|Year : 2019 | Volume
| Issue : 2 | Page : 146-153
Commercial herbal preparations ameliorate Plasmodium berghei NK65-induced aberrations in mice
Victor N Ogugua, Innocent U Okagu, Onyekachi M Onuh, Peter N Uzoegwu
Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria
|Date of Submission||25-Dec-2017|
|Date of Acceptance||15-Mar-2018|
|Date of Web Publication||31-Jul-2019|
Innocent U Okagu
Drug Discovery and Medical Parasitology Unit, Department of Biochemistry, University of Nigeria, Nsukka, Enugu State
Source of Support: None, Conflict of Interest: None
Background & objectives: The alarming failure in malaria treatment using conventional drugs calls for urgent search of alternatives; one of which is to exploit natural products such as plants. This study evaluated the effects of three selected commercial herbal preparations on albino mice infected with Plasmodium berghei NK65, a lethal strain of rodent malaria.
Methods: This study was conducted in the University of Nigeria, Nsukka between February and September 2017. A total of 30 adult albino mice were randomized into six groups of five mice each. Group 1 served as normal control. Mice in Groups 2-6 were parasitized with P. berghei. Group 2 mice were untreated while mice in Groups 3, 4, 5 and 6 were treated with 20 mg/kg body weight of artesunate; and 5 ml/kg body weight of the seleceted commercial herbal preparations designated as HA, HB and HC, respectively. The percent malaria parasitaemia, haematological parameters, lipid profile, liver function markers, antioxidant status and lipid peroxidation index were evaluated using standard protocol.
Results: It was observed that mice in Group 2 had significantly higher percentage of malaria parasitaemia when compared to mice in parasitized and treated groups. Also, haematological dysfunctions, dyslipidaemia, oxidative stress and hepatotoxicity seen in parasitized and untreated mice were restored in parasitized and artesunate- and herbal preparations-treated mice.
Interpretation & conclusion: Findings from the present study revealed that oxidative stress, characterized by low antioxidant status and high lipid peroxidation, contributes to complications in malaria. The results also indicate that the studied commercial herbal preparations possess good antimalarial and ameliorative effects on malaria-induced haematological, lipid, antioxidant and liver aberrations in mice. The acute toxicity profiles of the commercial herbal preparations suggested that they are tolerable and safe at the doses administered.
Keywords: Antioxidants; haematological indices; herbal preparations; lipid peroxidation; lipid profile; liver function enzymes; malaria; Plasmodium berghei
|How to cite this article:|
Ogugua VN, Okagu IU, Onuh OM, Uzoegwu PN. Commercial herbal preparations ameliorate Plasmodium berghei NK65-induced aberrations in mice. J Vector Borne Dis 2019;56:146-53
|How to cite this URL:|
Ogugua VN, Okagu IU, Onuh OM, Uzoegwu PN. Commercial herbal preparations ameliorate Plasmodium berghei NK65-induced aberrations in mice. J Vector Borne Dis [serial online] 2019 [cited 2019 Nov 17];56:146-53. Available from: http://www.jvbd.org/text.asp?2019/56/2/146/263722
| Introduction|| |
The World Health Organization (WHO) reported 216 million cases of malaria in 2016 which was higher than the 211 million cases reported in 2015. Similarly, the estimated number of malaria deaths in 2016 was 445,000 as compared with 446,000 in 2015, which indicates the high burden of malaria across the world. Malaria is a mosquito-borne disease caused by parasitic protozoans of the genus, Plasmodium. The disease is transmitted by infected female Anopheles mosquito. Five species of Plasmodium can infect humans. Plasmodium berghei is a species of malaria parasite that infects mammals other than humans. It is one of the four Plasmodium species that has been described in African murine rodents, the others being P chabaudi, P. vinckei and P. yoeli. These are not direct practical model for humans; though they act as model organisms in the laboratory for the experimental study of human malaria.
At present, about 100 countries or territories in the world are considered malarious, almost half of which are in Africa. In Nigeria, there is a uniform distribution and prevalence of the disease in both rural and urban areas. Malaria in Nigeria is said to be holoendemic, i.e. there is an intense all-year round transmission with greater intensity in the wet season than dry season. The severity of malaria infection can be determined by haematologi-cal, renal and hepatic malfunction. Liver dysfunction is a common complication that usually occurs in malaria infection; studies have reported sudden increase in activities of liver enzymes in the serum of malaria-infected individuals. This could be due to the invasion of liver cells by the sporozoites during exo-erythrocytic stage of malaria parasite life cycle. The changes caused in the hepatocytes by sporozoites can lead to the leakage of parenchymal and membranous enzymes of the liver into the circulatory system, which might be responsible for the increase in serum liver enzyme activities.
