Acute, sub-acute, sub-chronic and chronic toxicity studies of four important Nigerian ethnomedicinal plants in rats

Ethnomedicinal plants have been widely used in humans and animals as therapeutic remedies for the treatment, mitigation and prevention of diseases in traditional medicine for centuries in both developed and developing countries. The general perception is that medicinal plants are natural products devoid of synthetic preservatives and therefore, safe for discretional uses. Over 85% of disease conditions of humans and animals, ranging from bacterial infections to cancer and immunological disorders are treated with either natural products or compounds derived from natural products [1]. Natural medicines are fast becoming mainstay primary health-care alternatives worldwide, with approximately 50% of the USA population using natural medicines for the treatment and prevention of ill-health conditions [2]. About 70% of therapeutic compounds in use today or currently in clinical trials are obtained directly from natural products or synthesized from natural products [1]. This has led to the codification of the natural environment as a rich source of unique biodiversity for pharmaceutical lead-compound identification and drug discovery [3]. The drug discovery process is complicated and interwoven, requiring not only information about pharmacodynamics and pharmacokinetic parameters of the compound but also, more importantly, its safety [4].

Drug-induced liver injury and nephrotoxicity are the leading causes of pharmaceutical withdrawals of promising drug candidates in clinical trials [5, 6]. Whereas, aminotransferases and to a lesser extent, alkaline phosphatase, sorbitol dehydrogenase, glutamate dehydrogenase, gamma-glutamyltransferase, total bilirubin, total bile acids, and 5′-nucleotidase are the commonly evaluated biomarkers in drug safety assessment of hepatotoxicity; blood urea nitrogen, serum creatinine, sodium and phosphorous are the common endpoint indicators used to evaluate renal function [7]. Therefore, assessment of at least four serum parameters, involving a minimum of two for each of the hepatocellular and hepatobiliary serum biomarkers has been recommended for a safety study of xenobiotics [8]. The high susceptibility of kidneys to xenobiotics may not be unconnected with a copious volume of circulating blood (about 25% of cardiac output), which contains compounds (xenobiotics) or their metabolites delivered to the kidneys for filtration. Furthermore, the sole responsibility of nephrons in the processes of urine formation aggravates the concentration of toxicants contained in the blood in renal tubular fluid [5]. Although efforts are ongoing to identify specific urinary biomarkers to study the effects of potential drug candidate on kidneys and to monitor renal function with some successes being made, Food and Drug Administration of the United States of America still recommends the use of blood urea nitrogen and serum creatinine in rats as reliable indicators of toxic insult to the kidneys [9].

Azadirachta indica A. Juss. (Maliaceae), Anogeissus leiocarpus (DC.) Guill. & Perr. (Combretaceae), Khaya senegalensis (Desv.) A. Juss. (Maliaceae), and Tamarindus indica L. (Fabaceae) are important medicinal plants with wide ethnomedicinal and scientifically validated applications. T. indica is used to treat constipation, abdominal pain, wounds, diarrhoea and dysentery, fibril conditions and malaria [10] A. leiocarpus is used to treat tuberculosis, cough, toothache and dental caries, intestinal parasites, fungal infection, wound, malaria, diabetes and viral infections [11]. Similarly, K. senegalensis is reported to be effective against intestinal parasites, bacterial infections, diabetes, helminthosis, cancer and trypanosomiasis [12]. The ethnopharmacological applications of A. inidca include management of tuberculosis, malaria, cancer, diarrhoea, gastric ulcer, wound and fungal infection [13]. While medicinal plants are generally regarded as safe medicines with a wide margin of safety, nature has endowed them with metabolites to ward off potential attacks from animals and the environment. These metabolites are responsible for the pigmentation, fragrance and toxicity of some of these plants [14]. Recently, we reported the potential antitrypanosomal effects of stem-bark extracts of A. leiocarpus and K. senegalensis [15]. It is, therefore, necessary to study their safety margins. Thus, we aim to investigate A. indica, K. senegalensis, A. leiocarpus and T. indica for safety in short- (acute), medium- (sub-acute) and long- (sub-chronic and chronic) term uses in rat.

