Pistacia chinensis: A Potent Ameliorator of CCl4 Induced Lung and Thyroid Toxicity in Rat Model

In the current study protective effect of ethanol extract of Pistacia chinensis bark (PCEB) was investigated in rats against CCl4 induced lung and thyroid injuries. PCEB dose dependently inhibited the rise of thiobarbituric acid-reactive substances, hydrogen peroxide, nitrite, and protein content and restored the levels of antioxidant enzymes, that is, catalase, peroxidase, superoxide dismutase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase, γ-glutamyl transpeptidase, and quinone reductase in both lung and thyroid tissues of CCl4 treated rats. Decrease in number of leukocytes, neutrophils, and hemoglobin and T3 and T4 content as well as increase in monocytes, eosinophils, and lymphocytes count with CCl4 were restored to normal level with PCEB treatment. Histological study of CCl4 treated rats showed various lung injuries like rupture of alveolar walls and bronchioles, aggregation of fibroblasts, and disorganized Clara cells. Similarly, histology of CCl4 treated thyroid tissues displayed damaged thyroid follicles, hypertrophy, and colloidal depletion. However, PCEB exhibited protective behaviour for lungs and thyroid, with improved histological structure in a dose dependant manner. Presence of three known phenolic compounds, that is, rutin, tannin, and gallic acid, and three unknown compounds was verified in thin layer chromatographic assessment of PCEB. In conclusion, P. chinensis exhibited antioxidant activity by the presence of free radical quenching constituents.


Introduction
Free radicals are the major by-products produced by the cells of aerobic organisms and can start the autocatalytic reactions and spread the chain of damage by reacting with molecules and converting them into free radicals. Free radicals are mainly produced from these sources in the body: ubisemiquinone in the mitochondrial membrane, xanthine oxidase of endothelial cells, and myeloperoxidase and NADPH oxidase of neutrophils. But, xanthine oxidase and respiratory chain of mitochondria are the major sources of reactive oxygen species (ROS) [1]. Free radicals are extremely unstable and become stable by pairing an outer shell electron with biomolecules, for example, lipids, proteins, and DNA. At high concentrations, free radicals can cause damage to various cell structures, comprising proteins and nucleic acids, together with lipid peroxidation [2,3]. These injuries and damages are the major contributions towards aging, cardiovascular diseases, atherosclerosis, cancer, and inflammatory diseases [4]. Living cells contain a defensive system of antioxidants against the ROS which avoids their unnecessary production and inactivate them. Various antioxidants have been reported to protect the body from the damaging effects of oxidative stress. Nowadays, research has been increased in the area of finding novel natural antioxidants due to low side effects in comparison to synthetic antioxidants [5].
Carbon tetrachloride (CCl 4 ) is a commonly used xenobiotic to induce toxicity in animal models. It is well known as a hepatotoxin [6], nephrotoxin [7], and pulmonary toxin [8] and also produces injuries in other organs. It has been demonstrated that oxidative stress caused by CCl 4 is due to production of reactive free radicals such as hydrogen 2.2. Plant Collection. The barks of P. chinensis were collected from the campus of Quaid-i-Azam University, Islamabad, Pakistan, and were recognized by their local names and then confirmed by Prof. Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. Voucher specimen with Accession Number 27840 was deposited at the Herbarium, Quaid-i-Azam University, Islamabad. Plant sample (2 kg) was washed with distilled water and dried at room temperature in a shade for more than two weeks and grinded using electrical grinder.

Preparation of Extract.
For the preparation of ethanol extract of P. chinensis 4 litres of 80% ethanol was added to the 2 kg of powdered bark and sonicated for 2 h at 45 ∘ C. The extract was filtered by using Whatman filter paper number 45 after a week. Rotary vacuum evaporator was used to evaporate the solvent from filtrate and to obtain ethanol extract of P. chinensis (PCEB).