The use of conventional antimalarial drugs has not been very fruitful over the years due to the alarming rate at which malaria parasites have developed resistance to most used antimalarials such as chloroquine, hydroxychloroquine, primaquine and mefloquine, and even ar- temisinins. Also, synthetic and semi-synthetic antimalarial drugs that are in current use such as lumefantrine and artemisinin-based drugs, are inaccessible and unaf-fordable to poor rural dwellers; and these recommended antimalarial drugs have also been reported to be toxic. These necessitate a need to search for alternatives that are safe, cheap and available to even rural dwellers. Hence, this study was aimed to evaluate plant products with an- timalarial potential and ameliorative effect on complications caused by malaria infection.
| Material & Methods|| |
Collection of materials
This study was conducted at the University of Nigeria, Nsukka, Nigeria between February and September 2017. All the chemicals used in the study were of analytical grade and were obtained from commercial dealers in Nsukka and Onitsha cities, of Nigeria. Drugs used (arte- sunate and other three commercial herbal preparations — HA, HB and HC) were purchased from drug stores in Enugu State, Nigeria. According to the manufacturers, the compositions of the commercial herbal preparations studied are: HA, a product ofDr Igwodo Herbal Cleanser Nigerian Limited, Nigeria, is composed of a blend of Vernonica amygdalina (12%), Saccharum officinarum (11.5%), Allium sativum (13%), Cajanus cajan (11.5%), Zingiber officinale (0.5%), and Caramel (1.5%) in water; HB (Abllat Company Nigerian Limited, Lagos, Nigeria) is composed of a blend of Cymbopogon citrates (13%), Carica papaya leaves (12%), Mangifera indica bark (11%), Moringa oleifera leaves (11%), Citrus limonia (9%), Psidium guajava (9%), Zingiber officinale root (9%), and Allium sativum (6%) in water; and HC, an oral preparation manufactured by FESCO Herbal Mixture Nigerian Limited, Nigeria, is composed of a blend of various parts of plants such as Aloe vera, Cinnamonium aromaticum, Citrus aurantifolia, Aci- nos avensis, and Chenopodium murale in water (the percent composition of HC is not provided by the manufacturer). These preparations are indicated for both curative and prophylactic treatment of malaria and other diseases by the manufacturers and are widely sold and used in all parts of Nigeria and other African countries.
Management of experimental animals
Adult Wistar mice (n = 57) weighing 25-30 g were obtained from Animal House, Faculty ofVeterinary Medicine, University of Nigeria, Nsukka. These were acclimatized to laboratory conditions at the Animal House of Biochemistry Department, University of Nigeria, Nsukka for 14 days. They had free access to normal rat chow (Vital Rodent Feed Nigerian Ltd., Nigeria) and water ad libitum, and received humane care throughout the experimental period in accordance with the ethical rules and recommendations of the University of Nigeria Committee on the Care and Use of Laboratory Animals and the revised National Institute of Health Guide for Care and Use of Laboratory Animal (Pub No. 85–23, revised 1985) at the Animal House, Department of Biochemistry, University of Nigeria, Nsukka.
Acute toxicity study of herbal preparations
In total, 27 mice were used, i.e. nine mice per herbal preparation. In each case, nine mice were divided into three groups of three mice each. Those were orally administered with 1, 5 and 10 ml/kg body weight of the herbal preparation, respectively. The mice were observed for 24 h for behavioural changes and lethality.
Parasite inoculation and study design for antimalarial study
Donor mouse blood infected with P. berghei NK65 was obtained from the Department of Veterinary Parasitology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka and was used for inoculum preparation. Blood sample was drawn from a donor mouse by heart puncture and diluted serially in Alsever’s solution. The final suspension contains about 1*10 infected red blood cells (RBCs) in every 0.2 ml suspension. The experimental mice (n = 30) were divided randomly into six groups of five mice each. Group 1 served as normal control while Group 2 mice were untreated. The 0.2 ml suspension was injected intraperitoneally into the experimental mice in Groups 2–6 to initiate infection (parasitized). Infection for malaria was confirmed with microscopy after 72 h (Day 0) and treatment was initiated accordingly as shown in [Table 1]. Treatment with herbal preparations and artesunate was trough oral administration, once daily for 4 days (from Day 0–3).
