INTRODUCTION
Plants provide substitution for drugs in some rural areas. There is a rich
abundance of plants reputed in traditional medicine to possess protective and
therapeutic properties. The herbal medicine products are dietary supplements
that people take to improve their health. There are many herbs have been used
for a long time for claimed health benefits (Kayode and
Kayode, 2011). However, some are not effective and some may cause health
problems due to the increase and repeated use of them (Vaghasiya
et al., 2011), this occurs as result of the fact that many medicinal
plant contain toxic compounds like flavonoid, anthraquinones, tannins, triterpenes,
sterols, alkaloids and volatile oil (Amna et al.,
2011).
In tropical and subtropical countries, drought and the acute shortage of grass on pastures are frequent and these conditions may force animals to consume varying amounts of poisonous plants, which can cause damage to vital organs.
Abrus precatorius (Leguminosae) and Jatropha curcas (Euphorbiaceae)
contain the phytotoxin, toxalbumins, abrin and curcin, respectively (Lin
et al., 2003; Botha and Penrith, 2008). Ingestion
of both plants by sheep, calves, goats and chicks causes catarrhal enteritis
and hepatocellular necrosis (Joubert et al., 1984;
Omer et al., 1992; Abdel
Gadir et al., 2003), also it was reported that an ingestion of J.
tanjorensis leaves lead to toxicological effects expressed as changes in
weight, biochemical and ultrasonographic parameters of rabbits (Akhigbe
et al., 2009) while plant parts of A. precatorius reported
to be purgative, emetic and tonic (Arora et al.,
2011).
Trichodesma africanum L., a member of the family Boraginaceae, is locally
known as Hiraisha and is common in Western Sudan. Livestock grazing on Trichodesma
africanum when other pasture plants are scarce. Rhanterium epapposum
Oliv., a member of the family Asteraceae and locally known as Al-Arfaj, its
widely distributed in various regions of Sudan and other Afro-Asian countries
and used in local traditional medicine by people in rural areas as a remedy
for skin infections and gastrointestinal disturbances and as an insecticide,
Phytochemical analysis of the aerial parts of R. epapposum showed the
presence of flavonoids, tannins, sterols, triterpenes and volatile oils (Al-Yahya
et al., 1990).
Phytochemical analysis of the aerial parts of T. africanum demonstrated
the presence of alkaloids, sterols, triterpenes, tannins and anthraquinones
(El-Moaty, 2009) and pyrrolizidine alkaloid and other
compounds (EFSA, 2007).
Hence, it appears those pyrrolizidine alkaloids are among the most widely distributed
natural toxins affecting wildlife and livestock (Roeder,
2000).
The present study was planned to investigate the effects of various levels of aqueous and methanol extracts from the multipurpose medicinal plant, R. epapposum and the pasture plant, Trichodesma africanum, in Northern Kordofan State, on male Wistar rats for safety evaluation and nature of their effects.
MATERIALS AND METHODS
Plant material: R. epapposum and T. africanum aerial parts were collected from El-Hassahisa areas, Jazeera State and El Obeid areas, Northern Kordofan State, air-dried in the shade, ground separately by a mechanical grinder.
Animals and administration of plants material: Sixty, 2-month-old clinically healthy, male Wistar rats, were kept and fed with the premises of the Medicinal and Aromatic Plants Research Institute, National Research Center, Khartoum. The Rats were allotted at random to 9 groups, each of 6 rats.
Experimental design 1: Rats in group 1 served as untreated control. Rats in groups 2 and 3 were given aqueous extract of the aerial parts of the R. epapposum at 75 and 300 mg/kg/day via the oral route, respectively. The rats in groups 4 and 5 were given aqueous extract at 75 and 300 mg/kg/day via i.m. route.
The methanol extract dissolved in normal saline was administered to rats via
oral route at 75 mg/kg/day (group 6) or 300 mg/kg/day (group 7) and intramuscularly
at 75 mg/kg/day (group 8) or 300 mg/kg/day (group 9). Dosing was continued until
the rats died or were killed under diethyl ether anesthesia after 2 weeks of
treatment.
