Effects of Entandrophragma angolense aqueous extract on survival time of infected animals
Figure 1 summarizes the effects of plant extract on the mean survival time of animals for 28 days of experimentation. It appears that the intraperitoneal inoculation of 200 μL of 1×106 parasitized-erythrocytes to healthy mice significantly induced death in untreated animals from the day 10 to day 26 with the death of all mice in malaria control (p < 0.001) group compared to normal control. Contrarily, the administration the plant extract at any dose as well as chloroquine prevented death in infected animals (p < 0.001) (Figure 1).
Effects of E. angolense on the body weight evolution of infected-animals
Untreated malaria infected mice significant put off body weight by 26.22 % (p < 0.001) with regards to normal control group (Figure 2) at day 8 and over the 20 days of follow-up till the death of all animals at the day 26. The daily administration of the aqueous extract of E. angolense for five days, dose dependent prevented the decrease of body weight (p < 0.001) by 25.98%, 36.90% and 46.08% at the respective doses of 125, 250 and 500 mg / kg related to malaria control. The administration of the aqueous extract of E. angolense and chloroquine for 5 days then followed up for 20 days significantly curbed body weight (p < 0.001) loss as compared to the malaria control and normal control.
Effects of aqueous extract of Entandrophragma angolense on the parasitaemia
The effects of aqueous extract of E. angolense stem bark on parasitemia count of infected mice are shown in Figure 3. The intraperitoneal inoculation of P. berghei-parasitized-erythrocytes to healthy animals showed after 3 days post induction, an average parasitemia of 8.86 % that, without treatment rose up to 18.08% at the day 8 then 60.66% at the day 26, accompanying with the death of all animals in the malaria control group. The daily administration of the extract for 5 days resulted in the significant decrease in parasiteamia count by 65.44% (p < 0.05), 70.70% (p < 0.01) and 70.68% (p < 0.01) at the respective doses of 125, 250 and 500 mg / kg, compared to the malaria control. The percentage inhibition of parasite at the day 8 was lightly dose-dependent with 96.32%, 98.16% and 98.89% respectively at the dose 125, 250 and 500 mg/kg. The effective dose-50 (ED50) of aqueous extract of E. angolense was estimated at 25.32 mg/kg. A complete parasite clearance was achieved at the day 10, day 14 and day 16 at the respective dose of 500, 250 and 125 mg / kg.
Effects of the aqueous extract of E. angolense on some hematological parameters
The effects of the aqueous extract of E. angolense on some hematological parameters in P. berghei-infected animals after 8 and 28 days of experimentation are summarized in Table 1.
Table 1: Effects of Entandrophragma angolense on some heamatological parameters in infected mice
Parameters
|
Nor ctrl
|
Mal ctrl
|
CQ ctrl
|
EA125 mg/kg
|
EA250 mg/kg
|
EA500 mg/kg
|
|
|
|
Day 8
|
|
|
|
RBC (103 /µL)
|
5.90±0.28
|
3.31± 0.41a
|
5.40±0.45γ
|
6.53±0.23ß
|
6.08±0.13α
|
6.75±0.64γ
|
HGB (g/dL)
|
11.00± 0.50
|
5.15±0.52c
|
10.23±0.58 ß
|
11.20±0.52ß
|
10.70±0.50ß
|
12.55± 0.74γ
|
HCT (%)
|
31.58±1.69
|
17.06±1.83b
|
30.32±2.60µ
|