Determination of parasitaemia level in experimental animals
The level of parasitaemia in the experimental mice was determined haematologically, using microscopic technique. Thick blood smears were collected daily from tail blood, stained with Giemsa’s stain and examined under high power microscope (100 x oil immersion resolution) to determine the parasitaemia level. The level of parasitaemia was determined on Days 0, 1, 2 and 3. The percent parasitaemia was determined by counting the parasitized white blood cells (WBCs) out of total WBCs in random fields of the microscope using the formula:
Determination of haematological and biochemical parameters
On Day 5 (2-days post-treatment), after an overnight fasting, all the mice were sacrificed and their blood samples were collected and used for haematological and biochemical analyses. The haematological parameters included haemoglobin (Hb) concentration, packed cell volume (PCV), RBC, total white blood cell (TWBC) and differential white blood cell (DWBC) counts. Biochemical parameters evaluation included: serum concentrations of total cholesterol, serum high density lipoprotein (HDL) and triacylglycerol (TAG), low density lipoprotein (LDL), vitamin C, total bilirubin, vitamin E, malondialdehyde (MDA) concentrations; and activities of catalase (CAT), superoxide dismutase (SOD), glu- tathione peroxidase (GPx), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and alkaline phosphatase (ALP).
All the data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA using statistical products and service solutions (SPSS), version 18. Differences between means at 5% level (p <0.05) were considered statistically significant.
Ethical approval for the study was obtained from the Faculty of Biological Science Committee on Ethics and Biosafety (UNN-IRB/FBS/2016_0192). A total of 30 mice were used for antimalarial study while 27 were used for acute toxicity study.
| Results|| |
Acute toxicity profile of herbal preparations
Mortality and behavioural changes were not observed in any of the mouse used for acute toxicity investigation after 24 h of administration. The observation showed that the commercial herbal preparations are tolerable at the doses administered.
Percent malaria parasitaemia in experimental mice
Results of the percent malaria parasitaemia in the experimental mice measured after plasmodial inoculation are shown in [Table 2]. On Day 0, there was no significant (p >0.05) difference among the parasitaemia levels in all the parasitized experimental mice. On Day 1, it was observed that mice in Group 2 (parasitized and untreated) had a significantly (p <0.05) higher percent malaria parasitaemia compared to mice in Groups 3–6 (parasitized and artesunate- and herbal preparations-treated mice). Similar results were obtained on Days 2 and 3. From Days 0-3, the malaria parasitaemia of mice in Group 2 increased significantly (p <0.05) when compared to the trends in groups 3–6, which decreased. In the same vein, the percenta che- mosuppression of the artesunate was comparable with that of the herbal preparations post-treatment.
Haematological parameters of experimental mice
The results of the WBCs indices, PCV, Hb concentration and RBCs count of experimental mice are shown in [Table 3]. Mice in Group 2 had significantly (p <0.05) lower TWBC count compared to those in other groups (the normal unparasitized, parasitized artesunate- and herbal preparations-treated mice). There was no significant ( p >0.05) difference among the TWBC count of parasitized and treated, and normal unparasitized mice. Mice in Group 6 had significantly (p <0.05) lower neutrophil and lymphocyte percentages when compared to mice in other Groups. There was no significant (p >0.05) difference among the percent neutrophils of mice in Groups 1, 4 and 5, as well as between Groups 2 and 3. Similarly, there was no significant (p >0.05) difference among the percentage lymphocytes of mice in Groups 1, 4 and 5, as well as between Groups 2 and 3. Conversely, mice in Group 2 had significantly (p <0.05) lower PCV compared to those in other groups studied. There was no significant (p >0.05) difference among the PCV levels of animals in the treated and normal control groups. The Hb concentration of mice in Group 2 was significantly (p <0.05) lower than that of parasitized and treated, and normal control groups.
However, the Hb concentration of normal mice (Group 1) was significantly (p <0.05) higher than that of parasitized and treated groups. There was no significant (p >0.05) difference among the Hb concentrations of animals in the parasitized and treated groups (Groups 3, 4, 5 and 6). Mice in Group 2 showed a significantly (p <0.05) lower RBC count compared to those in the treated and normal control groups. Meanwhile, there was no significant (p >0.05) difference among the RBC count of mice in the parasitized and treated groups (Groups 3, 4, 5 and 6) and normal control.
Lipid profile of the experimental mice
As shown in [Table 4], mice in Group 2 (parasitized and untreated) had significantly (p <0.05) higher TC, LDL and TAG concentrations in comparison with mice in groups 3–6 (parasitized and treated mice). However, there was a significantly (p <0.05) lower HDL concentration in Group 2 compared to parasitized and treated mice. Also, there was no significant (p >0.05) difference among the TAG concentrations of mice in Groups 1, 3, and 4.
