Abstract
Cornelian cherry (Cornus mas) is a valuable source of phenolic antioxidants. The present study was aimed to investigate whether Cornus mas fruit hydro-methanolic extract (CMFE) can modulate the cisplatin-induced changes in liver antioxidant enzymes and histological structure. Forty Wistar rats were divided into a control group, cisplatin (Cis) group, CMFE group, CMFE 300 + Cis group, and the CMFE 700 + Cis group. After the intervention, blood and tissue samples were taken for biochemical and histopathological analysis. Cis caused reduction in the activity of liver antioxidant enzymes including SOD, GPx, TAC, and CAT and increased that of MDA. Moreover, exposure to Cis caused a reduction in serum level of AST, ALT, and ALP and a rise in serum level of GGT. Oral administration of CMFE for 16 days in the two different dosages at 300 and 700 mg/kg improved the Cis-induced changes of liver enzymes activity and serum enzymes level. Evaluating the histological structure of liver tissue, it was found that treatment by CMFE could ameliorate the Cis-induced changes to near normal histology. The results showed antioxidant and phenol contents in Cornus mas fruit could improve Cis-induced oxidative stress and liver histologic changes in rats.
Keywords: Cornus mas, Cisplatin, Antioxidant, Hepatotoxicity
Introduction
One of the pivot features which differentiate between antineoplastic drugs from other molecules is the intensity of adverse effects at therapeutic dose range. Most of anticancer drugs such as cisplatin (Cis) target directly on multiplying cells. The main mechanisms for Cis-induced toxicities attributed to the development of reactive oxygen species (ROS) and nitrogenous compounds by oxidative stress injury and diminish of antioxidant defense role [1]. Cisplatin-based chemotherapy is widely used for the treatment of tumors. However, the prevalence of Cis toxicity is high and liver toxicity is the most prominent side-effect of Cis [2].Oxidative stress appears to play an important role in Cis induced hepatotoxicity [3].
Natural antioxidants such as fruits and vegetables, which provide protection against free radicals, can decrease the incidence and mortality rates of cancer and heart diseases, in addition to their other health benefits [4, 5]. Recently, the consumption of herbs such as cornelian cherry (Cornus mas), with high levels of antioxidants and anthocyanins, has been increased. Cornus mas fruits are prescribed for gastrointestinal and excretory disorders [6], besides improving liver and kidney functions [7, 8]. This plant was used to treat diarrhea, intestinal inflammation, fever, malaria, kidney stones and kidney and bladder infections in traditional medicine. Cornus mas fruit has anthocyanins, flavonoids, and plenty of oxalic acid content [9]. It also contains antioxidant substances including butylated hydroxyanisole and butylated hydroxytoluene, and has the potential to fight cancer [10].
Considering the antioxidant efficacy of Cornus mas fruit, in the present study we investigated the effects of Cornus mas on liver functional enzymes, oxidative stress parameters, and histopathologic changes induced by Cis in rats.
Materials and Methods
Plant Material
Cornus mas plant and its fruits were authenticated by the Botany Department of Tabriz University, Iran and obtained from suburbs of Arasbaran protected jungle (East Azerbaijan, Iran). The physical and chemical safety of the fruits was authenticated by the experts of Drug Applied Research Center in Tabriz University of Medical Sciences (Tabriz, IRI.). The fruits were air-dried, protected from direct sunlight and powdered. The powder was kept in a closed container at 8 °C.
Extraction
A total of, 500 g of the powder was extracted with a mixture of methanol (Merck, Germany): water (7:3) at 25 °C. The mixture was then filtered through 0.45 µ pore size filters and the solvent was completely removed by a rotary vacuum evaporator (Hidolf, Germany) at 40 °C. Finally, the CME was frozen and stored in a deep freezer (− 70 °C) until use [11]. The yield of the extract was 50% with reference to dry starting material.
Analyses of CMFE
The DPPH assay method [12] was used to assay the antioxidant properties of the CME and RC50 (50% reduction capacity) was expressed as mg/ml. The control material, quercetin RC50, was 4 µg/ml. The total phenolic equivalent was determined by using Folin-Ciocalteu reagent, estimated using the gallic acid standard curve, and expressed as mg of gallic acid equivalents (GAE) per gram of extract [13]. The sum total of flavonoids was determined as described by Vador et al. (2012) using the spectrophotometric method [12, 14].
Animals and Treatment
Male albino Wistar rats (250–300 g) were purchased from Pasteur Institute (Tehran, Iran). The animals were housed in polypropylene cages in a temperature-controlled room (22 ± 2 °C) with relative humidity (44–55%) under 12/12 h light and dark cycles for 1 week before and during the experiments. Animals were provided with a standard rodent pellet diet and clean drinking water ad libitum. Animals were divided into seven groups of eight animals in each:
The normal control group; orally received distilled water (DW) daily by gavage needle for 16 days and an IP (intra-peritoneal) injection of sterile DW water on day 11.
