Abstract
Cyclophosphamide (CP)—also known as cytophosphan—is an alkylating agent that has many side effects in humans and rats. Rats were divided into 5 different groups to evaluate the protective effect of escin (ES) obtained from the horse-chestnut plant (Aesculus hippocastanum) against acute damage induce by CP. Groups: control group, ethanol group, ES group (100 mg/kg body weight (bw) ES for 14 days by gastric gavage), ES + CP group (100 mg/kg bw ES for 14 days by gastric gavage and 75 mg/kg bw CP i.p. on 14th day), and CP group (75 mg/kg bw CP i.p. on 14th day). After the experiment was completed, blood and tissue samples (liver, kidney, heart, brain, lung, and testis) were taken from the rats under anesthesia. When the CP group was compared with the control group, an increase was observed in the level of Malondialdehyde (MDA) in blood and all tissues except the lung, but when it was given together with escin, there was a decrease except kidney and lung (P < 0.05). Glutathione (GSH) level decreased in the blood and all tissues when CP was given, whereas an increase was observed in the heart, brain, and lung when given with escin (P < 0.05). There was no statistical change in the activities of superoxide dismutase and catalase enzymes in all tissues. ES reduced CP-induced damage in all tissues except the kidney. As a result, it was determined that ES had a protective effect against CP-induced tissue damage in rats due to its antioxidant properties.
Keywords: cyclophosphamide, escin, rat, oxidative stress, lipid peroxidation, histopathology
Graphical Abstract
Graphical Abstract.
Introduction
Cyclophosphamide; (N,N-Bis (2-chloroethyl) tetrahydro-2H-1,3,2-oxazaphosphorine-2-amine 2-oxide; CP), belongs to the nitrogenous mustards class, and is a cytotoxic bifunctional alkylating agent.1 CP is a chemotherapeutic agent and it is extensively used for the treatment of many cancer types such as lung, breast, ovary, lymphoma, and leukemia. It has been used in the treatment of autoimmune diseases such as rheumatoid arthritis at low doses. In addition, and used as immunosuppressant after bone marrow transplantations.2–4
Many side effects are related to the use of high-dose CP such as acute inflammation in the urinary bladder, kidney, and liver.5 It has been reported that CP causes oxidative stress in the liver and produces free radical species such as superoxide anion, hydroxyl radical, and hydrogen peroxide (H2O2), and suppresses antioxidant defense systems. These free radicals can attack soluble cell components as well as membranes, eventually causing cell dysfunction and cytolysis.3,6
The anti-cancer property of CP is mainly due to DNA alkylation. For the activation of CP in the liver, it revealed conversion into its metabolites, namely phosphoramide mustard and acrolein.7 It shows its main toxic effect as a result of the activity of its active metabolite, acrolein. Acrolein damages the antioxidant system by causing high free oxygen radical formation that is mutagenic for mammalian cells.8
The main active ingredient extracted from the fruit of the horse-chestnut tree is escin. Escin is a mixture of triterpene saponins and is widely used for the treatment of peripheral vascular disorders and in the cosmetic industry due to its anti-inflammatory and anti-edematous properties.9 It is known that free radicals and reactive oxygen species produced in the body play an important role in the development of various diseases. In recent years, there has been a tendency to use some plant-derived substances with antioxidant properties. One of them is β-escin and is obtained from the seeds of the horse-chestnut tree (Aesculus hippocastanum). It also has found an important place for the usage due to its antioxidant effect.10
In this study, it was aimed to investigate the possible protective effect of escin (ES) obtained from A. hippocastanum in blood and various tissues against oxidative damage caused by CP in male rats. For this purpose, lipid peroxidation levels, antioxidants measurement, and also histopathological examinations were performed.
Material and method
Material
Ethical Committee approval was obtained by Afyon Kocatepe University Experimental Animals Local Ethics Committee. For the study, 35 male Wistar albino rats were obtained from Afyon Kocatepe University Experimental Animals Application and Research Center and their care was also carried out in this center. In the present study, pure CP (Baxter, Halle, Germany) was used to induce toxicity. The chemicals and kits to be used to determine the parameters were obtained from the relevant companies.
