Approximately 150 g of Artemisia Judaica leaf were extracted twice with 70% ethanol using a 2 h. reflux extraction, and the extract was concentrated underreduced pressure. The concentrate was filtered, lyophilised, and subsequently stored at 4°C. The yield of the dried extract from starting crude materials was 16.22% (w/w).

Approximately, 100 g of Panax ginseng root was extracted twice with boiling water, filtered, evaporated in a rotary vacuum evaporator, and freeze-dried. The yield of the dried extract from starting crude materials was 21.07% (w/w). Approximately, 1500 g of Polygonum multiflorum root was extracted twice with 70% ethanol using a 2 h. reflux extraction, and the extract was concentrated under reduced pressure. The concentrate was filtered, lyophilised, and subsequently stored at 4°C. The yield of the dried extract from starting crude materials was 13.73% (w/w).

A total of fifty-four male rats (Sprague-Dawley) of 21 days old were utilised in this study and adapted in cages for one week. Rats were randomly divided into nine groups, each comprising six rats. The study was conducted for 30 days after 30 days age.

Homocysteine (Sigma-Aldrich®) (0.03 μmol/kg of b.w.) was administered subcutaneously, twice a day, from the 30th to the 60th day of the life of rats (Scherer et al., 2011). The rats were decapitated 12 h. after the last Homocysteine injection. The brain hippocampus weighed and kept frozen at -80°C until biochemical analysis. Group I: Controls received the same volume of saline solution (0.5 mL/100 kg of b.w.).

Group II: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/kg of b.w. daily for 30 days.

Group III: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/kg of b.w. + Artemisia judaica extract (50 mg/kg b.w. by oral feeding needle with tuberculin syringe) daily for 30 days.

Group IV: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/kg of b.w. + Panax ginseng extract (50 mg/kg per oral by oral feeding needle with tuberculin syringe) daily for 30 days.

Group V: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/g of b.w. + Polygonum multiflorum extract (400 mg/kg per oral by oral feeding needle with tuberculin syringe) daily for 30 days.

Group VI: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/g of b.w. + Artemisia Judaica + Panax ginseng with the same dose of previous group daily for 30 days.

Group VII: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/g of b.w. + Artemisia Judaica + Polygonum multiflorum with the same dose of previous group daily for 30 days.

Group VIII: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/kg of bodyweight + Panax ginseng + Polygonum multiflorum with the same dose of previous group daily for 30 days.

Group IX: served as hyperhomocysteinaemia and received homocysteine 0.03 μmol/kg of b.w. + Artemisia Judaica + Panax ginseng + Polygonum multiflorum with the same dose of the previous group daily for 30 days.

Preparation of the standard solution

MDA standard was prepared by dissolving 25 μL 1,1,3,3-tetraethoxypropane (TEP) in 100 mL of water to give a 1 mM stock solution. Working standard was prepared by hydrolysis of 1 mL TEP stock solution in 50 mL 1% sulphuric acid and incubation for 2 h at room temperature. The resulting MDA standard of 20 nmol/mL was further diluted with 1% sulphuric acid to yield the final concentration of 1.25 nmol/mL to get the standard for the estimation of total MDA Karatepe, 2004.

MDA activity was determined after extraction using following protocol: The samples were analysed on an Agilent HP 1100 series HPLC apparatus (USA). The analytical column Supelcosil C18 (5 µm particle and 80 Ao pore size) (250 x 4.6 ID). Mobile phase consists of 30 mmol KH2PO4 and methanol (65%-35%, H3PO4 by pH 4), and the mobile phase at a 1.5 mL/min. flow rate, wavelength 250 nm according to the method of Karatas et al. 2002 Karatas et al., 2002. Data Presented in Figure 3 . The resulting chromatogram identified MDA position and concentration from the sample as compared to that of the standard as previously reported in monoamine content calculation.

Nitrites and nitrate was determined according to the method of Papadoyannis, Samanidou, and Nitsos 1999 by HPLC Papadoyannis et al., 1999.

Preparation of the standard solution

Sodium nitrite and sodium nitrate used for the reference standard preparation with stock concentration 1 mg/mL. A standard mixture of nitrite and nitrate was used to determine the retention times and separation of the peaks. Nitrite and nitrate concentrations were equal in the mixture solution.

NO was determined after extraction using following protocol: The samples were analysed on an Agilent HP 1100 series HPLC apparatus (USA). The analytical column was anion exchange PRP-X100 Hamilton, 150 x 4.1 mm, 10 μm. The mobile phase was a mixture of 0.1 M NaCl - methanol, at a volume ratio 45:55. The flow rate of 2 mL/min., wavelength adjusted to 230 nm. Data Presented in Figure 4 . The resulting chromatogram identified each of nitrite and nitrate" positions and concentration from the sample as compared to that of the standards.

The activity of SOD was determined according to the method of Marklund and Marklund 1974 Marklund and Marklund, 1974. SOD activity was determined after extraction using following protocol: In a spectrophotometer Micro Cuvette, 0.985 of tris-HCL buffer was added to 0.010 mL of Pyrogallol solution and 0.005 mL of double distilled water. Absorbance was measured at 420 nm immediately and after one minute, the resultant A was considered as the experimental blank. The same procedure was carried out by the use of 0.005 mL of the prepared SOD containing solutions (standard) or sample instead of double distilled water.

