Effect of EEEA on Body Weight Changes in Control and Experimental Rats
Body weight changes have been used to evaluate the cause of the disease and response to drug therapies. Body weight gain by the animal is depending on the functional capacity of liver. Table 1 represents the initial and final body weight changes of rats from Group I – Group V. No significant change was found in the initial body weight among the five groups of rats. But the body weight was decreased significantly (P < 0.05) in DEN induced Group II rats when compared to the control Group I rats whereas in EEEA post treated Group III rats, it appeared near normal when compared with the Control Group I rats. This observation was in similar with Song et al., (2013) [27]. DEN brought a significant impairment in body growth. There were no significant (P < 0.05) changes observed among pre-treated Group IV and Standard treated Group V rats when compared with Control Group I rats. No death was observed in the experimental rats. This indicates the anticancerous effect of EEEA on hepatocellular carcinoma group.
Table 1
Effect of EEEA on Body Weight in Control and Experimental Animals
Organ (s)
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Weight of the rats before treatment (g)
|
180 ± 10.25
|
186 ± 11.74
|
188 ± 12.12
|
185 ± 11.02
|
190 ± 12.55
|
Weight of the rats after treatment (g)
|
228 ± 14.65ns
|
202 ± 10.32*
|
230 ± 13.21ns
|
227 ± 14.11ns
|
234 ± 12.95ns
|
Values are expressed as Mean ± SD for six rats
Data were analyzed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.001, ns = Non-significant (P < 0.05).
Effect of EEEA on Organ weight changes in Control and Experimental Rats
Table 2 shows the liver, kidney, spleen, brain, and heart weights of control and experimental rats. Due to carcinogenesis, Group II rats were observed to have a significant increase (P < 0.000) in the organ weight when compared with Control Group I rats. The post treated Group III rats show significant increase in liver, kidney, and spleen (P < 0.001), brain (P < 0.022) and significant decrease in heart (P < 0.014,) weights when compared with the Control Group I rat. Observation of current study was similar to the findings of Furuta et al., (2008) [28] where he was described the gradual increase in liver weight. It was also studied by Mohammed et al., (2014) [29] that the liver weight in HCC induced rat showed slight increase when compared to the control rats. However, when compared with Group II rats, a gradual increase in the weights of the organs were observed in Post treated Group III rats indicating reduced proliferation of cells in these groups which indicates the effect of EEEA on HCC. Pre-treated Group IV and Silymarin treated Group V rats show non-significant (P > 0.01) weight in the organs when compared with the Control Group I rats.
Table 2
Effect of EEEA on Organ Weight in Control and Experimental Animals
Organ (s)
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Liver (g)
|
3.97 ± 0.24
|
6.63 ± 0.28 *
|
4.82 ± 0.13$
|
4.09 ± 0.28ns
|
3.87 ± 0.10ns
|
Kidney (g)
|
1.06 ± 0.03
|
1.83 ± 0.17 *
|
1.32 ± 0.16 $
|
1.09 ± 0.09ns
|
1.11 ± 0.06 ns
|
Spleen (g)
|
0.32 ± 0.07
|
1.19 ± 0.06 *
|
0.68 ± 0.08 $
|
0.36 ± 0.08 ns
|
0.37 ± 0.09ns
|
Brain (g)
|
1.49 ± 0.10
|
1.20 ± 0.05*
|
1.28 ± 0.02#
|
1.55 ± 0.14ns
|
1.58 ± 0.11ns
|
Heart (g)
|
0.66 ± 0.10
|
1.04 ± 0.11 *
|
0.81 ± 0.02@
|
0.67 ± 0.06ns
|
0.65 ± 0.07ns
|
Values are expressed as Mean ± SD for six rats
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000, $P = 0.001, #P = 0.014, @P = 0.022 and ns = Non-significant (P < 0.05).
Measurement of Tumor Size
Tumor sizes for Group II rats were measured using vernier callipers on end of the experimental period which was depicted in the Table 3. An average size of tumor was found to be 7.32 mm at the end of the experiment (16th weeks) was observed in Group II DEN induced rats. Group III post treated rats, Group IV pre-treated rats and Group V Silymarin treated rats show reduced tumor size (0.25, 0.13 and 0.14) respectively, while Group I Control rats did not have tumor.
