Effect of Cucumis melo Fruit and Its Silver Nanoparticles on Haematological Parameters
The blood cells are the mobile units of the body’s protective system. Anemia is frequently occurring in cancer patients [21]. Usually, in cancer chemotherapy the main problems that are being encountered are of myelosuppression and anemia [22]. Haematological parameters showed a decrease in the levels of Hb, RBC, MCV, MCH, MCHC and Platelets, and concomitant increase in WBC in DEN induced cancer animals. Significant alterations in the hematological parameters were observed in EECMF, SNPs-AECMF and cyclophosphamide treated rats which indicate the protective effect of Cucumis melo fruit on the hemopoietic system. Table 1 shows the levels of hemoglobin, lymphocyte count and RBC count in the control and experimental groups of rats.
Table 1 Effect of Cucumis melo fruit on hematological parameters of DEN induced HCC
Groups
|
HB
|
PCV
|
WBC
|
RBC
|
Platelets
|
MCV
|
MCH
|
MCHC
|
Group I (Control)
|
14.00 ± 0.14
|
42.00 ± 0.42
|
6.51 ± 0.11
|
6.00 ± 0.14
|
8.48 ± 0.14
|
55.50 ± 1.87
|
21.83 ± 0.43
|
33.16 ± 1.16
|
Group II (DEN induced)
|
9.55 ± 0.36 a
|
28.33 ± 1.03 a
|
13.98 ± 0.19 a
|
3.98 ± 0.14 a
|
4.46 ± 0.21 a
|
47.33 ± 1.75 a
|
18.18 ± 0.42 a
|
27.08 ± 0.39 a
|
Group III (DEN + EECMF)
|
11.20 ± 0.21 b c
|
33.62 ± 0.63 b c
|
9.98 ± 0.54 b
|
5.50 ± 0.08 b
|
5.70 ± 0.17 b
|
50.00 ± 1.41 b
|
19.51 ± 0.20 b
|
30.71 ± 1.21 b
|
Group IV (DEN + SNPs-AECMF)
|
12.33 ± 0.38 b
|
37.73 ± 1.51 b c
|
7.73 ± 0.12 b
|
6.05 ±0.18 b
|
6.58 ± 0.16 b
|
52.72 ± 1.23 b
|
20.45 ± 0.64 b
|
32.5 ± 0.98 b
|
Group V (DEN+Cyclophosphamide)
|
13.03 ± 0.16
|
38.85 ± 0.25
|
7.83 ± 0.16
|
6.38 ±0.14
|
7.08 ± 0.23
|
53.11 ± 0.27
|
21.26 ± 0.16
|
34.05 ± 0.18
|
Values are expressed as mean ± S.D for six animals
Statistical comparison:
a- Group II vs Group I b- Group III vs Group II, Group IV vs Group II c - Group III vs Group V and Group IV vs Group. The letters a, b and c represent the statistical significance at p < 0.05.
Units : Hb – g / dl, PCV - %, WBC – Thousands / mm3, RBC – millions / mm3, Platelets – lakhs / mm3 , MCV – fL, MCH – Pg, MCHC–g / dl
The levels of hemoglobin and RBC were found to be significantly (p < 0.05) decreased whereas WBC levels were significantly increased in (Group II) cancer bearing animals when compared with (Group I) control animals. Co-administration of EECMF and SNPs-AECMF, significantly altered the hematological parameters when compared with hepatocellular carcinoma induced rats. Significant variations were not observed in SNPs-AECMF (Group IV) treated rats compared to standard drug (cyclophosphamide) rats. Our finding is similar with the study of Althaf and Sudaroli who observed the significant reduction in the total WBC count and increase in the levels of hemoglobin and RBC when treated with ethanolic extract of Vitex leucoxylon Linn leaves on hepatocellular carcinoma induced rats [23]. Increased WBC count indicates the decreased resistance of the body to toxicity induced by DEN. Decreased RBC count and hemoglobin also indicate the severity of hepatic damage induced by DEN. Decrease in the hemoglobin levels might be due to increased catabolism and degradation of hemoglobin. Reduction in hemoglobin content can be related to decrease in RBC number which in turn indicates anemic induction [24]. Increased RBC, hemoglobin levels and other parameters indicated the protective effects of EECMF and SNPs-AECMF on the hemopoietic system.
