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Toxicological study of Manihot esculenta Crantz (Euphorbiaceae) leaf extracts

Manihot esculenta , also named cassava, is a plant widely used as food but also in traditional medicine to treat many diseases such as anemia. However, consumption of M. esculenta is limited by its content in cyanogenic glycosides. Few studies have evaluated the toxicity of M. esculenta leaves. This study aims to evaluate the in vitro cytotoxicity of Artemia salina and the 28 days subchronic toxicity of M. esculenta leaves by oral administration of the extract to male Wistar rats. The A. salina assay showed that the alcoholic extract of M. esculenta presented some toxicity. The results of 28 days subchronic toxicity showed a significant increase in the relative weight of the liver. No changes in hematological parameters were observed apart from a significant increase in platelet number. Biochemical parameters such as SGOT, SGPT, glycemia, and ALP were not significantly changed. The histological sections showed no organ damage.


INTRODUCTION
Manihot esculenta is a worldwide plant belonging to Euphorbiaceae family's, extensively cultivated in tropical and subtropical regions 1 . Though, the main part of the plant used is its starchy tuberous root, its leaves are also consumed as vegetables 2, 3 and used for various medicinal purposes by local communities. Interestingly, cassava leaves are reported to have bioactivities such as anti-oxidant, anti-tyrosinase, anti-inflammatory, and hepatoprotective 2,4,5,6 . It is a source of calories in the human diet in the tropics 7 . The common use of these leaves is for disorders such as rheumatism, fever, headache, and loss of appetite. 8 In Nigeria, they are also utilized in the treatment of ringworm, tumor, conjunctivitis, sores, and abscesses. Apart from these, various literature have mentioned the use of plant leaves in hypertension, irritable bowel syndrome, aches, cancerous affections, affective excrescences of the eyes and tumors, antiseptic, cyanogenetic, demulcent, diuretic, dysentery, flu, marasmus, snake bites, prostatitis, and spasms, etc. 9,10,8 M. esculenta leaves are a good source of acids 11 . Additionally, it contains a cyanogenic glucoside, linamarin and lotaustralin 12 . Generaly, there is a detoxification mechanism which can avoid death when cyanide release is slow 13 . The consumption of large amounts of the bitter variety (containing higher cyanogenic glucosides) can cause severe toxicity resulting in Konzo, a paralytic disorder or even death. Routine ingestion of low levels of cyanide leads to chronic toxicity possibly developing into goiter (enlargement of the thyroid gland) or tropical ataxic neuropathy (a disorder involving the nervous system). However, scientific research is still needed to support these findings 9,8 . This study aims to evaluate the in vitro cytotoxicity of M. esculenta leaves on Artemia salina and its 28 days subchronic toxicity by oral administration of the extract on male Wistar rats.

Collection and extraction of plant material
Manihot esculenta leaves were collected from the Agronomic farmstead of University of Lomé (TOGO) after identification by the Botany and Ecology Department of the same University. A voucher specimen was registered in the herbarium under the number TG 15184.
Leaves were cleaned and dried under climatization (20°C). Then 500 g of dried and pulverized leaves were soaked in 7.5 L of ethanol-water (70-30) for 72 hours with occasional agitation. After double filtration with cotton and Whatman paper, the filtrate was evaporated using a rotating evaporator Heidolph 2 (Germany). The extract was dissolved in distilled water with 2% of Tween 80. The yield of this extraction was calculated according to the formula: R = (weight of extract / dry weight) x 100. Animal's care and handling were conformed to accepted guidelines 10,8

Phytochemical screening
The screening was performed to assess the presence of some chemical groups such as tannins and alkaloids, saponins, and flavonoids. Flavonoids, saponins, and tannins were checked as described by Karumi et al. 14 . For alkaloids, Bourchadat, Dragendorff, and Mayer reagents were used to determine their presence as described by Evans 15 .

Assessment of larval toxicity of the substance
Bioactivity of the extract was monitored by the brine shrimp lethality test 16 . Different concentrations of extract from 25 mg/mL to 0.049 mg/mL were prepared. Using a cone micropipette, a colony of 16 live larvae was brought into contact with a series of solutions of progressive concentrations of plant drug extracts. These media and controls were allowed to stir and live larvae were counted 24 hours after incubation. The mean percentage mortality was plotted against the logarithm of concentrations. The concentration (LC50), at which 50% of the larvae were killed, was determined from the graph 1, 17, 18 .

