative stress like cancer, heart failure, diabetes, Alzheimer and many other harmful diseases (Aro, 2003; Polidori, 2003; Ferrari et al., 2004).

Medicinal plants are considered as an important source of antioxidant compounds. Recently, there has been a considerable interest in finding natural antioxidants from plant materials to replace synthetic ones.

The family of Labiatae are generally known for their various effects such as analgesic and anti-inflammatory activity (Hernandez-Perez et al., 1995), antioxidant (Cuppett and Hall, 1998), hepatoprotective (Wasser et al., 1998) and hypoglycemic effects (Hosseinzadeh et al., 1998). Z. multiflora is a plant from that family that is distributed only in Iran, Pakistan and Afghanistan. It is greatly used for medicinal and condimental purposes in these countries. This plant with the vernacular name of Avishan shirazi in Iran has several traditional uses such as antiseptic, anesthetic and antispasmodic (Zargari, 1990). Our recent study confirmed its anti-colitis effect in experimental animals (Ashtaral-Nakhai et al., 2007). Our recent study provided evidence of the benefit effects of some plants in rats (Hasani et al., 2007; Shahriari et al., 2006) by reduction of blood Lipid Peroxidation (LP) and increased blood Total Antioxidant Power (TAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Regarding these reports, we hypothesized that Z. multiflora may have anti oxidative stress potential in rat.

MATERIALS AND METHODS

Plants material: Samples of Z. multiflora were collected from Shiraz, Iran, on 20 may 2006. The leaves of the plant was dried in shadow and stored in the Department of Botany of the Research Institute of Forests and Ranglands (TARI), Tehran. A voucher specimen (No. 58416) has been deposited at the Herbarium of TARI.

Preparation of total extract: Amount of 86.4 kg of plant powder was wet with a solvent (methanol) in a closed plastic container; then the wet powder put in a percolator and was macerated in 10 L methanol (100% v/v) for 24 h and subsequently, the solution was filtered and concentrated in a percolator by 100 drops per min. This procedure was repeated twice and three times with 10 L methanol (100% v/v), respectively. The extract was then concentrated under reduced pressure and appropriate temperature and the solvent was distilled in vacuum and finally 1.5 kg solid solvent was produced.

Materials: All chemicals were of highest purity (99.0%). Sodium acetate, 2,4,6-tripyridyl-s-triazine (TPTZ), 2-thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropan (MDA), Trichloroacetic Acid (TCA), glacial acetic acid, 1,1-diphenyl-2-hydrazyl (DPPH), FeCl3 . 6 H2O, HCl and n-butyl alcohol were purchased from Merck (Tehran). α-Tocopherol (Torolox) was purchased from ACROS organics, Belgium.

Animals and treatment: Experiments were performed on adult male Wistar rats from the Pasteur Institute of Tehran weighing 180-200 g. They were kept under standardized conditions (temperature 21-24°C and a light/dark cycle of 12/12 h and fed a normal laboratory diet. After 1 week of acclimatization, rats were divided into one control and four experimental groups with 6 animals in each group. The study protocol was approved by the Pharmaceutical Sciences Research Center (PSRC)/TUMS Ethics Committee.

The extract was dissolved in normal saline to provide a 20 mg mL-1 solution. Animals from group 1 to 3 received doses expressed on the basis of mg dry extract per kg body mass, namely 50, 100 and 200 mg kg-1 per day of the extract by intragastric intubation for 14 days. Group 4 received α-tocopherol (10 mg kg-1 per day) dissolved in saline by intragastric intubation as a reference antioxidant for comparison. The fifth group of animals was treated as control and received only saline.

Blood collection: About 4 mL of blood was collected through direct heart puncture from anesthetized rats. Intraperitoneal administration of pentobarbital (60 mg kg-1) was used to induce anesthesia in rats. The blood was centrifuged at 2000 g for 10 min to separate serum. The serum was kept at -20°C for subsequent determination of biochemical parameters.