Total Phenolic Content Estimation.
Spectrophotometric method [13] was used with minor modifications to determine total phenolic content. In short, 200 L of the sample (1 mg/mL) was mixed with 1 mL of the 1 : 10 Folin-Ciocalteu's reagent. To the mixture 2.5 mL of 7% Na 2 CO 3 was added after 5 min. The mixture was incubated at 23 ∘ C in the dark for 90 min. Absorbance was recorded at 765 nm. Total phenolic content was calculated from calibration curve of gallic acid. Estimation of total phenolic content was recorded in triplicate and presented as mg of gallic acid equivalents (GAE) per g of dry sample.

Total Flavonoid Content Estimation.
In a test tube, 0.3 mL of sample (1 mg/mL), 3.4 mL of 30% methanol, 0.15 mL of 0.5 M NaNO 2 , and 0.1 mL of 0.3 M AlCl 3 ⋅6H 2 O were thoroughly mixed. After 5 min, 1 mL of 1 M NaOH was added and mixed well. Absorbance was measured at 506 nm against the reagent blank. Total flavonoid content was estimated by using a calibration curve of rutin and expressed as mg rutin equivalents per g of dry sample [14].

Thin Layer Chromatography.
Thin layer chromatography was carried out for the presence of phenolics in the extract. Extract (50 mg) was dissolved in 1 mL of methanol. Precoated TLC plates of 20 × 20 cm and 0.25 mm thick were used in the experiment. For the activation of silica, plates were heated at 100 ∘ C for 45 min by placing them in oven. On the lower surface of plate, a line of 1 cm was drawn and 6 L of extract was plotted. After drying, the plates were run in the mixture of n-butanol, acetic acid, and water in a ratio of 4 : 1 : 5. Plates were removed when solvent reached the end of TLC. Solvent front was marked and plates were sprayed with ethanolic 2aminoethyle diphenyl borinate solution (1%) followed by 5% solution of ethanolic polyethylene glycol. Flavonoids were detected by observing its characteristic colors under UV light at 360 nm. Different bands were observed and their values were calculated by the following formula: value = Distance covered by band Distance covered by solvent . (1)

In Vivo Studies.
For in vivo studies CCl 4 was used as a source of free radicals to produce toxicity in lungs and thyroid gland of rat model. Lungs and thyroid tissues were investigated at biochemical and histological level.

Experimental Design.
Shah et al. [14] protocol was followed for the experimental design. Male Sprague Dawley rats (160-210 g) of seven weeks old were used as animal model in this study. They were maintained in cages at room temperature of 25 ± 3 ∘ C with a 12 h light/dark cycle and free access to water and feed. The study protocol was approved (No. 0246) by the ethical committee of Quaid-i-Azam University, Islamabad, Pakistan, for laboratory animal care and experimentation. Forty-two male rats were randomly distributed into 7 groups (6 rats/group). Group I remained untreated. Group II was treated with 30% CCl 4 in olive oil (1 mL/kg b.w.), intraperitoneally. Groups III, IV, and V were orally given silymarin (100 mg/kg b.w.), PCEB (200 mg/kg rat b.w.), and PCEB (400 mg/kg b.w.), respectively, after one hour of 30% CCl 4 injection. Groups VI and VII were given only PCEB (400 mg/kg b.w.) and olive oil (1 mL/kg b.w.), respectively, by oral route. Olive oil was used as vehicle for extract and silymarin treatment. Experiment was comprised of 60 days and treatments were given on alternative day.

Animal Dissection.
After last treatment, rats were unfed for 24 h. Chloroform was used to anesthetize animals and then dissected the animals from ventral side of the body. Blood was collected by piercing heart. Blood was collected in two types of tubes; that is, for serum analysis it was stored in small falcon tubes which was then centrifuged to obtain serum; the rest of the blood was collected in EDTA containing tubes for whole blood analysis. From the dissected animal, lungs and thyroid glands were removed and placed in saline solution. For histology, half of the lung and thyroid was stored in 10% formalin solution while the half was stored in liquid Nitrogen at −70 ∘ C for antioxidant enzymes and tissue stress marker examination.

