Aerial parts of Ajuga chamaecistus Ging. ssp. tomentella (Boiss.) Rech. f. were collected from east parts of Tehran, Iran, in June 2014, dried in suitable condition and then verified by Prof. G. Amin. A voucher specimen (THE-6697) was placed in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Hydroalcoholic extraction

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The well-dried and grounded (chopped) aerial parts of Ajuga chamaecistus Ging. ssp. tomentella (Boiss.) Rech. f. (195 gr) were extracted with 80 % ethanol three times (3×1200 ml) at room temperature. The solvent was evaporated on a rotary evaporator and in a vacuum oven to give a dark brown extract (42 g).

Fractionation of total extract

The extract (32 g) was suspended in 200 ml of 80% ethanol using an ultrasonic machine and partitioned between 80% ethanol, chloroform, and ethyl acetate. Removal of the solvents with a rotary evaporator resulted in the production of chloroform, ethyl acetate and residual hydro alcoholic fractions.

Free radical scavenging activity

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was done based on the method of (22) with some modifications according to the previous study (23). 

Animals

Male albino rat weighing 250–300 g were obtained from faculty of pharmacy, Tehran University of Medical Sciences and housed in groups of 6 with a 12h light–dark cycle and constant temperature (22°C). Animals were allowed to adapt to the laboratory for 30 min before the tests began. This study was approved by the ethics committee of the Pharmaceutical Science Research Center of TUMS

(Code no: IR.TUMS.PSRC.REC.1395.447 )

Drugs and Chemicals

Silymarin was purchased from Sigma (chemical St. Louis, MO, The USA), Carbon tetrachloride, was purchased from Merck (Germany). The assay Elisa kits were purchased from Zellbio© GmbH (Germany) Padginteb co.

Toxicity study

Carbon tetrachloride-induced liver damage in rats

Healthy albino rats were divided into 7 groups each containing 6 animals. Group 1 (blank) received normal saline. Group 2 (Control negative) received CCl4 (1 ml/kg body weight, i.p.) only for a single dose. Group 3-7 received ethyl acetate fraction dissolved in normal saline at three doses; 25, 50 and 100 mg/kg p.o, total extract (100 mg/kg p.o), standard drug Silymarin (100 mg/kg p.o) once in a day and CCl4 as mentioned above. Treatment duration was 7 days. Animals were sacrificed 24 h after the last administration.

Biochemical studies

Blood samples were taken from the left ventricle of the animals and after clotting at room temperature, the sera were isolated by centrifugation at 2500 rpm for 15 minutes, and until various biochemical parameters AST (Aspartate transaminase), ALT (Alanine transaminase), ALP (Alkaline phosphatase), LDH (Lactate dehydrogenase), and total protein content in the serum of CCl4-induced liver damage in rats, were stored at -80 ° C.

After collection of blood samples the rats were sacrificed and their livers excised, rinsed in ice-cold normal saline and were kept in falcon 50 ml and deep froze in -800C for the next experiments on homogenized liver tissue. Analysis of parameters such as Glutathione (GSH), superoxide dismutase (SOD) and Malondialdehyde (MDA) were measured spectrophotometrically in liver tissue samples using commercially available ZellBio kits (Germany).

Histopathologic examination

Samples taken from the liver were stable in 10% buffered formalin for 48 hours. The various stages of preparing tissue samples and paraffin blocks were made in the usual way. Finally, tissues with the thickness of 5?m sectioned by microtome cut and stained with hematoxylin-eosin and studied using light microscopy and were analyzed and photographed. In histopathology, liver sections were examined and other details were given ranks on their healing patent. The factors that were used for evaluation include bleeding, congestion, fibrin formation, existence and number of cells dilative single-core or multi-core formation of liver tissue (regeneration), various degrees of damage to liver cells, fibrosis and necrosis.

Statistical analysis:

 The statistical analysis was carried out by One-way Analysis of Variance (ANOVA) using sigma plot version 12.0. P values