31,24±1.70ß
|
31.76±0.53α
|
37.05±1.93γ
|
PLT (103 /µL)
|
257.20±3.85
|
222.40±8.76
|
244.75±5.50
|
266.80±5.49
|
202.80±1.81
|
234.20±7.15
|
WBC (103/µL)
|
3.26±0.23
|
5.00±0.34b
|
2.20±0.22µ
|
3.00±0.38α
|
3.47±0.72α
|
3.15±0.30α
|
LYM (103/µL)
|
2.67±0.05
|
1.00 ±0.17b
|
3.48±0.33γ
|
2.46±0.33α
|
2.50±0.10α
|
2.26±0.11α
|
GRA (103/µL)
|
0.41±0.04
|
0.99±0.08b
|
0.43±0.09ß
|
0.43±0.05ß
|
0.49±0.14α
|
0.45±0.11ß
|
MON (103/µL)
|
0.31±0.00
|
0.81±0.05c
|
0.31±0.07γ
|
0.34±0.01γ
|
0.33±0.02γ
|
0.24±0.00γ
|
|
|
|
Day 28
|
|
|
|
RBC (103 /µL)
|
5.56±0.97
|
Nd
|
4.13±
0.00
|
6.60±
0.00
|
5.09±
0.05
|
5.96±
0.20
|
HGB (g/dL)
|
12.68±1.39
|
Nd
|
9.41±0.00
|
12.57±0.06
|
11.13±0.54
|
11.5±0.31
|
HCT (%)
|
33.10±4.10
|
Nd
|
30.49±0.00
|
35.55±0.58
|
32.90±1.79
|
31.20±0.20
|
PLT (103 /µL)
|
342.40±6.62
|
Nd
|
253.00±0.00
|
212.00±0.00
|
229.50±2.05
|
209.00±0.00
|
WBC (103/µL)
|
3.44±0.28
|
Nd
|
2.80±0.00
|
3.22±0.26
|
3.15±0.30
|
3.20±0.66
|
LYM (103/µL)
|
3.07±0.03
|
Nd
|
2.70±0.21
|
2.49±0.00
|
2.74±0.18
|
2.52±0.01
|
GRA (103/µL)
|
0.38±0.04
|
Nd
|
0.34±0.00
|
0.42±0.00
|
0.40±0.00
|
0.41±0.00
|
MON (103/µL)
|
0.32±0.00
|
Nd
|
0.31±0.00
|
0.30±0.03
|
0.31±0.03
|
0.33±0.00
|
Value represents mean ± SD, n = [5-10], Nd = not determined, ap <0.05, bp <0.01, cp <0.001significant difference from the normal control (healthy mice receiving distilled water at 10 mL/kg, αp <0.05, ßp <0.01, γp <0.001 difference as compared to the malaria control (Mal ctrl) =infected mice treated with distilled water for 5 days; CQ ctrl = infected mice treated with chloroquine (10 mg/kg).EA = infected mice treated with E. angolense extract at the doses of 125mg/kg (EA 125 mg/kg), 250 mg/kg (EA 250 mg/kg) and 500 mg/kg (EA 500 mg/kg). Day 8 and day 28 = data recorded after the respective days 8 and 28 of the experiment. Nd = not determined. RBC = red blood cells, HGB = hemoglobin, HCT = hematocrit, PLT = platelets, WBC = white blood cells, LYM = lymphocytes, GRA = granulocytes, MON = monocytes.
It was noticed that, the administration of the plant extract restored hematological parameters with a significant increased in RBCs count of 97.28% (p < 0.01), 83.68% (p < 0.01) and 103.92% (p < 0.001); HGB level of 117.47% (p < 0.01), 107.76% (p < 0.01) and 143.68% (p < 0.001); HCT rate of 83.11% (p < 0.01), 86.16% (p < 0.05), and 117.17% (p < 0.001) and LYM of 9.80% ( p < 0.05), 7.84% (p < 0.01) and 10.68% (p < 0.05) at the respective doses of 125 , 250 and 500 mg/kg compared to malaria control. The extract has also induced a significant decrease in the total WBC count by 23.94%, 44.36% and 90.84% (p < 0.01); GRA count by 56.56% (p < 0.01), 50.50% (p < 0.05) and 54.54% (p < 0.01) and monocytes count (MON) by 58.02% , 59.25% and 70.37% (p < 0.001) compared to malaria control (Table 1). At the end of the follow up post treatment (day 28), no significant change in hematological parameters (RBC, HGB, HCT, WBC, PLT, LYM, GRA, and MON) was observed in animals treated with plant extract and normal control. However, a significant decrease in hemoglobin was recorded in chloroquine control (p < 0.05) compared to the plant extract. No significant change was observed in hematological parameters in test groups between the days 8 and 28 of experiment.