Lipid peroxidation assay: Thiobarbituric Acid Reactive Substances (TBARS) assay is the method of choice for screening and monitoring lipid peroxidation, a major indicator of oxidative stress. To precipitate the serum proteins, 2.5 mL of TCA 20% (m/V) was added into 0.5 mL of sample, which was then centrifuged at 1500 *g for 10 min. Then 2.5 mL of sulfuric acid (0.05 m L-1) and 2 mL TBA (0.2%) was added to the sediment, shaken and incubated for 30 min in a boiling water bath. Then, 4 mL n-butanol was added and the solution was centrifuged, cooled and the supernatant absorption was recorded at 532 nm using a UV-Visible spectrophotometer (Shimadzu, Japan). The calibration curve was obtained using different concentrations of 1,1,3,3-tetramethoxypropane as standard to determine the concentration of TBA-MDA adducts in sample (Satho, 1978).

Total Antioxidant Power (TAP) assay: The total antioxidant capacity of serum was determined by measuring its ability to reduce Fe3+ to Fe2+ by the FRAP (Ferric Reducing Ability of Plasma) test. The FRAP assay measures the change in absorbance at 593 nm owing to the formation of a blue colored Fe (II)-tripyridyltriazine compound from Fe(III) by the action of electron donating antioxidant. The FRAP reagent consists of 300 mmol L-1 acetate buffer pH = 3.6, 10 mmol L-1 TPTZ in 40 mmol L-1 HCl and 20 mmol L-1 FeCl3 . 6H2O in the ratio of 10:1:1. Briefly, 10 μL of serum was added to 300 μL freshly prepared and prewarmed (37°C) FRAP reagent in a test tube and incubated at 37°C for 10 min. The absorbance of the blue colored complex was read against a reagent blank (300 μL FRAP reagent + 10 μL distilled water) at 593 nm. Standard solutions of Fe2+ in the range of 100 to 1000 mmol L-1 were prepared from ferrous sulphate (FeSO4 . 7H2O) in water. The data was expressed as mmol ferric ions reduced to ferrous form per litter (FRAP value) (Benzie and Strain, 1996).

DPPH radical scavenging activity: In this test, serum ability to inhibit DPPH radical was measured (Yokozawa et al., 1998). DPPH is one of the few stable organic nitrogen radicals and has a maximum of absorption at 517 nm 20 μL of serum was added to 3 mL of DPPH solution (0.1 mmol L-1 in ethanol) and the reaction mixture was shaken vigorously. After incubation at room temperature for 10 min, the absorbance of this solution was determined at 517 nm. DPPH solutions without serum and with α-tocopherol were used as the control and reference, respectively.

Determination of LD50: In order to determine the acute toxicity (LD50) of Z. multiflora, doses of 10, 100, 1000 and 2000 mg kg-1 of the day extract were administrated to rats via intragastric tube. The animals were observed for 48 h and mortality was recorded at the end of this period (Hayes, 1988).

Statistical analysis: The values are reported as mean±SEM. One-way ANOVA and Tukey posthoc multicomparison tests were used for data analysis.

RESULTS AND DISCUSSION

The acute toxicity test (LD50) demonstrated that Z. multiflora extract is not lethal up to a dose of 2000 mg kg-1 and no sign of toxicity was observed and thus is considered non-toxic. Z. multiflora extract in doses of 50 and 100 mg kg-1 (p<0.05) increased the serum DPPH scavenging potential when compared to the control as follows: 50 (76.3%), 100 (135%). This value for α-tocopherol (10 mg kg-1) as compared to the control was 116% (p<0.05) (Fig. 1). Z. multiflora extract in the same doses (mg kg-1), significantly (p<0.05) increased the serum TAP when compared to the control as follows: 50 (63%) and 100 (74.5%) (Fig. 2). They also decreased the serum TBARS when compared to the control as follows: 50 (63%) and 100 (60.7%) (Fig. 3). This values for α-tocopherol (10 mg kg-1) in the TAP and TBARS assays were 67.7% (p<0.05) and 69.9% (p<0.05), respectively. Z. multiflora at dose of 200 mg kg-1 per day did not significantly alter the serum DPPH, TAP and TBARS.