Acute toxicity test
The limit dose of 5000 mg/kg was carried on three female Wistar rats according to the Organization for Economic Cooperation and Development (OECD) guidelines 19 . Each rat was sequentially dosed at the interval of 48 h. The animals were observed individually to check acute toxicity signs or behavioral changes, 30 minutes post-dosing during the first 24 h. The rats were observed at least once daily for 14 days.

Sub-chronic toxicity
Repeated-dose oral toxicity was achieved according to OECD guidelines 407 20 . The animals were divided into three groups of 6 each. The first group (group 1) received distilled water containing 2% of Tween 80 and served as control group. The second (group 2) and the third (group 3) groups received, respectively, the extract at 500 mg/kg and 1000 mg/kg body weight.
The extract was administrated daily for 28 days at the same time and the animals were observed at least twice daily for morbidity and mortality. All groups were administrated 1 mL/100 g body weight of solution. The body weights were recorded every day. The rats were observed for aggressiveness, mobility, diarrhea, appetite, and answer to sound stimulation. On the 29 th day, after 12 h of fast, the rats were anesthetized first by ether. Blood samples were collected from www.bjmhr.com Necroscopy of all rats was carried out and some organ weights (kidney, liver, spleen, heart, and testis) were recorded.

Statistical analysis
Results were expressed in mean ± standard error of the mean (SEM

Phytochemical screening and extraction of plant materials
The evaporation allowed us to obtain a yield of 8.15%. The results of the phytochemical screening have shown the presence of flavonoids, saponins, tannins, and the lack of alkaloids.
(data was not shown).
Brine shrimp toxicity screening were determined and were found to be 1.031 ± 0.11 mg/mL.

Acute toxicity
For a short (48 h) or long period of observation, the limit dose 5000 mg/kg of M. esculenta didn't lead to mortality or acute toxic effects of the three rats dosed. The LD50 was then over 5000 mg/kg.

Sub-chronic toxicity
In the clinical evaluation, no behavioural changes and death were observed at the end of treatment. Similarly, no significant differences in body weight were observed between the control and treated groups during this period (Table 1). The extract was administrated daily for 28 days. The body weights were recorded every day.
M. esculenta at 1000 mg/kg increased significantly (p <0.001) the relative weight of the liver as compared to the control group (Table 2).    The extract was administrated daily for 28 days. The haematological parameters were performed at the end of the administration. *** p <0.0001 significant difference as compared to the control  The LC 50 value of M. esculenta was 1.031 ± 0.11 mg/mL. Therefore, the hydroalcoholic extract of M. esculenta. leaves is not toxic according to Meyer et al. 1 , who classified crude extracts and pure substances into toxic (LC 50 value < 0.1 mg/ml) and nontoxic (LC 50 value > 0.1 mg/ml).
Regarding body weight, M. esculenta hydroethanolic extract had not induce significant changes in Wistar rat body weight. In our study, M. esculenta increased significantly (p <0.05) the liver relative weight. The assessment of the weight of organs such as the liver, kidney, spleen, testis, heart, pancreas, brain, and language is very essential in toxicological studies. The weight of a body or more, the relative weight is an important index used in physiology and toxicology 25 .
The increase in liver relative weight could be due to protein synthesis induction 26 or peroxisome proliferation or inflammation 25,27,28 No significant effect was observed on haematological and biochemical parameters. The dosage of transaminases (SGPT, SGOT), alkaline phosphatase, and glycemia is essential in the evaluation of liver toxicity 29,30 . As for creatinine and urea, they are used to assess renal toxicity 31  A similar study conducted by Soto-Blanco and Gorniak 33 , on goats fed directly with leaves whose cyanide hydroxide content was evaluated at 6 mg/kg and then decreased to 4.5 mg/kg, revealed hepatotoxicity. The difference with our study could be explained by the concentration of cyanogenic glycosides contained in the leaves, the animal material used and the duration of exposure.
Similarly, hepatic damage was found in cassava-treated dogs and hepatopathy was characterized by periportal hepato-laryngeal vacuolation 34 . Similar degenerative changes have been described in the livers of rabbits 35 and HCN-treated rats 36 , but in goats only minimal changes in the liver are observed 33 . Thus, it is possible that the liver is a target tissue for cyanide toxicity 37 . These results could explain the increase in the relative weight of the liver in our study.

CONCLUSION
In this study, we have evaluated the in vitro cytotoxicity of M. esculenta leaves on Artemia salina and its 28 days subchronic toxicity by oral administration of the extract on male Wistar