Effects of the aqueous extract of E. angolense on the blood glucose, liver and kidney functions
Table 2 summarizes the effects of E. angolense on blood glucose, some liver and kidney parameters functions in P. berghei-infected mice.
Table 2: Effects of Entandrophragma angolense on some biochemical parameters in infected mice
|
Normal ctrl
|
Malaria ctrl
|
CQ ctrl
|
EA125 mg/kg
|
EA250 mg/kg
|
EA500 mg/kg
|
Day 8
|
Blood glucose (mg/dL
|
114.50±1.80
|
103.00±2.21b
|
123.40±2.29a,γ
|
120.50±1.42γ
|
124.80±1.52b,γ
|
124.50±2.05b,γ
|
ALT (UI/L)
|
61.21±2.38
|
76.09±0.53a
|
59.31±2.39ß
|
55.92±3.11ß
|
56.84±6.53ß
|
48.24±4.23γ
|
AST (UI/L)
|
108.10±1.68
|
222.78±8.77b
|
129.61±8.85γ
|
171.00±2.10γ
|
140.00±7.11γ
|
131.00±7.10γ
|
Bilirubin (mg/dL)
|
1.18±0.01
|
3.23±0,14c
|
1.47±0,05γ
|
1.58±0.01γ
|
1.52±0.03γ
|
1.53±0.02γ
|
Creatinine (mg/dL)
|
0.51±0.04
|
1.96±0.04c
|
0.56±0.03γ
|
0.50±0.06γ
|
0.90±0.15γ
|
0.54±0.11γ
|
Proteins (mg/mL)
|
0.65±0.01
|
0.35±0.04 c
|
0.68±0.02γ
|
0.49±0.02a,µ,ᴨ
|
0.75±0.03γ
|
0.77±0.03γ
|
Day 28
|
Blood glucose (mg/dL
|
112.83±1.35
|
nd
|
118.00±0.00
|
113.50±0.15Ԑ*
|
116.80±1.64b
|
111.55±0.60ϕᴨ,θ
|
ALT (UI/L)
|
61.33±0.56
|
nd
|
55.81±3.50
|
58.41±2.12
|
57.02±3.36
|
62.61±2.33
|
AST (UI/L)
|
107±1.39
|
nd
|
114.71±5.94
|
111.99±4.10θ,#
|
116.45±1.39×
|
105.28±1.16×
|
Bilirubin (mg/dL)
|
1.36±0.11
|
nd
|
1.86±0.15b
|
1.45±0.02
|
1.57±0.09
|
1.22±0.02 ♯
|
Creatinine (mg/dL)
|
0.51±0.01
|
nd
|
0.56±0.09
|
0.61±0.06b,♯
|
0.72±0.07b
|
0.55±0.09
|
Proteins (mg/mL)
|
0.66±0.02
|
nd
|
0.75±0.05
|
0.59±0.01♯
|
0.66±0.02
|
0.72±0.04
|
Values represent mean ± SD, n = [5-10], ap <0.05, bp <0.01, cp <0.001 significant difference from the normal control (normal ctrl), ßp <0.01, γp <0.001 difference as compared to the malaria control (Malaria ctrl); µp < 0.05, Ԑp < 0.01, ϕp < 0.001 difference compared to chloroquine control (CQ ctrl), *p < 0.05, θp < 0.001 difference in given parameter between d8 and d28. Normal ctrl = healthy mice receiving distilled water (10 mL/kg; Malaria ctrl = infected mice treated with distilled water; CQ ctrl = infected mice treated with chloroquine (10 mg/kg). EA = infected mice treated with E. angolense extract at the doses of 125mg/kg (EA 125 mg/kg), 250 mg/kg (EA 250 mg/kg) and 500 mg/kg (EA 500 mg/kg). Day 8 and day 28 = data recorded after the respective days 8 and 28 of the experiment. nd = not determined. AST = aspartate aminotransferase, ALT = alanine aminotransferase.