Experimental design 2: The rats in group 1 were untreated controls. The aqueous extract from T. africanum aerial parts was given to rats orally at 75 mg/kg/day (group 2) and 300 mg/kg/day (group 3) and via i.m. route at 75 mg/kg/day (group 4) or 300 mg/kg/day (group 5).
The methanol extracts from plant aerial parts were administered in normal saline to rats via the oral route at 75 mg/kg/day (group 6) and 300 mg/kg/day (group 7) and via i.m. route at 75 mg/kg/day (group 8) and 300 mg/kg/day (group 9). Dosing was continued until the rats died or were killed under diethyl ether anesthesia after 2 weeks of treatment.
Average feed intake, body weight gain and feed efficiency were measured weekly for each group. Clinical signs and mortality rates were recorded. After one weeks of treatment, 3 randomly selected rats from each group were killed by decapitation. The remaining 3 rats/group were similarly killed after 2 weeks. Blood samples were collected from each of the killed rats for hematology and clinical chemistry. All rats were examined to identify gross lesions and specimens of the liver, kidneys, heart, spleen, intestines and muscles at site of injection, were fixed in 10% neutral buffered formalin and processed for histopathology.
Blood analysis: Hemoglobin (Hb) concentration, Packed Cell Volume (PCV),
Red Blood Cells (RBCs), White Blood Cells (WBCs), differential WBC count, Mean
Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular
Hemoglobin Concentration (MCHC) were estimated by standard methods (Schalm
et al., 1975).
Sera were analyzed for the activities of aspartate aminotransferase (AST),
alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and concentrations
of total protein, albumin, globulin, bilirubin, cholesterol and urea
by using commercial kits (Linear Chemicals, Barcelona, Spain).
Pathological examinations: At necropsy, all rats were examined to identify gross lesions and specimens of liver, kidneys, heart, spleen and intestines were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 6 μm and stained with hematoxylin and eosin (H and E) for histopathologic examinations.
Statistical analysis: All measured data were subjected to the analysis using SPSS version 11.5, the significance of differences between means were compared at each for all groups, p<0.05 was considered statistically significant.
RESULTS
Effects on Wistar rats of R. epapposum extracts given by different
routes of administration
Clinical findings: Depression, huddling together, reluctance to move, locomotor
disturbances, paresis of the limbs and recumbency were first observed in rats
receiving daily doses of water extract at 300 mg/kg/day orally (group 3, 7)
and 300 mg/kg/day i.m. (group 5, 9) 6 days post-dosing. One rat in group 3 died
10 days post-dosing. In rats receiving 75 mg/kg/day i.m. (group 4) and 75 mg/kg/day
orally (group 2), the symptoms were mild and no death occurred among the rats.
Control (group 1) had no clinical signs and was killed with survivors in other
groups on day 15.
Pathological changes: At necropsy, there was congestion or haemorrhage in the liver, kidneys and heart and varying degrees of muscular haemorrhage at the site of injection in groups 4, 5, 9 and 10. On microscopy, there was diffuse cytoplasmic vacuolation and necrosis of the hepatocytes, vacuolar degeneration, segmentation or necrosis of the glomerular tubules, acidophilic homogeneous material in affected renal tubules (Fig. 1) mild enteritis, myositis (Fig. 2) and accumulation of lymphocytes in vital organs.
Haematological changes: After treatment with water extract of the plant, the values of RBCs and PCV in groups 2 and 3 were lower (p<0.05) and MCV values were higher (p<0.05) in groups 3 and 4 than control and other groups. MCHC were significantly changed in all groups having higher values (p<0.05) compared to control. In two weeks, RBCs count were lower in groups 3, 4 and 5 compared to control and MCV was significantly higher in all groups shows significant increase in MCH groups 3 and 4 have lower p-value compared to control (Table 1).
