Medicinal plants have important role in cancer treatments and protect the vital organs from the adverse effect of chemotherapy treatments. This study was conducted to investigate the ameliorating effects of Rosemarinus officinalis leaves (ROLE) and Saussurea lappa roots (SLRE) extracts along with cisplatin (Cis) on hepato-renal and cardiac-toxicities induced in Ehrlich-bearing mice. Forty female Swiss albino mice were divided into 4 groups (n=10) as follows; the first group (Gp1) was served as control. Gp2-Gp4 were inoculated with 1 x 106 EAC-cells/mouse.  Gp2 was left without any treatment as EAC-bearing mice, and Gp3 had injected with Cis (2 mg/Kg) starting from day 3 to day7. Gp4 had injected with Cis as in Gp3, and then administered with ROLE/ SLRE from day 3 to day 13. At day 14, blood samples were collected and heart tissue homogenate was prepared for biochemical analysis. Serum hepatic enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST) ɤ-glutamyltransferase (ɤ GGT), bilirubin, creatinine and urea levels were significantly increased in EAC-bearing mice, however, the treatment of EAC-bearing mice with Cis/ROLE/SLRE decreased the mentioned biochemical parameters. The contents of troponin (TnI) and creatine-phosphokinase (CPK) in the heart tissue of EAC-bearing mice were also increased; however, the treatment with Cis/ROLE/SLRE exhibited a marked ameliorative effect on both Tn1 and CPK. These results suggested that administration of ROLE/SLRE along with Cis mitigated the toxicity in EAC-bearing mice

Cancer is one of the most health problems worldwide; breast cancer is the most frequent among women. Advanced breast cancer is considered incurable under current available therapies with distant organ metastases (Seidler and Huber, 2020). Chemotherapy of breast cancer have a number of bad side effects (Sorsa et al., 1985). In spite of early detection and various treatments over the last 10 years that caused a significant decrease in mortality, the frequency of breast cancer continues to rise for multifactor including family history (Britt et al., 2020). Presently, patients are treated with traditional therapies such as surgery, chemo- and radio-therapy, and newer types of treatment such as immune- and targeted therapy, depending on the type and stage of cancer (Baudino, 2015). Chemotherapy, in a cyclic duration, vigorously inhibit the development of malignant cells, produce palliative relief and symptomatic relaxation (Alam et al.,  2018).

Cisplatin (Cis) is a potent anticancer drug used to treat a variety of solid tumors, including testicular, bladder, lung, breast, head and neck cancers (Schoch et al., 2020). Other drugs such as cyclophosphamide can alleviate the accompanied side effects of tumors (El-Bolkiny, 2006). The clinical efficacy of Cis, however, is limited by serious effects specifically resistance of tumor cells and effectiveness, as well as dose-limiting hepato-renal and cardiac toxicities (Oun et al., 2018; Ghosh, 2019). They showed that the anti-cancer activity of cisplatin is occurred through various ways; the most accepted is generating DNA lesions and the activation of several signal transduction processes that ultimately lead to cell apoptosis and its side effects prevention is one of the main problems during cancer therapy (Kailnde et al., 2020). 

Medicinal plants were used to isolate active natural compounds for cancer therapy (Alenzi et al., 2010). Previous study showed that a total of 151 medicinal plants were used to treat different types of cancer, such as breast, liver, prostate, stomach and brain cancer (Agyare et al., 2018). Khoshia indica had anti-tumor activity in HepG2 cell line (Abdel-hamid et al., 2017). Rosemary (Rosemarinus officinalis L.) and costus (Saussurea lappa) are characterized with a plenty of biological activities (El-Naggar, et al., 2016; Bolkiny et al., 2019). The anti-proliferative activity of rosemary extract was due to three main active components; Carnosol (CS), Carnosic acid (CA) and Rosmarinic acid (RA) as shown by Tai et al. (2012). In the same line, Costus is rich in antioxidant, anti-tumor, anti-inflammatory and anti-microbial ingredients, which widely used for treating different types of disturbances worldwide (Bolkiny et al., 2019). To reduce side effects and adverse toxicity of Cis, we hypothesized that combinatory form of rosemary and costus may affect or modulate organs toxicities induced by Cis treatment in EAC-bearing mice. Therefore, this work aimed to investigate the role of rosemary leaves and costus roots extracts along with Cisplatin to alleviate the hepato- renal and cardio- toxicities induced in EAC-bearing mice.

Chemicals and reagents

Cisplatin, phosphate buffered saline (PBS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from El-HEKMA company, (El-Dokki, Egypt). Kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST) ɤ-glutamyltransferase (ɤ GGT) total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IDB), urea and creatinine kits were obtained from Biodiagnostic Company, Egypt. Creatinine phosphokinase (CPK) and troponin (Tn-1) kits were obtained from MyBioSource, (Ireland). 