Concentrations of vitamins C and E, and malondialdehyde and activities of antioxidant enzymes in experimental mice
Vitamin C concentration of mice in Group 2 was significantly (p <0.05) lower than those of mice in the parasitized and treated, and normal control groups. Mice in TA Group 6 had significantly (p <0.05) higher vitamin E concentration than those in other parasitized and treated, and normal control groups. There was no significant (p >0.05) difference among the vitamin E concentrations of mice in Groups 1, 2, and 3 as well as between Groups 4 and 5. However, the vitamin E concentration of mice in Groups 1, 2, 3 were significantly (p <0.05) lower than those of mice in Groups 4 and 5. The mean MDA concentration of mice in group 2 was significantly (p <0.05) higher when compared to other five groups. However, the MDA concentrations of parasitized and herbal preparations-treated mice (Groups 4, 5 and 6) were significantly (p <0.05) higher than those of mice in Groups 1 (control) and 3 (parasitized and artesunate-treated mice) [Table 5]. Also, the mean serum catalase activity of mice in group 2 was significantly (p <0.05) lower than other five groups. There was no significant (p >0.05) difference among the catalase activities of mice in Groups 1, 3 and 4, as well as between those of mice in Groups 5 and 6. Mice in Groups 1, 3, and 4 had significantly ( p <0.05) higher SOD activities compared to those in Groups 2, 4 and 5. Meanwhile, there were no significant (p >0.05) difference among the SOD activities of mice in Groups 1, 3, and 6, as well as Groups 2, 4 and 5. Also, there was significantly ( p <0.05) lower mean GPx activity in the parasitized and untreated mice (Group 2) in comparison to mice in other groups. However, there were no significant (p >0.05) difference among the GPx activities in parasitized and treated (3, 4, 5 and 6) and normal control mice (Group 1) [Table 5].
|Table 5: Concentrations of vitamins C and E, and MDA and activities of antioxidant enzymes in the experimental mice|
Click here to view
Activities of liver function enzymes and total bilirubin concentration of experimental mice
Mice in Group 2 (parasitized and untreated) had a significantly (p <0.05) higher AST, ALT and alkaline phos-phatase (ALP) activities when compared to those of mice in the parasitized and treated groups. However, there was no significant (p >0.05) difference between the AST activities of mice in Groups 3 and 4, as well as between 5 and 6. Similarly, there was no significant (p >0.05) difference among the ALT and ALP activities of mice in Groups 3, 4 and 6, as well as between that of Groups 1 and 5. Also, there was no significant (p >0.05) difference among ALP activities of mice in Groups 3, 4 and 6, as well as between Groups 1 and 5. The total bilirubin concentration of mice in Group 2 (parasitized and untreated) was significantly (p <0.05) higher than other groups. However, there was no significant (p >0.05) difference among the total bilirubin concentrations of mice in Groups 1, 5 and 6, as well as between groups 3 and 4 as shown in [Table 6].
|Table 6: Activities of liver function enzymes and total bilirubin concentration of experimental mice|
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| Discussion|| |
The present study evaluated the antimalarial and modulatory effects of selected commercial herbal preparations (HA, HB and HC) on Plasmodium berghei NK65- infected mice divided into different experimental groups. After 72 h of malaria parasite inoculation (Day 0), malaria infection was confirmed by microscopic examination and treatment was initiated. On Day 1 (after 24 h of the first treatment) it was observed that mice in Group 2 (parasitized and untreated) had a significantly (p <0.05) higher percent malaria parasitaemia compared to mice in other experimental groups (parasitized and treated, and normal control). Similar results were observed in Days 2 and 3. The percent malaria parasitaemia of mice in group 2 increased daily, which has also been observed in other studies,. A dose-dependent chemosuppression of para- sitaemia was observed in parasitized mice treated with the herbal preparations (Groups 4, 5 and 6) which were comparable to the observation made in the parasitized mice treated with the standard drug, artesunate (Group 3). Earlier reports have shown that the plant components used in the herbal preparations such as Aloe vera, Carica papaya,, and Allium sativum possess antimalarial effects. These components (synergistic action) might be responsible for the malaria chemosuppression observed in the present study.
