Procurement of Panax Ginseng:

The roots of Panax ginseng were purchased from the marketplace of Thi Qar city, Iraq.

Preparation of Roots

Washed the ginseng roots with distilled water to eliminate any contaminants. Next, dried the roots slowly at room temperature for 5-7 days in the shade until fully dry.

Cleaning and Grinding

Cleaned the dried roots to remove any remaining debris or dirt. Grind the cleaned roots into a fine powder using a grinder.

Extraction of Leaves:

50g of the powdered ginseng leaves were taken. This powder was measured and stored in a round-bottom flask.

Preparation of Extraction Solvent

The round-bottom flask with powdered ginseng leaves was treated with 200 mL of 70% ethanol.

Extraction Process

The mixture stood for 12 hours in a reflux extractor setup. This facilitated the effective extraction of bioactive compounds from ginseng leaves into the ethanol solvent.

Filtration

The mixture was filtered after the extraction period with Whatman filter paper (No. 31) in order to remove the extracted solution from the solid residue. The process facilitated the acquisition of a clear extract.

Drying of Extract

The filtered extract was then allowed to dry naturally in the shade in a petri dish that had been cleaned beforehand. By this method, the ethanol solvent in the extract evaporated, and what remained was the dried ginseng extract.

Collection and Storage

After drying, the ginseng extract is gathered and kept in a tightly sealed container at 4°C for future use. Adequate storage retains the stability and strength of the extract in the course of time.

Dosage Preparation

The desired dosage for the experiment was determined. In this study, doses of 100 mg/kg and 200 mg/kg of the ginseng extract were utilized.

Usage in Experiment

The prepared ginseng extract was then used in the experiment, as described in the methodology, to evaluate its protective efficacy against chlorine-induced adverse effects in female rabbits [10]. Treatment and Animal Housing Conditions: Thirty-two female rabbits, each weighing approximately (grams), were housed in the departmental Animal House in pairs within plastic cages. The animals were acclimated for 14 days prior to the experiment under controlled conditions (temperature: 25±2 °C, 12-hour light/dark cycle) with free access to food and water. The rabbits were randomly divided into four experimental groups, with eight rabbits per group, as follows:

• Group 1 (Negative Control): Rabbits received chlorine-free drinking water.
• Group 2 (Positive Control): Rabbits received tap water with chlorine estimated at 0.05 ppm/L.
• Group 3: Rabbits received water with chlorine at a concentration of 5 ppm/L.
• Group 4: Rabbits received water with chlorine at 5 ppm/L, along with ethanolic ginseng extract (200 mg/kg body weight), administered one hour after water intake.

According to Gabry et al. (2014), treatment continued for four weeks [11].

Blood samples, traits, and physiological assay:                                                

Hemoglobin (Hb) Quantification: Spectrophotometric determination of the hemoglobin concentration has been achieved through a method described in reference [12]. Packed Cell Volume (PCV): Packed cell volume, or hematocrit value, was determined through a spectrophotometric method following the procedure described in reference [12]. Red Blood Cell (RBC) Count: Red blood cell count was measured through spectrophotometry following the procedure described in reference [12]. Malondialdehyde (MDA) Measurement: The levels of malondialdehyde, the oxidative stress marker, were determined with the help of the Enzyme-Linked Immunosorbent Assay (ELISA) method as described in reference [13]. Transaminase Enzymes (AST, ALT) and Alkaline Phosphatase: Transaminase enzymes (aspartate transaminase – AST, alanine transaminase – ALT) and alkaline phosphatase levels were determined with a Reflotron apparatus and with commercially available kits following the manufacturer’s guidelines, as discussed in [14]. The above analysis methods were selected in order to examine several physiological parameters, such as blood characteristics and oxidative stress markers, in order to examine the effect of ginseng ethanolic extract against the toxic effects of chlorine exposure in female rabbits.                                                                                                                                 

Tissue Samples: liver tissues were collected from rabbits, washed with normal saline, prepared then fixed by using 10% formalin for histological examination.