Table 3
Effect of EEEA on Tumor Size in Liver of Control and Experimental Animals
Liver
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Size of Tumor (mm)
|
0
|
7.31 ± 0.47*
|
0.20 ± 0.05ns
|
0.13 ± 0.03ns
|
0.14 ± 0.02ns
|
Values are expressed as Mean ± SD for six rats (n = 6) and Data were analysed by Duncan’s multiple range test (DMRT). Statistically significant variation was derived by comparing Group II versus Group III, Group IV and Group V. *P = 0.05, ns = Non-significant (P < 0.05).
Effect of EEEA on Serum Antioxidant Status in Control and Experimental Rats
Table 4 shows the levels of serum antioxidant enzymes in control and experimental rats. In carcinogenesis lipid peroxidation plays a major role [30], is the most studied biologically relevant free radical chain reaction and measured as Malonaldehyde (MDA). A significant (P < 0.000) increase in the level of MDA and significant (P < 0.000) decrease in the level of GSH, SOD, CAT, GPx, Vitamin C, and Vitamin E were noticeable in DEN induced Group II rats when compared with the Control Group I rat. Our result agrees with the previous study [31]. Antioxidant levels were reverted back to near normal levels in Pre-treated Group IV and Standard drug treated Group V rats when compared with DEN induced Group II rats. Thus, the drug EEEA restored the changes to near normal by its antioxidant efficiency. The control Group I rats exhibit a near-normal value of these enzymes whereas post treated Group III rats show significant(P < 0.000) increase in MDA and significant (P < 0.001) decrease in Catalase and Vitamin E levels and significant (P < 0.05) decrease in GSH, SOD, GPx and Vitamin C levels when compared with Control Group I rats. Along with Vitamin E and Glutathione, Vitamin C also scavenges and detoxifies free radicals [32]. However, when compared with Group II rats, a significant recovery in the antioxidant status were observed in Post treated Group III rats. EEEA has the ability to restore the levels of SOD, CAT, GPx, Vitamin C, Vitamin E and increased GSH content and also its ability to decrease the levels of lipid peroxidation.
Table 4
Effect of EEEA on Serum Antioxidant in Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
MDA (nmol of MDA formed/L)
|
7.38 ± 0.68
|
14.68 ± 1.83 *
|
11.12 ± 0.46 *
|
8.04 ± 0.45ns
|
7.29 ± 0.56ns
|
GSH (mg/ dl)
|
8.45 ± 0.87
|
5.50 ± 0.84 *
|
6.71 ± 0.38 $
|
7.94 ± 0.49ns
|
8.14 ± 0.54ns
|
SOD (U/ml)
|
4.76 ± 0.57
|
3.24 ± 0.21 *
|
3.88 ± 0.13 $
|
4.56 ± 0.22ns
|
4.51 ± 0.38ns
|
Catalase (U/ml)
|
9.43 ± 0.55
|
6.34 ± 0.51 *
|
7.56 ± 0.18 *
|
9.02 ± 0.51ns
|
9.61 ± 0.83ns
|
GPx (U/ml)
|
9.19 ± 0.71
|
6.54 ± 0.39 *
|
7.80 ± 0.21 $
|
8.83 ± 0.63ns
|
9.03 ± 0.46ns
|
Vit-C (µg/dl)
|
4.51 ± 0.37
|
2.18 ± 0.30 *
|
3.20 ± 0.66 $
|
4.41 ± 0.56 ns
|
4.61 ± 0.36ns
|
Vit-E (µg/dl)
|
3.85 ± 0.26
|
2.16 ± 0.24 *
|
2.66 ± 0.19 *
|
3.46 ± 0.43ns
|
3.60 ± 0.45ns
|
Values are expressed as Mean ± SD for six rats |
Data were analysed by one-way ANOVA followed by post-hoc Bonferroni test. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000, $P = 0.001 and NS = Non-significant (P < 0.05). |
SOD (U) = 50% of NBT reduction/min; Catalase (U) = µmol of H2O2 consumed/min; |
GPx (U) = µmole of GSH utilized/min |
Effect of EEEA on Antioxidant in Liver Tissues of Control and Experimental Rats
Table 5 shows the levels of liver tissue antioxidant in control and experimental rats. A significant (P < 0.000) increase in the level of MDA and significant (P < 0.000) decrease in the levels of GSH, SOD, CAT, GPx, Vitamin C, and Vitamin E were noticeable in DEN induced Group II rats when compared with the Control Group I rat. It leads to further production of free radicals overwhelming the cellular antioxidant defence [33]. The decreased levels of these antioxidant vitamins and GSH observed in Group II rats during DEN administration might be due to the excessive utilization of these vitamins in scavenging free radicals. A similar finding has