In the current study protective effect of ethanol extract of
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) [
Carbon tetrachloride (CCl4) is a commonly used xenobiotic to induce toxicity in animal models. It is well known as a hepatotoxin [
The best source to get a range of novel herbal drugs is medicinal plants. Medicinal plants are local heritage with universal importance. Various medicinal agents have been derived from the natural sources; among them a lot of drugs are formulated due to their utilization in traditional medicine. Medicinal plants comprise vast range of organic substances which are capable to prevent most of the diseases related to oxidative stress [
CCl4, reduced glutathione (GSH), glutathione reductase,
The barks of
For the preparation of ethanol extract of
Spectrophotometric method [
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 NaNO2, and 0.1 mL of 0.3 M AlCl3·6H2O 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 [
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
For
Shah et al. [
Forty-two male rats were randomly distributed into 7 groups (6 rats/group). Group I remained untreated. Group II was treated with 30% CCl4 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% CCl4 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.
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.
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 T3 and T4 were analysed by using MicroLISA kits.
For the estimation of triiodothyronine (T3) or thyroxine (T4) specific amount of anti-T3 or anti-T4 antibody is coated on the microtiter wells. A measured amount of serum and a constant amount of T3 or T4 conjugated with horseradish peroxidase are added to the microtiter wells. During incubation, T3 or T4 present in the serum and conjugated T3 or T4 compete for the limited binding sites on the anti-T3 or anti-T4 antibody. After 60 min incubation, the wells are washed to remove unbound T3 or T4 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 T3 or T4 in the sample. Series of standards are run in the same way to quantify the concentration of T3 or T4 in sample. Standards, samples, and controls (50
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 5-part hematology analyzer (Abbott diagnostics, Germany).
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 CCl4 in rats.
For histopathological studies, lung and thyroid tissues were fixed in 10% formalin and embedded in paraffin, sectioned at 4
The values were expressed as means ± standard deviation (SD) of six observations in each group. Multiple comparison test (Tukey HSD) by Statistix 8.1 software at 0.05% level of probability among different groups was performed and expressed in the form of homogenous group.
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.
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
For visualization of flavonoids and phenolics present in ethanol bark extract of
Table
TLC of bark ethanol extract of
Color |
|
Compound |
---|---|---|
Dark brown | 0.18 | Unknown |
Purple blue | 0.43 | Unknown |
Yellow | 0.57 | Rutin |
Light blue | 0.78 | Tannin |
Fluorescent yellow | 0.75 | Unknown |
Fluorescent blue | 0.81 | Gallic acid |
The present experiment was carried out to determine the protective role of
CCl4 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
Preventive effect of PCEB on serum (a) protein, (b) albumin, and (c) globulin; mean ± SD (
The protective effect of PCEB on total leukocyte count, neutrophils, lymphocytes, eosinophils, monocytes, and haemoglobin levels against CCl4 induced toxicity is shown in Table
Protective effect of PCEB on hematological parameters.
Treatment | Leukocyte |
Neutrophil |
Lymphocyte |
Eosinophil |
Monocyte |
Hemoglobin |
---|---|---|---|---|---|---|
Control | 5.3 ± 0.02a | 61.6 ± 2.1b | 36.3 ± 1.5d | 1.5 ± 0.3d | 1.7 ± 0.01d | 13.7 ± 0.20a |
CCl4 1 mL/kg | 4.6 ± 0.32d | 57.3 ± 1.5d | 48.6 ± 3.1a | 2.6 ± 0.2a | 2.8 ± 0.10a | 11.9 ± 0.15d |
CCl4+Silymarin | 5.1 ± 0.55b | 59.5 ± 0.4c | 37.0 ± 1.0c | 1.6 ± 0.1c | 1.8 ± 0.07c | 13.7 ± 0.40a |
CCl4+PCEB 200 mg/kg | 4.9 ± 0.25c | 67.7 ± 0.2a | 41.3 ± 1.5b | 1.9 ± 0.1b | 2.1 ± 0.32b | 13.2 ± 0.10c |
CCl4+PCEB 400 mg/kg | 5.2 ± 0.47bc | 65.7 ± 0.2a | 37.5 ± 1.5c | 1.7 ± 0.1bc | 1.9 ± 0.50c | 13.4 ± 0.20b |
PCEB 400 mg/kg | 5.2 ± 0.17b | 59.0 ± 1.0b | 36.9 ± 0.2d | 1.4 ± 0.1e | 1.8 ± 0.04cd | 13.6 ± 0.40a |
Vehicle control | 5.3 ± 0.18a | 61.2 ± 0.4b | 35.8 ± 1.0bd | 1.5 ± 0.1d | 1.7 ± 0.05d | 13.5 ± 0.01ab |
Mean ± SD (
In this study, protective effect of PCEB against CCl4 induced pulmonary toxicity was estimated. In order to characterize the protective effect of PCEB, change in antioxidant enzyme level was evaluated after CCl4 treatment. Table
Protective effect of PCEB on lung CAT, POD, and SOD.