The apparent lack of morbidity and absence of mortality in the determination of median lethal dose (LD₅₀) of the four plants show that their LD₅₀ is exceeds 5000 mg/kg. According to Lorke [19] and Tauheed et al [20], a chemical or drug candidate with an LD₅₀ of 5000 mg/kg or greater is acutely safe and can be used for therapeutic purposes. The apparent safety level recorded in the present study underscores non-safety dose optimisation usage of medicinal plants in folkloric medicine. This agrees with the finding of Tauheed et al [20], who observed that the use of a wide range of doses of decoction and infusion of medicinal plants in traditional medicine could be due to their wide margin of safety.

Bodyweight changes and organo-somatic index (OSI) have been used to predict the toxic effects of exogenous compounds [21]. Because of the importance of the liver and kidneys in drug metabolism and excretion, ratiometric differences of these organs were compared with the bodyweights of treated rats. The significantly lower weight gain observed in the rats treated with T. indica may not be due to obvious physiological perturbation since the plant demonstrated protective rather than toxic effects in all the parameters evaluated. The ability of some medicinal plants to decrease weight gain has been used to manage obesity [22]. Although clinical and histological findings from the present study showed varying degrees of toxicity, wet OSI of liver and kidneys did not reveal significant changes. Our results agree with the earlier results of Onu et al. [23] who reported non-significant change in the OSI of kidney and heart of rats subjected to sub-chronic administration of K. senegalensis and Tantulo and Fotedar [24] who demonstrated no difference in OSI of black tiger prawn juveniles. It follows that significant changes in OSI may occur following extensive insult to the body. OSI may, therefore, not be a sensitive parameter for toxicological studies and its interpretation should be made with caution.

A. leiocarpus and K. senegalensis significantly increased serum levels of ALT, AST and ALP at various time intervals, thus, indicating toxic insults to the liver. Unlike AST and ALP which are found in high proportion in other organs and tissues of the body (eg, skeletal muscle, heart, liver, kidney, pancreas and erythrocyte), ALT is predominantly found in the cytosol of hepatocytes and is the most sensitive biomarker of hepatocellular injury used for the assessment of functional integrity of the liver or otherwise toxic insult to the liver [7]. Higher values of aminotransferases (ALT and AST) recorded in our study may indicate hepatocellular injury, while higher values of ALP show hepatobiliary injury. Leakage of transaminases to the extracellular compartment occurs following damage to the liver which is rapidly detected in the serum. Whereas elevated values of ALT may be seen in enzyme induction in rats [25] and muscle injury, elevated AST often occurs following damage to other organs/tissues. However, the magnitude of ALT elevation is often greater than AST when both enzymes are elevated due to liver damage. This has been attributed to the longer half-life of ALT and the binding of a sizable proportion of AST to mitochondria [26]. ALP is the most widely used biomarker of hepatobiliary injury in common preclinical species. Serum ALP levels increase when the patency of the bile duct is reduced and is used in a nonclinical and clinical marker of cholestatic liver injury [25, 27]. The Aminotransferases play a critical role in amino acid metabolism by retaining amino groups during amino acid degradation for subsequent synthesis of new amino acids [28]. The significantly higher values of the transaminases, especially in the A. leiocarpus-treated group may explain the lower weight gain recorded in this group. High values of aminotransferases in the serum mean that the enzymes are being lost, which negatively affects amino acid synthesis, and, thus promotes muscle wastage and consequently, lowers bodyweight gain. Whereas K. senegalensis significantly decreased TP in the acute study only, A. leiocarpus produced a significant reduction in TP in the AS, SAS, SCS and CS. Thus, it may be inferred that A. leiocarpus exhibited a sustained depressant effect on the liver and produces prolonged inhibition of protein synthesis. Low protein levels have been shown to be due to extensive liver damage [29].

