INTRODUCTION

It is estimated that by the year 2025 there will be approximately 20 million patients with cancer [1-4]. Therefore, attention is focused on preventive procedures as a definitive cancer management strategy [5, 6]. Current estimates point out that two-thirds of cancer-related deaths can be prohibited via way of lifestyle changes, particularly through nutritional means [6, 7].

Cancer side effects for the body and psychosocial stats can represent the most important and dangerous part of a cancer patient's life. So, nutritional factors especially fruits and vegetables to be taken very seriously by increased care as a preventative and lower considerable side effect of cancer chemical drugs [8].

A lot of research has detected that regularly eating a rich plant's diet on the top fruits and vegetables is closely linked to lowering the risk of cancer by containing them on an abundance of phytochemicals that have strong anti-cancer properties [9-11].

Many new studies confirmed that pomegranate fruit is considered a strong nutritional anticancer factor [12, 13]. Pomegranate fruit (Punica granatum L.) has been discovered to extend therapeutic activity such as antioxidants, anti-carcinogens, anti-inflammatory, antimicrobials, and anti-parasites [14-17]. Scientific evidence references that phytochemicals (phenolic compounds, anthocyanins, tannins, and others) in plants have therapeutic actions by their antioxidant characteristic [18]. Pomegranate fruit has polyphenols as antioxidants which possess a wide range of impact towards various kinds of free radicals as compared to different known antioxidants actions such as ascorbic acid, vitamin E, and β-carotene [19]. Anthocyanin in pomegranate fruit is one type of polyphenols that have high significant antioxidant activity compared to the famous antioxidants in green tea and red wine [20].

 Beetroot (Beta vulgaris L.) is a known vegetable that contains a large number of carbohydrates, fat, minerals, vitamins, and phytochemical ingredients with bioactive properties [21]. The phytochemical compounds of plants involve carotenoids, flavonoids, polyphenols, saponins, the water-soluble pigments like betalain, which interact in the cells to produce bioactive actions [22, 23]. The antioxidant power of the beetroot plant makes it at the pinnacle of ten vegetables because it has a high percentage of these phytochemicals [24, 25].

Several previous researchers have elucidated the anticancer activities of pomegranate and beetroot plant ingredients in a chain of human cancer cells [26-30].

Despite the development in the field of pharmaceutical industries, but it has a lot of cumulative interference on human health that leads to death in the long term for its use. As there is a great need to raise the immunity system against diseases, especially cancers, the idea was for this research. And because of what it contains pomegranate and beetroot plants and their mixture as natural foods, popular, accessible to everyone and its actions as protective chemical activities at the top anthocyanins and betalain, it was used against carcinogen of Ehrlich ascites carcinoma (EAC) as a tumor model in female mice.

MATERIALS AND METHODS

Materials

Animals

Female CD-1 mice (8 weeks of age) were obtained from Taif University KSA. Public Health Guide for the care and use of laboratory animals was applied by the guidelines of Ethics. Mice laboratory conditions were adapted by leaving a week before the experiment [31]. Ehrlich Ascites Carcinoma (EAC) was obtained from National Cancer Institute-Cairo, Egypt, and transplanted in mice as the ascitic form. Preparing the solution of phosphate buffer saline (PBS) at 2 ×10⁶ viable cells/ml was performed to induce tumor cell and injected intraperitoneally (i.p.) at dose 0.25×10⁶ tumor cells/mice suspended in 0.1 ml [32].

Chemicals and Plants

Cisplatin (cis-diamminedichloroplatinum) and other kits used in the analysis were purchased from Sigma–Aldrich, Co., USA. The plant materials pomegranate and beetroot were purchased from the local market. Fresh juices of pomegranate pulp seeds and beetroot were obtained after compressed and filtered directly by the electric mixer.

