Evaluation of the biochemical, haematological and histopathological parameters of female Wistar rats fed with aqueous and ethanol extracts of Aspilia africana leaves

Introduction The spread of diseases and infections in the world today has continued to rise despite availability of orthodox drugs. Factors such as high cost of orthodox medicine, unfavorable drug side effects and lack of success in treating different ailments have contributed to the rapid spread of diseases in Nigeria (1,2). This has led to the search for better therapeutics. The unconventional use of plants products alone or in combination with modern regimens to treat different diseases is gaining popularity in recent times. This paradigm shift in the doctrine of healing is not unconnected with bioavailability, larger safety margin, and http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.33


Introduction
The spread of diseases and infections in the world today has continued to rise despite availability of orthodox drugs. Factors such as high cost of orthodox medicine, unfavorable drug side effects and lack of success in treating different ailments have contributed to the rapid spread of diseases in Nigeria (1,2). This has led to the search for better therapeutics. The unconventional use of plants products alone or in combination with modern regimens to treat different diseases is gaining popularity in recent times. This paradigm shift in the doctrine of healing is not unconnected with bioavailability, larger safety margin, and bioaccumulation of phytoconstituents which can block or inhibit disease progression (3,4). The advantages of herbal products over orthodox drugs have led to indiscriminate use of plant-based medicine over a long period of time without proper dosage monitoring and understanding the danger associated with their use (5,6). To this end, there is increasing evidence that these phytoproducts may perturb homeostatic balance in animals if not monitored; as they may trigger off liver and kidney degenerations, oxidative stress, inflammatory response, metabolic malfunction, genotoxicity and carcinogenesis (2,7,8).
Several parts of the plants have been recommended by traditional medicine practitioners for use in the treatment and management of various diseases and infections in Nigeria without proper dosage monitoring and prescription. This has led to severe organ degenerations which go on unnoticed over time. Despite various documented haematological and biochemical changes associated with the intake of plant-based therapies, herbal dealers have continued to advertise their medicinal benefits among rural dwellers; thus, increasing their widespread use in developing countries. One of such medicinal plants with broad spectrum applications in ethnomedicine is Aspilia africana. The plant is used to stop bleeding, heal wound, remove corneal opacities, and manage various stomach complaints (21)(22)(23). In our previous studies, we identified various bioactive compounds using gas chromatography combined with mass spectrometry (GC-MS) in the leaves of A. africana; including: 9,12,15-Octadecatrienoic acid, methylester, (z,z,z)-, stigmasterol, hexadecanoic acid methylester (z)-, methyl linolelaidate, and octadecynoic acid as the most predominant active ingredients while phytol, 3,7,11,15-Tetramethyl-z-hexadecen-1-ol, and vitamin E were the least (22). Therefore, the present study is aimed at evaluating the impact of aqueous and ethanol leaf extracts of A. africana on female Wistar rats by determining specifically the biochemical (liver function test, renal function tests, and lipid profile), histopathological (kidney and liver) and haematological parameters of the female Wistar rats.

Materials and Methods
Collection and identification of plant material Fresh samples of Aspilia africana leaves were collected from the vegetation area of Abia State University, Uturu, Abia State, Nigeria. The plant was identified and authenticated at the Forestry Herbarium, Ibadan, Nigeria and a voucher specimen (Accession No: JONES FHI 6637) was deposited there for future reference.

Sample preparation
The leaves were separated, cleaned and washed with distilled water and dried under shade. The dried leaves were ground to fine powder with an electric blender.

Aqueous extract
One hundred grams of fine powdered A. africana leaf sample was extracted with 2000 mL distilled water by continuous refluxing for 2 hours at 100 o C. The greenish liquid obtained was filtered with muslin cloth and the filtrate further evaporated to dryness in water bath at 100 o C.

Ethanol extract
Fifty grams of fine powdered A. africana leaf sample was extracted with 500 mL ethanol for 24 hours using Soxhlet apparatus at 65 o C. The extract was evaporated under reduced pressure using rotary evaporator and further concentrated in a water bath at 65 o C.
Qualitative phytochemical analysis The test carried out was based on procedures outlined by (24,25).