Serum Analysis.
After dissection of animals, blood was collected in falcon tubes and centrifuged at 4000 rpm for 20 min at 4 ∘ C, after 30 minutes, to collect the serum samples. Total protein, albumin, and globulin, in serum samples, were estimated with the help of AMP diagnostics company kits, while T 3 and T 4 were analysed by using MicroLISA kits.
(1) Total Protein Estimation. Estimation of total protein is based on Biuret reaction which measures the amount of colored complex formed when protein reacts with alkaline solution in the presence of copper salt. An aliquot of 10 L of serum sample was added to 1 mL reagent (NaOH 53 mM, CuSO 4 6 mM, potassium iodide 6 mM, potassium sodium tartarate 21 mM). After incubation of 10 min at 37 ∘ C, optical density was measured at 550 nm spectrophotometrically. Distilled water plus reagent was used as a blank and albumin was used as a standard. For the calculation of total protein the following formula was used: where is concentration of standard (mg/dL).
(2) Albumin Estimation. Albumin estimation is based on the principle that in acidic medium (pH 3.8) albumin reacts with BCG (bromocresol green) to produce a colored complex, which is measured spectrophotometrically. An aliquot of 10 L of serum sample was added to 1.5 mL reagent (bromocresol green 0.25 mM, citrate buffer 100 mM, Triton X-100 10 g/L). Optical density was measured at 628 nm spectrophotometrically. Distilled water plus reagent was used as a blank and albumin (40 g/L) was used as a standard. For the calculation of albumin the following formula is used: where is concentration of standard (mg/dL).
(3) Globulin Estimation. The following formula was used for globulin estimation in serum and urine samples:

Triiodothyronine (T 3 ) or Thyroxine (T 4 ) Estimation.
For the estimation of triiodothyronine (T 3 ) or thyroxine (T 4 ) specific amount of anti-T 3 or anti-T 4 antibody is coated on the microtiter wells. A measured amount of serum and a constant amount of T 3 or T 4 conjugated with horseradish peroxidase are added to the microtiter wells. During incubation, T 3 or T 4 present in the serum and conjugated T 3 or T 4 compete for the limited binding sites on the anti-T 3 or anti-T 4 antibody. After 60 min incubation, the wells are washed to remove unbound T 3 or T 4 conjugate. A solution of TMB substrate is added which results in formation of blue color. The color development is stopped by addition of 2 N HCl and absorbance is measured spectrophotometrically at 450 nm. The intensity of color is directly proportional to the amount of enzyme present and inversely related to the unlabeled T 3 or T 4 in the sample. Series of standards are run in the same way to quantify the concentration of T 3 or T 4 in sample. Standards, samples, and controls (50 L) are added into appropriate wells and 100 L of enzyme conjugate is also added. After mixing the microtiter plate is incubated at room temperature for 60 minutes. After incubation wells are washed 5 times with 1x washing buffer and 100 L of TMB substrate is added in each well. After 20 min incubation 100 L of stop solution is added and optical density is measured at 450 nm.

Haematological
Analysis. Anticoagulated blood samples were used for the determination of total leukocyte count, neutrophils, lymphocytes, eosinophils, monocytes, and haemoglobin level by using cell DYN ruby automated 5part hematology analyzer (Abbott diagnostics, Germany).