Experimental Design

Mice were divided into six groups nine mice per group. Gr.1: served as a negative control (healthy mice) injected by PBS (10 mg/mice i.p.). Gr.2:6 mice were injected (i.p) by EAC at dose 0.25 × 10⁶ cells/mice after treated with oral administration of juices for 15 days. Gr. 2: served as EAC positive control group. Gr. 3: served as a reference of chemically treated drug group by injected Cisplatin (10 mg/mice i.p.) two times from 5-7 days of injection of the EAC. All mice were feed by oral administration of pomegranate and beetroot juices before 15 days of injected EAC as nutritional treatment as following: Gr. 4: was take a single oral administration of pure pomegranate juice (1ml/mice) on an empty stomach daily. Gr. 5:  was take a single oral administration of beetroot juice on an empty stomach daily. Gr. 6: was take a mixture of pomegranate and beetroots juice as a single oral administration (1ml/mice) on an empty.

At the end of the experiment period, mice were fasted overnight then weighed and blood samples were drawn from the optic vein of the mice's eye and sacrificed by cephalic separation of the mice spinal cord. Biochemical analysis was performed by clear serum after the Eppendorf tube centrifuged at 3000 rpm. for 15 minutes.

Biological Evaluation

The animal diet was restricted daily as feed intake then body weight was recorded weekly. The biological evaluation was calculated by determination of body weight gain % (BWG %) feed efficiency ratio (FER) according to the method of Herawati et al. [33]. The liver of all mice groups was removed, washed with saline solution, dried with filter paper, and weighted according to the method of Drury and Wallington to calculate a relative liver weight [34].

Biochemical Analysis

Separated mice serum was taken to determine liver function as total protein and albumin according to Yatzidis then globulin calculated by formula= TP- Albumin [35].  Liver enzymes as 'Aspartate aminotransferase (AST), alanine aminotransferase (ALT) according to Anyanwu et al. [36], and alkaline phosphatase (ALP) according to Young et al. [37]. The serum blood lipid concentration was estimated as total cholesterol and HDL and LDL and VLDL were calculated by the method of Alsoodeeri et al. [38], triglyceride according to Fossati et al. [39].

Tumor Cell Count

The ascetic fluid was withdrawn from EAC-bearing mice after sacrificed by using a 10ml plastic syringe containing 5 mL of cold saline. EAC cells were separated individually from the ascetic fluid by centrifugation at 300 xg for 2 minutes and carefully transfer the supernatant fluid into a tube. PBS was used in the cells washing twice to eliminate the blood cells. Then, saline solution was re-suspended in a fixed volume for the washed packed cells. The tumor cell suspensions were prepared and enumerated using trypan blue dye exclusion and hemocytometer [40].

Statistical Analysis

All data results found in tables are expressed as means ± standard deviation (S.D.), P values <0.05 and 0.001 were considered significant which analyzed by one-way analysis of variance (ANOVA) using LSD test. The SPSS computer Program (v.16) were statistically analyzed by using methods of Armitage and Berry [41].

RESULTS ANDDISCUSSION

Biological Evaluation

Results in Table 1 show the effect of P, BR juices and their mixture on body weight gain (BWG%), feed intake (FI), feed efficiency ratio (FER), and the relative liver weight of mice injected with EAC, and CIS compared to the normal healthy mice. A relatively significant increment was observed in the body weight gain at (P < 0.05) in the P+ EAC and PB+ EAC groups (7.33±0.87 and 6.16±1.41, respectively). Feed efficiency ratio (FER) demonstrated a highly significant rise in the P juice group (1.98±0.53) as compared to the normal group. No significant change in the feed intake was found in any of the P, BR, and PB mix juice groups, also no significant increase in relative liver weight was recorded in oral administrated groups as compared to the normal group.

Data in the table recorded as mean ± SD of nine mice. * Significant P-value < (0.05) **Significant P-value <0.001compared to positive control (EAC) with LSD post-test.  EAC: Ehrlich Ascitic Carcinoma, CIS: cisplatin, P: Pomegranate, BR: Beet Root, PB: Pomegranate+ Beet Root.

