Sub-acute Toxicological Evaluation of Methanol Whole Plant Extract of Tapinanthus dodoneifolius (D.C) Danser in Wistar Rats

1. INTRODUCTION

The increasing use of medicinal plants in the treatment of many ailments necessitates the toxicological screening to ascertain their safety among users [1]. Toxicity is a term that describes the level of an adverse effect caused by the interaction of a toxicant and cell(s) of a living organism [2]. Medicinal plants are essential part of healthcare delivery system in resource-poor areas of the world. About 80% percent of the world’s population uses herbal medicine in their healthcare delivery system [3]. The greater demands for resources and time in evaluating the safety and standardizing the use of medicinal plants have led to much emphasis of screening them for efficacy based on traditional claims rather than safety. Toxicological evaluation of medicinal plants is as important as pharmacological validation of ethnomedicinal claims [4]. Other reason that may have led to the shift of attention more on pharmacological validation may stem from the general belief of safety and acceptability of medicinal plants.

Tapinanthus dodoneifolius is a bushy parasitic plant that grows on a wide range of trees around the savannah zone. The plant belongs to the family Loranthaceae. T. dodoneifolius is an important plant use in the treatment of many diseases such as stomach ache, diarrhoea, dysentery, diabetes, epilepsy, hepatitis, hypertension, wounds, cancer etc among the Hausa and Fulani tribes of northern Nigeria. The local names of the plants among different tribes in Nigeria are Etu-lonchi (Nupe), Elozie (Ibo), Kauchi (Hausa) and Afomu Igba (Yoruba) [5]. Other ethnomedicinal uses of Tapinanthus dodoneifolius are treatment of inflammation, fever, infection, dizziness, energy loss, irritability, vertigo and headache. The has a broad spectrum of activity against multidrug resistant bacteria and fungal isolates of farm animals [6, 5]. T. dodoneifolius is also reported to have larvicidal and molluscidal effects. Related Africa mistotle Agelanthus dodonefolins has been reported to have antiplasmodial activity [7]. Despite the dependence on medicinal plants most especially in resource poor areas, there is still sketchy information about their safety [8]. Tapinanthus dodoneifolius is one of such plant. This study was undertaken to evaluate sub-acute toxicity profile of methanol whole extract of Tapinanthus dodoneifolius in Wistar rats.

2. MATERIALS AND METHODS

Collection of Plant

The whole plant of Tapinanthus dodoneifolius was collected from the bush in Sabon Gari Local Government Area of Kaduna State, Nigeria in the month of June, 2018. It was identified and authenticated by Mr. Namadi Sanusi of the Department of Botany, Ahmadu Bello University, Zaria. A voucher number; 0370 was obtained by comparing with existing specimen.

Preparation of Plant Extract

The whole plant of Tapinanthus dodoneifolius was washed, air-dried under shade after which it was grounded to coarse powder using pestle and mortar. About 900 g of the powdered plant material was extracted with 7 L of 70 %^v/v methanol for three days using cold maceration with intermittent shaking. The mixture was then filtered using Whatman filter No 1. The filtrate was concentrated and subjected to air drying in crucible. Percentage yield of the methanol extract was calculated and the obtained extract was stored in a desiccator and subsequently referred to as Methanol Extract of Tapinanthus dodoneifolius (METD). Fresh concentrations of the extract were prepared on each day of the experiment by reconstituting in distilled water.

Experimental Animals

Wistar rats of both sexes were obtained from the Animal House Facility of the Department of Pharmacology and Therapeutics Ahmadu Bello University, Zaria. Animals were housed in cages with rodent diet and water ad libitum. Ethical approval with an approval number: (ABUCAUC/2019/006) was obtained from the Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC).