The Cis group, orally received DW daily for 16 days and an IP injection of 5 mg/kg Cis on day 11.
The CMFE group, orally received 700 mg/kgBW CMFE daily for 16 days and an IP injection of DW on day 11.
The CMFE 300 + Cis group, orally received 300 mg/kgBW CMFE daily for 16 days and an IP injection of 5 mg/kgBW CIS on day 11.
The CMFE 700 + CIS group, orally received 700 mg/kgBW CMFE daily for 16 days and an IP injection of 5 mg/kgBW Cis on day 11 [15, 16].
At the end of the experimental period under anaesthetic condition the blood samples were taken from each animal by cardiac puncture and then all the rats were sacrificed and liver tissues were excised. Tissue samples were fixed in 10% neutral buffered formalin during 24 h and embedded in paraffin.
Histopathological Evaluation
Histopathological evaluation was performed on liver tissues. Paraffin-embedded specimens were cut into 5-μm thick sections and stained with hematoxylin and eosin for examination under a light microscope (BH-2; Olympus, Tokyo, Japan).
The score of liver damage severity was semi-quantitatively assessed as follows: necrosis, inflammatory cells infiltration, fatty change, and focal congestion and haemorrhage. The microscopic score of each tissue was calculated separately for each parameter, as also the sum of the scores given to each criteria. Scores were given as absent (0), slight (1), moderate (2), and severe (3) for each criterion. The maximum score is noted as 12.
Biochemical Analyses
Of the taken liver samples 100 mg was homogenized in 1 mL ice cold phosphate buffer solution (PBS) (pH, 7.4) using a glass tissue homogenizer. Then, the homogenized samples were centrifuged for 10 min at 10000 g and 4 °C temperature. The supernatants were removed and stored in − 70 °C freezer until use.
The supernatant superoxide dismutase (SOD), glutathione peroxidase (GPx), and total antioxidant capacity (TAC) were determined by commercial kits (Randox, Italy). The catalase (CAT) activities of the supernatants were assayed by Cayman kit (USA). The malondialdehyde (MDA) contents of the supernatants were analysed by the barbituric acid method. Alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (GGT) of the blood serum were measured by the enzymatic methods (Parsazmun, Iran) with the automated biochemistry analyzer (Abbott, U.S.A.). The automated Abbott biochemistry analyser (Alcyon 300, USA), after calibrating and validating the biochemical tests, was used for performing the tests. All the supernatants’ results were divided and normalized with the supernatants’ protein contents, which were assessed by a commercial kit (Parsazmun, Iran) and expressed as per milligram tissue protein.
Animals’ Rights and Ethics
All protocols were approved by the Animal Research Ethics Committee of Tabriz University of Medical Sciences (certificate no.: 5-4-9723) and were performed in accordance with the related guidelines.
Statistical Analysis
All results are expressed as mean ± standard error of the mean one way analysis of variance followed by multiple comparison. Chi square test with the Tukey post hoc and independent samples T test was used to compare different parameters. P < 0.05 was considered to be significant.
Results
Antioxidant Indices of Cornus mas Hydromethanolic Extract
Composition of CMFE in terms of total phenolic and flavonoid compounds and antioxidant activity are represented in Table 1.
Table 1.
Composition of C. mas fruit extract (CMFE) (three measurements average)
| Sample | Antioxidant activity (RC50; µg/ml) | Total phenolic content (mg GAE/g extract) | Total flavonoid (%) |
|---|---|---|---|
| CMFE | 246.2 ± 0.2 | 131.5 ± 0.3 | 0.07 ± 0.01 |
Effect of CMFE on Liver and Serum Antioxidant Enzymes
The activity of liver antioxidant enzymes including SOD, GPx, TAC, and CAT in Cis group was found to be lower in the Cis group than in the control group. The activity of these enzymes in treatment groups were reversed when compared with Cis group that shown in Table 2. Effect of Cis and CMFE on liver MDA is shown in Table 2. Administration of Cis to rats significantly increased level of MDA, the indicator of lipid peroxidation. Treatment with CMFE protected against the Cis induced oxidative stress by reducing the lipid peroxidation. Moreover; a reduction in blood serum concentration of AST, ALT, and ALP and an increase in GGT level were found which were reversed in the control levels in the CMFE co-treated groups (Fig. 1).
Table 2.