Treatment | CAT (U/min) | POD (U/min) | SOD (U/mg protein) |
---|---|---|---|
Control | 4.7 ± 0.16b | 11.1 ± 0.61c | 3.7 ± 0.18a |
CCl4 1 mL/kg b.w. | 1.1 ± 0.17d | 3.1 ± 0.28f | 0.9 ± 0.43e |
CCl4+Silymarin | 5.3 ± 0.50a | 19.4 ± 0.84a | 3.4 ± 0.10ab |
CCl4+PCEB 200 mg/kg b.w. | 2.1 ± 0.15c | 10.1 ± 0.58d | 1.4 ± 0.55d |
CCl4+PCEB 400 mg/kg b.w. | 5.4 ± 0.53a | 17.2 ± 0.73b | 3.1 ± 0.58bc |
PCEB 400 mg/kg b.w. | 4.7 ± 0.16b | 10.5 ± 0.59cd | 3.2 ± 0.19bc |
Vehicle control | 4.2 ± 0.14b | 11.0 ± 0.01c | 3.3 ± 0.25b |
Mean ± SD (
The protective effects of PCEB on GST, GSH, and GSH-Px in lung tissue are shown in Table
Protective effect of PCEB on lung GST, GSH, and GSH-Px.
Treatment | GST (nM/min/mg protein) | GSH (nM/min/mg protein) | GSH-Px (nM/min/mg protein) |
---|---|---|---|
Control | 177.0 ± 2.9c | 21.7 ± 1.31a | 144.3 ± 2.78a |
CCl4 1 mL/kg b.w. | 94.3 ± 4.3f | 14.8 ± 1.25c | 78.7 ± 3.44e |
CCl4+Silymarin | 206.7 ± 6.5a | 18.5 ± 1.11b | 137.7 ± 4.29b |
CCl4+PCEB 200 mg/kg b.w. | 163.0 ± 4.6d | 16.8 ± 1.25bc | 115.3 ± 4.65d |
CCl4+PCEB 400 mg/kg b.w. | 189.3 ± 5.0b | 17.2 ± 1.24b | 120.3 ± 5.82c |
PCEB 400 mg/kg b.w. | 169.0 ± 6.3c | 20.8 ± 1.10a | 137.0 ± 1.34b |
Vehicle control | 171.4 ± 2.7c | 21.3 ± 1.12a | 141.5 ± 1.36a |
Mean ± SD (
Table
Protective effect of PCEB on lung
Treatment |
|
GSR (nM/min/mg protein) | QR (nM/min/mg protein) |
---|---|---|---|
Control | 402.7 ± 10.3a | 233.7 ± 6.3b | 217.5 ± 4.0a |
CCl4 1 mL/kg b.w. | 102.0 ± 5.5d | 191.7 ± 4.4e | 129.3 ± 2.0e |
CCl4+Silymarin | 375.0 ± 5.6ab | 215.3 ± 2.3d | 204.5 ± 1.2b |
CCl4+PCEB 200 mg/kg b.w. | 108.7 ± 6.4d | 176.3 ± 4.4f | 158.4 ± 4.3f |
CCl4+PCEB 400 mg/kg b.w. | 124.6 ± 6.7d | 244.3 ± 2.2a | 197.2 ± 2.4d |
PCEB 400 mg/kg b.w. | 325.0 ± 7.6c | 222.0 ± 3.2c | 200.4 ± 1.1c |
Vehicle control | 361.0 ± 5.9bc | 210.9 ± 2.5d | 209.6 ± 2.3b |
Mean ± SD (
The protective effects of PCEB on protein, TBARS, nitrite content, and H2O2 against CCl4 induced alterations are shown in Table
Protective effect of PCEB on lung Protein, TBARS, nitrite, and H2O2.
