Abstract: Background and Objective: The influence of orthodox drugs-plants based combination therapy on the organs function remains the basis of toxicity study. This study evaluated the effects of ethanol leaf extract and bio-active fraction of Tithonia diversifolia administered singly and in combination with chloroquine on selected organs using chloroquine resistant Plasmodium yeolii (P. yeolii R) infected mice. Materials and Methods: Ninety-six mice were divided into 12 groups of 8 mice each (n = 8). Groups II to XII mice were infected with P. yeolii R and treated with varying doses of crude ethanol extract and bio-active fraction of T. diversifolia leaf singly and in combination with chloroquine. Liver marker enzymes, kidney function test and organs’ histology of the experimental animals were assayed using standard protocol. Data obtained were analyzed using one way analysis of variance, followed by post hoc multiple comparison test using SPSS software. Results: The infected mice treated with antimalarial drugs (chloroquine and artemether) and varying doses of the crude extract and bioactive fraction (C70:M30) singly and in combination with therapeutic dose of chloroquine showed significant (p<0.05) reduction in alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) activities compared with the infected mice administered 0.2 mL of distilled water. Co-administration of bioactive fraction and chloroquine to parasitized mice showed significant (p<0.05) higher total protein concentration compared with the parasitized mice administered 0.2 mL of distilled water. The infected mice administered 0.2 mL of distilled water showed significant (p<0.05) increase in total protein, urea and creatinine level compared with the normal control mice. Histological examination of the liver revealed that, combination of low dose of extract with chloroquine showed a mild hepatitis with mild degeneration of the liver cells. However, hemozoin pigment deposition tends to be more in the low dose compared with the high dose of extract treated mice. The examined sections of the kidney showed the normal renal histo-architecture. Conclusion: This study showed the therapeutic effects of synergistic treatment of T. diversifolia and chloroquine in alleviating hepatotoxicity resulting from chloroquine resistant P. yeolii.
INTRODUCTION
Malaria has remained one of the most devastating diseases in tropical and sub-tropical regions of the world despite the global fight against the disease1. Malaria in pregnancy increases the infant risk of low birth weight, abortions and still birth in addition to the maternal burdens of anaemia and mortality2.
Malaria has been reported to be one of the factors responsible for acute renal failure among children in malaria endermic areas3. Renal tubular changes have been reported to be associated to P. falciparum infection more than glomerular changes and complication may range from minor blood urea, hyper-nareamia, hyperkalemia, low urine specific gravity, metabolic acidosis, tubular necrosis and acute renal failure accompanied by frequent oliguria and hyper catabolism4.
Liver involvement in severe malarial infection is commonly a significant cause of morbidity and mortality among humans5. This could result from invasion of the liver cells by sporozoites during malarial parasite life cycle. The changes caused in the hepatic cell by sporozoites can lead to the leakage of parenchymal and membranous enzymes of the liver into the circulatory system; the severity of malaria infection has been linked with the increase in the level of liver enzymes in the blood4,6.
Chemotherapy remain the kernel of malaria control and the previously efficacious chloroquine has substantially failed as a prophylactic and therapeutic anti-malarial which have paved way for the use of other anti-malarial drugs including sulphadoxine-pyrimethamine, mefloquine and halofantrine7. These drugs are not only expensive than chloroquine but also parade heterogeneous levels of toxicity and may invoke poor compliance in patients7. Investigation of anti-malarial potentials of orthodox drugs in combination with plants used in traditional therapy of malaria has been one of the means of searching for new anti-malarial drugs that will liberate human race from the burden of resistant malaria.