Experimental animals
Female Wistar rats of approximately the same age and the same weight were obtained from animal farm, University of Nigeria Nsukka and used for this study. They were housed in metallic cages after being certified clinically healthy and kept under standard environmental temperature (27°C ± 2°C) during the period of acclimatization. The animals had free access to clean water and were fed with standard grower feed obtained from a market feed store within the University. The principles of laboratory animal care were strictly adhered to and all activities carried out were approved by the University Ethics Committee.
Sub-acute toxicity study (LD 50 ) After being allowed to acclimatize for 2 weeks, the animals were divided into five groups of 3 animals each according to their body weight. Group-I was identified as the control group and received food and 0.5 mL normal saline, while the other groups (group II-V) identified as experimental groups received aqueous and ethanol extracts of A. africana orally at different doses in addition to food and water ad libitum, thus; group-II (500 mg/kg.bw), group-III (1000 mg/kg.bw), group-IV (2500 mg/kg.bw), and group-V (5000 mg/kg.bw). The experimental animals were monitored for behavioral changes, toxicity signs and mortality for 24 h according to OECD guideline (26).
Acute toxicity study and dose selection A total of eighteen female Wistar rats were divided into 3 groups. Group-I identified as the control group, received food and 0.5 mL normal saline, while the experimental groups received aqueous and ethanol leaf extracts of A. africana orally at different doses of 250 and 500 mg/kg.bw, respectively in addition to food and water ad libitum for 14 days (26).
Biological specimen collection Before collecting blood samples (by cardiac puncture), animals were anesthetized by diethyl ether and then blood samples were collected into plain and ethylenediamine tetraacetic acid (EDTA) bottles for and haematological and biochemical analyses. Then, the bottles were centrifuged for 15 minutes at 12 000 rpm. The plasma and serum were decanted and transferred into another set of test tubes and stored in a refrigerator for further analysis. Then, each rat in was quickly sacrificed by cervical dislocation. Each animal was placed in the supine position, the limbs were fixed to make the autopsy of the organs of interest easy. The liver and kidneys of each animal were visually examined for the possible signs of gross lesions. Then, for histopathological studies, the considered organs were removed from each rat and blotted on the filter paper. Each organ was weighed, rinsed in normal saline, and the sections were prepared and placed in bottles containing 10% neutral buffered formalin.

Histopathological examinations
The kidney and liver were dissected out, washed in normal saline, and fixed in 10% buffered formalin, processed, embedded in paraffin wax, sectioned at a thickness of 4-5 μm, stained and observed under light microscope at ×200 and ×400 to examined the histological changes (27).

Statistical Analysis
Results were expressed as mean ± SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) with GraphPad Prism statistical software package, version 6. Where there was significant difference, a Post Hoc multiple comparison was done using Tukey's honestly significant different (HSD) test. A P value of <0.05 was considered statistically significant.

Preliminary phytochemical screening of the A. africana leaves
The phytochemical screening of aqueous and ethanol leaf extracts of A. africana showed existence of flavonoids, phenols, cardiac glycosides, alkaloids, saponins, hydrogen cyanide (HCN) and tannins (Table 1).
Acute and sub-acute toxicity studies Because of the complex content of plant-based therapies we hypothesize that A. africana may pose, at least some level of toxicities to the experimental animals. To this end, we carried out the acute and sub-acute toxicity studies of the extracts using female Wistar albino rats at various concentrations and observations were made after 1 hour. The results showed that the plant extracts were less toxic even at the highest dose of 5000 mg/kg body weight (b.w) orally (Table 2). No mortality was recorded throughout the 24 hours of exposure, however, few clinical toxicity signs: restlessness, scratching of mouth, jumping up and loss of appetite were observed. All the experimental groups treated with plant extracts at doses of 250 and 500 Changes in organosomatic indices of female rats treated with extracts of A. africana leaves for 2 weeks. Relying on information obtained from sub-acute toxicity study only, sometimes cannot provide enough qualitative evidence on the overall influence of plant-based extract in animals. In this study, we orally administered aqueous and ethanol extracts of A. africana leaves to female rats for 2 weeks, while routinely recording their mean body weights. The mean body weight and relative liver and kidney weights of the rats fed with aqueous extract of A. africana leaves increased at 250 and 500 mg/kg b.w. At the same, we observed a corresponding decrease in the organosomatic indices of rats treated with ethanol leaf extract of the plant (Table 3), thus highlighting that ethanol extract of A. africana may pose more harm to the animals.
Changes in haematological parameters of female rats treated with A. africana for 2 weeks Effects of the extract of A. africana leaves on blood volume of female rats post 2 weeks of treatment. Acute and sub-acute toxicity studies and measurement of organosomatic parameters are insufficient to rely upon in toxicological studies of foreign materials. Hence, understanding the interaction of these plant extracts becomes imperative. An abnormality in blood volume is an indication of toxicity. To this end, we measured the overall effect of aqueous and ethanol extracts of A. africana leaves on blood volume of female Wistar rats. The results revealed that both extracts at the tested doses significantly altered the blood levels of RBC, PCV and platelet (decrease), and WBC (increase). The ethanol extract (Figure 1). of the plant was a mild anaemic and anti-coagulating culprit (P < 0.05) than its aqueous counterpart (Figure 2), which significantly varied the blood volume of the animals.

