In vivo antioxidant and anti-inflammatory effects of Balanites aegyptiaca (l) del (Balanitaceae) galls and leaves

Balanites aegyptiaca is a plant with extensive medicinal properties. The present study aims to evaluate the anti-inflammatory effect of B. aegyptiaca trough the turpentine-induced inflammation. The aqueous acetone extracts of galls and leaves have been used to test the anti-inflammatory effects on adult male Wistar-Bratislava albino rats. Total leucocyte count was performed with an optical microscope (Olympus), using a Burcker-Turk counting-chamber. The nitric oxides synthesis (NOx), the total oxidative status (TOS) and total antioxidant response (TAR) of the serum were measured using a colorimetric assay. The results have showed that all the extracts reduced significantly (p<0.0001) the total leukocytes and total nitrites and nitrates levels in the rats serum. The oxidative stress evaluation showed that the treatment with any dose of B. aegyptiaca extracts was significantly decreased (p<0.0001) the total oxidative status. TAR increasing did correlate to TOS decrease by comparison with the inflammation group. In general, an interesting anti-inflammatory effect was found in this study with the greatest activity found in the gall extracts. B. aegyptiaca could then be a potential source of natural antioxidants and anti-inflammatories.


Introduction
Balanites aegyptiaca (B. aegyptiaca), also known as 'Desert date' in English is one of the most common but neglected wild plant species of the dry land areas of Africa and South Asia [1]. Different parts of this plant are traditional used in several African folk medicines [1][2][3][4].
B. aegyptiaca is well known for its multiple pharmacological properties. Literature has revealed that the barks are anthelmintic [5], the leaves and galls have antibacterial properties [6] andthe fruits are effective against Aedes aegypti larvae [7,8].
The antioxidant potentialities of the barks, the galls and the leaves [2,4,9], analgesic activity of the leaves [10] and the anti-inflammatory activity of the galls and leaves [6,11] have been demonstrated in vitro. It is also proved that the galls and the leaves of this plant inhibit xanthine oxidase and acetylcholinesterase [4]. Anti-inflammatory activities of aerial part B. aegyptiaca have been demonstrated using the carrageenin-induced edema in the rat method [12].
To complete these pharmacological data we undertook to evaluate the anti-inflammatory effect of B. aegyptiaca trough the turpentine-induced inflammation. For that the aqueous acetone extracts of its galls and leaves have been used to test the anti-inflammatory effects on adult male Wistar-Bratislava albino rats. www.ssjournals.com

Plant materials
The galls and the leaves samples and the botanical identification of B. aegyptiaca have been well documented in the previous article [6].

Extraction
The aqueous acetone extracts were obtained using 50 g of dried and powdered of each sample [6].

Experimental design
The Wistar-Bratislava albino rats weighing 200-250 used for the experiments were bred in the Animal Facility of Iuliu Hatieganu University of Medicine and Pharmacy. The study protocol has been described by [13]. Turpentine oil (6 mL/kg BW) was used to induce the inflammation by i.m. injection. The anti-inflammatory control groups treated with meloxicam (3.2 mg/kg BW) and diclofenac (20mg/kg BW) by i.p. injection [13,14]. One group was tested by i.p. injection with galls sample (250 mg/kg BW) and three groups with leaves samples at a dose of 250, 500 and 750 mg/kg BW, respectively.

Evaluation of anti-inflammatory effect
Different methods were used to assess the antiinflammatory activity of the galls and the leaves from B. aegyptiaca. Total leucocyte count was performed with an optical microscope (Olympus), using a Burcker-Turk counting-chamber and the Griess reaction was used to indirectly determine NO synthesis (NOx) of the serum using a colorimetric assay [13].

Evaluation of antioxidant effect
The total oxidative status (TOS) and total antioxidant response (TAR) of the serum were measured using a colorimetric assay [13]. The oxidative stress index (OSI) expressed in arbitrary unit is an indicator of the degree of oxidative stress and is calculated by the ratio of the TOS to TAR.

Statistical analysis
The data were expressed as Mean ± Standard deviation (SD) of three determinations. Statistical analysis (ANOVA with a statistical significance level set at p<0.0001 and linear regression) was carried out with XLSTAT 7.1.

Anti-inflammatory activity
The effects of the B. aegyptiaca gall and leaf extracts on the total leukocyte count are presented in the figure 1 (A). The inflammation increased significantly the total leukocyte count compared to the control (p<0.0001). Diclofenachas prevented considerably the inflammation effect (p < 0.0001). The test samples were presented good anti-inflammatory activities. The gall extracts (250 mg/kg BW) and leaf extracts (500 mg/kg BW and 750 mg/kg BW) reduced more the total leukocyte count levels than the diclofenac (p<0.0001). The similar effect (p>0.0001) was found with the diclofenac and the leaf extracts (250 mg/kg BW). The gall extracts decreased more the total leukocytes than leaf extracts at the dose 250 mg/kg BW.
The NOx in the turpentine oil-treated group serum was highest (p<0.0001) compared to serum from the control rats ( figure 1 (B)). NOx production was decreased considerably (p<0.0001) by diclofenac and meloxicam. It was found that the gall and leaf extracts were also reduced NOx production in the inflamed rat serums. The NOx reduction by the leaf extracts was dose-dependent, with the highest dose showing the best inhibition effect (p<0.0001). The gall extracts decreased twice more the NOx than the leaf extracts at a dose of 250 mg/kg BW.