Rosemary leaves and costus roots extracts preparation

Rosemarinus officinalis leaves (Rosemary) and Saussurea lappa (Costus) roots were purchased from a local market, Alexandria City, Egypt. The plant materials were identified and authenticated by a taxonomist at Botany Department, Faculty of Science, Tanta University. R. officinalis leaves and S. lappa roots were grinded and then 50 g of each powder was mixed with 500 mL of 80% ethanol. After 3 days, R. officinalis leaves extract (ROLE) and S. lappa roots extract (SLRE) were collected and weighted to calculate yield extract percentage (%) from each plant parts (El-Naggar et al., 2016). 

Animals

Forty healthy adult female CD-1 mice (20 ± 2 g) were obtained from Cairo 

University, Egypt. Mice were maintained in suitable cages with standard hygienic circumstances. 12-hour Light/Day cycles and a balanced commercial diet with free access of water were provided. All the experiments were done at Zoology Department, Faculty of Science, Tanta University under coding number IACUC-SCITU-0099.

Mouse inoculation and experimental design

Ehrlich ascites carcinoma (EAC) cell line was obtained from the National Cancer Institute (Cairo University, Egypt). EAC cells (0.25 X l06 cells) were inoculated intraperitoneally injection (i.p.) in 200 µl of PBS. Mice were left for 10 days, then tumor cells were harvested under sterile conditions for the in vivo study (Geran et al., 1972). Mice were randomly divided equally into six groups (n=10). Group 1 (Gp1) was kept as a negative control. Mice in Gp2 - Gp4 were inoculated intraperitoneally with 0.25X106/mouse. Gp3 had treated with Cis. Gp4 had injected with a combination of Cis/ROLE/SLRE according to Salem et al. (2016).

Determination of biochemical parameters

The hepatic ALT and AST activities were determined according to Reitman and Frankel (1957). ɤ-GT activity was assessed according to Szasz et al. (1974). Serum bilirubin and urea was assessed according to Rand and Pasqua (1962) and Patton and Crouch (1977), respectively. Serum creatinine was determined based on the method of Bowers and Wong (1980). Creatine Phosphokinase (CPK) and Troponin I (Tn-1) were determined in the heart tissue by competitive ELIZA according to Hamilton et al. (2020) and Wilkinson and Grand, (1978), respectively.

Statistical analysis:

The obtained data were expressed as means ± standard deviation of the mean (X + SD). Differences between groups were determined using one-way ANOVA followed by Dunnette test and post-hoc test, Student’s t test where appropriate. Significant differences between means were indicated by p-values < 0>.

hepatotoxicity in EAC bearing mice.The tumor inoculation in mice induced significant increase in ALT, AST and ɣ-GT activities in EAC bearing mice as compared to naïve group (Gp1) (Table 1).

Treatment of EAC bearing mice with Cis (Gp3) led to a significant decrease in the activities of these enzymes as compared to Gp2 (p ≤ 0.05). Combinatorial treatment with Cis/ROLE/SLRE (Gp4) led to further significant decrease in the previous enzymes activities compared EAC-bearing mice that treated with Cis alone (Gp3). In addition, there was a high significant increase in TB, DB and IDB levels in EAC-bearing mice compared to the control. In Gp3, that treated with Cis, these levels of TB, DB and IDB decreased significantly compared to EAC-bearing mice (p ≤ 0.05). The levels of TB, DB and IDB were further decreased after treatment with ROLE/SLRE in EAC/Cis (Gp3) as shown in table (2).

The treatment with Cis/ROLE/SLRE lowered the renal-toxicity in EAC bearing mice.

The serum levels of creatinine and urea showed high significant increase (p ≤ 0.05) in EAC-bearing mice as compared to control. These levels of creatinine and urea in Gp3 decreased as compared to EAC-bearing mice when treated with Cis. As compared to Gp3, the levels of creatinine and urea were significantly lowered post-treatment with ROLE/SLRE in EAC/Cis mice group (Gp3) (Table 3).

Cis/ROLE/SLRE treatment reduced cardiotoxicity in EAC bearing mice.

There was a high significant increase in tissue creatin-phosphokinase (CPK) and troponin (Tn-1) in EAC-bearing mice as compared to the control. Treatment with Cis alone (Gp3) led to high significant decrease in the value of CPK and Tn-1 as compared to EAC-bearing mice. The CPK and Tn-1 were further reduced after treatment with ROLE/SLRE in EAC/Cis (Gp3) as shown in (Table 4).