Data obtained by DPPH, FRAP and TBARS assays indicate that Z. multiflora effectively inhibits oxidative stress in vivo. The composition of the essential oil of Z. multiflora was studied by GLC, Column Chromatography (CC), NMR and GLC/MS (Ebrahimzadeh et al., 2003; Mohagheghzadeh et al., 2000; Shaiq et al., 1999; Shafiee and Javidnia, 1997). Regarding above studies, Zatarinal, β-sitosterol, stigmasterol, oleanolic acid, betulinic acid, hexadecanoic, luteolin, α-tocophorolquinone and Rosmarinic Acid (RA) were reported as the composition of Z. multiflora essential oil. On the other hand, phytochemical screening of ethanolic extract of the plant supported the presence of monoterpen phenolic compounds in Z. multiflora (Ali et al., 2000; Ramesh et al., 1998; Martinez-Vazquez et al., 1996), mainly carvacrol, p-cymene, thymol, linalool and γ-terpinene (Mohagheghzadeh et al., 2000). RA as a flavonoid from Z. multiflora extract is has significant antioxidant and chelating properties. This positive effect can result in reduction of free radical-induced damages in the body. In supporting this idea, there is evidence that flavonoids have anti phosphodiesterase activity and thus could elevate intracellular levels of cyclic nucleotides (Abdollahi et al., 2003a).

Image for - On the Anti Oxidative Stress Potential of Zataria multiflora Boiss (Avishan shirazi) in Rats
Fig. 1: Antioxidant potential of Z. multiflora in DPPH assay compared to α-tocopherol in rat blood. Data are mean±SEM of 6 animals in each group. *Different from the respective control (p<0.05). α-Tocopherol (α-toco) was administered at a dose of 10 mg kg-1 per day

Image for - On the Anti Oxidative Stress Potential of Zataria multiflora Boiss (Avishan shirazi) in Rats
Fig. 2: Antioxidant potential of Z. multiflora in TAP assay compared to α-tocopherol in rat blood. Data are mean±SEM of 6 animals in each group. *Different from the respective control (p<0.05). α-Tocopherol (α-toco) was administered at a dose of 10 mg kg-1 per day

Image for - On the Anti Oxidative Stress Potential of Zataria multiflora Boiss (Avishan shirazi) in Rats
Fig. 3: Antioxidant potential of Z. multiflora in TBARS assay compared to α-tocopherol in rat blood. Data are mean±SEM of 6 animals in each group. *Different from the respective control (p<0.05). α-Tocopherol (α-toco) was administered at a dose of 10 mg kg-1 per day

Recent studies well indicate that both cAMP and cGMP can diminish oxidative stress in many biological systems and diseases (Aghababaeian et al., 2005; Milani et al., 2005; Radfar et al., 2005; Abdollahi et al., 2003b; Abdollahi et al., 2003c). Therefore, the beneficial effects of Z. multiflora in oxidative stress mainly back to its strong antioxidant potential.

Results indicated that Z. multiflora does not act dose-dependently and dose of 200 mg kg-1 was ineffective. In explanation, it has to be mentioned that the extract has several compounds that some of them at higher doses may produce an unknown condition leading to hiding of antioxidant effects. This will be elucidated by examination of each components of this extract separately.

This preliminary study indicates the interesting antioxidative stress potential of Z. multiflora in vivo that is comparable to that of α-tocopherol and further supports our recent findings about anti colitis effects of Z. multiflora in mice. Further studies are needed to elucidate whether Z. multiflira could be useful in the management of human diseases resulting from oxidative stress.

ACKNOWLEDGMENT

This study was financially supported by a grant from Pharmaceutical Sciences Research Center, TUMS.

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