Phytochemical Screening

Preliminary phytochemical screening of the methanol whole extract of Tapinanthus dodoneifolius was carried out according to methods described by Trease and Evans and Ayoola [9, 10]

Acute Toxicity Study

The acute toxicity study was carried out according to the Organization for Economic Co-operation and Development (OECD) guidelines 423[11] and fixed dose studies was adopted with 5000 mg/kg body weight as the limit dose. Three rats were fasted for 3-4 hours, and each rat was administered 5000 mg/kg of methanol whole plant extract of Tapinanthus dodoneifolius. Food was withheld for 1 hour post drug administration. Each rat was observed individually for the first 30 minutes and periodically during the first 4 hours and then daily for 14 days for any sign of toxicity, such as tremor, convulsion, salivation, lacrimation, diarrhea, lethargy, sleep, respiratory, behavior pattern, onset and recovery from toxicity and death.

Sub-acute Oral Toxicity Study

The method described by the Organization for Economic Co-operation and Development (OECD) test guideline 407 was adopted for the study [12]. Twenty-four rats (12 males and 12 females) were weighed and grouped into 4 of 3 male and 3 female rats in each group (the males were separated from the females). Group 1, 2, 3 and 4 received 10 mL/kg distil water, 250, 500 and 1000 mg/kg per oral of the extract respectively for 28 consecutive days. Animals were sacrificed under chloroform anaesthesia after being deprived of pellet but had free access to water for 24 hours. Blood samples were collected through cardiac puncture into EDTA and non-heparinized containers for haematological and biochemical analysis respectively. The liver, heart, spleen, stomach, kidney and lungs were collected, weighed and stored in 20 mL of 10 % formyl saline sample bottles (Axiom, Zhanjiang Gong Jong medical technology Co. Ltd, China) and thereafter processed for histopathological studies.

Body Weight

The body weights of the animals were taking at weekly interval during the course of the 28-days treatment. The weight changes were calculated in respect to the initial body weights on day zero.

Organ Weight Index

The heart, lungs, stomach, liver, spleen and kidney were collected, freed of connective tissues and rinsed in normal saline, blotted with filter paper and weighed. The relative organ body weight (ROW) was calculated as: ROW= (organ weight/total body weight) × 100.

 Haematological Analysis

Blood samples were collected and assayed. The parameters assayed include haematocrit (HCT), Hemoglobin (HB), red blood cell (RBC) count, platelet count (PLT), white blood cell (WBC) count, monocytes (MON), lymphocytes (LYMPH), granulocytes (GRAN) using automated haematology analyzer (Mythic 18 by Orphee, Switzerland).

Biochemical Analysis

Serum blood samples were analyzed for alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), albumin (Alb), total protein (TP), urea (Bu), creatinine (Crea) using ( Gesan Chem 200, USA)  automated machine.

Histopathology

The kidney, liver, heart, lungs, stomach, and spleen were placed into 10 % formalin fixative for histological examinations. Tissue slides were viewed at a magnification of x250 and photomicrographs of the tissues were obtained with the aid of a consultant histopathologist

Statistical Analysis:

Data were expressed as mean ± SEM. One-way and spilt plot analysis of variance (ANOVA) were performed appropriately to compare the differences between means followed by Bonferoni post hoc tests using SPSS version 20.0. A mean difference was considered significant when p ≤ 0.05.

3. RESULTS

The percentage yield of the powdered plant was 14.4%.

Phytochemical Constituents

The preliminary phytochemical screening of METD revealed the presence of alkaloids, cardiac glycosides, saponins, tannins, flavonoids, steroids/triterpenes, terpenoids and carbohydrates. However, anthraquinones was absent.

Acute Toxicity Study

The methanol whole plant extract produced no adverse effect in rats at the dose of 5000 mg/kg. There were no changes in behavior, nature of stool and saliva. There was no mortality observed in the different groups of rats after 7 days. The oral median lethal dose (LD₅₀₎ of the plant was estimated to be ≥ 5000 mg/kg.