Preparation of plant extract
The Horse Chestnut (A. hippocastanum L.) used in the study was obtained from the garden of the Uşak Forestry Management Directorate and identified at Uşak University. It was collected in September 2018 and after drying in the room without sunlight, the extraction process was carried out according to the patent numbered “United States Patent—3,609,137” “Process for the production of an escin rich concentrate of active material from Horse-chestnut seeds.” To remove the oil contained in the horse-chestnut fruits, which were cut into small pieces in the extraction of the plant, the plant part, which was separated by filtration after 1.5 h with 2% acetic acid solution, was extracted at room temperature and with stirring, using a 50% ethanol-water solvent system. The obtained extract was concentrated under a vacuum and turned into powder. The amount of active substance contained in this extract was determined by high-performance liquid chromatography (HPLC; Agilent 1100 Series, Waldron, Germany) method. The HPLC analysis was carried out on a system with a quaternary pump, DAD detector system, autosampler, and column oven. This equipment has a degasser system. The chromatographic technique was developed using a Nemesis C-18 (3 μ, 150 × 4.6 mm; Phenomenex, Cheshire, United Kingdom) column. The mobile phase was a mixture of 1.0% phosphoric acid in water and methanol (42/58, v/v) at a flow rate of 1.0 mL min−1. Eluent detection was carried out at a wavelength of 210 nm using an ultraviolet detector in 13 min. Total amount of escin detected as 58.28%.
Experimental protocol
Healthy male Wistar albino rats weighing 250–300 g were purchased from the Animal Breeding Laboratories of the Experimental Animal Research and Application Center (Afyon, Turkey). Animals were fed a standard rat diet and provided ad libitum access to water. The environment in which the rats were housed was kept on a 12-h light/dark cycle, at room temperature (25°C) and relative humidity (50–55%). In this study, a total of 35 male rats, 7 rats in each group, were randomly divided into 5 groups. All animals fasted overnight before the experiment.
The groups were created as follows:
(i) Control group (standard rat food and drinking water for male rats in the control group).
(ii) Ethanol group (0.2 mL 37% ethanol for 14 days via gastric gavage).
(iii) Escin group (100 mg/kg bw horse-chestnut extract containing escin dissolved in 20 mL + 37% ethanol via gastric gavage for 14 days).
(iv) Escin + CP group (100 mg/kg bw horse-chestnut extract containing escin dissolved in 20 mL + 37% ethanol via gastric gavage for 14 days + CP 75 mg/kg bw i.p. on day 14th).
(v) CP group (i.p. distilled water was given for 14 days + CP 75 mg/kg bw was dissolved in distilled water and given i.p. on day 14th).
After 12 h of fasting, blood and tissue samples were obtained from animals under general anesthesia (ketamine (65 mg/kg, i.p.) − xylazine (7 mg/kg, i.p.)).
Preparation of blood, erythrocytes, and homogenate
Blood samples from each group were taken into heparinized tubes. Part of the blood was separated and the other part was prepared for superoxide dismutase (SOD) and catalase (CAT) activities. Within 30 min after blood collection, it was centrifuged at 3,500 rpm for 15 min at 4°C. Thus, erythrocyte and plasma were separated. The precipitated erythrocytes were washed 3 times with isotonic saline and the fluffy coat was removed. Then, isotonic saline and erythrocytes were added in the same volume and stored in a deep freezer at −20°C. The erythrocyte suspension was destroyed by osmotic pressure using 5 volumes of cold deionized water. SOD and CAT activities were measured from the formed erythrocyte lysate.11 After the dissection of rats, liver, kidney, heart, brain, lung, and testis tissues were taken and were immediately washed with ice-cold 0.9% NaCl. Each tissue was cleared of foreign tissue and rinsed in chilled 0.15 M Tris–HCl buffer (pH 7.4). These tissues were homogenized in 0.15 M Tris–HCl buffer (pH 7.4) to obtain 10% (w/v) homogenate. Tissues were centrifuged at 2,500 rpm for 10 min at 4°C. Supernatants obtained after centrifugation were stored at −20°C until analysis.12
Measurement of Malondialdehyde
Malondialdehyde (MDA)—an important marker for lipid peroxidation—was measured using the Draper and Hadley method in whole blood and the method of Ohkawa et al. in tissue homogenates.13,14 The principle of the method is based on the spectrophotometric measurement of the color resulting from the reaction of MDA and thiobarbituric acid (TBA). The absorbance of this color obtained at the end of the analysis was measured spectrophotometrically at a wavelength of 532 nm. The MDA concentration was calculated by the absorbance coefficient of the MDA-TBA complex and expressed as nmol/g in tissue.