Antioxidant Enzymes Estimation.
Tissue homogenate was prepared by homogenizing 100 mg of lung and thyroid tissue in 1 mL of 100 mM potassium phosphate buffer containing 1 mM EDTA at pH 7.4. Supernatant was collected in clean falcon tubes after centrifugation for 30 min at 12000 ×g at 4 ∘ C and was used for further analysis. The following assays were carried out to analyze the pharmacological activity against the toxicity induced with CCl 4 in rats.
(1) Catalase (CAT) Activity. Measurement of CAT activity is based on the methodology of Shah et al. [14], which relies on decomposition of H 2 O 2 . An aliquot of 25 L of tissue homogenate was added to 100 L of 10 mM H 2 O 2 and 625 L of 5 mM EDTA buffer (pH 8.0). The disappearance of H 2 O 2 in the reaction mixture by catalase was measured spectrophotometrically at 230 nm. CAT activity was expressed as U/mg protein.
(2) Peroxidase (POD) Activity. POD activity was measured spectrophotometrically by the method of Khan and Ahmed [15]. An aliquot of 25 L of tissue homogenate was added to 1 mL of pyrogallol solution and 125 L of H 2 O 2 was added and mixed. After one minute change in absorbance was measured at 430 nm. Change in absorbance/min is defined as one unit POD activity.
(3) Super Oxide Dismutase (SOD) Activity. Measurement of SOD activity was carried out according to Kakkar et al. [16] by using sodium pyrophosphate buffer and phenazine methosulphate. Tissue homogenate was centrifuged for 10 min at 1500 ×g followed by 10000 ×g for 15 min. Supernatant was collected and used to determine SOD activity. An aliquot containing 150 L of supernatant was added to 600 L of 0.052 mM sodium pyrophosphate buffer (pH 7.0) and 50 L of 186 M phenazine methosulphate. 100 L of 780 M NADH was added to start enzymatic reaction. Addition of 500 L of glacial acetic acid after 1 minute stops the reaction. Optical density was determined at 560 nm to measure the color intensity. Results are expressed in units/mg protein.

(4) Gamma Glutamyl Transpeptidase ( -GT) Activity.
To determine the activity of -GT, methodology of Fossati and Prencipe [17] was followed. Two reagent solutions were prepared in Tris buffer (50 mM/L, pH 8 at 37 ∘ C). Reagent 1 contains, per litre of Tris, 116 mM of glycyl glycine, 3.6 mM of 2, 5-dimethylphenol, 12 kU of ascorbate oxidase, and 5 g of Triton X-100 surfactant. Reagent 2 contains 20 mM of glycylglycine and 24 mM of -glu-DBHA per litre of Tris buffer. An aliquot of 50 L of tissue homogenate was added to mixture containing 1 mL of reagent 1 and 200 L of reagent 2. Enzyme activity was measured at 37 ∘ C by recording the absorbance changes for 180 s after a 2 min lag phase at 620 nm.
(5) Reduced Glutathione (GSH) Activity. GSH activity was measured according to method of Jollow et al. [18]. 500 L of tissue homogenate was precipitated by addition of 500 L of sulfosalicylic acid (4%). After 1 hour incubation at 4 ∘ C, samples were centrifuged for 20 min at 1200 ×g. 33 L of supernatant was collected and added to aliquots containing 900 L of 0.1 M potassium phosphate buffer (pH 7.4) and 66 L of 100 mM DTNB. Reduced glutathione reacts with DTNB and forms a yellow colored complex. Absorption was measured at 412 nm. M GSH/g tissue represents GSH activity.
(6) Glutathione Peroxidase (GSH-Px) Activity. Glutathione peroxidase (GSH-Px) was determined colorimetrically according to the method of Ozdemir et al. [19] using NADPHcoupled reduction of GSSG catalysed by Glutathione reductase which can be measured at 340 nm. Using molar coefficient of 6.23 × 10 3 /M/cm, GSH-Px activity was determined as amount of NADPH oxidized/min/mg protein.
(7) Quinone Reductase (QR) Activity. Quinone reductase activity is measured by a method of Benson et al. [20], which is based on reduction of dichloro phenol indophenol complex. An aliquot of 33.3 L of tissue homogenate was added to 233 L of bovine serum albumin, 6.6 L of 0.1 mM NADPH, 33.3 L of 50 mM FAD, and 710 L of 25 mM Tris-HCL buffer (pH 7.4). Optical density was measured at 600 nm. Using molar extinction coefficient of 2.11 × 10 4 /M/cm, QR activity was determined as nmoles of DCPIP reduced/min/mg protein.
(8) Glutathione Reductase (GSR) Activity. GSR activity was determined by using NADPH as substrate [21]. An aliquot of 50 L of tissue homogenate was added to 50 L of 0.5 mM EDTA, 50 L of 0.1 mM NADPH, 25 L of 1 mM oxidized glutathione, and 825 L of 0.1 M sodium phosphate buffer (pH 7.6). With the help of spectrophotometer, decomposition of NADPH is measured at 340 nm at 25 ∘ C, using molar extinction coefficient of 6.23 × 10 3 /M/cm. GSR activity was determined as amount of NADPH oxidized/min/mg protein.
(9) Glutathione-S-Transferase (GST) Activity. GST assay was based on the formation of CDNB conjugate [22]. An aliquot of 150 L of tissue homogenate was added to 100 L of 1 mM reduced glutathione, 12.5 L of 1 mM CDNB, and 720 L of sodium phosphate buffer. Optical density was measured at 340 nm, using a molar coefficient of 9.61 × 10 3 /M/cm. GST activity was measured as amount of CDNB conjugate formed/min/mg protein.
(10) Lipid Peroxidation Assay (TBARS). Using TBA, TBARS (thiobarbituric acid reactive substances) were measured in the tissue homogenate [23]. An aliquot of 100 L of tissue homogenate was added to 100 L of 100 mM ascorbic acid, 10 L of 100 mM FeCl 3 , and 290 L of sodium phosphate buffer (pH 7.4). Reaction solution was incubated for 1 hour in a shaking water bath at 37 ∘ C. Addition of 500 L of 10% TCA stopped the reaction. Place reaction tubes were placed in boiling water bath for 15 minutes after adding 500 L of 0.67% TBA. After 15 min tubes were shifted on a crushed ice for 5 minutes and centrifuged for 10 minutes at 2500 ×g. Optical density of supernatant was measured at 535 nm to determine the amount of TBARS formed. Using molar extinction coefficient of 1.560 × 10 5 /M/cm, lipid per oxidation activity was measured as an amount of TBARS formed/min/mg tissue.