Tithonia diversifolia (Hemsl) A gray, is commonly referred to as Mexican sun flower or tree marigold, it is a bushy perennial weed commonly find on the fields and road sides of Nigeria8. Tithonia diversifolia is administered in several forms; oral decoction of the leaves for the treatment of hepatitis, diabetes, malaria, pain, chemoprevention and anti-helicobacter pylori9. Exploitation of the anti-malarial actions of plant extracts in combination with monotherapies like chloroquine with a view to enhance drug efficacy in resistance malaria infection is currently generating scientific interest without consideration of the cytotoxic effects. This study therefore; aimed to assess the effects of ethanol leaf extract and bio-active fraction of T. diversifolia administered with chloroquine on pathology of vital organs (liver and kidney) of albino mice infected with chloroquine resistant Plasmodium yeolii parasites.
MATERIALS AND METHODS
Animals: Adult male albino mice of 3-4 months old weighing 20-34 g were obtained from the Animal House of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka. The animals were acclimatized for 7 days under standard environmental conditions, with a 12 h light and dark cycle maintained on a regular feed and water ad libitum. The animal experiments were conducted as per the University of Nigeria Scientific Research Committee in Nigeria. The experimental protocol was approved by the Faculty of Science Animal Ethics committee of University of Nigeria with reference number pg/12/64667.
Preparation and extraction of plant material: The plant material for this study (Tithonia diversifolia leaves) was collected randomly from trees within the International Institute of Tropical Agriculture (IITA) Ibadan, Oyo State in July, 2015 and authenticated by the plant taxonomist, Mr. Esimekhuai Donatus of the Department of Botany, University of Ibadan, Oyo State, Nigeria. The extraction and fractionation procedure was carried out as reported by Omoboyowa et al.10.
The Fraction Chloroform 70 and methanol 30% (C70: M30) was observed to show the highest significant percentage parasitaemia chemo-suppression in the activity guided study reported in Omoboyowa et al.10. The bioactive fraction and the crude ethanol extract were combined with chloroquine for this study.
Chemicals/Reagents: All the chemicals used in this study were of analytical grade and supplied by sigma incorporated, USA.
Experimental design: Ninety-six mice were divided into 12 groups of 8 mice each (n = 8) and treated according to the experimental design below:
| • | Group I: Uninfected control given normal saline only |
| • | Group II: Infected control given normal saline only |
| • | Group III: Infected mice treated with 10 mg kg–1 b.wt., of chloroquine |
| • | Group IV: Infected mice treated with 5 mg kg–1 b.wt., of artemether lumefantrine |
| • | Group V: Infected mice treated with 100 mg kg–1 b.wt., of crude ethanol extract |
| • | Group VI: Infected mice treated with 200 100 mg kg–1 b.wt., of crude ethanol extract |
| • | Group VII: Infected mice treated with 100 mg kg–1 b.wt., of crude ethanol extract in combination with 10 mg kg–1 b.wt., of chloroquine |
| • | Group VIII: Infected mice treated with 200 mg kg–1 b.wt., of crude ethanol extract in combination with 10 mg kg–1 b.wt., of chloroquine |
| • | Group IX: Infected mice treated with 100 mg kg–1 b.wt., of active (C70:M30) fraction |
| • | Group X: Infected mice treated with 200 mg kg–1 b.wt., of active (C70:M30) fraction |
| • | Group XI: Infected mice treated with 100 mg kg–1 b.wt., of active (C70:M30) fraction in combination with 10 mg kg–1 b.wt., of chloroquine: |
| • | Group XII: Infected mice treated with 200 mg kg–1 b.wt., of active (C70:M30) fraction in combination with 10 mg kg–1 b.wt., of chloroquine |
Procurement of parasitaemia: Chloroquine resistant Plasmodium yeolii used for this study was obtained from the MR4/ American type culture collection (ATCC), Manassas, USA maintained by serial passage in mice. Preparation of inoculum was from donor mouse with Plasmodium yeolii parasitaemia established by microscopic examination of thin blood film under oil immersion at X100 magnification and measured as percentage erythrocytes7. Each mouse was infected with standard inoculums of approximately 107 parasitized erythrocytes suspension in normal saline (0.2 mL) from donor mouse.