Effects of the extract of A. africana leaves on red cell indices of female rats post 2 weeks of treatment.
On further analysis, we noticed that there was a dosedepended alteration in red cell indices (Hb, MCV, MCH, and MCHC) following oral administration of aqueous and ethanol extracts of A. africana (Figures 3-4, respectively). The extracts of the plant caused a decrease in the level of Hb (P < 0.05) when compared to the control. Conversely, there was a non-significant change in the level of MCV, MCH and MCHC especially at 250 mg/kg b.w when compared to the control, suggesting that aqueous extract In phase 1 acute toxicity study, the aqueous and ethanol leaf extracts of A. africana were administered to the animals at moderate doses and the signs of toxicity were observed for 1 h. In the absence of any mortality and abnormal behavioral changes, we moved on to phase 2.
In phase 2, the doses of the plant extracts were raised up to 5000 mg/kg body weight and also observed for possible signs of toxicity.     The result showed significant decrease (P < 0.05) in serum total protein when compared to the control, while serum levels of ALT, AST, ALP and bilirubin increased (P < 0.05) significantly in a dose dependent manner, implying that both extracts may be toxic to the liver of the animals. Furthermore, the administration of aqueous and ethanol extracts of the A. africana leaves altered the serum levels of urea, creatinine, and electrolytes (Figures 9 and  10, respectively). While serum urea, creatinine, sodium, potassium and chloride ions increased significantly (P<0.05), a non-significant variation in serum level of bicarbonate ion was observed when compared to the control.
Changes in histopathological indices of female rats treated with A. africana for 2 weeks. The aqueous and ethanol extracts of A. africana leaves presented no signs of toxicity, moderate alterations in organ to body weight ratio, haematological, and biochemical parameters. Relying on these data to predict the safety of the plant is inadequate. To validate our findings, a histopathological study was done to ascertain the impact of aqueous and ethanol extracts of A. africana leaves on the liver and kidney. The light microscopic study of the cross section of liver tissues of the control revealed normal architecture of hepatocytes with cytoplasm. The shape and size of the nucleus with nuclear membrane was appropriate ( Figure 11A). Groups treated with aqueous extract of A. africana leaves showed several distortions in liver architecture ranging from sinusoidal dilatation, microvesicular degeneration, hydropic change, and degenerating aggregate of inflammatory cells ( Figure 11B);    The extract at both doses slightly altered serum levels of total protein, ALT, AST, ALP, and total bilirubin, stat. test (One-way ANOVA; a P < 0.05 versus control; b P < 0.05 versus treatment), error bars represent standard deviation. microvesicular degeneration, necrosis and aggregation of inflammatory cells as well as loss of liver parenchyma ( Figure 11C). Furthermore, ethanol extract of A. africana leaves brought about changes in liver architecture including congested central vein, sinusoidal obliteration, few vesicular degeneration and tissue infiltration ( Figure   11D); necrosis and mild vesicular degeneration ( Figure  11E). The histological study of the cortical sections of the kidney following a 2-week administration of the aqueous and ethanol extracts of A. africana leaves is presented in Figure 12. The cortical view of the control kidney tissue showed normal renal corpuscles, proximal convoluted tubules, distal convoluted tubules and collecting ducts ( Figure 12A). Widespread coagulative necrosis, and vesicular degeneration of both glomerular tuft and tubules ( Figure 12B) as well as widespread necrosis and vesicular degeneration affecting the renal corpuscles and the tubules ( Figure 12C) were observed in grouped treated with 250 and 500 mg/kg bw aqueous extract of A. africana leaves. Ethanol extract of the plant also brought about alterations in the architectural framework of the kidney. Some of these changes include obliterated tubular lumen, enlarged glomerular tuft and areas of necrosis ( Figure  12D); degenerating glomerular tuft, and mild tissue infiltrations ( Figure 12E).