EFFECT OF Cucumis melo Fruit ON LIVER FUNCTION MARKERS
The serum biomarkers and biochemical indicators can be an excellent tool or index to evaluate both functional and metabolic aspects of liver [25]. Several enzymes are produced in the liver and are normally distributed within the cells of the liver. Elevation of serum enzyme is taken as the sensitive biomarker of liver toxicity. The determination of various liver enzymes, alanine amino transferase (ALT), aspartate amino transaminase (AST), alkaline phosphatase ALP), ¡-glutamyl transpeptidase (¡-GT), lactate dehydrogenase (LDL) in serum helps to understand the functional status of liver and to detect liver injury. In the present study, DEN induced hepatocellular cancer is clearly evidenced by the marked elevation in serum SGPT, SGOT, GGT and ALP and a decreased level of these enzymes in the liver tissue, these biochemical marker enzymes are indicators of tumor response [26]. Serum GGT levels increased linearly with increases in small tumor mass, ALP levels are elevated in association with small tumors and further increases with increasing tumor mass [27].
ALP is used as a specific tumor marker during diagnosis in the early detection of cancer. It is well established that (ALT) levels signifies the presence of active disease and increases risk, particularly if the ALT is persistently or intermittently elevated over the years [28].
LDH is a cytosolic enzyme that catalyses the reversible oxidation of L-lactate to pyruvate. Their increased activity in serum confirms increased hepatocyte membrane permeability and cellular leakage. The levels of LDH is strongly correlated with tumor bulk as high rate of glycolysis takes place in cancerous condition, which is the only energy producing pathway for the malignant cells [29].
¡-GT is also believed to be an important indicator and the early enzyme marker of hepatocarcinogenesis. It is located in the outer membrane of the hepatic cells and their elevation reveals cholestasis and bile duct necrosis [30].
5’-nucleotidase (5’NT) is present at the bile canalicular and sinusoidal surface of the plasma membrane of hepatocytes. 5’NT hydrolyze nucleotides with a phosphate group on carbon atom of the ribose sugar and it was found to be elevated in cancer bearing animals. In addition, 5’NT is also used as a diagnostic tool for liver injury [31]. In the present investigation, an increased activity of 5’NT activity was observed in hepatocellular carcinoma bearing animals. The elevation of the marker enzyme may be correlated with the progression of the malignancy and also due to the hepatic cell damage, which may cause leakage of 5’nucleotidase into the circulation.
The activity of AST in the DEN induced carcinoma rats was found to be significantly increased in serum when compared to the control (Group I) rats. Similar trend was observed in the activities of ALT, ALP, LDH, GGT and 5’NT in serum of cancer bearing animals. Co-administration of EECMF and SNPs-AECMF treated rats caused a significant decrease in the levels of serum liver marker enzymes when compared to DEN induced rats (Group II). EECMF treated rats (Group III) was found to be significantly improved when compared with cyclophosphamide treated rats whereas significant difference was not observed in silver nanoparticles of aqueous extract of Cucumis melo fruit treatment.
Liver cancer induced rats (Group II) showed significantly (p < 0.05) elevated the levels of these enzymes when compared to control rats. Administration of EECMF and SNPs-AECMF treated rats significantly lowered the levels compared to cancer bearing animals. Significant changes were not observed in the silver nanoparticles treated rats (Group IV) when compared with standard drug cyclophosphamide treated rats. The activities of liver marker enzymes viz AST, ALT, ALP, LDH, GGT and 5’NT in liver of control and experimental rats is presented in Table 2.