Drugs: The monotherapeutic drug administered for the control and treatment groups were chloroquine (CLARION, Nigeria) and the ACT drug administered was Lokmal®a brand of artemether lumefantrine (EMZOR, Nigeria). These were dissolved in normal saline to obtain fixed dose of 10 and 5 mg kg–1, respectively and used in the assay.
Biochemical assay: The activity of aminotransferases (AST and ALT) was determined by the method of Reitman and Frankel11. Plasma alkaline phosphatase activity was assayed spectrophotometrically according to the method described by King and King12. The concentration of bilirubin was determined by the method of Malloy and Evelyn13. The plasma urea concentration was carried out according to the method outlined by Watt and Chrisp14, the creatinine concentration was determined according to the method described by Peters15 and the total protein concentration was evaluated using the method outline by Lowry et al.16 as described in Randox assay kits.
Liver histology: The animals were sacrificed and the abdominal cavity of each rat opened, the liver and kidney were taken out. The organs were fixed in 10% formalin. After complete fixation the blocks was embedded in paraffin and sections cut at 5 μm (micron) which were then stained with haematoxylin and eosin and mounted in Canada balsam. Microscopic examinations of the sections were then carried out under a light microscope17.
Statistical analysis: The data obtained were analyzed using one way analysis of variance. The results were expressed as Mean±SD. Post hoc multiple test comparison was used to compare the group means after each treatment with control measurements. Significant differences were obtained at p<0.05.
RESULTS
The results of the liver function parameters (AST, ALT, ALP and Total bilirubin) of P. yeoliiR infected mice treated with ethanol extract and C70:M30 fraction of T. diversifolia singly and in combination with chloroquine is shown in Table 1. The P. yeoliiR-infected mice without treatment showed significant (p<0.05) decrease in ALP activities and total bilirubin level and significant (p<0.05) increase in ALT activity compared to the normal control mice. The infected mice treated with 10 mg kg–1 b.wt., of chloroquine showed non-significant (p>0.05) reduction in ALT, ALP activities and significant (p<0.05) reduction in ALT compared to the parasitized mice administered 200 mg kg–1 b.wt., of C70 M30 fraction in combination with chloroquine.
The results of the selected kidney function parameters (urea, creatinine and total protein) of P. yeoliiR infected mice treated with ethanol leaf extract and C70:M30 fraction of T. diversifolia singly and in combination with 10 mg kg–1 b.wt., of chloroquine are shown in Table 2. The P. yeoliiR infected mice without treatment showed significantly (p<0.05) higher urea, creatinine and lower total protein concentration compared to the normal control mice.
| Table 1: | Effect of Co-administration of Ethanol leaf Extract of T. diversifolia and chloroquine on liver function parameters of mice infected with Plasmodium yeoliiR (pr1) |
| CQ: Chloroquine, C70 M30: Chloroform 70% Methanol 30%, ap<0.05 significant compared to the normal control mice, bp<0.05, significant compared to the infected mice administered 0.2 mL of distilled water, cp<0.05, significant compared to the infected mice treated with 10 mg kg–1 b.wt., of chloroquine | |
| Table 2: | Effect of co-administration of ethanol leaf extract of T. diversifolia and chloroquine on kidney function parameters of mice infected with Plasmodium yeoliiR (pr1) |
| CQ: Chloroquine, C70 M30: Chloroform 70% Methanol 30%, ap<0.05 significant compared to the normal control mice. bp<0.05 significant compared to the infected mice administered 0.2 mL of distilled water, cp<0.05, significant compared to the infected mice treated with 10 mg kg–1 b.wt., of chloroquine | |
| Table 3: | Histopathological effects on the liver |
| +: Mild, ++: Moderate, +++: Severe, -: Not detected, CQ: Chloroquine, C70 M30: Chloroform 70% Methanol 30% | |
The parasitized mice treated with 10 mg kg–1 b.wt., of chloroquine and 5 mg kg–1 b.wt., of artemether showed non-significant (p>0.05) reduction in urea, creatinine and total protein concentration compared to the infected mice without treatment. The mice infected with P. yeoliiR, treated with 100 mg kg–1 b.wt., of C70:M30 fraction in combination with 10 mg kg–1 b.wt., of chloroquine showed non-significant (p>0.05) higher urea, creatinine and total protein concentration compared to the parasitized mice treated with 10 mg kg–1 b.wt., of chloroquine only.