Results
Many medicinal characteristics are found in the medicinal plants and are commonly used in herbal drug. As synthetic drugs have many side effects, scientists had moved towards natural medicines. Nowadays, all over the world scientists are exploring the scientific basis of effectiveness of traditional medicine and in this context many herbal drugs have been analyzed and their phytochemicals have been isolated and presented to the pharmaceutical industries for drug formulation.

Estimation of Total Flavonoid and Phenolic Contents.
Phenolics and flavonoids are most potent constituent of plants that is responsible for the important antioxidant behavior. Therefore their quantification is an important step to estimate its quantity. Total flavonoids contents were estimated as 325 ± 10.5 mg rutin/g of dry extract while total phenolic content was estimated as 226 ± 11.5 mg gallic acid equivalent/g of dry extract.

In Vivo Studies.
The present experiment was carried out to determine the protective role of P. chinensis extract against CCl 4 induced lung and thyroid toxicity at biochemical and histological level in rats. 4 Toxicity on Serum Protein, Albumin, and Globulin. CCl 4 treatment in rats caused significant change in the serum protein profile. To analyze the protective effect of PCEB, the fluctuations in serum total protein, albumin, and globulin were analyzed (Figure 1). A significant difference was observed in CCl 4 treated group in comparison to control group. PCEB treatment showed protective role by significantly ( < 0.05) increasing its level in the serum.

Estimation of Haematological Parameters.
The protective effect of PCEB on total leukocyte count, neutrophils, lymphocytes, eosinophils, monocytes, and haemoglobin levels against CCl 4 induced toxicity is shown in Table 2. A significant ( < 0.05) decrease in total leukocyte count, neutrophils, and hemoglobin level was observed in CCl 4 treated rats while the number of lymphocytes, eosinophils, and monocytes significantly increased due to CCl 4 intoxication. Toxicity of CCl 4 was ameliorated with cotreatment of PCEB which significantly increased the total leukocyte count and neutrophils and hemoglobin level while decreased the number of lymphocytes, eosinophils, and monocytes in a dose dependent manner.