Glutathione measurement
The method defined by Beutler et al. was used for the measurement of glutathione (GSH) concentration in whole blood and tissue homogenates.15 The method determines the reaction between glutathione and 5,5′-dithiobis-2-nitrobenzoic acid resulting in thiolate formation. Optical density was measured at a wavelength of 412 nm in a spectrophotometer. Results were expressed as nmol/g of tissue.
Measurement of SOD activity
Antioxidant enzyme activity of SOD in erythrocyte and tissue homogenate Sun et al. were measured according to the method.16 The absorbance obtained by reducing the nitroblue tetrazolium in the medium to blue-colored formazone by superoxide radicals was determined spectrophotometrically at a wavelength of 560 nm. SOD was expressed as U/mgHb in erythrocyte and U/μg for protein in the tissue.
Measurement of catalase activity
CAT activity in erythrocyte lysate was determined according to Luck17 method, and tissue homogenate was determined according to Aebi 18 method. These methods are based on the breakdown of H2O2 by catalase. The reaction contains 50-mM phosphate buffer, 10-mM H2O2, and a sample, and the mixture should be pH 7.0. The reduction rate of H2O2 was monitored for 45 s at 240 nm at room temperature. One unit of CAT corresponds to the amount of enzyme that breaks down 1 μmol of H2O2 per minute at pH 4.5 at 25°C. CAT activity (k; nmol/min) was expressed as k/μg for protein in the tissue.
Measuring protein concentrations
The protein content in the tissue was tested according to the colorimetric method of Lowry et al.19
Measuring hemoglobin concentrations
Hemoglobin (Hb) was determined colorimetrically by the cyanomethemoglobin method according to Drabkin and Austin.20 All spectrophotometric measurements were performed using a Shimadzu 1601 UV–vis spectrophotometer (Tokyo, Japan).
Preparation of tissues for histopathological analysis
At the end of the study, liver, kidney, heart, brain, lung, and testis tissues were placed in 10% formalin solution for histopathological analysis. The fixation of tissues was done for 48 h. Tissues were dehydrated by passing through graded alcohol (70–100%). After cleaning the tissues in xylene, they were embedded in paraffin. The sections of 5–6-μm thickness were obtained. After that, these tissues were stained with hematoxylin–eosin (H&E). As a result, each section was examined under a light microscope (Olympus BX51, and Microscopic Digital Picture Analysis System with DP20 attached, Tokyo, Japan). Also, histopathological evaluations were statistically analyzed and presented in Table 5.
Table 5.
Histopathological evaluation of 75 mg/kg bw dose of CP and 100 mg/kg bw of escin administration.
| Tissue | Histopathological findings | Control | Escin | Ethanol | CP | CP + Escin |
|---|---|---|---|---|---|---|
| Brain | Hyperemia | 0.00 ± 0.00b | 0.00 ± 0.00b | 0.07 ± 0.06b | 2.60 ± 0.54a | 0.18 ± 0.44b |
| Areas of degeneration and focal gliosis in neurons | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.26 ± 0.41a | 0.37 ± 0.56b | |
| Lung | Hyperemia | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.09 ± 0.07c | 2.27 ± 0.40a | 0.45 ± 0.15b |
| Thickening of interalveolar septal tissue | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.27 ± 0.98a | 0.57 ± 0.16b | |
| Edema in alveoli | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.60 ± 0.54a | 0.55 ± 0.20b | |
| Areas of MNC infiltration in the interstitial region | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.11 ± 0.89a | 0.46 ± 0.17b | |
| Heart | Hyaline degeneration | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 1.93 ± 0.41a | 0.44 ± 0.14b |
| Myocardial bleeding areas | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.10 ± 0.88a | 0.56 ± 0.17b | |
| Liver | Hyperemia | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.10 ± 0.51a | 0.43 ± 0.18b |
| Sinusoidal dilation | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.07 ± 0.05c | 2.43 ± 0.82a | 0.44 ± 0.14b | |
| Areas of MNC infiltration in the periportal areas | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.10 ± 0.30a | 0.51 ± 0.20b | |
| Kidney | Enlargement of Bowman’s space in glomeruli | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.27 ± 0.41a | 0.49 ± 0.13b |
| Vacuolization formations in the glomeruli | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.10 ± 0.63a | 0.60 ± 0.10b | |
| Bleeding area in the interstitial region | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.60 ± 0.54a | 0.89 ± 0.22b | |
| Testis | Vacuolization formations in the TSC lumen | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 2.10 ± 0.63a | 0.58 ± 0.21b |
| Reduction in spermatozoa in TSC lumen | 0.00 ± 0.00c | 0.00 ± 0.00c | 0.00 ± 0.00c | 1.60 ± 0.54a | 0.44 ± 0.18b |
a ,b,cValues with different letters on the same line are statistically significant (P < 0.05).
