In vivo antisalmonella effect
The establishment of infection was clearly reflected by some changes in animals physiology including the excretion of watery stool, the presence of blood and mucus in the stool, the reduction of activity and the exponential increase in the rate of
S. typhimurium in the feces of rats after administration of infectious load. This suggested that the bacteria proliferated in the organs after having invaded the system, and challenged the non-specific defense mechanism of rats. The decrease of the bacterial load observed during treatment may be due to the combined action of the extract and immune system given the fact that this decrease was also noted in the negative control group (infected and untreated). Animals treated at therapeutic healing dose (55 mg/kg) recovered in the same period as those treated with multiples of the therapeutic dose. This result suggests that our sample may have a higher activity in vivo. due to their metabolism. The phytochemical screening revealed the presence of several classes of compounds in
V. paradoxa leaf extract among which: phenols and polyphenols, flavonoids, alkaloids, tannins, saponins, cardiac glycosides and anthocyanins. Some of these secondary metabolites (Flavonoids, alkaloids) have already shown several pharmacological properties including antibacterial properties [
24‐
26]. Since these metabolites are present in our extract, some of them could be associated to the antibacterial activity observed. The increase in total serum cholesterol in negative control group and treated groups might be attributed to the decreased activity of cholesterol 7α-hydroxylase, due to the combined effect of infection and extract administration. This enzyme catalyzes the conversion of cholesterol to bile acid, which is a major route of elimination of cholesterol [
27]. This could also be explained by its high production in the liver and its low uptake in the genitals as a precursor of androgen [
28]. Infection could have stimulated its hepatic production and the same time inhibited its transfer to genital organs. Despite this increase in total cholesterol level, the extract may have no risk of cardiovascular disease since it did not affect the arterogenic index. Indeed, Jafri et al. [
27], has shown a great positive correlation between excess of bad cholesterol (LDL) and/or lack of good cholesterol (HDL) are risk factors for cardiovascular diseases.
Increased serum creatinine followed by a decrease in urinary creatinine observed in infected an none treated group indicates kidney damage [
28]. This shows that infection might have result to glomerular alteration, leading to a decrease in its elimination in the urine. This hypotheses could be supported by glomeruloscleroses noted on kidneys histological section. The significant (
p < 0.05) increase in transaminases in negative control and test groups may be due to animals liver injury [
29,
30]. Moreover, animals treated with doses ≥ 220 mg/kg have ALT and AST levels relatively lower than the negative control group (untreated group). These results suggest that at these doses, the extract would induce sudden correction on liver cell damage and would result in an hepatoprotective effect, but significant decreases were observed in liver and kidney relative weight and their protein content, suggesting that this extract might have neither hepatoprotective nor kidneyprotective potential effect. Their protein content could have moved to the blood stream due to the injury of these tissues. This hypothesis can be supported by the presence of flavonoids in our extract. According to Uche et Aprioku [
24], flavonoids have anti-inflammatory, antiallergic and hepatoprotective properties. The increase of bilirubin in infected animals could be due to endogenous production of oxidizing substances that have a role in antioxidation (glutathione, uric acid, bilirubin, protein thiol groups, polyamines etc.). The histological sections of liver revealed histopathological changes in infected and untreated animals (negative control) as well as those treated with doses ≥ 220 mg/kg. The presence of inflammatory foci follow by dilation of sinusoids capillaries observed on liver cross sections of test animals might be due to invasion of the liver and destruction of hepatocytes by
salmonella. At dose 440 mg/kg liver cross section shows a normal appearance, similar to that of neutral group. The extract at this dose would not only treat typhoid, but might had corrected damages caused by the infection.
Kidney cross section shows that apart from a slight tubular clarification observed negative control animals, treatment had resulted in a dose-dependent increase of tubular clarification, glomerular degeneration follow by mezangial espansion in animals treated at a dose 440 mg/ml. This result suggests that, V. paradoxa aqueous extract may cause kidney injury. This hypothesis is further supported by the significant increased observed in serum bilirubin and urinary protein.
Acute toxicity
Overall, acute toxicity study did not reveal any negative behavioural change at lower doses (≤8 g/kg) in mice, as compared to the controls. However, a reduced reaction to noise was observed in mice receiving the extract at doses ≥ 8 g/kg, suggesting that it may have a depressant or sedative effect on the central nervous system [
29] at high doses. A reduced reaction to pinch was observed as from 16 g/kg, indicating the effect of the extract on the perception of pain, which may either be due to its nociceptors, to the inhibition of the production of algogenic substances (e.g. prostaglandins, histamines), or to the inhibition of the painful message transmission at the central level [
29]. At high doses (≥16 g/kg), the stool was watery, indicating that the extract may cause diarrhoea at these doses. These results suggest that, at higher doses, the extract may have an irritating action on the smooth muscle of the intestinal wall causing a change to fluid and electrolyte permeability [
31]. The extract may as well cause an acceleration of the intestinal transit, acting as a laxative [
32].
Forty eight hours after administration of different extracts, we found total mortality at the dose 20 g/kg. Convulsion and coma observed few minutes before the death of animals may be due to a decrease in oxygen affinity to hemoglobin because its sites were occupied by the metabolites therein. According to Delongeas et al. [
33], the water leaf infusion of
V. paradoxa can be classified among none toxic substances since the LD
50 is > 5 g/kg in both sexes.