|
| Fig. 1: |
Vacuolar degeneration and necrosis of renal tubules with acidophilic
homogeneous substance in affected tubules of a rat receiving 300 mg/kg/day
of methanol extract of R. epapposum via i.m. route for 6 days, H
and E x200 |
|
| Fig. 2: |
Myositis at site of injection in a rat receiving 300 mg/kg/day
of methanol extract of R. epapposum via i.m. route for 6 days, H
and E x100 |
| Table 1: |
Haematological changes in rats given R. epapposum aqueous
and methanol extracts by different routes of administration for 2 weeks |
|
| Values are Mean±SE, Means within rows with no common
letter(s) are significantly different (p<0.05), Hb: Hemoglobin, RBC:
Red blood cells, PCV: Packed cell volume, MCV: Mean corpuscular volume,
MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration,
WBC: White blood cells |
|
After treatment with water extract of the plant, the values of RBCs and PCV
in groups 2 and 3 were lower (p<0.05) and MCV values were higher (p<0.05)
in groups 3 and 4 than control and other groups. MCH and MCHC were higher value
in all groups compared to control. lymphocytes in groups 7 and 9 were slightly
lower than other groups. In two weeks PCV value in groups 7, 8 and 9 has lower
p value than other groups, group 7 shows the most significant increase in MCV
and the lowest p value for WBCs count.After one week of treatment, by methanolic
extracts the values of RBCs in groups 8 and 9 and PCV in group 8 were lower
(p<0.05) than control and other groups. MCV values were higher.
Changes in serum constituents: These data are presented in Table 2. After one week of treatment with aqueous extract, the activities of AST and ALP in groups 2, 3, 4 and 5 and ALT in group 3 were higher (p<0.05) than control and other groups. The concentration of albumin in group 4 was lower (p<0.05) and that of globulin was higher (p<0.05) in groups 2,3,4 and 5 than other groups. After 2 weeks of treatment, the activities of AST were higher (p<0.05) in groups 2-5. The activity of ALP was lower in groups 2, 4 and 5 and total protein concentration was higher (p<0.05) in group 2 than other groups. Globulin concentration was higher (p<0.05) in groups 3 and 4 than other groups. Urea was higher (p<0.05) in groups 2, 3 and 5 than other groups.
After one week of treatment with methanol extract, the activities of AST and
ALP in groups 6-9 and of ALT in groups 8 and 9 were higher (p<0.05) than
control and other groups. The concentrations of globulin were higher (p<0.05)
in groups 6, 8 and 9 than control. Cholesterol concentration in group 6 was
the lowest (p<0.05) among all groups.
| Table 2: |
Changes in serum constituents of rats given R. epapposum
aqueous and methanol extracts by different routes of administration for
2 weeks |
|
| Values are Mean±SE, Means within rows with no common
letter(s) are significantly different (p<0.05), AST: Aspartate aminotransferase,
ALT: Alanine aminotransferase, ALP: Alkaline phosphatase |
After 2 weeks of treatment, the activities of AST in groups 6-9 and those
of ALT in groups 7-9 were higher (p<0.05) than control and other groups.
The concentration of total protein and globulin were higher (p<0.05) in all
groups compared to the control. Group 6 shows the most significant decrease
in urea concentration.
Effects on Wistar rats of T. africanum extracts given by different
routes of administration
Clinical findings: Huddling together, depression,
inability to move, locomotors disturbances, paresis of the limbs and recumbency
were first seen in the rats receiving daily doses of aqueous extract at 300
mg kg-1 orally (group 3) and 300 mg kg-1 i.m. (group 5)
5 days post-dosing. Two rats from group 3 died on day 10. The clinical signs
were mild in rats of groups 2 and 4 receiving the aqueous extract of the plant
at 75 mg kg-1 per os or 75 mg/kg/day i.m. Survivors were killed
15 days post-dosing.
One rat receiving 300 mg/kg/day orally of methanol extract (group 7) died on day 10 but none of the rats receiving 300 mg/kg/day i.m. of methanol extract (group 9) died during this period and manifested obvious clinical signs of locomotor disturbances, paresis of the limbs and depression. The rats given 75 mg/kg/day orally (group 6) or 75 mg/kg/day i.m. of methanol extract (group 8) for 15 days showed less marked clinical disturbances. Survivors were killed 15 days post- dosing.