Statistical analysis
One-way ANOVA test was used with the SPSS 20.0 statistical package program. Duncan test was used to evaluate different results statistically. Data were expressed as “mean ± standard deviation.” P < 0.05 was considered as significant value.
Results
Effect on lipid peroxidation and reduced glutathione
Malondialdehyde level is widely used as a marker of free radical-mediated LPO. Compared to the control rats treated with CP, a significant increase in MDA levels was observed in all tissues measured except the lung (P < 0.05). On the other hand, in the group given escin together with CP, the MDA level was found to be significantly decreased in tissues other than kidney and lung (Table 1; P < 0.05). Glutathione is a non-enzymatic antioxidant substance in detoxification and reduces the toxic effect of xenobiotic metabolites. It was observed that GSH levels decreased significantly in all tissues measured in the CP group (P < 0.05; Table 2). In the group given escin together with CP, GSH levels increased in heart, lung, and brain tissues (P < 0.05), but there was no significant change in other tissues.
Table 1.
Effect of 75 mg/kg bw of CP and 100 mg/kg bw of escin on whole blood and tissue MDA levels in male rats.
| Groups | Blood (nmol/mL) | Liver (nmol/g tissue) | Kidney (nmol/g tissue) | Heart (nmol/g tissue) | Brain (nmol/g tissue) | Lung (nmol/g tissue) | Testis (nmol/g tissue) |
|---|---|---|---|---|---|---|---|
| Control | 2.45 ± 0.36c | 3.37 ± 0.40c | 3.32 ± 0.41b | 2.71 ± 0.43c | 1.89 ± 0.16c | 3.28 ± 0.55 | 3.39 ± 0.55c |
| Ethanol | 2.51 ± 0.16c | 3.27 ± 0.17bc | 3.12 ± 0.52b | 3.09 ± 0.38c | 2.16 ± 0.14bc | 3.93 ± 0.40 | 3.50 ± 0.52c |
| Escin | 2.75 ± 0.10bc | 3.42 ± 0.32bc | 3.65 ± 0.47b | 3.19 ± 0.35c | 2.22 ± 0.42bc | 3.58 ± 0.27 | 4.07 ± 0.56bc |
| Escin+CP | 2.92 ± 0.35b | 3.69 ± 0.43b | 4.68 ± 0.97ab | 3.63 ± 0.20b | 2.42 ± 0.36b | 3.69 ± 0.15 | 4.24 ± 0.79b |
| CP | 4.19 ± 0.41a | 4.23 ± 0.19a | 5.44 ± 1.11a | 4.05 ± 0.31a | 3.57 ± 0.45a | 4.06 ± 0.90 | 5.38 ± 0.35a |
Mean ± standard deviation; n = 7.
a ,b,c,dValues with different letters in the same column are statistically significant (P < 0.05).
Table 2.
Effect of 75-mg/kg bw of CP and 100 mg/kg bw of escin on whole blood and tissue GSH levels in male rats.