The concomitant increase in the levels of urea, creatinine, Na⁺ and K⁺ at various levels of treatment with the plants could be attributable to renal impairment, leading to the inability of the kidneys to regulate the amount of these substances that are excreted from the body. Although even a marked increase in serum urea may not point towards primary renal dysfunction since increased urea excretion is a sequel to increased protein metabolism, a marginal increase in serum creatinine has been reported to have a grave long-term consequence on the survival of the patient [30]. Creatinine is a late indicator of acute renal injury [31] and has been used as one of the primary indicators of acute kidney injury [32].

The plants used in the present study are not haemotoxic since they maintained normal blood parameters in all the studies. The ability of the tested plant extracts to maintain adequate haematological parameters may account for their apparent wide margin of safety and thus, maybe the reason why preparations made from these plants, and perhaps other medicinal plants, are used freely.

Histological examination of tissues and organs is used to corroborate physical and clinical findings and aids definitive diagnosis. The observed histopathological lesions, ranging from nonlethal changes (eg, degeneration, congestion) to lethal changes (eg, necrosis) indicate some levels of toxic insults to the chief metabolizing and detoxifying (liver) and excretory (kidneys) organs in the body. Whereas degenerative changes often indicate nonlethal injuries to cells, necrosis indicates terminal, lethal injury to cells [33]. Our findings showed that acute studies produced predominantly degenerative changes in the hepatocytes. This implies that acute studies are associated with mild, reversible damage to the liver in the form of cellular degeneration, which corroborates our clinical findings. We reported significantly higher values of aminotransferases, particularly, ALT; a liver-specific enzyme. Clinical and histopathological findings in this study further revealed salient insult to the liver which is often missed during routine acute toxicity study in the determination of median lethal dose. In routine LD₅₀ determination, animals are dosed once and observed for 14-day before they are sacrificed for clinical and histological examinations. Indeed, mild insults like the transient elevation of aminotransferases in the serum and congestion and degenerative changes in the histological section might have resolved during the 2-week observation period and no obvious lesions may be seen on day 14. Nonetheless, routine LD₅₀ determination is still useful since only a fraction of the LD₅₀ is used for therapeutic evaluation. This is the reason why several authors have reported earlier that compounds with LD₅₀ of 5000 mg/kg is acutely safe [19, 20, 34] and the recommendation by the WHO that such compounds can be used for therapeutic studies [35]. Furthermore, we observed that intermediate and prolonged administrations for 2 and 12–14 weeks, respectively, are associated with lethal cellular injuries in the form of necrosis and fibrosis.

Only K. senegalensis showed a nephrotoxic effect. It is possible that K. senegalensis is concentrated in the renal tubules and thus provokes a cellular response in the form of congestion, cellular infiltration and necrosis.

We conclude that Anogeissus leiocarpus, Khaya senegalensis, Azadirachta indica and Tamarindus indica (and perhaps, other medicinal plants) are not devoid of toxic effects, especially when large doses are used. The obvious lack of morbidity and mortality does not rule out pathology but because rats are not immediately sacrificed (eg, keeping animals for 14 days) after administration (single dose in the case of acute toxicity study) or last administration (for continuous dosing in the case of long-term studies) of plant extract(s) or pharmaceutical agents, the transient rise in the traditional biomarkers of liver (transaminases and other enzymes) and kidney function tests are reversible and might have resolved before the animals were sacrificed. Single large dose administration in short-term use produces greater reversible toxic effects on important organs of metabolism and excretion than one-quarter of the same dose used over a prolonged period of medium- (4 weeks) and long-term (12–14 weeks) uses. The ability of these plant extracts to maintain adequate hematological parameters, bodyweight and absence of mortality may be responsible for free usage of preparations made from these plants in folkloric medicine. Finally, the study showed that A. leiocarpuss and K. senegalensis are more toxic than A. india and T. indica.