Pulmonary Toxicity.
In this study, protective effect of PCEB against CCl 4 induced pulmonary toxicity was estimated. In order to characterize the protective effect of PCEB, change in antioxidant enzyme level was evaluated after CCl 4 treatment. Table 3 shows the protective effect of PCEB on lung tissue CAT, POD, and SOD. In comparison to control group, the levels of CAT, POD, and SOD in lung tissues were considerably ( < 0.05) decreased after the CCl 4 treatment. PCEB cotreatment reversed the activity of these enzymes towards normal by increasing their level in a dose dependent way.
The protective effects of PCEB on GST, GSH, and GSH-Px in lung tissue are shown in Table 4. In comparison to control group, the activity of GST, GSH, and GSH-Px was decreased   Mean ± SD ( = 6). Means with different letters (a-e) indicate significance at < 0.05.
( < 0.05) with CCl 4 treatment. Toxicity of CCl 4 on GST, GSH, and GSH-Px was erased with cotreatment of PCEB in a dose dependent manner. Table 5 shows the change in level of GSR, -GT, and QR after different treatments. Treatment of CCl 4 decreased ( < 0.05) the level of these enzymes in lung tissue versus the control group of rats. Treatment with PCEB recovered the normal level of these enzymes in a dose dependent manner.
The protective effects of PCEB on protein, TBARS, nitrite content, and H 2 O 2 against CCl 4 induced alterations are shown in Table 6. After CCl 4 treatment, a significant decrease ( < 0.05) in the level of lung protein as well as increase ( < 0.05) in the TBARS, nitrite, and H 2 O 2 content was observed. Ethanol extract of P. chinensis bark reversed the level of protein, TBARS, nitrite content, and H 2 O 2 in lung tissue towards the normal level in a dose dependent manner.
It has been proved that lungs can be damaged by administration or ingestion of drugs and chemicals, but it can also be damaged by the environmental toxicant primarily through inhalation. After hematoxylin and eosin staining, various histological features of lung tissue of all experimental groups were observed as shown in Figure 2. Control and vehicle control group showed normal cellular structure and normal morphology. Thin walled alveoli and distinct alveolar septa were observed and junctions of alveolar walls and fibroblasts were also noticeable. The shape of terminal bronchiole was normal and Clara cells were pointed towards the cavity of bronchiole. A clear difference in histology was observed in lung tissue of CCl 4 treated group. Various injuries like rupture of alveolar walls and bronchioles, aggregation of fibroblasts, and disorganized Clara cells were also observed. Terminal bronchiole had constricted inner epithelium and reduced lumen, and ultimately shortened air passage. The lung sections of rats treated with PCEB showed somewhat normal histological morphology in a dose dependent manner with high dose (400 mg/kg b.w.) showing more protective effect than the low dose (200 mg/kg b.w.). The structure of terminal bronchioles was quite normal but inner epithelium was slightly constricted showing minor effects of CCl 4 . Clara cells were normal and organized. The fibroblasts were also 8 BioMed Research International

Thyroid Toxicity.
Thyroid gland was also examined for CCl 4 toxicity and PCEB protection against oxidative stress. Figure 3 depicts the protective effect of PCEB on thyroid hormones, that is, T 3 and T 4 . CCl 4 intoxication decreased ( < 0.05) the level of T 3 and T 4 . However, PCEB cotreatment restored the normal level of T 3 and T 4 in a dose dependent manner. Normal histology of thyroid tissue with oval and round thyroid follicle was observed in the control group (Figure 4). The lumen of follicles was filled with colloid, lined by a cubical to columnar epithelial cells. Blood vessels were also in normal shape. Hypertrophy and colloid depletion in thyroid follicle were observed in CCl 4 treated rats. Hyperplasia in follicles was prominent due to change in shape of follicles and congestion of blood vessels. Normal histology was observed in silymarin treated group. Coadministration of PCEB significantly protected thyroid tissue from CCl 4 injuries in a dose dependent manner but a mild congestion in blood vessels was observed, showing the effects of CCl 4 toxicity.