| Groups | Blood (nmol/mL) | Liver (nmol/g tissue) | Kidney (nmol/g tissue) | Heart (nmol/g tissue) | Brain (nmol/g tissue) | Lung (nmol/g tissue) | Testis (nmol/g tissue) |
|---|---|---|---|---|---|---|---|
| Control | 23.11 ± 1.30a | 6.83 ± 0.81a | 5.98 ± 0.58a | 3.08 ± 0.76a | 4.74 ± 0.66a | 3.42 ± 1.11a | 4.32 ± 0.94a |
| Ethanol | 22.40 ± 2.85ab | 6.72 ± 0.83a | 5.94 ± 0.64a | 3.64 ± 0.47a | 4.78 ± 0.58a | 3.98 ± 1.03a | 4.92 ± 0.80a |
| Escin | 22.32 ± 1.26ab | 6.44 ± 1.16c | 4.88 ± 0.47ab | 3.06 ± 0.55a | 4.33 ± 0.54a | 4.00 ± 0.53a | 3.86 ± 0.59ab |
| Escin+CP | 20.40 ± 1.24bc | 6.29 ± 0.59b | 3.68 ± 0.36b | 2.23 ± 0.47b | 3.42 ± 1.17b | 2.92 ± 0.51b | 3.34 ± 0.65bc |
| CP | 19.05 ± 1.22c | 5.70 ± 0.54b | 3.62 ± 0.49b | 1.44 ± 0.15c | 2.96 ± 0.93c | 2.46 ± 0.47c | 3.07 ± 0.23c |
Mean ± standard deviation; n = 7.
a ,b,c Values with different letters in the same column are statistically significant (P < 0.05).
Effect on antioxidant enzymes
The antioxidant enzymes SOD and CAT activities were determined in the erythrocytes, liver, kidney, heart, brain, lung, and testis tissues of the rats as shown in Tables 3 and 4. It was observed that escin did not cause a statistical change in SOD and CAT enzyme activities against oxidative stress caused by CP (P ˃ 0.05).
Table 3.
Effect of 75 mg/kg dose of CP and 100 mg/kg escin on erythrocyte and tissue SOD activities in male rats.
| Groups | Erythrocyte U/g hb | Liver U/μg protein | Kidney U/μg protein | Heart U/μg protein | Brain U/μg protein | Lung U/μg protein | Testis U/μg protein |
|---|---|---|---|---|---|---|---|
| Control | 10.00 ± 0.11 | 6.76 ± 3.45 | 5.80 ± 0.4 | 5.52 ± 2.74 | 13.41 ± 2.87 | 7.02 ± 0.69 | 10.15 ± 1.42 |
| Ethanol | 11.42 ± 0.21 | 6.34 ± 4.28 | 4.81 ± 1.84 | 8.10 ± 1.90 | 12.01 ± 3.59 | 5.92 ± 0.97 | 11.72 ± 1.92 |
| Escin | 11.32 ± 0.14 | 3.16 ± 2.84 | 5.71 ± 1.02 | 7.15 ± 1.18 | 11.07 ± 2.12 | 6.71 ± 1.14 | 10.85 ± 2.63 |
| Escin+CP | 11.87 ± 0.10 | 3.57 ± 2.28 | 5.41 ± 0.84 | 8.08 ± 1.19 | 9.77 ± 1.62 | 5.59 ± 1.32 | 9.32 ± 1.53 |
| CP | 12.30 ± 0.27 | 2.61 ± 1.42 | 6.01 ± 0.53 | 8.45 ± 3.94 | 10.53 ± 1.60 | 6.03 ± 1.59 | 11.57 ± 2.11 |
Mean ± standard deviation; n = 7.
Table 4.
Effect of 75 mg/kg bw of CP and 100 mg/kg bw of escin on erythrocyte and tissue CAT activities in male rats. k; nmol/min.
| Groups | Erythrocyte k/g hb | Liver k/μg protein | Kidney k/μg protein | Heart k/μg protein | Brain k/μg protein | Lung k/μg protein | Testis k/μg protein |
|---|---|---|---|---|---|---|---|
| Control | 230.01 ± 11.80 | 3.95 ± 0.60 | 3.73 ± 0.18 | 4.20 ± 1.12 | 7.65 ± 1.94 | 4.74 ± 0.53 | 5.75 ± 1.01 |
| Ethanol | 231.12 ± 13.11 | 4.42 ± 0.87 | 4.15 ± 0.55 | 5.58 ± 0.56 | 5.04 ± 2.13 | 3.98 ± 0.65 | 6.55 ± 1.12 |
| Escin | 227.21 ± 4.88 | 4.63 ± 1.75 | 4.23 ± 0.72 | 4.51 ± 1.17 | 6.15 ± 0.56 | 4.42 ± 0.94 | 6.63 ± 0.98 |
| Escin+CP | 233.34 ± 11.15 | 3.12 ± 0.26 | 3.87 ± 0.44 | 4.39 ± 0.52 | 6.31 ± 0.84 | 4.09 ± 0.87 | 5.64 ± 0.74 |
| CP | 242.52 ± 12.61 | 3.58 ± 0.68 | 4.16 ± 0.46 | 4.54 ± 1.96 | 6.50 ± 0.92 | 4.24 ± 1.01 | 5.81 ± 0.92 |
Mean ± standard deviation; n = 7.