As Table 3 showed the pathological effects of the synergistic treatment on the liver of malaria infected mice.
| Table 4: | Histopathological effects on the kidney |
| +: Mild, ++: Moderate, +++: Severe, -: Not detected, CQ: Chloroquine, C70 M30: Chloroform 70% Methanol 30% | |
| Fig. 1: | Section of the liver histo-architecture of the mice in the normal control group that were given distilled water without P. yeolii infection. Liver from this group showed normal hepatic histo-architecture, it showed normal hepatocytes arranged in radiating chords around the central veins, diverging towards the portal areas with normal bile ducts, hepatic arteries and hepatic veins. P: Portal area |
| V: Central vein. H&E X100 | |
Hepatic necrosis, kupffer cell hyperplasia and haemozoin decomposition were observed to be severe (+++) in the parasitized mice without treatment compared to mild (+) effects observed in the P. yeoliiR infected mice administered 200 mg kg–1 b.wt., of C70: M30 fraction and therapeutic dose of chloroquine.
The histopathological effects of synergistic treatment of T. diversifolia and chloroquine on P. yeoliiR infected mice. Tubular nephrosis and perivascular interstitial mononuclear cell infiltration were observed to be severe in P. yeoliiR infected mice without treatment which were moderate and mild respectively in infected mice treated with 200 mg kg–1 b.wt., of C70:M30 fraction and therapeutic dose of chloroquine in Table 4.
Liver histology: Histopathological studies revealed some damages caused by the chloroquine resistant malaria parasite and extent of reduction of these damages by the administered drugs, crude extract and bioactive fraction singly and in combination with chloroquine, for example, death of cells of the liver (hepatic necrosis) and deposition of haemozoin in the hepatic cells as shown in Fig. 1-12.
Kidney histology: Histopathological studies revealed minor damages caused by the chloroquine resistant malaria parasite and effect of administered crude extract and bio-active fraction of Tithonia diversifolia singly and in combination with chloroquine, on the renal system of P. yeoliiR infected mice as shown in Fig. 13-23.
| Fig. 2: | Section of the liver from mice infected with Plasmodium yeoliiR without treatment showed severe diffuse hepatocellular vacuolar degeneration with widespread haemozion pigment deposition (white arrow) as well as mild multifocal and periportal infiltration of inflammatory leucocytes (black arrow) |
| P: Portal area. H&E X100 | |
| Fig. 3: | Section of the liver from Plasmodium yeoliiR infected mice administered 10 mg kg–1 b.wt., of chloroquine showed mild Hemozoin pigment deposition (black arrow). The pigments appears to be most present in the Kupffer cells |
| P: Portal area, V: Central vein. H&E X100 | |
| Fig. 4: | Section of the liver from Plasmodium yeoliiR infected mice administered 5 mg kg–1 b.wt., of artemether showed severe widespread vacuolar hepatocellular degeneration with slight periportal deposition of inflammatory leucocytes (black arrow). Hemozoin pigments appear to be widespread, in Kupffer cells (white arrow) |
| V: Central vein, P: Portal area. H&E X100 | |
| Fig. 5: | Photomicrograph of the liver of Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia showing hemozoin pigment deposition in cytoplasm of hepatocytes and Kupffer cells (black arrow) and mild deposition of mononuclear inflammatory leucocytes around the portal triad (white arrow) |
| B: Bile duct, C: Central vein. H&E X100 | |
| Fig. 6: | Examined section of the liver from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia showed mild hepatocellular degeneration and necrosis, widespread hemozoin (malaria pigment) deposition in the cytoplasm of Kupffer cells (white arrow), multifocal aggregates of mononuclear inflammatory cells as well as moderate periportal infiltration of mononuclear infiltrates (black arrow) |
| P: Portal area, V: Central vein. H&E X100 | |
| Fig. 7: | Section of the liver from Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine showed a moderate widespread vacuolar degeneration, mild haemozoin pigment deposition (white arrow) and mild periportal infiltration of inflammatory leucocytes (black arrow) |
| P: Portal area, V: Central vein. H&E X100 | |