Pathologic changes: Post-mortem findings in rats receiving T. africanum
water or methanol extract by different routes of administration included
congestion or haemorrhage in the liver, kidneys, heart and intestines and muscular
haemorrhage at the sites of injection and atrophy in aqueous extract-dosed rats
of groups 3, 4, 5 and methanol-dosed rats in groups 7-9.
|
| Fig. 3: |
Severe lymphocytic infiltration in a rat injected i.m. with
300 mg/kg/day of T. africanum methanol extract for 2 weeks, H and
E x100 |
|
| Fig. 4: |
Liver of rats receiving T. africanum oral doses of
aqueous extract 300 mg kg-1 for 2 weeks, showing, fatty cytoplasmic
vacuolation of the centrilobular hepatocytes, necrosis and hemorrhage, H
and E x200 |
On microscopy, there was haemorrhagic myositis with lymphocytic accumulation
(Fig. 3) between the muscle fibres that showed necrosis particularly
in groups 4, 5, 7 and 9. Diffuse cytoplasmic vacuolation and necrosis of the
hepatocytes (Fig. 4), vacuolar degeneration and necrosis of
the renal tubules, splenic haemosiderosis, catarrhal enteritis and accumulation
of lymphocytes in vital organs were observed. These changes were less marked
in groups 2 and 6. Control rats (group 1) showed no significant lesions.
Haematological changes: After one week of treatment with aqueous extract,, the values of RBCs and PCV were lower (p<0.05) in groups 2, 4 and 5 than control and other groups. The values of MCHC in groups 2, 4 and 5 were higher (p<0.05) than control. The values of WBCs in groups 4 and 5 were lower (p<0.05) than other groups. After 2 weeks of treatment, values of RBCs were lower (p<0.05) in groups 2, 4 and 5 and PCV values were lower (p<0.05) in groups 2-5 than other groups. The values of MCV were higher (p<0.05) in groups 2 and 4 (Table 3).
| Table 3: |
Haematological changes in rats given T. africanum aqueous
and methanol extracts by different routes of administration for 2 weeks |
|
| Values are Mean±SE, Means within rows with no common
letter(s) are significantly different (p<0.05), Hb: Hemoglobin, RBC:
Red blood cells, PCV: Packed cell volume, MCV: Mean corpuscular volume,
MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration,
WBC: White blood cells |
After one week of treatment with methanol extract, PCV values were lower (p<0.05) in groups 8 and 9 than other groups. MCHC values were higher (p<0.05) in all groups compared to the control.
After 2 weeks of treatment with methanol extract of T. africanum, the values of RBCs were lower (p<0.05) in groups 6, 8 and 9. The values of PCV were lower (p<0.05) in group 9 and MCV values were higher (p<0.05) in all groups. Group 7 shows the lowest p-value in neutrophils and consequently in lymphocytes. The values of WBCs were lower (p<0.05) in groups 6 and 7 and those of lymphocytes were higher (p<0.05) in groups 6, 7 and 8 than other groups.
Changes in serum constituents: After one week of treatment with aqueous extract of the plant, the activities of AST in groups 2 and 3 and those of ALT in groups 4 and 5 were higher (p<0.05) than other groups. The activity of ALP was lower (p<0.05) in groups 2, 4 and 5 than control and other groups. The concentration of total protein was significantly increased in group 2 and that of globulin was higher (p<0.05) in all groups (Table 4).
After 2 weeks of treatment, the activities of AST were higher (p<0.05) in all groups. The activity of ALP was lower in groups 2, 3 and 5. Bilirubin concentration was lower in groups 3 and 4. Urea was lower (p<0.05) in groups 2-5 compared to the control.
