ACUTE AND SUB-ACUTE DERMAL TOXICITY STUDIES OF MORINDA CITRIFOLIA L. FRUIT EXTRACT IN SPRAGUE DAWLEY RATS

 Objective: Morinda citrifolia is one of the most significant plants that are used in traditional medicine. However, details of the dermal toxicity of M.citrifolia are still undiscovered. The objective of this study is to investigate the in vivo acute and subacute dermal toxicity of ethanolic extract of M.citrifolia fruit extract at doses 2000 and 5000 mg/kg in acute and 500, 1000 and 2000 mg/kg body weight in rats.Methods: In acute experiment, a total of 40 female rats were divided into eight groups, each group had five rats, and a total of 66 male rats weredivided into 11 groups of six rats in sub-acute experiment.Results: The extract at a single dose of 2000 and 5000 mg/kg of body weight did not produce treatment-related signs of toxicity or mortality inany of the animals tested during the 14-day observation period. In the repeated dose 28-day study, the application of 500, 1000 and 2000 mg/kg ofbody weight/day of fruit extract revealed no significant change (p>0.05) in bodyweight, hematological and biochemical parameters compared withthe control group. Similarly, grosspathology and histopathology examinations of liver, spleen, kidneys, and skin did not reveal any morphologicalalteration.Conclusion: Overall, the results recommend that the close application of M. citrifolia fruit extract did not deliver any critically dangerous impact inrats. Subsequently, the concentrate can be employed for pharmaceutical plans.Keywords: Morinda citrifolia, Acute dermal toxicity, Fruit extract, Sub-acute dermal toxicit

Effects of Morinda citrifolia on early stage of leukaemia in rats

We have studied the effects of Morinda citrifolia on haemogram parameters in rats with early stage of leukaemia. A total of 32 male Sprague Dawley rats were divided into four groups (n=8) namely control, Morinda citrifolia (MC), N-methyl-N-nitrosourea (MNU) and MC+MNU group. Leukaemia induced rats were provided with died M. citrifolia fruits incorporated into rat diet at a dose of 3000 mg/kg body weight were given daily to the MC and MC+MNU groups. Results showed there were no significant differences in the leukon and erythron parameters in all groups. Although lymphocytosis was not observed in the MNU and MC+MNU groups, examination of the blood morphology showed a typical morphology of leukaemic cell. Seventy-five and sixty percent of rats in the MNU and MC+MNU groups, respectively, had leukaemia. Our results indicate that examination of blood smear provides an important tool for diagnosis of early stage of leukaemia and daily supplementation of M. citrifolia at the dose of 3000 mg/kg reduced the incidence of early stage of leukaemia in rats

Expression of circulating CD146 associated with endovascular dysfunction in adenine-induced chronic renal damage in rats using an EvaGreen real-time RT-PCR assay

The integrity of the endothelial layer of the blood vessel, intima, is critical since damage can lead to atherosclerosis. Assessment of its integrity is therefore very important although this can not be done through routine diagnostic analysis. An alternative non-invasive method should be established to assess its integrity. The objective of this study was to develop a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) using an EvaGreen dye for the assessment of vascular endothelial damage-associated with chronic renal damage in rats via quantification of the expression level of CD146, which is one of the blood biomarkers of endovascular dysfunction and/or damage. Comparison of the sensitivity of the EvaGreen qRT-PCR and the conventional RT-PCR was also performed in this study. For this purpose, a total of 16 male Sprague Dawley rats were divided equally into two groups, namely the control and adenine. Results showed that rats fed with diet containing 0.75% adenine for six consecutive weeks developed chronic renal damage through elevations in the blood urea nitrogen (BUN) and blood creatinine (Cr) levels, increased urine protein to creatinine ratio (UPC) and decreased creatinine clearance (CrCl). The confirmatory diagnosis of chronic renal damage was made through gross and histopathological examinations of the kidneys. The results were consistent with lesions of chronic renal damage. The expression of circulating CD146 was mildly increased in rats with chronic renal damage analysed using the EvaGreen qRT-PCR or the conventional RT-PCR, suggesting a mild degree of endovascular damage. The intensity of the EvaGreen qRT-PCR products, however, was brighter than the conventional RT-PCR, indicating that the EvaGreen qRT-PCR is more sensitive compared to conventional RT-PCR, which is further recommended for analysis of CD146 expression in rats