4. DISCUSSION

The use of medicinal plants like Tapinanthus dodoneifolius in the treatment of human diseases has enjoyed global acceptability [13]. Comprehensive knowledge about the safety of the plant is yet to be examined despite numerous pharmacological beneficial activities. Therefore, the present study was carried out to ascertain the acute and sub-acute toxicity of Tapinanthus dodoneifolius in experimental animals. The methanol whole plant extract of Tapinanthus dodoneifolius was found to be non-toxic since there was no sign of toxicity and mortality at 5000 mg/kg. The phytochemical constituents such as alkaloids, cardiac glycosides, saponins, tannins, flavonoids, steroids/triterpenes, terpenoids and carbohydrates present in the extract may be responsible for the observed pharmacological activity [14]. A non-significant increase in body weight (Figure 1).was observed which may be due to the nutritional benefit of the plant extract [15]

Haematological parameters are used to ascertain the level of safety of compounds or extract when exposed to human [16, 17] and has been used to explain haematologically-related functions [18]. Changes in haematological parameters are necessary to risk examination due to their predictive values of toxicity [19]. The non-significant changes in RBC, HCT, Hb, and PLT level by METD (Table 1) show that the plant does not affect erythropoiesis and osmotic fragility of the red blood cells [20]. The decrease in the level of monocytes and granulocytes could suggest that the extract may exert toxic effect on the immune system and cause agranolocytosis at dose of 1000 mg/kg and above. The white blood cells (WBC) are the first line of defence which responds to infectious agents, inflammatory process or tissue injury.

The methanol whole plant extract of Tapinanthus dodoneifolius did not show significant changes in AST and ALT enzymes (Table 2), ALP was decreased significantly at dose of 1000 mg/kg suggesting inhibition of the enzyme by the extract.

The serum biochemical assay is used to assess renal and hepatic functions in order to find possible pathological changes due to the extract. Renal and hepatic function analysis is highly useful in the toxicity screening of compounds including medicinal plants as both are important for the excretion and metabolism of drugs by an organism [21]. The liver contains host of enzymes (ALT, AST, ALP) that serve as biochemical markers of liver damages. Once there is a liver damage, these enzymes leak into the serum and show increased activities [22].

The extract at all doses tested could not significantly alter the level of total protein (Table 2). This implies that it is non-toxic to the kidneys and liver. Serum protein and albumin are sensitive indicators of liver function. They are synthesized and metabolized in the liver and serve as a means of assessing hepatocellular injury [23]. Total protein measurement reflects nutritional status and may be used to screen and help diagnose kidney, liver disease and many other conditions. Low total protein levels suggest a liver, kidney disorder or any other condition in which protein is not digested or absorbed properly. High total protein level may be seen with chronic inflammation or liver infections [24].

Measurements of urea, creatinine and uric acid are used to assess renal dysfunction and their normal values indicate absence of renal damage [25]. From the result obtained (Table 3), there was no significant difference among the extract treated groups compared to the control. This suggests that the extract does not seem to possess toxic effect on the kidney.  Decrease in organ weight is an expression of toxicity resulting from exposure to toxic substances [26]. The liver; kidney, lungs, stomach, spleen and heart were not adversely affected compared to the control group, throughout the 28 day treatment period (Table 4). Hence, the whole plant extract is nontoxic to the organs in the sub-acute study.

There were no any adverse abnormalities in their gross examinations of the heart, kidney, stomach, spleen, lungs and liver isolated in various group.  The histological studies on these organs by the extract did not reveal any pathological changes after treatment compared to the control when administered for 28 days as seen in plate figures 2, 3, 4, 5, 6 and 7.

5. CONCLUSION

The methanol whole plant extract of T. dodoneifolius does not seem to possess the toxic effects that could affect its ethnomedicinal use.

ACKNOWLEDGEMENT

The authors are grateful to the entire staff of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria for providing the enabling environment to carry out this work.

CONFLICT OF INTERESTS

There are no conflicts of interest.

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