The results showed that P. berghei-infected mice induced after 8 days a significant decrease of glycaemia by 11.16% (p < 0.01) and proteins level by 46.33% (p < 0.001) and significant increase in ALT and AST activities by 10.58% (p < 0.05) and 51.47% (p < 0.001), respectively, in bilirubin level by 63.50 % (p < 0.001), in creatinine level by 73.95% (p < 0.001) compared to the normal control (Table 2).
However, the daily administration of the extract for five days resulted in significant increase of blood glucose level by 16.99%, 21.16% and 20.87% (p < 0.001) compared to the malaria control. In comparison to the normal control, it was observed a significant increase in glycaemia in infected animals treated with extract at the dose 250 mg/kg (p < 0.01) and 500 mg/kg (p < 0.05). After the 20 days of follow up (day 28), a significant increase in the blood glucose level was noted in the animals treated with the extract at 250 mg/kg (p < 0.01) while that of doses 125 and 500 mg/kg decrease compared to the normal control. Interestingly, the blood glucose level significantly decreased at the dose of 500 mg/kg at the day 28 (p < 0.001) compared to the day 8. No significant change was observed between test groups and chloroquine control group.
The daily intake of extract significantly prevented the rise of ALT activities by 28.35%, 27.17% (p < 0.01) and 38.19% (p < 0.001) and AST activities by 23.24% (p < 0.05), 41.19% and 66.52% (p < 0.001) at the respective doses of 125, 250 and 500 mg/kg as compared to the malaria control. The AST concentration was significantly low in animals treated with the extract at the dose of 250 mg/kg and 500 mg/kg (p < 0.01) compared to those receiving the dose 125 mg/kg. At the end of follow up, no change was observed in ALT activities amongst animals. However, a significant low AST activity of 34.50%; (p < 0.001), 16.82% and 19.84% (p < 0.05) was recorded with the extract at the respective doses of 125, 250 and 500 mg/kg at the day 8 compared to the day 28.
A significant low creatinine concentration (p < 0.001) by 74.31%, 60.03% and 72.37% was recorded at the days 8, respectively, at 125, 250, and 500 mg/kg compared to the malaria control. After the follow up period of 28 days, significant an increase in the creatinine concentration (p < 0.01) was observed at 125 mg/kg compared to normal control. It was also noted an increase in the creatinine concentration (p < 0.01) at the same dose between day 8 and day28.
The bilirubin concentration significantly decreased at the day 8, in the treated mice with plant extract by (p < 0.001) whatever the extract dose compared to the normal control. No change was recorded compared to chloroquine control.
The test groups showed significant increase in protein levels (p < 0.001) by 53.33% and 54.54% at the respective dose of 250 mg/kg and 500 mg/kg compared to the malaria control. Proteins level was significantly enhanced in the extract at 250 and 500 mg/kg (p < 0.001) compared to the dose 125 mg/kg.
In comparison to the chloroquine control, it was observed a significant decrease in protein (p < 0.05) at 125 mg/kg. At the day 28, protein concentration was restored in all treated groups and no change was observed among them.