been made in the seaweed Acanthophora spicifera [34]. Antioxidant levels were reverted back to near normal levels and non-significant (P < 0.05) changes in Pre-treated Group IV and Silymarin treated Group V rats when compared with DEN induced Group II rats. The Control Group I rats exhibit a near-normal value of these enzymes whereas post treated Group III rats show significant (P < 0.002) increase in MDA and significant (P < 0.002) decrease in Catalase, GSH, SOD, GPx, Vitamin E, and Vitamin C levels when compared with Control Group I rats. In the antioxidant system, SOD is the first line of defence against the oxidative damage by superoxide radicals [35]. However, when compared with DEN induced Group II rats, a significant recovery in the antioxidant status were observed in Post treated Group III rats. The present investigation highlights the chemopreventive potential of Enhalus acoroides against DEN induced HCC by quenching lipid peroxidation and increasing the antioxidant status in the RBC through free radical scavenging and has the potential of protecting the endogenous enzymatic and non-enzymatic antioxidant activities.
Table 5
Effect of EEEA on Antioxidant in Liver Tissues of Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
MDA (nmol of MDA formed/L)
|
10.45 ± 0.45
|
15.63 ± 0.58 *
|
12.63 ± 0.65 $
|
9.51 ± 0.59ns
|
10.28 ± 0.59 ns
|
GSH (mg/ dl)
|
6.52 ± 0.39
|
4.87 ± 0.43 *
|
5.66 ± 0.13 $
|
6.35 ± 0.25ns
|
6.55 ± 0.38ns
|
SOD (U/ml)
|
3.10 ± 0.37
|
2.02 ± 0.10 *
|
2.36 ± 0.09 $
|
2.94 ± 0.24ns
|
3.11 ± 0.27ns
|
Catalase (U/ml)
|
4.55 ± 0.42
|
2.29 ± 0.37 *
|
3.53 ± 0.38 $
|
4.21 ± 0.10ns
|
4.54 ± 0.39ns
|
GPx (U/ml)
|
8.03 ± 0.20
|
5.08 ± 0.26 *
|
6.79 ± 0.21 $
|
7.59 ± 0.46ns
|
7.94 ± 0.35ns
|
Vit-C (µg/dl)
|
5.41 ± 0.33
|
3.16 ± 0.24 *
|
3.85 ± 0.27$
|
4.92 ± 0.52ns
|
5.16 ± 0.25ns
|
Vit-E (µg/dl)
|
4.20 ± 0.24
|
2.48 ± 0.36 *
|
3.45 ± 0.15 $
|
3.95 ± 0.15ns
|
4.10 ± 0.34ns
|
Values are expressed as Mean ± SD for six rats
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P < 0.000, $P < 0.002, and ns = Non-significant (P < 0.05).
3.6 Effect of EEEA on Tumor Markers in Control and Experimental Rats
Tumor markers produced by the tumor and when present in elevated levels, indicate the presence of carcinoma as intracellular substances in tissues or may be released into the circulation and found in serum [36]. AFP, DNA, RNA CEA and liver weight are considered to be most important references, broadly used in animal studies to diagnose and observe the development of hepatocellular carcinoma [37]. Table 6 indicates the effects of EEEA activity of Tumor markers such as DNA, RNA, AFP, and CEA of control and experimental rats. The levels of tumor markers were significantly (P < 0.000) elevated in DEN induced Group II rats compared with the Control Group I rat. AFP, a tumour associated fetal protein, has long been employed as a serum fetal tumour marker to monitor disease progression [38]. RNA levels were found to be increased in the cancerous condition as DNA and RNA are directly related to each other, an abnormally increased content of DNA may lead to an increased transcription, which in turn increased RNA content in tumor cells. Present findings are similar to the Pakkir et al., (2011) [39] study. Tumor markers were reverted back to near normal levels and non-significant (P < 0.05) in pre-treated Group IV and Silymarin treated Group V rats when compared with Control Group I rats. Present findings are in concordance with Nermin et al., (2008) [40] study. Post treated Group III rats show significant (P < 0.009, P < 0.003, and P < 0.002) elevation in RNA, AFP, and CEA levels respectively when compared with the Control Group I rat. However, a significant decrease when compared with DEN induced Group II rats and significant increase when compared with the Control Group I rat in the levels of these tumor markers indicate a significant antitumor activity of ethanolic extract of Enhalus acoroides.