Liver and serum antioxidant enzymes in control and treated groups (n = 8)
| Parameter#\group | Control | Cis | CMFE | CME 300 + Cis | CME 700 + Cis | P value |
|---|---|---|---|---|---|---|
| Serum antioxidant enzymes level | ||||||
| AST (u/l) | 190.67 (12.95) | 142.50 (24.30) | 156.33 (16.42) | 185.17 (44.23) | 185.00 (50.15) | 0.065 |
| ALT (u/l) | 110.83 (15.27) | 66.50 (10.89)£ | 79.50 (10.48) | 116.33 (19.96) | 114.20 (32.95) | < 0.001 |
| ALP (u/l) | 469.67 (140.15) | 145.17 (25.54)§ | 384.00 (82.87) | 328.83 (193.70) | 331.60 (102.13) | 0.003 |
| GGT (nmol/mg prot/min) | 4.33 (.51) | 7.67 (3.38) | 5.83 (2.63) | 5.17 (1.32) | 5.80 (2.49) | 0.181 |
| Liver antioxidant enzymes activity | ||||||
| SOD (U/mL) | 36.36 (3.22) | 26.07 (5.89)ŧ | 37.35 (4.38) | 37.61 (5.35) | 31.64 (9.85) | 0.011 |
| GPx (U/mL/100) | 71 (23) | 56 (5) | 80 (14) | 63 (13) | 62 (17) | 0.130 |
| TAC (µmol trolox equivalent/mL) | 170 (2) | 155 (5)ε | 161 (3) | 171 (5) | 173 (10) | 0.002 |
| CAT (nmol/min/mL) | 42.30 (8.82) | 27.17 (9.75) | 37.05 (11.13) | 38.27 (6.84) | 38.69 (8.18) | 0.086 |
| MDA (nmol/mL) | 9.88 (1.51) | 18.15 (8.04)η | 8.71 (4.08) | 8.06 (1.82) | 8.62 (1.16) | 0.002 |
AST Aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphatase, GGT gamma-glutamyl transferase, SOD superoxide dismutase, GPx glutathione peroxidase, TAC total antioxidant capacity, CAT catalase, MDA malondialdehyde
#Data are expressed as Mean (SD) and P.value based on Anova followed by tuky post hoc test
£P value < 0.05 compared with control, CDDP300, and CDDP700 group
§P value < 0.05 compared with control and CMFE group
ŧP value < 0.05 compared with control, CME, and CDDP300 group
εP value < 0.05 compared with control, CDDP300 and CDDP700 group
ηP value < 0.05 compared with control, CME, CDDP300, and CDDP700 group
Fig. 1.
Liver, rat. Inflammatory cell infiltration (arrows) associated with hepatic necrosis (arrow heads) and hemorrhage (H) in CMFE group
Effect of CMFE on Liver Histopathology
Liver histopathologic change indexes in control, Cis, and treated groups are shown in Table 3. Administration of Cis caused hyperemia, inflammatory cell infiltration, and hepatocyte necrosis (Fig. 2). Treatment with CMFE ameliorated the changes to near normal histology (Figs. 3, 4).
Table 3.
Liver histopathologic changes index in control and treated groups (n = 8)
| Parameter | Control | CMFE | Cis | CMFE300 + Cis | CMFE700 + Cis |
|---|---|---|---|---|---|
| Necrosis | 0.00 | 0.50 | 1.40* | 0.83 | 0.50 |
| Inflammation | 0.00 | 0.17 | 0.60 | 0.17 | 0.00 |
| Fatty change | 0.00 | 0.33 | 1.40* | 0.33 | 0.33 |
| Congestion and hemorrhage | 0.00 | 0.50 | 1.80* | 0.83 | 0.67 |
| Total pathologic changes | 0.00 | 1.50 | 5.20* | 2.17* | 1.50 |
CMFE Cornus mas fruit hydromethanolic extract, Cis cisplatin. The results are expressed as means of scores
*Significantly different when compared with the control group (P < 0.01)
Fig. 2.
Liver, rat. Hyperemia (strikes), inflammatory cell infiltration (arrows) and hepatocyte single necrosis (arrow heads) in Cis group
Fig. 3.
Liver, rat. Hyperemia (strikes), focal necrosis with inflammatory cell infiltration (arrows) and hepatocyte single necrosis (arrow heads) in CMFE300 + Cis group
Fig. 4.
Liver, rat. Hyperemia (strike) and hepatocyte single necrosis (arrow heads) associated with Kupffer cells hyperplasia (arrows) in CMFE700 + Cis group
Discussion
Effect of CMFE on Serum and Liver Antioxidant Enzymes
Using an experimental rat model, we evaluated the liver antioxidant enzymes activity and serum antioxidant enzymes level in Cis-treated rats with and without CMFE co-administration. The Cis caused difference in the antioxidant profile when compared to the control group. According to our findings, exposure to Cis caused a decrease in serum AST, ALT, and ALP levels; however, an increase was found in GGT level. Moreover, Cis-treated rats indicated decreased levels of antioxidant enzymes SOD, GPx, TAC, and CAT and decreased level of MDA, as a marker of lipid peroxidation. Taking a look at Table 2 it can be understood that supplementation with CMFE could reverse the changes in liver antioxidant enzymes activity and plasma antioxidant enzymes level caused by Cis. These results indicate oxidative stress induced by Cis, which can protects by CMFE administration.