Effects of aqueous extract of Entandrophragma angolense on some oxidative stress parameters
The figure 4 shows the effects of the aqueous extract of E. angolense on some anti-oxidative factors as superoxide dismutase (SOD) and catalase activities, reduced glutathione (GSH), nitrites (NO) and malondialdehyde (MDA) concentration in the liver and kidney of P. berghei-infected mice, treated for five days (day 8) and monitored for 20 days (day 28). P. berghei-infected mice displayed significant breakdown in SOD by 91.46% (p < 0.001) in liver and 87.43% (p < 0.01) in kidney, in reduced glutathione (p < 0.001) by 90.03% and 60.31%, in catalase activity by 65.78% and 60.64 % (p < 0.001) in liver and kidney, respectively, in nitrites level by 50% (p < 0.001) in liver while the MDA concentration increased (p < 0.001) compared to the normal control after eight days. The single daily dose administration of the E. angolense bark extract resulted in a significant dose-dependent increase in SOD activity at the day 8, by 91.42% and 92.43% (p < 0.001) in liver, by 50.41% and 55.66% (p < 0.01) in kidney at the respective doses of 250 and 500 mg/kg compared to malaria control (Figure 4A). A significant enhancement in SOD activity (p < 0.001) was observed in liver of animals treated with plant extract at 250 and 500 mg/kg compared to the chloroquine control. The SOD activity significantly decreased in liver (p < 0.001) and in kidney (p < 0.01) of animals treated with extract at 125 mg/kg compared to those receiving extract at 250 and 500 mg/kg. After the follow-up period of 20 days, no significant change in SOD activity was observed in the liver as well as in kidneys of animals treated with the extract.
The extract induced significant increase in catalase activity ( p < 0.001) by 60.60%, 55.17% and 66.66% in the liver, by 47.61% (p < 0.05), 69.44% (p < 0.001) and 57.69% (p < 0.001) in the kidney, at doses of 125, 250 and 500mg/kg respectively, compared to the malaria control (Figure 4B). In comparison to the chloroquine control, a significant decrease in catalase activity was observed in kidney (p < 0.01) of animal treated with extract at 125 mg/kg. Likewise, it was noticed a significant enhancement in catalase activity in the liver of animal treated with extract at 500 mg/kg (p < 0.05). A significant decrease in renal catalase activity was recorded in animals treated at 125 mg/kg (p < 0.001) and 500 mg/kg (p < 0.05) compared to those treated at 250 mg/kg. At the day 28, a significant decrease in catalase activity (p < 0.05) was observed in animals treated with plant extract compared to the chloroquine control.
The plant extract administration resulted in a significant restoration in glutathione level by an increase of 91.05%, 90.71% and 90.90% (p < 0.001) in the liver and by 41.75% (p < 0.01), 63.57% and 59.50% (p < 0.001) in the kidney at the respective doses of 125, 250 and 500 mg/kg compared to the malaria control (Figure 4C). It was observed a significant increase in glutathione concentration in the liver of animals treated with plant at the dose of 125 mg/kg (p < 0.01), 250 mg/kg (p < 0.05) and 500 mg/kg (p < 0.001) compared to the chloroquine control. After 20 further days of the follow-up, no change in glutathione level was observed among the experimental groups.
The extract absorption led to significant increase in hepatic nitrites level by 33.33% (p < 0.05) and 55.50% (p < 0.001) at the respective doses of 250 and 500 mg/kg compared to malaria control (Figure 4D). Compared to the normal control, treated animals with chloroquine (10 mg/kg) or plant (125 mg/kg) resulted in significant decrease in nitrites level by 37.50% (p < 0.05) and 40.01% (p < 0.01), respectively, in the liver. Among the animals treated with plant extract, significant increase in nitrites concentration was observed at the hepatic level in group receiving 500 mg/kg compared to those treated with the 125 and 250 mg/kg (p < 0.001) and chloroquine. No significant change in nitrites rate was observed in kidney of experimental animals. After additional 28-days follow-up, the nitrite levels were restored in all experimental groups.
The daily administration of a single dose of E. angolense extract for five days induced a significant decrease in MDA concentration in both liver and kidney by 59.02%, 61.02% and 63.83% (p < 0.001) in the liver and by 79.91%, 76.75% and 74.42% (p < 0.001) in the kidneys, at the respective doses of 125, 250 and 500 mg/kg compared to the malaria control (Figure 4E). A significant decrease in MDA concentration in the liver and kidney was recorded in chloroquine control (p < 0.001) compared to the malaria control. A significant increase in MDA liver (p < 0.01) was observed at the extract dose of 500 mg/kg compared to chloroquine control. No change in MDA level was observed within animals treated with plant extract. The hepatic and renal MDA levels did not changed among the experimental animals after 28 days of follow-up.