This current study aimed to investigate the therapeutic role of ROLE and SLRE in a combinatorial form against cisplatin induced toxicity in EAC model. Present data showed a high significant increase in ALT, AST and GGT activities in EAC-bearing mice that in accordance with Hozayen et al. (2014). When treated with Cis slightly changes in serum ALT, AST and GGT activities compared to EAC- bearing mice alone as previously shown by Niu et al. (2017).  When treated with ROLE/SLRE together normalize the value near to the control, this may due to the complimentary effect of their antioxidant activity between two plants, reduce ROS formation, reduce hepatic injury and restore liver functions near to the control. In addition, our results revealed high levels of serum bilirubin (total, direct, indirect) in EAC-bearing-mice and those treated with Cis and decreased significantly after ROLE/SLRE administration compared to control. These results agree with Alexandre et al. (2000) and Wyld et al. (2003) who showed relationship of serum bilirubin level with survival rate in metastatic breast cancer patients. Present results are also in line with other data that assessed normal or increased level of bilirubin was observed during chemotherapy (Damodar et al., 2014). 

The current results showed an increase in the serum creatinine and urea levels in both EAC and EAC/Cis mice; however, the treatment with ROLE/SLRE improved these creatinine and urea levels toward normal. Our results are in agreement with Salem et al. (2011) who informed renal dysfunction in EAC-bearing mice and increased serum creatinine and urea levels. Moreover, our results agree with Coca and Parikh (2008) who concluded that increased creatinine and BUN in Cis treated animals indicated reduction in the glomerular filtration rate (Schrier et al., 2004). However, post-treatment with Rosmarinic acid (RA) is markedly ameliorated these pathological changes. These results agree with Tousson et al. (2019) who indicated a protective and medical role of rosemary in many organs. They also observed that results of creatinine and urea values were around negative control group. Other investigators showed nephron-protective properties of S. lappa on toxic effects of paracetamol due to the high concentration of flavonoids and alkaloids they contain as antioxidant and/or free-radical scavenging activities (GIRI et al., 2019).

The presence of the high level of urea in EAC-bearing mice comes in line with Abd Eldaim et al. (2019); El-Naggar & El-Said (2020) and agree with Aldubayan et al. (2019) who reported that elevation in serum kidney functions in Ehrlich solid tumor. Also, Cis reduced the elevated levels of serum urea and creatinine compared to EAC alone (El-Ablack et al., 2020). In the same line, significant decreases in the levels of blood urea and creatinine were observed in Cis treated mice after administration of ROLE/SLRE extracts. Urea reduction may due to its ability of ROLE to reduce renal dysfunction. Correspondingly, Dizaye (2012) suggested that R. officinalis extract contains constituents having nephron-protective and antioxidant properties with a potent scavenging ability of free radicals to prevent the toxic effects of Cis.

Cardiac tissue troponin (Tn) and creato-phosphokinase (CPK) are essential for prognosis in myocardial infarction and other cardiac diseases as cited by Ahmed et al. (2019). Our data showed that a significant increase in both Tn1 and CPK serum levels not only in EAC-bearing mice heart tissue, but also after treatment with Cis. Treatment of EAC/Cis mice with ROLE/SLRE together led to a marked improvement of Tn1 and CPK restoring it around normal levels. In a similar study, EAC causes the suppression of the mammalian target of rapamycin, a main signaling protein responsible for cell growth maintenance. This suppression is the major cause of cardiomyopathy associated with this type of cancer (Manne et al., 2013). Treatment with CP may increase heart weights and ventricular hypertrophy due to the pressure overload, which causes systolic and diastolic dysfunction (Liu et al., 2005). Similar findings are obtained in a recent study that demonstrated cardiotoxicity of a chemotherapy, doxorubicin (Saleh et al., 2020). 

The suggested mechanism of Cis-induced cardiotoxicity could be elucidated according to two elements; ROS generation and superoxide anion (HO) in mitochondria (Conklin and Nicolson, 2008). The generated radicals can extensively damage cardiac tissue by reacting with cell membrane lipids and proteins with subsequent nucleic acids’ damage and release free radicals and cardiac enzymes such as TnI and CPK-which considered as delicate markers of cardiac tissue injuries (Conklin & Nicolson, 2008). Consequently, attenuation of cardiotoxicity and protection of the heart against oxidative stress using free radical scavengers have previously been suggested (Swamy et al., 2011). The present concomitant usage of natural antioxidants ROLE / SLRE can play a therapeutic role to protect against or prevent Cis-induced cardiotoxicity in mice by lowering both Tn and CPK in the heart tissue. This suggests a protective and therapeutic role of rosemary and costus on the myocardium, reducing the myocardial damage, thereby restricting the leakage of these enzymes in serum. It can be concluded that administration of ROLE/SLRE extracts along with Cis treatment can ameliorate hepato-renal- and cardiac-toxicities induced toxicity in EAC- bearing mice.

All authors have approved this article and declare no conflicts of interest.