Histopathological changes
The histopathological changes in the organs of animals in the experimental groups were described in detail and shown in Fig. 1. Neuronal degeneration with focal gliosis in the brain tissues of the animals in the CP group (Fig. 1A4), thickening of the interalveolar septal tissue in the lungs (Fig. 1B4), areas of hyaline degeneration, and necrobiotic changes in the heart muscle cells (Fig. 1C4), sinusoidal dilatation in the livers and necrobiotic changes in the hepatocytes (Fig. 1D4), decrease in spermatozoid density in tubules seminiferous contortus (TSC) lumens, and necrobiotic changes in Sertoli cells were observed in testis tissues (Fig. 1F4).
Fig. 1.
The ES on CP-induced damage in the brain (A), lung (B), heart (C), liver (D), kidney (E), and testis (F) of rats. Representative figures were stained with H&E. The original magnification was 20× and the scale bars represent 100 μm. 1-Control; 2—Escin; 3—Ethanol; 4—CP; and 5—CP + Escin. Neuronal degeneration and focal gliosis with neuronal degeneration in the brain tissues of the animals in the CP group (A4), thickening of the interalveolar septal tissue in the lungs (B4), areas of hyaline degeneration in the heart muscle cells and necrobiotic changes (C4), sinusoidal dilatation and hepatocytic changes in their livers (C4), and a decrease in the density of spermatozoids in the tubulous seminiferous contortus (TSC) lumen and necrobiotic changes in the Sertoli cells (E4) were observed in testicular tissues. Less histopathological changes were observed in the brain, lung, heart, liver, and testis tissues in the CP and escin groups compared to the CP group (A–F5, respectively).
Less histopathological changes were observed in the brain, lung, heart, liver, and testis tissues in the CP and escin groups compared to the CP group (Fig. 1A–F5, respectively). No significant histopathological changes were observed in the organs of control, ethanol, and escin groups (Fig. 1A–F-1, 2, and 3, respectively).
Discussion
The antioxidant role of escin has been reported in many studies conducted in recent years.21,22 Although CP has extensive usage for the treatment of cancers, it causes severe cytotoxicity in humans and experimental animals as a result of long-term use.23 Studies have shown that CP reduces ant-oxidative activities and creates oxidative stress in the liver.24 Cengiz et al. reported liver damage with CP with a single-dose injection of 200 mg/kg i.p. dose rate of CP in rats. They stated that after giving 10-mg/kg escin, a decrease in MDA level and an increase in GSH level was observed in the escin group compared to the CP group in the blood and liver tissue. It was concluded that escin has anti-oxidative.25 In another study, it was stated that lipid peroxidation increased significantly in the examination made of damaged liver tissue homogenate of rats with CCl4, and escin given at a dose of 3.6 mg/kg reduced this increase. It has also been stated that it has an antioxidant effect.22 Kucukkurt et al. also stated that the application of extract containing escin reduced oxidative stress and also significant improvement in the pathological findings was reported. It was concluded that this effect of the escin-containing extract may be due to the positive effect on the antioxidant system stated in previous study.26 In a study conducted by inducing damage to rats by ischemia/reperfusion in the liver, it was stated that the level of MDA was increased, whereas escin caused a significant decrease in the MDA level.27 In our study, horse-chestnut extract containing escin was given to the rats by gastric gavage at a dose of 100-mg/kg bw for 14 days. At the end of this study, tissue and blood samples were taken by applying CP, 75-mg/kg bw i.p. By the literature, it was found that CP application caused significant damage by increasing lipid peroxidation and decreasing glutathione levels in tissues, thus increasing oxidative stress. On the other hand, it was determined that escin inhibited this CP-induced lipid peroxidation except in kidney and lung tissues and increased glutathione levels in heart, lung, and brain tissues. In an in vitro study, Zhao et al. used different free radical screening tests to measure the antioxidant potential of escin and they stated that escin has a free radical scavenging ability.28
Escin consists of a complex mixture of triterpene ester saponins (>30 different saponins) and is considered the main active ingredient of it. Aesculus hippocastanum extract has been found to have the potential to act as a scavenger of reactive oxygen species (ROS) and is 20 times more potent against superoxide than ascorbic acid.29 A study was carried out on the protective effect of Centella triterpene saponins (EXT) in liver tissue that caused hepatotoxicity with CP. In the study, EXT (250 mg/kg/day) was given to rats in whom toxicity was caused by CP (10 mg/kg/day). It was determined that lipid peroxidation increased with CP application and decreased with EXT application. Significant increase in the level of glutathione was reported.30 Zhang et al. investigated the protective effect of saponin obtained from Panax ginseng root and leaves against CP-induced oxidative damage in mice. Researchers stated that a single dose of 100 mg/kg administered CP decreased the GSH level and SOD and CAT enzyme activities. However, it is stated that P. ginseng saponins at different doses (low-dose: 25 mg/kg, medium-dose: 50 mg/kg, high-dose: 100 mg/kg) given together with CP inhibited this decrease and even significant increase in the level of these enzyme has been reported.31 These studies suggest that antioxidant properties of escin may be related to its saponin content.