Effects of Entandrophragma angolense aqueous extract on some organ architecture
Effects on the liver
The figure 5 illustrates micrography of the liver section in infected mouse treated with the aqueous extract of E. angolense for 5 days (day 8) then followed up for 20 days (day 28). On the days 8 and 28, the liver section of healthy mice presents a normal parenchyma with a well differentiated portal vein, bileduct, hepatic artery, hepatocytes separated by sinusoidal capillaries where Kupffer cells are well observed (Figures 5A and A’). The liver section of malaria control mice at the day 8 shows major damage in the tissue with aclarified parenchyma, an inflammatory focus spread along the peripheral portal space surrounded by leukocyte infiltrations and vascular congestion in the hepatic artery, Kupffer cells were stained by malarial pigment (Figure. 5B). The micrograph of the liver of chloroquine control presented a portal vein, a bile duct, a hepatic artery, hepatocytes, sinusoidal capillaries, Kupffer cells containing malarial pigment and an inflammatory zone along centrilobular vein (Figure 5C), which were no longer observed at the 28th (Figure 5C’). It was observed at day 8 in the liver of infected mice treated with the aqueous extract of E. angolense (125 mg/kg), an undisclosed inflammatory focus with localized leukocyte infiltration and Kupffer cells containing malarial pigment (Figure 5D). These changes disappeared at the day 28 (Figure 5D’). The micrography of the liver of infected animal receiving the plant extract at the dose 250 and 500 mg/kg presented no change in the tissue with the architecture quite similar to that of a normal control, as well as on day 8 (Figure 5E) and on day 28 (Figure 5E’).
Effects on the kidney tissue
The figure 6 shows the effects of E. angolense extract on the photomicrography of the kidney section of P. berghei-infected mouse at day 8 and day 28. It was observed in the kidney section of healthy mouse presents a normal appearance of the renal parenchyma where glomerulus, Bowman's space, proximal and distal tubules and the collector tubes are well differentiated (Figure 6A). In the infected mouse, it was noted some alteration in kidney tissue marked by the absence of urine space, clarification in the tubules and some inflammatory sites (Figure 6B). The treatment of infected mouse with chloroquine (10 mg/kg) or with plant extract (125, 250 and 500 mg/kg) protected against anatomic damage as observed in the kidneyofmalaria control. The kidney section shows normal appearance of the renal parenchyma with glomerulus, Bowman's space, proximal and distal tubules are distinctly observed both on day 8 (Figures 6 C, D, E, F) and on day 28 (Figures 6 C’,D’, E’, F’).
Effects on the spleen tissue
The effects of the aqueous extract of E. angolense on the spleen of P. berghei-infected mouse and followed for 8 and 28 days are presented in Figure 7. The micrography of the spleen from healthy mouse shows normal parenchyma with distinctly white and red pulp, trabecula, central artery and splenic artery (Figure 7A). In the malaria-infected mouse, it was noted major disorganization in the parenchyma marked by the absence of differentiation of the white and red pulp; dilatation of splenic arteries and the presence of malarial pigment in parasitized red blood cells (Figure 7B). Spleen micrography of infected mice treated with chloroquine (10 mg/kg) (Figure 7C) or with aqueous extract of E. angolense at 250 and 500 mg/kg (Figures 7 E and F) present normal architecture with white and red pulp quite distinct as similar to the normal control, however the presence of malarial pigment both on day 8 (Figures 7 C, E, F) and on day 28 (Figures 7 C’, E’,F’) while spleen section of animals treated with the extract at 125 mg/kg shows less pronounced disruption in the structure of the organ with the presence of malarial pigment (Figure 7 D).