Discussion
Since time immemorial, man has continued to depend on plant-based products to treat and manage diseases without understanding the mechanisms of action and adverse effects of some pharmaceutically bioactive principles (28). Available information over the years revealed that improper intake of herbal products can cause severe liver and kidney injuries, acute and chronic abnormalities, anaemia, cirrhotic transformation, and necrosis (6,29). Hence, our investigation into biochemical and histological alterations associated with oral administration of aqueous and ethanol extracts of A. africana leaves in female Wistar rats becomes imperative.
The oral administration of the aqueous and ethanol extracts of A. africana leaves did not produce lethality in rats up to 5000 mg/kg bw although there were mild signs of toxicity. This observation is in agreement with Figure 9. Effect of aqueous extract of Aspilia africana leaves on kidney function parameters of female rats. The extract at both doses slightly altered serum levels of urea, creatinine, sodium ion (Na + ), potassium ion (K + ) chloride ion (Cl -) and bicarbonate (HCO 3 -), stat. test (One-way ANOVA; a P < 0.05 versus control), error bars represent standard deviation Figure 10. Effect of ethanol extract of Aspilia africana on kidney function parameters of female rats. The extract at both doses slightly altered serum levels of urea, creatinine, Na + , K + , Cland HCO 3 -, stat. test (Oneway ANOVA; a P < 0.05 versus control), error bars represent standard deviation.
the findings of Taziebou et al (30) who documented acute and sub-acute toxicity of A. africana leaves in male and female mice with LD 50 of 7500 mg/kg and effective dose of 1000 mg/kg. To understand the extent of this toxicity, we investigated changes in organosomatic indices of the rats following oral administration of the plant for 2 consecutive weeks. Organosomatic indices are imperative when monitoring the effect of medicinal plant on the body of animals (6,31). A dose which causes ≥10% reduction in the body weight of an animal is considered to be toxic (32,33). In this study, ethanol extract of A. africana leaves at different doses decreased the mean body to organ weight ratio when compared to the control and group treated with aqueous extract of the plant. The relative liver weight and relative kidney weight for all groups treated with aqueous and ethanol leaf extracts of the plant increased in a dose dependant manner when compared to control. Alterations in liver and kidney weights suggest that the aqueous extract of the plant at 250 and 500 mg/ kg bw may have caused hepatic and renal hypertrophies (30,34). Based on this, we infer that both extracts may contain irritating compounds that are capable of causing damage to major organs of the body.
Evaluation of haematological parameters are not only used to determine the extent of deleterious effect of herbal extracts, but also to explain functions of plant extracts or their products on the blood of animals (35,36). In this study, the aqueous and ethanol extracts of the A. africana leaves significantly (P < 0.05) decreased the levels of platelets, RBC, PCV, Hb, and MCV of test animals when compared to the control group. The fall in RBC count, PCV, and haemoglobin content may be correlated with the induction of anemia, defective haematopoesis, inhibited erythropoiesis, and increased destruction of red blood cells (6,37,38).
Liver function tests (total protein, ALT, AST, ALP, and total bilirubin) provide information about the state of the liver by describing its functionality, cellular integrity and link with the biliary tract (5,39,40). Alanine amino transferase (ALT), a cytosolic enzyme whose activities increase as a result of cellular membrane damage, as well as aspartate amino transferase (AST) are used for the detection of hepatocellular damage in animals (41,42). The result of our present study revealed that serum ALT and AST levels were significantly (P < 0.05) elevated in treated animals, indicating onset of hepatocellular injury at higher doses of aqueous and ethanol extracts of A. africana leaves in rats. This observation is in line with the work of Kifayatullah et al (43) and Olaniyan et al (44) who reported the toxic effect of extracts of Pericampylus glaucus (in BALB/c mice) and Nelsonia campestris (in rats), respectively. Alkaline phosphatase (ALP) is a standard biomarker of biliary tract obstruction (45,46). In the present study, we observed a significant (P < 0.05) increase in serum level of ALP following ingestion of aqueous and ethanol extracts of A. africana leaves by female Wistar rats. Significant reductions (P < 0.05) in total protein were attributed to several causes including massive necrosis of the liver, deterioration of liver function, hepatic resistance to insulin and glycogen impairment of oxidative phosphorylation (47). Presently, the level of total protein decreased significantly (P < 0.05) in groups treated with aqueous and ethanol extracts of A. africana leaves. Bilirubin is the metabolic product of the breakdown of heme derived from senescent red blood cells. The degree of increase of serum bilirubin has prognostic significance in chronic liver injuries, but not in mild liver injuries (48). Accordingly, levels of total bilirubin significantly (P < 0.05) increased with dose suggesting the onset of tissue and cellular damage. Similarly, we observed significant decrease in total cholesterol, triacylglycerol, and low-density lipoprotein with a corresponding increase in high density lipoprotein in groups treated with aqueous and ethanol extracts of the plant. Calculated cholesterol/ HDL-C ratio for both doses was lower in aqueous extract (1.03) and ethanol extract (1.43) at 500 mg/kg bw when compared to the control, which had a value of 2.09. This indicates antilipidemic property of the plant extract.