The oxidative stress induction by Cis has been proven to be involved in Cis-induced toxicity [17, 18]. Oxidative stress, inflammation, fibrogenesis, and apoptosis are involved in the in vivo mechanism of Cis-induced changes and injuries. ROS can directly act on cell components, such as lipids and proteins, and destroy their structure. Cis can increase the presence of the produced ROS through all these pathways, resulting in the pathogenesis of Cis-induced adverse effects [19]. The results showed the protective effects of CMFE on oxidative stress biomarkers against hepatotoxicity induced by Cis in rats.
The restoration of liver and plasma enzymes in the Cornus mas co-treated rats suggest an improvement in the liver and plasma antioxidant status, which may be a function of the antioxidant properties of the Cornus mas phenolic and antioxidant contents. According to the previous study Cornus mas has high antioxidant and polyphenol contents [20]. The findings of present study were in accordance with other similar trials. Alavian et al. [21] showed that treatment with CMFE in the two different dosages at 200 and 500 mg/kg for 14 days remarkably prevented CCl4 induced changes of serum antioxidant enzymes activity. Somi et al. [22] in a similar research revealed that oral administration of CMFE to rats for 16 days, afforded hepato-protection against CCl4-induced changes in serum marker enzymes activities and liver antioxidant enzymes activities.
Considering the results of present study, administration of Cis resulted in significant elevation in liver MDA concentration, which is one of the end products of lipid peroxidation in the liver tissue, indicating elevation of lipid peroxidation along with histopathological injury. Treatment by CMFE markedly modulated the MDA concentration. Similarly Somi et al. [22] revealed that the evaluated level of MDA content in homogenate of rat liver was modulated in Cornus mas treatment groups at 200 and 500 mg/kg compared with the toxic group.
The hepato-protective effect CMFE may be due to its antioxidant components, leading to normalization of fluctuated biochemical profiles induced by Cis exposure. Therefore, plant extract compounds effect on the liver is to keep its normal function.
Effect of CMFE on Liver Histopathology
The current study was focused to investigate the protective effect of CMFE on Cis-induced hepatotoxicity in rat models. Cis is one of the most potent cytotoxic drugs commonly used for the treatment of variety of cancers such as testicular, ovarian, endometrial, lung, germ, bladder, head and neck, and cervical carcinoma. However, besides of its clinical efficiency, it has severe side effects such as nephrotoxicity, liver toxicity, bone marrow suppression, myelo-suppression, neurotoxicity, gastrointestinal toxicity, ototoxicity, and hypersensitivity reactions [23, 24]. Several studies have focused on the alternative approach to protect Cis-induced hepatotoxicity using natural products-derived antioxidants [2]. Our histopathology findings in the liver tissue of the rat revealed hyperemia, inflammatory cell infiltration and hepatocyte necrosis after the execution of a single dose of cisplatin. The histopathologic changes including liver necrosis, inflammation, fatty change, and congestion and hemorrhage were significant in Cis group compared with control group; however, treatment with CMFE improved the hepatotoxic changes induced by Cis (Table 3). The results of present study were in accordance with the study conducted by Alavian et al. showed that oral administration of CMFE by rats for 14 days provided a significant hepato-protection by decreasing elevated serum level of enzymes and liver lipid peroxidation content.
Considering the previous evidences the beneficial effect of CMFE might be due to the presence of antioxidant compounds [22]. The Cornus mas is a rich source of the antioxidants like anthocyanins and vitamin C. Previous investigations have shown the protective effects of the anthocyanin-rich and vitamin C-rich extracts from some natural products on the chronic hepatotoxic biochemical changes in the rats. Administration of the extract in two different dosages at 300 and 700 mg/kg improved liver histopathologic changes in Cis treated groups. Purification of the active component(s) of Cornus mas to determine the exact protective effects on hepatocytes is recommended for further studies.
Conclusion
In conclusion, the findings of the present study demonstrate that Cornus mas fruit extract can provide protection against cisplatin-induced changes in serum and liver antioxidant enzymes and liver histological structure. According to in vivo studies no interfere between natural antioxidants and cancer therapeutics has been reported. Moreover, co-administration of natural antioxidants, such as Cornus mas, and chemotherapy drugs, could enhance the effectiveness of the treatment and reduce the unwanted side effects.
Acknowledgements
The authors would like to thank Student Research Committee of Tabriz University of Medical Sciences for their financial support and the Drug Applied Research Center for technical support.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no competing interests.
Ethics Approval and Consent to Participate
The study was cleared by the Institutional Animal Ethical Committee of Liver and Gastrointestinal Diseases Research Center (LGDRC) (No 91/232 – LGDRC).
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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