Chemical implementation:

Chlorinated water at concentration (5 ppm/L) prepared previously before each use for animal groups was treated with chlorinated water [15]. Tap water quality criteria are investigated to assess the liberated chlorine levels (ppm/L) by gathering samples of water and using a pocket digital colorimeter that analyzes water samples for liberated chlorine by following the instructions for use. Likewise, the device is modern technology with high sensitivity, having a range from 0.01 up to 1.00 ppm/L [16]. The Statistical Analysis: Mean values of serum indices were analyzed by one-way analysis of variation (ANOVA), obtained mean differences and standard deviations (Mean ± SD) between treated and control groups, P-values (P<0.05) are considered statistically significant. Table 1 serum MDA and blood parameters in control group and treated groups with Ginseng extract plus chlorine in female rabbits. Data are expressed as mean ± SD (n=8). The different letters refer to significant difference (P< 0.05). 

Results

Figures & Tables

Discussion

As shown in table (1) the results indicate that the exposure to Tap water 2ndgroup and Chlorine  5ppm water 3rd group induced remarkable alteration in blood indices, there is a significant reduction p≤0.05 in the average of RBC, Hb and PCV compared with control 1st group, While the treatment with ethanolic extract ginseng and chlorine resulted in notable increase in  RBC, Hb and PCV values comparison with group exposed to chlorine alone the decline in these components was mostly observed in the second and third groups could attributed to the high portions of chlorine and due to disinfection the water by chlorine and overlapping or suppression the excretion of erythropoietin hormone from the kidney and with a low portions from the liver, which the first and most responsible for synthesis of erythrocytes from bone marrow, may be due to hematotoxicity caused by chlorine treatment which lead to decrease synthesis of red blood cells in bone marrow. Chlorine caused a significant decrease in the levels of RBC count, Hb and PCV, indicating anemia. This anemia may come in part from the effects of free radicals generated by the chlorine on RBC. Several studies have suggested that RBC are particularly vulnerable to oxidative stress because they are exposed to higher concentrations of oxygen and, thus, Hb can easily be oxidized [17]. On the other hand, the activity of ALAD (d-aminolevulinic acid dehydratase which represents a key regulatory enzyme of heme synthesis pathway) is highly sensitive to the presence of stressful chemical;  ALAD is a sulfhydryl-containing and its inhibition can be attributed to the binding of chlorine with sulfhydryl groups), the inhibition of ALAD enzyme and the reduction of Hb and RBC led to decreased heme synthesis and ultimately anemia and therefore lowering in Hb, PCV estimates this agreed and confirmed with [1,18], increased in estimates of blood traits Hb and PCV table (1) in the treated with ethanolic extract ginseng and chlorine group could be attributed to  The extract of P. ginseng possesses antioxidant properties was explained by the presence of ginsenosides which are the main active compounds in ginseng, that reducing oxidative stress [18]. This protective effect may be due to the higher ability to eliminate free radicals and protect the cell membranes from the lipid peroxidation [19], The data illustrated in table (1) revealed remarkable elevation in MDA level to Tap water(2nd group) and Chlorine 5ppmwater (3rd group) compared to normal rabbits, whereas the receiving animals ethanolic extract ginseng and chlorine (group 4th) prominent reduction in MDA level as compared to the chlorine group.