Table 6
Effect of EEEA on Tumor Markers in Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
DNA
(mg/g wet tissue)
|
1.99 ± 0.20
|
3.14 ±. 0.56*
|
2.67 ± 0.19ns
|
2.05 ± 0.12ns
|
2.04 ± 0.99ns
|
RNA
(mg/g wet tissue)
|
2.32 ± 0.75
|
3.75 ± 0.14*
|
3.23 ± 0.29#
|
2.46 ± 0.36ns
|
2.49 ± 0.29ns
|
AFP (ng/ml)
|
0.09 ± 0.03
|
0.64 ± 0.15*
|
0.36 ± 0.07$
|
0.15 ± 0.06ns
|
0.14 ± 0.05ns
|
CEA (ng/ml)
|
0.05 ± 0.01
|
0.46 ± 0.13*
|
0.21 ± 0.05@
|
0.07 ± 0.01ns
|
0.06 ± 0.03ns
|
Values are expressed as Mean ± SD for six rats
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000, $P = 0.003, #P = 0.009, @P = 0.002 and ns = Non-significant (P < 0.05).
3.7 Effect of EEEA on Serum Liver Markers in Control and Experimental Rats
Table 7 indicates the effects of EEEA activity of the serum liver marker enzymes such as AST, ALT, ALP, GGT and non-enzymatic liver markers such as Bilirubin, Protein, Albumin, and Globulin of control and experimental rats. DEN induced Group II rats’ exhibit significant change (P > 0.000) in the activity of these liver markers when compared to Control Group I rats. Rocchi et al., (1997) [41] stated that there was an elevation in the levels of transaminases in serum of HCC patients. In concurrent with the above reports an elevated serum aminotransferase were observed in Group II rats bearing HCC, whereas they appeared to be neutralized to near normal and non-significant (P < 0.05) in Group IV EEEA pre-treated rats. Due to the development of tumor, tissue gets damaged which leads to the elevation of ALP into circulation [42] and this enzyme level have been increased in serum of the tumor bearing rats and this elevation is significantly suppressed by the supplementation of EEEA. Standard drug treated Group V rats do not show noticeable changes in these parameters and non-significant (P < 0.05) when compared with the Control Group I rats. Post treated Group III rats show significant changes in GGT (P < 0.000), ALT, AST, ALP, Bilirubin, and protein (P < 0.001), albumin (P < 0.038) levels when compared with the Control Group I rat. The A/G ratio is primarily used to evaluate the liver function. The decrease in A/G ratio in post- treated Group III and pre-treated Group IV rats after the treatment was in similar pattern as that of control Groups I rats. This indicates the protective effect of EEEA over liver and improvement in its functional efficiency.
Table 7
Effect of EEEA on Serum Liver Markers in Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
ALT (IU/L).
|
28.18 ± 1.23
|
59.61 ± 1.39 *
|
37.67 ± 1.51$
|
25.58 ± 2.17ns
|
27.24 ± 1.57ns
|
AST (IU/L).