Discussion
Regardless of the enormous advancement in the field of pharmacology and conventional chemistry in creating effective medicines, still plant kingdom is a reservoir of natural therapeutics and offers a valuable source of novel drugs and medicinal entities. Medicinal plants and their phytochemicals are the main source of herbal drugs that can affect the physiological system of animals either directly or indirectly. Plant-based medicines have minimal or no side effects; therefore, these medicines are acknowledged for treatment of number of diseases. Various plants derived antioxidant-based therapeutic medicines are being in use for the prevention and cure of many diseases such as Alzheimer's disease, diabetes, stroke, atherosclerosis, and cancer [27]. The current study mainly concentrates on the role of P. chinensis in ameliorating the CCl 4 induced oxidative stress in rats. It is evident from the results that P. chinensis ethanol extract possess protective action against lung and thyroid injuries induced by carbon tetrachloride induced oxidative stress. Results suggest that the extract was effective in dose-dependent manner and maximum protective activity of the extract was obtained when administered at the dose of 400 mg/kg body weight.
Quantitative pharmacological screening exhibited high amount of total phenolic and flavonoid contents. Similar results were obtained by Shah et al. [14] in phytochemical analysis of S. cordata.
The screening of bioactive substances present in the plants is the most important job in pharmaceutical research. For this purpose, chromatographic study of plant extracts proved to be very reliable and useful. The TLC of PCEB confirmed the presence of different important phenolic compounds. Decrease in the level of serum protein, albumin, and globulin was observed after the CCl 4 treatment in rats. The oxidative damage of some amino acids is considered as the major cause of metabolic dysfunction in CCl 4 induced damage [28]. PCBE at 400 mg/kg ameliorated the synthesis of proteins, albumin, and globulin.
In the current study, CCl 4 administration greatly affected haematological parameters. A decrease in neutrophils and total leukocyte count was observed which might be due to leucopoenia. The depletion in haemoglobin level in current study could be attributed to destruction of red blood cells, enhanced removal from circulation or decrease in their formation, and disturbed hematopoiesis. Ballinger [29] reported that reduction in haemoglobin level results in iron deficiency anaemia which is characterized by a microcytic hypochromic blood picture. The blood picture was improved in rats cotreated with PCEB, which points towards the protective role of P. chinensis against CCl 4 induced microcytic hypochromic anaemia. These results suggest that ethanol bark extract of P. chinensis significantly protected the destructive effects of CCl 4 in the treated rats.
Various studies have confirmed that free radicals are involved in various metabolic alterations and diseases. Radical reactions are mainly responsible for the in vivo toxic effects of CCl 4 . Due to the bioactivation of CCl 4 , various free radicals are produced including CCl 3 OO • and CCl 3 O • radicals, which induce lipid peroxidation and decrease the activities of endogenous antioxidant enzymes, ultimately resulting in organ damage [30]. CCl 4 treatment causes injuries in liver, kidney, and lung tissue in experimental 10x 40x 10x 40x animals. According to Khan [31], reactive oxygen species produced from CCl 4 cause oxidative damages in lungs of rats, possibly by altering the antioxidant enzymes status. Various antioxidant enzymes for example, catalase, peroxidase, and superoxide dismutase, play an important role in protecting lung tissues from free radical induced damages [32]. In the present study, the decrease in level of antioxidant enzymes and GSH in lung and thyroid tissues was observed in CCl 4 treated group. Coadministration of PCEB ameliorated the level of antioxidant enzymes in a dose dependent manner, suggesting protective role of antioxidant enzymes against CCl 4 generated free radicals. Similar results were observed by Shah et al. [14] on antioxidant enzymes of liver. Glutathione system is involved in the xenobiotic and drug metabolism. For functional and structural maintenance, level of GSH is very important. GSH is an important thiol protein that functions in the catalysis of numerous metabolites and manages the cellular defence system against oxidative stress generated by free radicals. The