There was an observed total restoration in RBC count in the parasitized and herbal preparations-treated mice (Groups 4–6) relative to normal mice (Group 1), suggesting that the commercial herbal preparations possess erythropoietic and anti-anaemic effects and hence, can be used for the treatment of anaemia. In addition, some plant components of the herbal preparations have been shown to boost blood cells production. There was a significant (p <0.05) decrease in the mean WBC count in the infected and untreated mice compared to that of the parasitized herbal preparations- and parasitized artesunate-treated mice. A similar trend of decrease in WBC was observed by Odeghe et al in parasitized mice when compared with control. Bakhubaira and Francis et al in their separate studies observed a similar result in human malaria. The significant (p <0.05) increase in WBC displayed by parasitized and treated mice indicates an improved ability of the mice to combat the infection. These findings agree with those of Richards et al; and Lee et al.
Higher TC, LDL and TAG concentrations in parasitized and untreated mice vs. parasitized and treated, and normal mice suggests that the herbal preparations and artesunate possess some ameliorative effects on malaria- induced lipid aberrations. The observed lower mean HDL concentration in parasitized and treated mice compared to those of normal mice is consistent with results of earlier studies that reported increased serum lipoprotein fractions in malaria-infected human subjects when compared with apparently-healthy control subjects. Results from this study showed that increase in plasma lipids in malaria subjects is consistent with the degree of parasitaemia. This is in agreement with the report of Visser et al, wherein they found that increase in plasma lipid is directly proportional to increase in malaria parasitaemia.
The observed variation in vitamins C and E concentrations in parasitized and treated mice may be due to difference in the vitamin contents of the herbal preparations as well as their ability to act as antioxidants, preventing some free-radical associated complications in malaria. On the other hand, elevation in MDA concentration, an indicator of oxidative stress via lipid peroxidation in the parasitized and untreated mice when compared to control suggests the existence of oxidative stress in malaria. Oxidative stress may have resulted from the release of free-radical intermediates of malaria, which attack on cellular components, and breakdown products of haemoglobin such as haeme and free iron metal during erythrocytic stage of Plasmodium life cycle. These products are free-radical and free-radical-producing agents, respectively. Other reports have also suggested the existence of oxidative stress in malaria. Further, malaria parasite in the exo-erythrocytic stage attack hepatocytic membrane, causing lipid peroxidation which could be responsible for high concentration of MDA as seen in the parasitized and untreated mice.
Decrease in the activities of SOD and CAT with increase in MDA level in parasitized and untreated mice when compared to the normal control mice provides evidence of existence of oxidative stress leading to enhanced lipid peroxidation, tissue damage and collapse of the antioxidant defence mechanism against free-radicals. However, administration ofherbal preparations and artesunate restored the activities of these enzymes, showing buffering effects on the stress posed to the antioxidant defence system by malaria infection. This suggests that the herbal drugs used in this study can boost the potential of the antioxidant defence system through the phytochemical contents of the plant components as earlier reported. Reports have shown that antioxidants ameliorate symptoms of malaria; hence, the use of these herbal preparations as both antimalarials and antioxidants may be helpful in management of malaria and prevention of complications associated with malaria.
Evidence from the present study showed that malarial infection can induce changes in the liver enzyme activities in serum and increases haemolysis of RBCs leading to elevation in bilirubin concentration. Studies have shown that malaria parasitaemia engenders increased RBC haemolysis, which is associated with increase in bilirubin biosynthesis, hepatocellular damage, biliary tract obstruction and jaundice. Parasitized and untreated mice had significantly (p <0.05) higher AST, ALT and ALP activities and total bilirubin concentration when compared with parasitized and treated mice. Treatment of parasitized mice with herbal preparations and artesunate significantly (p <0.05) restored the activities of the liver enzymes and total bilirubin concentration in serum relative to the normal mice. These suggest that longer treatment may totally restore the hepatotrophy in the parasitized mice. These findings are consistent with the findings of other studies that showed elevated activities of liver function enzymes in infected and untreated subjects,,. In general, this study has shown that the polyherbal preparations are potentially applicable in treating malaria and restoring malaria-induced haematological and biochemical changes.
| Conclusion|| |
Findings from the present study indicates that studied commercial herbal preparations possess good antima-larial effects at the doses administered. They also have ameliorative effects on the haematological, antioxidant, liver and lipid aberrations associated with malaria infection in mice. However, the degree of action varies among the different commercial herbal preparations studied which could be attributed to the differences in the plant compositions. Also, result of the acute toxicity profile of the commercial herbal preparations suggests that they are tolerable at the doses studied. Though, further studies are needed to ascertain the doses that will give total malaria parasite suppression.
Conflict of interest
The authors declare no conflict of interest.
| Acknowledgements|| |
The authors wish to thank Dr Simeon Egba of Shalom Biochemical and Research Laboratory, Nsukka for his technical assistance and Dr P.E. Joshua of Department of Biochemistry, University of Nigeria, Nsukka for statistical analysis.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]