| Fig. 8: | Examined section of the liver from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine showed mild hepatocellular degeneration and necrosis, widespread haemozoin (malaria pigment) deposition in the cytoplasm of Kupffer cells (white arrow), multifocal aggregates of mononuclear inflammatory cells as well as severe periportal infiltration of mononuclear infiltrates (black arrow) |
| P: Portal area . H&E X100 | |
| Fig. 9: | Examined section of the liver from Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of C70: M30 fraction of Tithonia diversifolia showed mild hepatocellular degeneration and necrosis, widespread haemozoin (malaria pigment) deposition in the cytoplasm of Kupffer cells and mild periportal infiltration of mononuclear infiltrates (black arrow) |
| P: Portal area, V: Central vein. H&E X100 | |
| Fig. 10: | Examined section of the liver from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of C70: M30 fraction of Tithonia diversifolia showed mild hepatocellular degeneration and necrosis, widespread hemozoin (malaria pigment) deposition in the cytoplasm of Kupffer cells (white arrow), multifocal aggregates of mononuclear inflammatory cells as well as periportal infiltration of mononuclear infiltrates (black arrow) |
| P: Portal area, V: Central vein. H&E X100 | |
| Fig. 11: | Examined sections of the liver from Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of C70: M30 fraction of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine showed mild hepatocellular degeneration and necrosis, widespread hemozoin (malaria pigment) deposition in the cytoplasm of Kupffer cells (white arrow), multifocal aggregates of mononuclear inflammatory cells as well as moderate periportal infiltration of mononuclear infiltrates (black arrow) |
| P: Portal area. H&E X100 | |
| Fig. 12: | Sections from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of C70: M30 fraction of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine showed widespread, mild to moderate hepatocellular vacuolar degeneration (black arrow). Mild periportal hepatitis with mild deposition of hemozoin pigments in Kupffer cells (white arrow) |
| C: Central vein, P: Portal area. H&E X100 | |
| Fig. 13: | Section of the kidney from mice infected with Plasmodium yeoliiR without treatment |
| G: Glomerulus. H&E X400 | |
| Fig. 14: | Section of the kidney from Plasmodium yeoliiR infected mice administered 10 mg kg–1 b.wt., of chloroquine |
| G: Glomerulus, H&E X400 | |
| Fig. 15: | Section of the kidney from Plasmodium yeoliiR infected mice administered 5 mg kg–1 b.wt., of artemether |
| G: Glomerulus, H&E X400 | |
| Fig. 16: | Photomicrograph of the kidney of Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia |
| G: Glomerulus. H&E X400 | |
| Fig. 17: | Examined section of the kidney from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia |
| G: Glomerulus | |
| Fig. 18: | Section of the kidney from Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine |
| G: Glomerulus, H&E X400 | |
| Fig. 19: | Examined section of the kidney from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of ethanol leaf extract of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine |
| G: Glomerulus, H&E X400 | |
| Fig. 20: | Examined section of the kidney from Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of chloroform 70: methanol 30 fraction of Tithonia diversifolia |
| G: Glomerulus, H&E X400 | |
| Fig. 21: | Examined section of the kidney from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of chloroform 70: methanol 30 fraction of Tithonia diversifolia |
| G: Glomerulus, H&E X400 | |
| Fig. 22: | Examined section of the kidney from Plasmodium yeoliiR infected mice administered 100 mg kg–1 b.wt., of chloroform 70: Methanol 30 fraction of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine |
| G: Glomerulus, H&E X400 | |
| Fig. 23: | Section of kidney from Plasmodium yeoliiR infected mice administered 200 mg kg–1 b.wt., of chloroform 70: Methanol 30 fraction of Tithonia diversifolia and 10 mg kg–1 b.wt., of chloroquine |
| G: Glomerulus, H&E X400 | |
DISCUSSION
The intake of medicinal plants as alternative medicine has been supported because of their relatively cheap availability. This is coupled with the belief that they could significantly improve human health in terms of prevention and control of various ailment and infections. They also exhibit far less frequent side effects when compared with modern medicine18.