maintenance of GSH activity in cell is dependent on the level of glutathione reductase and NADH [33]. Significant decrease in activities of GSH was observed in the present study. The decreased activity of glutathione system in lung tissue of CCl 4 intoxicated rats might be due to the increased lipid peroxidation or inactivation of the antioxidative enzymes. Administration of PCEB ameliorated the CCl 4 toxicity, thereby increasing the activity of antioxidant enzymes and GSH. Khan [31] reported similar observations during administration of Launaea procumbens methanol extract against CCl 4 induced oxidative stress. In vivo studies carried out by other workers also point out that CCl 4 reduces the level of GSH in lung and thyroid tissues [34]. Similar results were obtained for the silymarin treated group which is a known antioxidant and commonly used as hepatoprotective agent [35]. Thus, activation of these enzymes by the administration of P. chinensis bark extract clearly shows that PCEB through its free radical scavenging activity could exert a beneficial action against pathophysiological alterations caused by free radicals.
Treatment of rats with CCl 4 causes oxidative damage to pulmonary and thyroid proteins and lipids which are the main cause of producing toxicity in humans by CCl 4 [3]. Lipids confined in the cell membranes are very sensitive to the oxidative stress. Lipid peroxidation converts polyunsaturated fatty acids into small and more reactive elements. CCl 4 is a toxic chemical which produces various free radicals causing lipid peroxidation. Increase in lipid peroxidation is calculated in terms of thiobarbituric acid reacting substance (TBARS) which measures the damage caused to the membranes by free radicals [36]. So, TBARS level and H 2 O 2 content are supposed to be main markers of CCl 4 -induced oxidative stress [37]. The nitrite ion is a ligand which binds to the metal centres and causes vasodilation by producing nitric oxide. In the current study, decrease in protein content was observed while TBARS level, nitrite, and H 2 O 2 increased due to CCl 4 treatment which was restored to normal level after treating with PCEB. Oxidative stress induced by CCl 4 was also evident on lung and thyroid histomorphological level. In current study PCEB reduced the CCl 4 induced toxicity in lung tissues as shown by normal structure of terminal bronchioles and Clara cells while marked reduction in interstitial infiltration was seen. In vivo infection and inflammation in the lung tissue of CCl 4 treated rats show the deleterious effects of CCl 4 . Edema and inflammatory responses of lungs are positively linked with the function of lungs, together with the oxygenation index and the airway pressure [38]. Lung injuries and inflammation can be attenuated by inhibiting the production of ROS. PCEB treated rats showed marked reduction in epithelial degeneration and decrease in number of alveolar macrophages with suppression of inflammatory cellular infiltration. The protective effect of PCEB might be due to the presence of tannin in the extract, as it has been reported to have anti-inflammatory properties [39]. In the current study, the in vivo protective effect of PCEB against CCl 4 induced thyroid toxicity was also investigated. Free radicals produced by bioactivation of CCl 4 increase lipid peroxidation and decrease the level of antioxidant enzymes resulting in damage to thyroid tissue. In this study, CCl 4 treatment caused hypothyroidism as evident by the decrease in level of serum T 3 and T 4 . T 3 and T 4 are the two main hormones of the thyroid gland and are continuously required by body for normal growth. Decrease in level of these hormones depicts malfunctioning of thyroid gland. PCEB treatment ameliorated the CCl 4 induced toxicity and increased the level of T 3 and T 4 in serum of rats. Our results are partially consistent with other reports where decrease in T 3 was recorded, while T 4 level was not significantly changed [40]. However, other studies are completely comparable to these findings where treatment of rats with CCl 4 and allyl alcohol significantly decreased T 3 and T 4 level [41,42].

Conclusions
It can be concluded that P. chinensis bark extract contains antioxidant activity as it prevented the oxidative stress and increased the antioxidant effect in lungs and thyroid tissues of male rats. Our results demonstrate the PCEB protective role against CCl 4 generated free radicals damages and suggest for further study to isolate the bioactive component in pure form from P. chinensis bark.