Although there was no significant difference in SOD and CAT enzyme activities in our study, some studies seem to affect these enzyme levels. A single dose of 200 mg/kg i.p. It was stated that 10 mg/kg of escin caused a significant increase in the activities of these enzymes, despite a significant decrease in SOD and CAT enzyme activity in the heart tissue of CP-given mice.32 In a study conducted by inducing damage to rats by ischemia/reperfusion in the liver, it was stated that despite the decrease in SOD and CAT enzyme activities, escin increased the level of these values and protected against the negative effects of ROS.27
Cengiz et al. reported that escin application reduces the histopathological disorders caused by CP.25 Daban et al. reported that histological damage was reduced in a study investigating the protective ES in rats with ischemia/reperfusion injury in the liver.24 In another study, it was stated that escin ameliorated the pathological changes in CCl4-induced liver damage.22 Jiang et al. studied the antioxidant ES against acute liver injury. In the damaged liver tissue of mice injected with lipopolysaccharide (LPS; 40 mg/kg); granulocyte infiltration, deterioration in cellular morphology, severe obstruction in hepatic sinusoids, and hepatocellular necrosis has been reported. Escin has been reported to significantly reduce these toxic effects.33 Similarly, in this study, it was observed that the escin-containing extract attenuated the histopathological damage caused by CP.
Conclusion
It has been concluded that the A. hippocastanum extract containing escin has a protective effect against CP-induced tissue damage in rats. İt revealed this protective effect both by inhibiting LPO and increasing the activities of the antioxidant defense system. At the same time protects cells against CP-induced toxicity and increases regeneration against tissue damage.
Acknowledgments
This study was supported by Afyon Kocatepe University Scientific Research Council, Afyonkarahisar, Turkey (Project no: 17.KARIYER.189). All authors express their gratitude to this institution. We also thank Biologist Dr Mehtap Dönmez Şahin for identifying the Horse Chestnut (Aesculus hippocastanum L.) used in the study.
Contributor Information
İsmail Küçükkurt, Department of Biochemistry, Faculty of Veterinary Medicine, 03200 Afyon Kocatepe University, Afyonkarahisar, Turkey.
Erten Akbel, Usak Health Training School, Usak University, 64100 Usak, Turkey.
Sinan İnce, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, 03200 Afyon Kocatepe University, Afyonkarahisar, Turkey.
Damla Arslan Acaröz, Bayat Vocational School, Afyon Kocatepe University, Bayat, 03200 Afyonkarahisar, Turkey.
Hasan Hüseyin Demirel, Bayat Vocational School, Afyon Kocatepe University, Bayat, 03200 Afyonkarahisar, Turkey.
Fahriye Kan, Department of Biochemistry, Faculty of Veterinary Medicine, 03200 Afyon Kocatepe University, Afyonkarahisar, Turkey.
Funding
This study was supported by Afyon Kocatepe University Scientific Research Council, Afyonkarahisar, Turkey (Project no: 17.KARIYER.189).
Conflict of Interest: There is no real or potential conflict of interest to declare.
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