The result of the liver function parameters of P. yeoliiR infected mice treated with crude extract and bioactive fraction of T. diversifolia singly and in combination with therapeutic dose of chloroquine shown in Table 1 revealed that ALT activity was significantly (p<0.05) higher in the infected mice administered 0.2 mL of normal saline compared with normal control mice while the infected mice treated with anti-malarial drugs (chloroquine and artemether) and varying doses of the crude extract and bioactive fraction (C70:M30) of the leaf singly and in combination with therapeutic dose of chloroquine showed reduction in liver enzyme activities compared with the infected mice administered 0.2 mL of normal saline, suggesting that the administration of the crude extract and bioactive fraction singly and in combination with chloroquine interact to ameliorate the hepatic damage caused by activities of the parasite. The result of the liver enzymes is in agreement with report of Martey et al.19. The result of the total bilirubin revealed significantly (p<0.05) higher concentration of the parameter in the mice infected with P. yeoliiR administered 0.2 mL of normal saline and 10 mg kg–1 b.wt., of chloroquine compared with the infected mice treated with varying doses of the crude extract and bioactive fraction singly and in combination with chloroquine. This suggests ameliorative impact of the treatment on hepatobiliary function of the liver. The rapid haemolysis of the red blood cell results in increased plasma bilirubin levels when the hepatic cells cannot excrete the bilirubin as rapidly as it is been formed, there is an increase in the plasma level of bilirubin20,21. Obstruction of the bile ducts as it occurs on gallstone or liver damage such as hepatitis may not affect the rate of bilirubin formation but the bilirubin formed cannot pass from the blood into the intestine21,22.
The mean concentration of urea, creatinine and total protein of chloroquine resistant P. yeolii infected mice treated with anti-malarial drugs; crude extract and bioactive fraction of T. diversifolia singly and in combination with chloroquine are shown in Table 2. The results revealed that, the infected mice administered 5 mg kg–1 b.wt., of artemether, varying doses of the crude extract and bioactive fraction only significantly (p<0.05) lower the mean total protein concentration compared with the infected mice administered 0.2 mL of normal saline. Co-administration of bioactive fraction and chloroquine to parasitized mice showed significantly (p<0.05) higher total protein concentration compared with the parasitized untreated control mice. The infected mice administered 0.2 mL of normal saline showed significant (p<0.05) increase in total protein, urea and creatinine level compared with the normal control mice. The higher value observed in these parameters may be attributed to impairment in renal function associated with P. yeoliiR infection. These results are consistent with the report of Ogbadoyi and Gabi23, who observed increase in these parameters in P. falciparum infected patients. High level of proteinuria is characteristics features of renal dysfunction24. In healthy kidneys, protein are normally completely filtered from the blood stream and then re-absorbed allowing no protein or small amounts of protein into the urine25. Therefore, that the amounts of protein in the plasma of malaria infected mice were significantly (p<0.05) higher than the normal control mice is indicative of some level of renal dysfunction which may be attributed to the effect of the malaria infection. The result obtained is consistent with the report of Akanbi4. In acute renal failure, plasma urea increases more rapidly than plasma creatinine concentration. Despite all these consideration, plasma urea levels do not reflect the performance of the kidneys as compared to creatinine. This is because; urea production is also affected by dehydration, food intake and tissue catabolism25. Thus, an increase in plasma creatinine concentration in the infected mice suggests that the normal functioning of the kidney have been compromised.