Tissues thiocyanate (SCN) concentration and liver pathology of sheep and goats fed on cassava forages

Cassava leaves are good sources of protein which have a potential to substitute grain concentrate in livestock feed. However, a major constraint in using cassava fodder as animal feed is the presence of hydrogen cyanide (HCN). A study was conducted to compare the cumulative effects of thiocyanate (a product from the detoxification of hydrogen cyanide) at 4 mg and 7 mg HCN/ kg body weight on sheep and goats. Thiocyanate was sourced from the detoxification of hydrogen cyanide in cassava. The tissue thiocyanate concentrations were found to be significantly (p<0.05) higher in liver (2.29 μg/mL/g tissue) of goats as compared to that of sheep. Meanwhile, histological examination of the liver revealed the presence of periportal necrosis. In spite of detoxification process of hydrogen cyanide to thiocyanate, it could be concluded that at 7 mg HCN/kg body weight, considerable amount of thiocyanate was retained in the body and accumulated in the liver

Some chemical carcinogens for leukaemia induction and their animal models

Animal models have been providing invaluable contributions to the better understanding of mechanisms of cancer (including leukaemias) development and effectiveness of most of the treatments. Chemical carcinogens are generally used to study the biology of cancers including leukaemias in many animal models, including rats and mice. The studies in most cases are aimed at the development and evaluation of cancer treatments and preventions. Some of the most common chemical carcinogens used in animal models for leukaemias include N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), dimethyl benz(a)anthracene (DMBA) and benzo(a)pyrene (BaP). This review provides highlights on different animal models of leukaemia induced by the chemical carcinogens mentioned earlier, at the same time discussing the contributions of these models to the leukaemia diagnosis in laboratory animal models for subsequent development of treatment

Subacute oral toxicity assesment of ethanol extract of Mariposa christia vespertilionis leaves in male sprague dawley rats

The term Butterfly tea refers to decoction of Mariposa christia vespertilionis leaves which is widely consumed by cancer patients throughout Malaysia and has gained a huge popularity among Malaysians, not only cancer patients but also researchers to discover the real potential of this plant. Herein, the study is aimed at evaluating the possible toxicity in 28-day subacute oral toxicity of ethanolic extract M. christia vespertilionis in male Sprague Dawley rats. The 28-day subacute toxicity study was conducted to detect the no-observed adverse effect level (NOAEL). In this study, a total of 30 rats were divided into the control, 5% DMSO (vehicle), low dose (75 mg/kg), medium dose (125 mg/kg) and high dose (250 mg/kg) groups. The extract was administered daily from day 1 until day 28. At the end of the study, the animals were humanely sacrificed and assessed for the effect extract of Mariposa christia vespertilionis leaves on body weight and relative organ weights and haematological, biochemical and histopathological parameters. The haematological and serum biochemical parameters for the assessment of kidney and liver injuries were carried out. Results of haematological and serum biochemistry results showed no changes in the control and treated groups. In the histopathology, evaluation of kidney tissues in all treated groups showed no significant (p > 0.05) lesions. In contrast to kidney, liver tissues showed significant differences (p < 0.05) in lesions observed in low dose (430 mg), medium dose (700 mg) and high dose (1480 mg) groups with very mild, mild and mild to moderate lesion of hepatic necrosis, in the respective groups, and very mild hepatic degeneration and hepatitis were scored in all three groups

Subchronic oral toxicity study of Morinda citrifolia (mengkudu) in Spraque Dawley rats