Catalase (CAT) activity was measured by the method of Del Maestro and McDonald 1987. The method is based on the rate of H₂O₂ degradation by the action of CAT contained in the examined samples followed spectrophotometrically at 230 nm in 5 mM EDTA, 1 M Tris-HCl solution, pH 8.0.

The procedure used for the determination of acetylcholinesterase activity in the brain hippocampus samples of rabbit is a modification of Ellman et al. 1961 Ellman et al., 1961 method as described by Gorun et al. 1978 Gorun et al., 1978.

AChE activity was determined after extraction using following protocol: The brain hippocampus tissue samples were weighed and homogenised in a 20-mmol-phosphate buffer, pH 7.6 (5 % w/v). The following reagents were pipettes in a cuvette: 0.14-mL phosphate buffer 20 mmol (pH 7.6), 0.05 mL of 5-mmol acetylthiocholine iodide and 0.01 mL of tissue homogenate or serum. After 10 min. of incubation at 38°C, the reaction was stopped with 1.8 mL of DTNB – phosphate ethanol reagent. The colour was read immediately at 412 nm using Shimadzu spectrophotometer UV –1601. Omitting the enzyme from the incubation mixture made the control samples. After addition of the colour reagent, appropriate amount of tissue homogenates or serum was added to the control. The cholinesterase activity was determined as µmol SH from a standard curve.

IL-6 levels in the rat brain were estimated using a rat-specific immunoassay kit (Rat IL-6 ELISA) from Glory Science (Del Rio, Texas, USA) according to the manufacturer's protocol. The intensity of the coloured product was directly proportional to the concentration of rat IL-6, as evaluated using a micro plate reader (Biotech ELx800; Biotech Instruments) set at 450 nm. The sample concentration was determined against a standard curve and is expressed in nanograms of IL-6 per gram of brain tissue.

BDNF levels were estimated using a rat-specific immunoassay kit (Rat BDNF ELISA) from Glory Science, according to the manufacturer's protocol. The intensity of the coloured product was directly proportional to the concentration of rat BDNF, as determined using a micro plate reader (Biotech ELx800) set at 450 nm. The sample concentration was determined against a standard curve and is expressed in nanograms.

Comet assay of DNA was estimated according to the classic alkaline single-cell electrophoresis protocol Xu et al., 2013. Samples were stained with SYBR Green I (Sigma-Aldrich, St Louis, MO) and analysed by Comet Score 1.5 software. Percent of DNA in comet tails was considered as the marker of genotoxic effect.

The values were expressed as the mean ± SE for the 6 male rats in each group. Differences between groups were assessed by one way analysis of variance (ANOVA) using SAS (2004) software for Windows (version 13.0). Statistical analysis of the obtained data was performed using the general linear model (GLM). Significant differences among means were evaluated using Duncan’s (1955) Duncan, 1955. Multiple Range Test.

The following linear model was applied: Yij = μ + αi + ξij, Yij = Observation measured, Μ = Overall mean, αi = Effect of treatment. ξij = Experimental error assumed to be randomly distributed ( σ2 = 0 ).

Data presented in Figure 1 , Figure 2 , Figure 3 and Figure 4 records the effect of homocysteine oral administration on antioxidant parameter (CAT, MDA, NO and SOD) of hippocampus brain area at the end of experiment. Homocysteine induces a significant decrease (p<0.05) in the hippocampus CAT and SOD and significant increase (p<0.05) of NO and MDA compared to control group. On the other hand, PG, PM, AJ+PG, PG+PM and AJ+PG+PM showed significant differences (p<0.05) against homocysteine group and rounded than the control group. Also, AJ+PM showed ameliorative response and nearly rounded to control for CAT, NO, MDA and SOD compared with homocysteine group but AJ only didn’t show any improvement than homocysteine group.

As showed in Figure 5 , Figure 6 , and Figure 7 , records the effect of homocysteine oral administration on IL-6, BDNF and SOD of hippocampus brain area at the end of experiment. Homocysteine caused elevation in inflammatory markers (IL-6), and AChE of hippocampus brain area. Homocysteine also induces a significant decrease (p<0.05) in the hippocampus neurotrophic factor (BDNF) as compared to control group. On the other hand, all treated group except AJ only showed significant (p<0.05) ameliorative effect of SOD, IL-6, and BDNF against homocysteine group and rounded than the control group, but AJ only didn’t show any improvement than homocysteine group at the same parameters.

Concerning the brain genotoxic potential of homocysteine using the comet essay, there was a significant increase in the tail length of DNA, tail intensity (DNA%) and tail moment in the homocysteine-treated rats compared to the control. On the other hand, the PG and PM treated groups significantly decreased DNA tail length, intensity and moment as compared to the homocysteine-treated rats, while the AJ treated groups showed no pronounced effect ( Table 1 and Figure 8 ).