The predominant histological changes observed in malarial hepatopathy involve vacuolar (fatty) degeneration, inflammatory cellular infiltration and hemozoin (malarial pigment) deposition in cytoplasm of tissue macrophages (Kupffer cells) and connective tissues. These lesions are used to qualify the severity of the malarial hepatopathy. The P. yeolii R infected mice treated with 10 mg kg–1 b.wt., of chloroquine and 5 mg kg–1 b.wt., of artemether showed better liver histology. However, that of chloroquine treated animals appears more normal than the artemether treated mice. It could imply that chloroquine has less hepatotoxic effect compared with Artemether. Treatment of parasitized mice with 100 mg kg–1 b.wt., of crude extract shows relatively normal hepatocytes but evidence of severe hepatitis was observed. The liver histology of infected mice without treatment showed hepatocellular damage with severe hepatic necrosis, kupffer cell hyperplasia and haemozoin decomposition. This result is consistent with the hepatotoxicity of malaria parasite reported by Otegbade et al.26. Hemozoin deposits appear to be higher with the level observed in parasitized mice treated with 200 mg kg–1 b.wt., of the crude extract which also showed mild hepatitis. Co-administration of 100 mg kg–1 b.wt., of the crude extract and 10 mg kg–1 b.wt., of chloroquine showed a mild hepatitis with mild degeneration of the liver cells when combined administration of 10 mg kg–1 b.wt., of chloroquine with the 200 mg kg–1 b.wt., of crude extract to parasitized mice revealed severe lesions. Its possible implication is that, a combination of the drug and crude extract may be detrimental to the liver. Treatment of parasitized mice with 100 mg kg–1 b.wt., of the bioactive fraction showed a better liver histology compared with the result observed when treated with 200 mg kg–1 b.wt., of the fraction with respect to inflammation. Treatment of parasitized mice with 100 and 200 mg kg–1 b.wt., of the bio-active fraction combined with 10 mg kg–1 b.wt., of chloroquine showed mild hepatocellular damage and inflammation. However, haemozoin pigment deposition tends to be more in the co-administration of 100 mg kg–1 b.wt., of the fraction with 10 mg kg–1 b.wt., of chloroquine compared with administration of 200 mg kg–1 b.wt., in combination with 10 mg kg–1 b.wt., of chloroquine. The sections of the kidney collected from the animals in all the groups did not show significant change in the renal histo-architecture as shown in Fig. 13-23. This implies that neither the infection, nor the treatments had an effect on the histo-morphology of this organ. Nonetheless, this does not negate the possibility of biochemical lesions in the organ as these may occur without obvious morphological changes depending on the level of intoxication.
CONCLUSION
The present study has demonstrated the protective effect of combine treatment of chloroquine and active fraction of T. diversifolia leaf extract to reverse organs’ damage initiated by chloroquine resistant P. yeolii. Further studies might define the bioactive compounds responsible for this observed therapeutic effect of T. diversifolia.
SIGNIFICANCE STATEMENT
This study discover the interaction between the crude extract and bioactive fraction (C70 M30) of Tithonia diversifolia leaf administered with therapeutic dose of chloroquine in ameliorating the hepatic and nephrotic damage resulting from activities of chloroquine resistant Plasmodium yeolii parasite during malarial infection that can be beneficial for the use of this plant to enhance the efficacy of chloroquine against resistant malaria. This study will help researchers to uncover the critical areas of herbal therapy that many researchers were not able to explore. Thus, a new theory on safety of orthodox drugs-plants combination therapy may be arrived at.
ACKNOWLEDGMENT
The authors wish to acknowledge MR4/American Type Culture Collection for providing the malaria parasite Plasmodium yeolii contributed by Wallace Peters and Brian L. Robinson used in the study.