The evaluation of oxidative stress
The serum TOS level was increased (p<0.0001) inthe turpentine-induced inflammation group compared to the control group ( figure 2 (A)). Importantly, diclofenac and meloxicam were reduced (p<0.0001) the TOS level. Meloxicam has reduced more TOS levels than diclofenac (p<0.0001) and similar to control group (p>0.0001). The treatment with any dose of B. aegyptiaca gall and leaf extracts was significantly decreased the TOS levels (p<0.0001). Similar TOS reducing levels was found with the leaves extracts (250 mg/kg BW) and meloxicam (p>0.0001). Moreover the gall extracts (250 mg/kg BW) were more potent reducing than meloxicam (p<0.0001). These results also showed that the gall extracts are better than leaf extracts in the total oxidative stress reduction.
Turpentine-induced inflammation reduced the TAR (p<0.0001) compared to the control while the meloxicam treatment significantly increased the TAR ( figure 2 (B)). Our results demonstrated that this decrease in the TAR has been prevented by the treatment of B. aegyptiaca gall and leaf extracts (p<0.0001). Diclofenac didn't prevent the turpentine-induced inflammation (p>0.0001). TAR increasing did correlate to TOS decrease by comparison with the inflammation group.
In the inflammation group, the OSI was significantly elevated (p<0.0001) compared to control group ( figure 2 (c)). The treatments with meloxicam and leaf extracts (250 mg/kg BW) have presented the same OSI (p>0.0001). The gall (250 mg/kg BW) and leaf (500 and 750 mg/kg BW) extracts were most effective (p<0.0001).

Discussion
Inflammation is appreciated as general, nonspecific response to tissue injury in many diseases [15]. Neutrophils, macrophages, endothelial, and other cells at the site of inflammation may produce reactive oxygen species (ROS) and reactive nitrogen species, which play a modulating role in the inflammatory response [16].
This study showed that B. aegyptiaca gall and leaf extracts proved to have anti-inflammatory effect through a mechanism involving the inhibition of total leucocytes and a reduction in oxidative stress.The total leucocytes count results obtained shown that B aegyptiaca gall and leaf extracts have decreased to half the turpentine-induced inflammation in the rats and have been more effective than the diclofenac used as standard molecule. As for NOx results, all the extracts reduced the nitric oxides levels in inflamed rats. Concerning the evaluation of oxidative stress through TOS, TAR and OSI measurements, interesting results were also found. The gall and leaf extracts prevented considerably the turpentine-induced inflammation by reducing the levels of TOS and increasing the TAR in the serum. OSI assesses the global oxidant/antioxidant balance in the living organisms [17]. The TOS decrease and TAR increase after B. aegyptiacagall and leaf extracts treatments reduced more the OSI than the meloxicam treatment. www.ssjournals.com The antioxidant and anti-inflammatory properties of B. aegyptiaca extracts have been already study in vivo. The fruits extract exhibited a good total antioxidant capacity [18]. The methanol and butanol extracts and isolated saponins from bark [2] as well as the ethanolic and petroleum ether extracts of dried aerial parts [10] reduced significantly the rat paw edema induced by carrageenan. Indeed, turpentine oil is like carrageenan a non-antigenic inflammatory stimulus [13]. This activity could be due to capacity of B. aegyptiaca extracts to reduce total leukocytes and NOx levels in the rat serum as demonstrated in ours study. The inhibition of NOx synthesis is an important mechanism of anti-inflammatory effect [13,19]. The latest explanation of antioxidant therapy failure comes from the finding that antioxidants do not inhibit oxidative stress and the associated inflammation at the same time [17]. In this study, the OSI has been well correlated to NOx and leukocytes count levels after treatment of B. aegyptiaca gall and leaf extracts proving that oxidative stress and inflammation are interlinked processes.
Chronic inflammation can last for several months and even years and can eventually cause several diseases such as rheumatoid arthritis, atherosclerosis, asthma, heart disease, ulcerative colitis, and some cancers [26]. B. aegyptiaca gall and leaf extracts could then be exploited for the chronic inflammation disorders.

Conclusion
Aqueous acetone extracts of Balanites aegyptiaca galls and leaves were used to evaluate their effects against the rat turpentine-induced inflammation. The results obtained showed that all the extracts prevented the inflammation by reducing the total leukocytes count and the NOx. The TOS decrease and TAR increase after treatments reduced more the OSI than the meloxicam a standard antiinflammatory molecule. B. aegyptiaca could then be a potential source of natural antioxidants and antiinflammatories that could have great importance as a therapeutic agent in the prevention of inflammation, cancer, aging, rheumatism and neurodegenerative diseases.