Table 2 Effect of Cucumis melo fruit on the activities of hepatic marker enzymes in serum of DEN induced HCC
Groups
|
AST
|
ALT
|
ALP
|
LDH
|
GGT
|
5’NT
|
Group I
(Control)
|
72.14 ± 0.94
|
142.77 ± 1.53
|
17.44 ± 0.62
|
358.78 ± 1.84
|
21.28 ± 1.57
|
3.28 ± 0.21
|
Group II
(DEN induced)
|
41.06 ± 1.53a
|
129.13 ± 1.56 a
|
32.45 ± 1.32a
|
432.46 ± 2.06 a
|
36.20 ± 1.27 a
|
7.50 ± 0.14 a
|
Group III
(DEN + EECMF)
|
46.68 ± 2.38 b c
|
136.40 ± 2.07 b c
|
28.02 ± 1.11 b c
|
401.95 ± 5.11b c
|
31.64 ± 0.98 b c
|
6.58 ± 0.19 b c
|
Group IV
(DEN + SNPs-AECMF)
|
56.00 ± 0.87 b
|
147.41 ± 3.42 b
|
26.46 ± 2.08 b
|
363.80 ± 2.00 b
|
28.40 ± 1.68 b
|
5.45 ± 0.18 b
|
Group V
(DEN+Cyclophosphamide)
|
57.40 ± 1.06
|
149.30 ± 1.41
|
25.94 ± 1.67
|
365.90 ± 2.97
|
29.87 ± 1.08
|
5.25 ± 0.22
|
Values are expressed as mean ± S.D for six animals
Statistical comparison:
a- Group II vs Group I b- Group III vs Group II, Group IV vs Group II c - Group III vs Group V and Group IV vs Group V. The letters a, b and c represents the statistical significance at p < 0.05.
Units: AST, ALT, LDH - µ moles of pyruvate liberated / min / mg protein, ALP - µ moles of phenol liberated / min / mg protein, GGT - µ moles of p – nitroanilide liberated / min / mg protein, 5’ NT - µ moles of phosphorus liberated / min / mg protein.
Polyphenolic extract of Blighia sapidaarilli fruits at various doses were administered to DEN-induced carcinoma rats showed the reduced activities of ALP, ALT and AST in liver and serum enzymes are reported [32]. Our result agrees well with elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin and simultaneous fall in the levels of the marker enzymes in the liver tissue induced by DEN was altered after the administration of ethanolic leaf extract of the medicinal plant Cassia fistula Linn [33]. The structural integrity of the cells has been damaged in cancer induced animals, and this results in cytoplasmic leakage of enzyme in to the blood stream, leads to the elevated levels of these enzymes in blood with subsequent fall in the tissues [34].
Hence, EECMF and SNPs-AECMF could have exerted their therapeutic effect against DEN induced HCC probably by preventing membrane damage, loss of integrity as well as by repairing hepatic tissue damage caused by tumor induction, thus inhibiting the release of these marker enzymes into the serum, indicating that Cucumis melo fruit has the ability to prevent further development of HCC.
EFFECT OF Cucumis melo fruit ON THE LEVELS OF CANCER MARKERS
For hepatocellular carcinoma (HCC), the common method of screening high risk patients by alpha-fetoprotein (AFP) and ultrasonography has been shown to result in earlier detection [35]. AFP is a 70-kD glycoprotein consisting of 591 amino acids and 4 % carbohydrate residues, encoded by a gene on chromosome 4q11-q13. Normally produced during gestation by the fetal liver and yolk sac, AFP is highly elevated in the circulation of newborns with concentrations decreasing during the next 12 months to 10-20 μg / L. Only when serum levels of AFP rise above 500 ng / ml, it is sufficiently specific for hepatocellular carcinoma (HCC) [36]. The serum marker, alpha fetoprotein (AFP) has been used as a diagnostic test for HCC [37]. AFP, as a specific sign of tumor, could provide the important referential value for cancer diagnosis, histopathological classification and prognosis. AFP content in tumor patient was remarkably higher than the normal tissue near carcinoma and the precancerous lesion tissue [38].
Carcino Embryonic Antigen (CEA) is an important tumor-associated antigen, and it’s over expression has been used to identify or diagnose early colorectal, gastric, pancreatic, ovarian cancer and liver cancer. Over expression of CEA in serum always existed in metastatic HCC, and not in primary HCC. CEA are considered cDNA of fatty acid synthesis , tumor necrosis factor specific biomarkers for liver cancer and it is synthesized (TNF) and the house keeping gene-actin as a control, mainly in the fetal stage; practically no production of this were analyzed and the results of gene expression was marker occurs in the normal adult [39].