A subchronic oral toxicity study was conducted to evaluate the safety of Morinda citrifolia in Sprague-Dawley (SD) rats. For this purpose, the fruit of Morinda citrifolia were oven dried and ground into powder form before incorporating into diet and fed to SD rats (10 males and 10 females per group) at dose levels of 2000 (low dose) and 5000 (high dose) mg/kg body weight/day for 13 weeks. Clinical observations were recorded, while body weight and feed consumption were measured throughout the study. At the end of the study, all the rats were subjected to a full necropsy. Their blood samples were collected for clinical pathology, whereas selected organs were weighed and tissues were preserved from all the animals. Total protein was found to be significantly lower (p<0.05) in male rats of all the treatment groups. Meanwhile, total white blood cells (3.96 ×10³/µl) and spleen weight (0.14%) were found to be significantly lower (p<0.05) in female rats of the low dose group. Nevertheless, the differences observed were within the normal range of normal healthy rats that were considered to be not toxicological significance. It was concluded that the no-observed-adverse-effect level (NOAEL) for Morinda citrifolia was 5000 mg/kg body weight/day

Histopathological features of peripheral T-cell lymphoma in Sprague Dawley rats induced with N-methyl-N-nitrosourea

This study described the histopathological features of peripheral T-cell lymphoma in male Sprague Dawley rats following intraperitoneal (i.p.) injections of N-methyl-N-nitrosourea (MNU) at a dose of 60 mg/kg body weight per injection, administered twice weekly for 2 consecutive weeks, and followed by a five-month’s observation period. Control rats were injected with normal saline, i.p. All the rats treated with MNU had enlargement of lymph nodes, with 30% had hepatosplenomegaly and 7% had enlarged kidneys at necropsy. Malignant lymphoma was observed in the lymph nodes, spleen, liver, lung, heart, and kidneys. The neoplastic cells were characterised as undifferentiated, and small to large size with bizarre pleomorphic nuclei. The severity was further described as mild, moderate and severe, based on the diffuseness of the lesions. Nonetheless, similar lesions were not observed in the thymus of the rats. Immunohistochemistry staining of the organs was positive for CD3 antibody, which is consistent with T-cell lymphoma

Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay

Bcl-2 is an anti-apoptotic gene that is involved in the apoptosis process. Suppression of apoptosis by anti- apoptotic gene can contribute to the occurrence of diseases such as leukaemia. The objectives of this study were 2-folds: first, to compare the sensitivity of an EvaGreen quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) with a conventional RT-PCR for the amplification of the Bcl-2 gene; second, to determine the expression of the Bcl-2 gene in N-methyl-N-nitrosourea (MNU)-induced leukaemiain rats using the EvaGreen qRT-PCR assay. A total of 32 male Sprague Dawley rats were assigned into two groups (n=16), namely, control and MNU groups. In particular, MNU was administered intraperitoneally (i.p) at a dose of 60 mg/kg body weight per injection at two times per week for 2 consecutive weeks. The rats were sacrificed after five months and blood samples were collected for RNA extraction and haemogram. The RNAs were converted into cDNA and amplified using both the EvaGreen qPCR and the conventional PCR assays. All the results were normalised with a housekeeper gene, i.e. glyceraldehyde 3-phosphate dehydrogenase (GADPH). The products of amplification were run on gel electrophoresis and all the results were then compared. Based on the relative intensity of the bands, the EvaGreen qRT-PCR assay was highly sensitive compared to the conventional RT-PCR assay as the Bcl-2 gene could not be amplified using the conventional RT-PCR. Interestingly, the results in this study showed that the expression of Bcl-2 was higher in rats with marked lymphocytosis as compared to the leukaemic rats with normal to mildly increase in lymphocyte count. In conclusion, EvaGreen qRT-PCR assay is more sensitive compared to the conventional RT-PCR, and Bcl-2 gene is abundantly expressed in leukaemic rats with marked lymphocytosis compared to the leukaemic rats with normal to mildly increase in lymphocyte number