Serum urea and creatinine increased significantly in female Wistar rats administered aqueous and ethanol extracts of A. africana. Urea and creatinine are compounds derived from proteins which are eliminated by the kidney. Elevation in serum urea and creatinine as obtained in this study could be attributed to altered renal functions associated with decrease circulating blood volume within the kidney, loss of normal excretory function of creatinine and muscular cell damage as described by  Taziebou et al (28) and Debebe et al (6). Electrolytes are important biomarkers for monitoring the impact of herbal extract on the integrity and functionality of the kidneys (49,50). Common electrolytes of clinical importance are sodium, potassium, chloride and bicarbonate ions as they are relevant for the following physiological functions: regulation of total amount of water in the body and generation of electrical signals for communication in the brain, nervous system and muscles (sodium ion), regulation of heartbeat and the function of the muscle (potassium ion), maintenance of normal balance of fluid (chloride ion) and act as buffer to maintain the normal levels of acidity in blood and other fluids in the body (bicarbonate ion). These investigations revealed significant (P < 0.05) increases in the levels of sodium, potassium, and chloride rats administered aqueous and ethanol extracts of A. africana leaves. Alteration in the normal electrolytes level may be due to interference with respiratory function, kidney diseases, metabolic and associated conditions.
Relying on biochemical and haematological dose dependent biomarkers to predict the onset of toxicity without appropriate histopathological assessment will mask tissue damage due to the ingestion of herbal extracts by animals (48). Liver and kidney microscopic pathologies serve as important tools for identifying and characterizing liver and kidney injuries respectively, whether or not biochemical, haematological and macroscopic changes are also identified (6). The followings are possible patterns of liver and kidney injuries during hepatorenal toxicity: zonal necrosis, and vascular lesions, glomeruli hypercellularity, tubular necrosis, loss of individual lining cells and sloughing off of cells into lumina (6,29,51). The result of our present study revealed histopathological changes in the liver and kidney tissues of the rats administered with aqueous and ethanol extracts of A. africana leaves at 250 and 500 mg/kg bw. Such changes include sinusoidal dilatation, microvesicular degeneration, hydropic change, degenerating aggregate of inflammatory cells, necrosis, aggregation of inflammatory cells and loss of liver parenchyma (liver), widespread necrosis and vesicular degeneration affecting the renal corpuscles and the tubules (kidney). These findings are in line with the observations of Tanuja et al (29) and Debebe et al (6) who investigated the toxic effects of aqueous leaf extract of Phyllanthus niruri in Swiss albino mice and Albizia gummifera seeds in Albino Wistar rats, respectively. The alterations in biochemical, haematological and histopathological indices of the female Wistar rats following exposure to aqueous and ethanol leaf extracts of the A. africana may be due to the preponderance of hydrogen cyanide (HCN). Although, some beneficial phytochemicals were present in the extracts of the plant, but the concentration of HCN was very high. This work further corroborates the findings of Smith et al (52), who opined that excess intake of HCN containing foods may lead to death within a short time. HCN, occurring in plants in the form of cyanogenic glycosides is a potent toxicant which yields hydrogen cyanide on hydrolysis (53,54).

Conclusion
In the light of our findings of sub-acute and acute toxicity studies, we may conclude that the aqueous and ethanol extracts of A. africana leaves, despite their antilipidemic property, are not devoid of toxic effects as they significantly (P < 0.05) altered haematological, liver, kidney and histopathological indices. Our results provide current evidence on the toxicity profile of aqueous and ethanol extracts of A. africana leaves at higher doses, and as a result the decoction of A. africana leaves using water or ethanol as a solvent should be taken with precautions. However, further studies are needed to identify, isolate and characterize the bioactive compounds responsible for the overall biochemical, hematological and histopathological insults.

Authors' contributions
UOA and GCC designed the study and interpreted the results. CI did statistical analysis of the results. UOA, KON, CAN, OAC, UAO, JKA, and CU did the experiments. UOA, GCC and CI wrote the manuscript. All authors read and confirmed the last edition of the manuscript and confirmed it for publication.