Biochemical Analysis

Data in Table 2 illustrated results of liver enzymes as aspartate aminotransferase (AST) (U/L), alanine aminotransferase (ALT) (U/L), and alkaline phosphatase (ALP) (U/L) of all groups compared to the EAC control group. The levels of AST, ALT, and ALP recorded a significant increment at (P < 0.001) in the CIS group (35.00±3.74,37.83±1.16 and 332.27±3.93 U/L, respectively) as compared to the EAC control group.

On the other hand, daily oral administration juice with P group induced a significant reduction at (P < 0.001) in the level of liver enzymes (AST, ALT, and ALP) (15.33±2.25, 11.50±1.04 and 170.79±1.79 U/L, respectively), furthermore ALT and ALP revealed to a significant decrement (P < 0.001) of PB juice group (12.67±0.81 and 166.69±2.95 U/L, respectively, also, BR group demonstrated a significant diminish at (P < 0.001) with ALP enzyme (194.96±3.63 U/L) as compared to the EAC control group. It was noted that the best results were with the oral administration of P and PB juice.

Data in the table recorded as mean ± SD of nine mice. * Significant P-value < (0.05) **Significant P-value <0.001compared to positive control (EAC) with LSD post-test.  EAC: Ehrlich Ascitic Carcinoma, CIS: cisplatin, P: Pomegranate, BR: Beet Root, PB: Pomegranate+ Beet Root.

Results found in Table 3 showed total protein (TP), albumin, and globulin as liver functions of all groups compared to the EAC control group. The levels of TP and albumin recorded a significant increment at (P < 0.001) and (P < 0.05) in all of daily oral administration juices groups (6.59±0.26, 6.50±0.11, 7.07±0.26, 3.36±0.19, 3.31±0.15 and 3.51±0.22 g/dL, respectively) as compared to the EAC control group. It is also, noticed that daily oral administration of juices led to a noticeable rise of TP and albumin, especially when compared to CIS which leads to a marked decrease of the two function results. On the other hand, the globulin level recorded a non-significant increment in PB mixture juice as compared to the EAC group.

Data in the table recorded as mean ± SD of nine mice. *Significant P-value < (0.05) **Significant P-value <0.001compared to positive control (EAC) with LSD post-test.  EAC: Ehrlich Ascitic Carcinoma, CIS: cisplatin, P: Pomegranate, BR: Beet Root, PB: Pomegranate+ Beet Root.

In Table 4 the results review the total cholesterol (TC) (mg/dL), triglycerides (TG), high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very-low-density lipoproteins (VLDL) of all groups compared to the EAC control group.

The levels of TC, TG, LDL, and VLDL recorded a high significant raise at (P < 0.001) in the CIS group (144.21±5.89, 106±3.91, 97.08±6.12, and 21.38±1.07mg/dL, respectively). In contrast, the HDL level had a significant decrease (P < 0.001) in the CIS group (25.75±2.97 mg/dL) as compared to the EAC control group.

Daily oral administration with PBmix juices induced alow significant diminishes at (P < 0.001) in all levels of lipid profile as compared to the EAC control group. Followed by juice which revealed a low significant reduction at (P < 0.001) in the level of lipid profile except HDL level showed reduced at (P < 0.05) (38.51±1.28 mg/dL). At the end of the juice group effect, BR juice occurred a low significant least in TG and VLDL levels (110.41±4.99 and 22.00±1.32 mg/dL) at (P < 0.001), then in other data lower at (P < 0.05) as compared to the EAC control group.

These results proved that plant juices and their mixture led to a raise of benefit lipids as HDL and lower bad lipids when compared to EAC or CIS as a treated chemical drug.

Data in the table recorded as mean ± SD of nine mice. *Significant P-value < (0.05) **Significant P-value <0.001compared to positive control (EAC) with LSD post-test.  EAC: Ehrlich Ascitic Carcinoma, CIS: cisplatin, P: Pomegranate, BR: Beet Root, PB: Pomegranate+ Beet Root.