The levels of alpha feto protein and carcinoembryonic antigen in the serum of control and experimental rats are given in the Figure 1.
Values are expressed as mean ± S.D for six animals
Group I- Control; Group II- DEN induced HCC; Group III- DEN + EECMF; Group IV- DEN + SNPs-AECMF; Group V- DEN + Cyclophosphamide
Statistical comparison:
a- Group II vs Group I; b- Group III vs Group II, Group IV vs Group II; c – Group III vs Group V and Group IV vs Group V. The letters a, b and c represents the statistical significance at p < 0.05.
Hepatoma bearing rats (Group II) possessed increased levels of alpha feto protein and CEA when compared to control rats. Treatment with EECMF (Group III) and SNPs-AECMF (Group IV) decreased the levels of AFP and CEA when compared to DEN induced HCC rats (Group II). Treatment with the EECMF and SNPs-AECMF caused a significant depletion in the levels of AFP and significant difference was not recorded when compared to standard drug treated rats. Administration of limonin isolated from orange and lemon seeds, decrease the tumor marker AFP levels might against
N-nitrosodiethylamine (DEN, 200 mg / kg) induced phenobarbital promoted experimental hepatocellular carcinoma in male wistar albino rats. Morin (3, 5, 7, 2’, 4’-pentahydroxyflavone), a plant-derived flavonoid significantly lowered the AFP and CEA levels in DEN-induced cancer rats [40].
Exposure of rats to certain carcinogens like DEN causes an elevation of circulating AFP level. CEA is cleared from the circulation by the liver with significant traces taken up by the spleen and lungs. An increase in serum CEA levels upon DEN treatment was presumably associated with production rates of tumor, its location and stage, size, differentiation and vascularity [41]. Upon liver injury these enzymes enter into the circulatory system due to altered permeability of the membrane. The observed reduction in the levels of AFP and CEA in EECMF and SNPs- AECMF treated animals might be due to a decrease in the rate of tumor development, indicating their anticancer activity because of the presence of alkaloids, flavanoids, terpenoids and other phytochemicals of Cucumis melo fruit extract [42].
Histopathological Investigations of Liver and Kidney
Microscopic observations of Cucumis melo fruit treated DEN-induced rat liver are given in Figure 2 shows the histopathological examination of liver and kidney. Control (Group I) rats revealed normal liver hepatocytes with granulated cytoplasm, small uniform nuclei, and central vein surrounded by cords of hepatocytes and normal architecture. Group II (DEN-induced) rats showed loss of architecture and lobules of neoplastic hepatocytes with a focal area of fatty change. Group III (EECMF) rats exhibited DEN showed a moderate cancerous change, fatty change, and hydropic degeneration. Groups IV (SNPs-AECMF) and Group V (Standard drug) rats showed fewer neoplastically-transformed cells and the hepatocytes maintained near-normal architecture.
The histological study of rat kidneys showing Group I renal glomerular tubules and normal architecture. Group II (DEN induced HCC rats) showing blood vessel congestion and inflammatory infiltration. Group III (EECMF treated rats) shows moderate renal glomeruli congestion of intersitial blood vessels and hemorrhage with minimal inflammatory infiltration. Group IV (SNPs-AECMF) and Group V (Cyclophosphamide) treated rats shows mild glomeruli congestion with narrow focal dilation and mild inflammatory infiltration.
Histopathological investigation of liver showed loss of architecture and lobules of neoplastic hepatocytes with a focal area of fatty change in DEN induced liver cancer rats. These changes were found to be reduced tremendously in EECMF, SNPs-AECMF and standard drug cyclophosphamide treated animals. Kidney of cancer induced rats showed tubular damage and portal inflammation within due to glomerular damage. The kidney of treated rats with EECMF and SNPs- AECMF showed the mild portal inflammation. Treatment with Cucumis melo fruit extract showed the improvement in the architecture of liver and kidney of cancer rats which might be due to the reduction in the cancerous condition of the organs.