|
57.64 ± 1.42
|
82.61 ± 2.20 *
|
65.08 ± 1.86$
|
59.53 ± 1.83 ns
|
60.09 ± 1.77ns
|
ALP(IU/L)
|
60.63 ± 1.91
|
71.67 ± 1.73 *
|
65.85 ± 0.88 $
|
59.98 ± 2.81ns
|
59.12 ± 1.83ns
|
GGT (IU/L)
|
19.03 ± 1.39
|
35.58 ± 2.43 *
|
29.24 ± 1.48 *
|
20.38 ± 1.59 ns
|
21.40 ± 1.46ns
|
Bilirubin (mg/dl)
|
0.89 ± 0.16
|
1.86 ± 0.06 *
|
1.42 ± 0.15$
|
1.07 ± 0.19ns
|
0.98 ± 0.04ns
|
Protein (gm/dl)
|
7.38 ± 0.31
|
4.54 ± 0.30 *
|
6.08 ± 0.33$
|
6.98 ± 0.36ns
|
7.33 ± 0.62ns
|
Albumin (gm/dl)
|
3.79 ± 0.42
|
2.89 ± 0.31 *
|
3.18 ± 0.17 @
|
3.78 ± 0.39ns
|
4.03 ± 0.32ns
|
Globulin (gm/dl)
|
3.59 ± 0.55
|
1.65 ± 0.43 *
|
3.04 ± 0.33ns
|
3.20 ± 0.40ns
|
3.30 ± 0.58ns
|
A/G ratio
|
1.09 ± 0.27
|
1.75 ± 0.47*
|
1.12 ± 0.19ns
|
1.00 ± 0.10ns
|
1.25 ± 0. ns
|
Values are expressed as Mean ± SD for six rats |
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000, $P = 0.001, #P = 0.011, @P = 0.038 and ns = Non-significant (P < 0.05). |
Effect of EEEA on Serum Kidney Markers in Control and Experimental Rats
Table 8 indicates the effects of EEEA activity of the serum kidney marker such as Urea and Creatinine of control and experimental rats. Induction of oxidative stress by DEN altered the functions of kidney markers in rats. DEN induced hepatic rats showed impairment in kidney function which was indicated by the significantly increased levels of serum urea, and creatinine. In concurrent with the above statement, our results indicated that the exposure of rats to Group II DEN induced rats cause significant (P < 0.000) increase in the levels of urea and creatinine when compared with the Control Group I rat. Our results were similar to that of who showed that Tabernaemontana coronaria caused a marked reduction in the levels of blood urea and serum creatinine in DEN induced rats [43]. Kidney markers were reverted back to near normal levels and non-significant (P < 0.05) in pre-treated Group IV and Silymarin treated Group V rats when compared with Control Group I rats. Post treated Group III rats show significant (P < 0.001 and P < 0.041) increase in urea and creatinine levels respectively when compared with the Control Group I rat. However, a significant decrease when compared with DEN induced Group II rats and significant increase when compared with the Control Group I rat in the levels of these kidney marker enzymes shows the potential renal functions of EEEA.
Table 8
Effect of EEEA on Serum Kidney Markers in Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Urea (mg/dl)
|
26.19 ± 1.85
|
40.71 ± 1.28 *
|
31.91 ± 1.18$
|
25.35 ± 1.98ns
|
26.39 ± 1.68ns
|
Creatinine (mg/dl)
|
0.70 ± 0.03
|
0.81 ± 0.02*
|
0.74 ± 0.02#
|
0.71 ± 0.02ns
|
0.69 ± 0.03ns
|
Values are expressed as Mean ± SD for six rats
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000, $P = 0.001, #P = 0.041 and ns = Non-significant (P < 0.05).
Effect of EEEA on Serum Lipid Profile in Control and Experimental Rats
Table 9 indicates the effects of EEEA activity of Lipid parameters such as Triglycerides (TG), Total cholesterol (TC), HDL, and LDL of control and experimental rats. The levels of total cholesterol, triglycerides, and LDL in serum of control and experimental rats were significantly (P < 0.001) increased, and HDL level was significantly (P < 0.001) decreased in Group II DEN induced HCC rats when compared with the control Group I rats. The treatment with EEEA showed significantly altered the levels of lipid profile when compared with DEN induced Group II rats. EEEA post treated Group III rats showed significantly decreased levels of total cholesterol and triglycerides when compared with DEN induced rats. No significant (P < 0.000) changes were observed in EEEA pre-treated Group IV and Silymarin treated Group V rats compared to Control Group I rats indicated the effects of EEEA in maintaining the normal status of lipid profile in the serum.