The outcomes of this study (Table 1) revealed significantly (p<0.05) higher in malondialdehyde concentration especially in female rabbit serum that treated Tap water and  Chlorine 5ppmwater compared with another groups could be attributed to the portability of (chlorine) to activating the oxidation and producing the free radicals and increasing fat peroxidation leading to oxidative stress, therefore increasing malondialdehyde concentration as an important production of peroxidation or (chlorine) may be  cause activating to enzymes that in change of unsaturated fatty acid oxidation, which lead to producing hydrogen peroxide and therefore disorder in balance between production of free radicals and antioxidant defenses which caused tissues damage and increased MDA production, this outcomes agreed with [20,21]. The present study showed that the co-administration of ginseng combined with chlorine may have beneficial role and protective effects against chlorine intoxication. That role played by ginsenosides free phenolic fractions have been shown to induce the cytosolic antioxidant enzyme superoxide dismutase by enhanced nuclear protein binding to its gene regulatory sequences. Ginseng contains polyphenols, flavonoids, saponins, these properties effects might be due to the potential antioxidant, free radical scavenging radical mechanism and detoxification effected, these results similar to those [7, 9]. Data obtained from table (2) the outcomes exhibited that the oral exposure of Tap water (2nd group) and Chlorine 5ppmwater (3rd group) induced prominent increase p≤0.05 in levels of liver enzymes (AST and ALT) compared to the corresponding control, whereas the animals received ethanolic extract ginseng and chlorine (group 4th) produced a notable decrease in levels of the above enzymes comparison to chlorine. The progressively increased in the outcomes of (AST and ALT) in second and third groups could be attributed to the oxidative stress in liver and the injury done to tissues that contains those enzymes especially liver cells resulting in production free radicals (ROS) that impact cellular activity especially cell wall functions, as considered chlorine as toxic and harmful impact materials to cell functions from through the elevation in the biomarkers of liver (Transaminases  activity) indicator to cellular damage of liver hence released these enzymes in to circulation, the oxidative stress and hepatic injury induced by this by- products, similar results were agreed and confirmed by some researches [22]. On the other hand, co-administration of ginseng extract combined with chlorine restored the levels of enzymes in the serum of the rabbits as an indication of protective effect of ginseng extract against liver damage induced by chlorine. This is consistent with [26], who stated that ginseng has a productive effect on hepatotoxicity. The hepatoprotective effects of  ginseng extract by diminishing AST and ALT activities in serum due to three of its components (falcarinol, octadecadienoic acid and acetate [23,24].                                                                                

Histopathological Study of Liver

A histopathological assessment in the liver of the control groups were evaluated, consist of a repeated hexagonal unit called hepatic lobules, each lobule is occupied by center vein, hepatocytes and sinusoid are arranged in cords radiating from the central hepatic (Fig. 1A) the inter lobular in the portal vein where the branches of a hepatic artery, hepatic portal vein and bile ducts, blood sinusoids were lined with Kupffer cells and endothelial cells (Fig. 1A).                                                                                              

After 4 weeks from orally given chlorine, the liver of rabbits showed alteration in structure, dilation and congestion of central vein and sinusoid capillaries, moderate enlargement of hepatic cell, which containing to the portal triad to the portal triad could be revealed to the formation of fibrosis, infiltration of mononuclear inflammatory cells in the portal area and central vein, thickening in the lining endothelial layer, some nuclei chromatin was fragmented and cytoplasm contained many vacuoles and necrotic in some area (Fig 1B,1C). When rabbits treated with  ginseng extract (200mg/kg) plus chlorine the liver examination revealed amelioration section from treated chlorine 4 weeks alone clear central and portal veins were filled with erythrocytes, all hepatocytes had eosinophilic cytoplasm, some of these vacuolated cytoplasm and sinusoid were narrowed and irregular shaped (Fig 1D.), This results clarified as the hepatotoxicity was artificially in normal, healthy rabbits with metabolic activity stable, so any effect of chlorine or the indirect influence of liver enzymes on hepatocytes activity. Previous suggest on showed clear histological changes included damaged of the hepatocytes, wider sinusoid spaces and presence of binucleated cells indicating proliferation and regeneration. inflammatory cells in the hepatic tissue and necrosis due to chlorine exposure may suggest that chlorine might interact with proteins and enzymes of liver interstitial tissue interfering with the antioxidant defense mechanism and leading to generation ROS, which in turn may imitate an inflammatory. The ameliorating effects of ginseng which significantly inhibits liver fibrosis by inhibiting activation, proliferation and expression of collagen, transforming growth factor and tissue inhibitor of metalloproteinase-1 in hepatic stellate cell, the major cause of liver fibrosis [20-24]. However, the ginseng extract regulating inducible hepatic enzymes, and might be associated with modulating liver cytochrome P450 activation and protein 152 phosphorylation, similar have also been reported by [6]. 

The authors declare that there is no conflict of interest regarding the publication of this paper.

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