Treatment | Protein ( |
TBARS (nM/min/mg protein) | Nitrite ( |
H2O2 (nM/min/mg tissue) |
---|---|---|---|---|
Control | 2.2 ± 0.16a | 3.5 ± 0.16e | 44.9 ± 1.58e | 1.4 ± 0.17e |
CCl4 1 mL/kg b.w. | 0.8 ± 0.14e | 6.1 ± 0.18a | 71.8 ± 2.25a | 2.9 ± 0.15a |
CCl4+Silymarin | 2.1 ± 0.15c | 3.6 ± 0.35d | 52.2 ± 1.19cd | 1.5 ± 0.11d |
CCl4+PCEB 200 mg/kg b.w. | 1.6 ± 0.38d | 4.3 ± 0.15b | 67.3 ± 0.43b | 2.3 ± 0.52b |
CCl4+PCEB 400 mg/kg b.w. | 2.2 ± 0.15bc | 3.7 ± 0.36c | 55.2 ± 0.10c | 1.6 ± 0.15c |
PCEB 400 mg/kg b.w. | 2.2 ± 0.14b | 3.6 ± 0.92e | 53.0 ± 1.66d | 1.5 ± 0.15d |
Vehicle control | 2.2 ± 0.13a | 3.6 ± 0.16e | 46.9 ± 1.15e | 1.4 ± 0.75e |
Mean ± SD (
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
Microphotograph of rat lungs (H & E stain). (a) Control group; (b) CCl4 group; (c) Silymarin+CCl4 group; (d) PCEB (200 mg/kg b.w.) + CCl4 group; (e) PCEB (400 mg/kg b.w.) + CCl4 group; (f) PCEB (400 mg/kg b.w.); (g) vehicle control group. TB: terminal bronchiole; CC: Clara cells; AS: alveolar septum; AM: alveolar macrophages; A: arteriole.
Thyroid gland was also examined for CCl4 toxicity and PCEB protection against oxidative stress. Figure
Protective effect of PCEB on thyroid hormones: (a) T3 and (b) T4; mean ± SD (
Microphotograph of rat thyroid (H & E stain) at 40x. (a) Representative section of thyroid from the control group showing normal histology with normal shaped follicles cells containing follicles; (b) CCl4 group; (c) Silymarin+CCl4 group; (d) PCEB (200 mg/kg b.w.) + CCl4 group; (e) PCEB (400 mg/kg b.w.) + CCl4 group; (f) Only PCEB; (g) vehicle control group. C: Colloid; FC: follicular cells.
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 [
Quantitative pharmacological screening exhibited high amount of total phenolic and flavonoid contents. Similar results were obtained by Shah et al. [
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 CCl4 treatment in rats. The oxidative damage of some amino acids is considered as the major cause of metabolic dysfunction in CCl4 induced damage [
In the current study, CCl4 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 [
Various studies have confirmed that free radicals are involved in various metabolic alterations and diseases. Radical reactions are mainly responsible for the
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 [
Treatment of rats with CCl4 causes oxidative damage to pulmonary and thyroid proteins and lipids which are the main cause of producing toxicity in humans by CCl4 [
In the current study, the
It can be concluded that
The authors declare that there is no conflict of interests regarding the publication of this paper.