After one week of treatment with methanol extract, the activities of AST and
ALP were higher (p<0.05) in all groups but only group 7 and 8 shows significant
change in regard to ALT.
| Table 4: |
Changes in serum constituents of rats given aqueous and methanol
extracts of T. africanum by different routes of administration for
2 weeks |
|
| Values are means±SE, Means within rows with no common
letter(s) are significantly different (p<0.05), AST: Aspartate aminotransferase,
ALT: Alanine aminotransferase, ALP: Alkaline phosphatase |
The activity of ALP was higher (p<0.05) in group 6 and lower (p<0.05)
in groups 7-9 than other groups. The concentrations of total protein and globulin
in all groups were higher (p<0.05) than control. Urea concentration was higher
(p<0.05) in group 6. After 2 weeks of treatment, the activities of AST and
ALT were lower P value (p<0.05) in all groups. The activity of ALP was lower
(p<0.05) in groups 7, 8 and 9 than other groups. The concentration globulin
in all groups were higher (p<0.05) than control.
DISCUSSION
In the present study, the effects on rats of the aqueous and methanol extracts
prepared from R. epapposum and T. africanum aerial parts and given
at different dose levels by different routes of administration were investigated
similar to other finding in regard to toxicity of R. epapposum to Wistar
rats (Younis and Adam, 2008). The results indicated
that the plant extracts are toxic and lethal to rats by whatever route (orally
and i.m.) it was given. Previous phytochemical investigations of T. africanum
have demonstrated the presence of pyrrolizidine alkaloid trichodesmine, β-amyrin,
β-methyl oleanate, potassium nitrate, a nitrogen containing toxin, β-sitosterol
and stigmasterol (Omar et al., 1983). R. epapposum
contains flavonoids, tannins, sterols, triterpenes and volatile oils (Ageel
et al., 1987; Al-Yahya et al., 1990).
The characteristic features of the extracts from aerial parts of R. epapposum and T. africanum were hepatonephrotoxicity, leukocytosis due to lymphocytosis or leukopenia due to neutropenia and anaemia. The anaemia was macrocytic normochromic, as indicated by the high MCV and normal MCHC values. These changes were evidenced by alterations in AST, ALT and ALP activity and in total protein, albumin, globulin, cholesterol and urea concentrations. The occurrence of myositis brought about by the plant methanol extract given via i.m. route probably contributed to the increase in AST activity.
The presence of haemosiderin deposits in red pulp of the spleen might have
resulted from slight destruction of RBCs by unknown toxic constituent (s) in
aerial parts of T. africanum. It seems likely that splenic haemosiderosis
might have been caused by saponin present in the extract prepared from the plant
aerial parts. Iron was believed to be released from lysed RBCs to the circulation
and passed to urine, perhaps bound to transferrin (Abu Damir
et al., 1993).
Nakhla et al. (1991, 1992)
conducted toxicologic studies on Balanites aegyptiaca and Trigonella
foenum-graecum, popular medicinal plants in Sudan and other countries and
found that injection by different routes of administration of the saponin in
chicks caused hepatonephropathy and anaemia. Neither Trigonella saponin
nor Balanites saponin caused bilirubinaemia probably due to the presence
of lesions in the hepatic centrilobular zone of the test chicks. Tariq
et al. (1985) injected rats via intraperitoneal route with ethanol
and chloroform extracts from T. anguina and observed that both extracts
produced sedation accompanied by rapid respiration.
Galal et al. (1991) were able to prepare ethanol
extract as well as other fractions from the bark of Albizia anthelmintica
to compare their toxicity in rats. These authors found that the butanolic fraction
was more toxic and lethal to rats than ethanol extract and that both extracts
caused varying degrees of hepatonephropathy.
Further studies are necessary to isolate and characterize the constituents in plants aerial parts and elucidate their exact modes of action and interaction.
CONCLUSION
The aerial parts of R. epapposum were toxic but not lethal, to Wistar rats at the concentrations in the test diets. The toxicity from T. africanum aerial parts was severe as evidenced by inefficiencies of food utilization, extensive tissue damage, significant serobiochemical and haematological changes and death among the rats 2 weeks after treatment.
The study also described the comparative toxicity to rats of R. epapposum and T. africanum aerial parts aqueous and methanol extracts and confirmed the development of vital organ lesions as well as myositis at site of injection being more marked in case of T. africanum extract.
RECOMMENDATIONS
Studies are, therefore, needed to elucidate the mechanism responsible for the development of vital organ lesions in rats and other animals.
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