Table 9
Effect of EEEA on Serum Lipid Profile in Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Triglycerides (mg/dl)
|
109.34 ± 3.56
|
141.34 ± 2.91*
|
124.53 ± 3.15*
|
112.40 ± 2.38ns
|
110.83 ± 2.43ns
|
Total cholesterol (mg/dl)
|
89.90 ± 2.82
|
150.37 ± 2.40*
|
133.71 ± 2.43*
|
93.70 ± 1.76ns
|
91.46 ± 2.89ns
|
HDL (mg/dl)
|
35.31 ± 1.92
|
22.69 ± 1.31*
|
26.14 ± 1.82*
|
33.50 ± 2.17ns
|
31.21 ± 2.01ns
|
LDL (mg/dl)
|
32.72 ± 3.73
|
99.42 ± 2.60*
|
82.67 ± 2.35*
|
37.72 ± 1.74 ns
|
38.09 ± 4.68ns
|
Values are expressed as Mean ± SD for six rats
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000 and ns = Non-significant (P < 0.05).
Effect of EEEA on Blood Haematological Profile in Control and Experimental Rats
Table 10 shows the levels of RBC, WBC, Hb, PCV, MCV, MCH, and MCHC in control and experimental rats. Mean value of haemoglobin in Group II rats decreased when compared to normal control Group I rats because of induction of hepatocellular carcinoma. This was similar with the reports of Ge et al., (2011) [44] who also stated that significant decrease in haemoglobin in human patients with cancer in the GI system including hepatic carcinoma. Hassan et al., (2018) [45] stated that DEN induced hepatic carcinoma rats was found to be slight decrease in the RBC count. Our finding was similar to the above reports that the haematological parameters were reverted back to near normal levels and non-significant (P < 0.005) in Pre-treated Group IV and standard drug treated Group V rats when compared with Group I control rats. Post treated Group III rats show significant (P < 0.001) changes (Hb and RBC levels were decreased with a concomitant increase in WBC) and significant (P < 0.005) increase in PCV and MCV were observed when compared with the Group I control rats. However, when compared with group II rats, a significant recovery in the haematological levels were observed in Group III rats shows the hepatoprotective potential of EEEA.
Table 10
Effect of EEEA on Blood Haematological Profile in Control and Experimental Animals
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Haemoglobin
(gm/dl)
|
14.62 ± 0.79
|
9.47 ± 0.27 *
|
11.49 ± 0.33 *
|
14.02 ± 0.34ns
|
14.57 ± 0.38ns
|
WBC(x103/L)
|
7.70 ± 0.26
|
12.55 ± 0.24 *
|
11.40 ± 0.40 *
|
8.05 ± 0.31ns
|
7.55 ± 0.32ns
|
RBC(x106/L)
|
6.35 ± 0.32
|
4.55 ± 0.39 *
|
5.05 ± 0.69 *
|
6.35 ± 0.31ns
|
6.55 ± 0.45ns
|
PCV (%)
|
46.31 ± 2.25
|
64.40 ± 1.95 *
|
51.20 ± 1.96 $
|
48.82 ± 1.26ns
|
45.45 ± 1.36ns
|
MCV (pg)
|
73.09 ± 5.17
|
142.25 ± 11.42 *
|
103.00 ± 14.89 $
|
77.06 ± 4.60ns
|
69.72 ± 5.93ns
|
MCH (fg)
|
23.05 ± 1.45
|
20.93 ± 1.82ns
|
23.14 ± 3.39ns
|
22.14 ± 1.53ns
|
22.31 ± 1.16ns
|
MCHC (%)
|
31.60 ± 1.73
|
14.71 ± 0.58 *
|
22.48 ± 1.12 $
|
28.73 ± 1.15ns
|
32.08 ± 1.17ns
|
Values are expressed as Mean ± SD for six rats
Data were analysed by one-way ANOVA followed by the individual comparison was obtained by Post-hoc Bonferroni test, a multiple comparison procedure by the SPSS. Statistically significant variation was derived by comparing Group I versus Group II, Group III, Group IV and Group V, *P = 0.000, $P = 0.001, #P = 0.010, and ns = Non-significant (P < 0.05).
Mormphometric Analysis of Liver
The efficacy of any hepatic drug is essentially dependent on its ability in minimising the harmful effects or maintaining the normal hepatic physiology that has been distributed by a hepatotoxin. Figure 1 shows the effect of EEEA on morphological changes in liver of control and experimental rats. Gross liver pathology of the control Group I rats showed normal architecture with normal size and shape and brownish red liver with pale red tinge. Group II rats after DEN treatment showed grayish-white visible multi-nodules on the outer surface, about 1 mm in diameter. The liver showed a significant widening of intercellular spaces between hepatocytes, many invaginations of the cell membrane and irregularly shaped biliary canaliculi when compared with Control Group I rats. Our findings are similar to Hassan et al., (2018) [45]. Post treated Group III rats showed a noticeable recovery in the liver architecture with normal size and shape when compared with DEN induced Group II rats. Pre-treated Group IV rats showed perceptible recovery in the liver architecture with normalizing of cell surface, cell membrane, reduced number of nodules, size and shape when compared to DEN induced Group II rats. No significant changes appeared between the Control Group I and Silymarin treated Group V rats.
Histopathological Studies
Liver
Figure 2 represent the photomicrographs of liver sections stained with hematoxylin and eosin (40X) from control and experimental rats of EEEA. Group I Control rats show normal liver tissue with hepatocytes, portal triad showing prominent central vein. DEN induced Group II rats show liver tissue with ballooning degeneration of the hepatocytes, nucleomegaly, kupffer cell activity, regular nuclear membrane and focal collection of inflammatory cells around portal triad with fibrosis. These observations were similar to the findings reported by Youssef et al., (2012) [46]. Mohammed et al., (2014) [28] who showed that treatment with DEN leads to vacuolated hepatocytes, dilated blood sinusoids, massive portal leukocyte infiltration and disordered arrangement of dysplastic hepatocytes. Post treated Group III rats show liver tissue with mild inflammation, degeneration and congested sinusoids. Pre-treated Group IV rats show liver tissue with normal architecture and central vein and Silymarin treated Group V rats show liver with normal histological arrangement and kupffer cell activity when compared with control rats. The present study confirmed the reliability of histopathological methods and biochemical indices in ascertaining liver integrity and functionality of the Enhalus acoroides.
Kidney
Plate showing histopathological observation (40X) of kidney tissue which shows Group I (a) Control rats show normal kidney with Bowman’s capsules, Proximate convoluted Tubules (PCT), Distal convoluted Tubules (DCT) and Intersitium appears normal. The Group II (b) DEN induced rats distraction of bowman’s capsules and glomeruli, congestion and sever degeneration of renal tubules when compared to control Group I. Group III (c) post treated rats show mild degeneration of bowman’s capsules and glomeruli with congestion otherwise normal PCT and DCT and Group IV (d) Pre-treated rats shows normal Glomeruli with normal tubules. Group V (e) rats show kidney with normal Bowman’s capsules and renal tubules.
Spleen
Plate showing histopathological observation (40X) of spleen tissue which shows Group I (a) Control rats show spleen with thin capsule, prominent red and white pulp. The Group II (b) DEN induced rats show spleen with congestion and moderate degeneration of red and white pulp when compared to control Group I. Group III (c) post treated rats show spleen with mild congestion and Group IV (d) Pre-treated rats’ shows spleen with normal architecture. Group V (e) rats show spleen with normal histological structure.
Heart
Figure 5 depicted the photomicrographs of Heart sections stained with Hematoxylin and Eosin (40X) from control and experimental rats of EEEA. Group I Control rats’ shows cardiac myocytes with normal striated muscle, homogenous sarcoplasm. DEN induced Group II rats show heart with broken cardiac myocytes and irregular striated muscles. EEEA post treated Group III rats show heart with mild distraction of cardiac myocytes, intra muscular wall and centrally placed plump oval nuclei. EEEA pre-treated Group-IV rats show cardiac muscle with normal oval nuclei and Silymarin treated Group-V rats show heart with normal muscle fibres with normal nuclei when compared to the Control Group I rats.
Brain
Plate showing histopathological observation (40X) of brain tissue which shows Group I (a) Control rats show brain tissue with glial cells. The Group II (b) DEN induced rats show brain tissue with reactive gliosis, perivascular oedema and congestion when compared to control Group I. Group III (c) post treated rats show brain tissue with proliferation of neuroglial tissue, mild oedema, and prominent vessels and Group IV (d) Pre-treated rats’ shows normal brain tissue. Group V (e) rats show brain with normal architecture of tissue with glial cells.