In vitro and in vivo antiplasmodial assays of selected Nigerian commercial herbal formulations

Introduction Malaria remains a major public health problem especially in tropical and sub-tropical countries of the world (13). This acute infection is caused by five species (vivax, falciparum, ovale, malariae and knowlesi) of the protozoan genus Plasmodium. However, P. falciparum causes the most serious complications and significant number of deaths. The parasite is transmitted to humans through the bite of the female anopheles mosquito which thrives in humid, swampy areas and breeds in stagnant water (4). In 2017, nearly 50% of the world’s population were at risk of the disease (3). An estimated 1.2 billion people are at high risk of transmission (≥1 case per 1000 population), half of which live in the African regions; 80% of such cases are concentrated in 13 countries, and over half in Nigeria, Congo, Ethiopia, Tanzania and Kenya (5). So, Africa has the highest incidence rate of malaria disease and the highest death rate in the world. This is attributed to the http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.47


Introduction
Malaria remains a major public health problem especially in tropical and sub-tropical countries of the world (1)(2)(3). This acute infection is caused by five species (vivax, falciparum, ovale, malariae and knowlesi) of the protozoan genus Plasmodium. However, P. falciparum causes the most serious complications and significant number of deaths. The parasite is transmitted to humans through the bite of the female anopheles mosquito which thrives in humid, swampy areas and breeds in stagnant water (4). In 2017, nearly 50% of the world's population were at risk of the disease (3). An estimated 1.2 billion people are at high risk of transmission (≥1 case per 1000 population), half of which live in the African regions; 80% of such cases are concentrated in 13 countries, and over half in Nigeria, Congo, Ethiopia, Tanzania and Kenya (5). So, Africa has the highest incidence rate of malaria disease and the highest death rate in the world. This is attributed to the prevalence of P. falciparum and the effective mosquito vector, Anopheles gambiae, in Africa (6). Mortality has risen in recent years, probably due to increasing resistance to antimalarial medicines (7,8).
One of the major challenges faced in the fight against malaria is the ability of the most virulent causative agent, P. falciparum, to develop resistance to antimalarial drugs and how quickly the resistance spread. In some parts of the world, including Nigeria, chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) are no longer considered drug of choice for treating malaria due to their recorded high failure rate (9,10). Although vaccines are anticipated to be the best long-term control option, they are still undergoing clinical trials (11)(12)(13)(14)(15).
Interestingly, most malaria endemic countries are found in areas of the world whose populations still rely heavily on traditional medicines of plants' origin for their primary healthcare needs. Due to factors such as: high cost of western medicine, the common belief that medicines of plant origin are cheap, easily available, safe and effective (16,17); several antimalarial formulations of plants origin have flooded the Nigerian market. It seems logical then to assess the therapeutic claims of these formulations. In view of the general acceptability of the selected herbal formulations, this study was set to assess their therapeutic claims using both in vivo and in vitro methods and compare their effects with standard antimalarial drugs. According to ethno-botanical survey in Nigeria (18), it was reported that some 98 species from different families of plants are used in traditional medicine singly or in combination to treat malaria infection. These plants used for malaria treatment or prevention represent more than half of Nigeria medicinal species (19).
Till date, no data are available for validating the antimalarial use of these herbal formulations (Ruzu bitters, Deep root, African Iba, M&T capsule and Blood purifier) but most of the extracts have been used singly for assessment of antimalarial properties in vitro and/or in vivo (20,21). Alstonia bonnie, Markhamia tomentosa, Moringa oleifera, Mangifera indica, Cymbopogon citratus, Psidium guajava which have different species widely distributed in Africa, Asia and America have been reported to be effective in treatment of several diseases including Malaria (19,(22)(23)(24). In this study, the antiplasmodial activities of six commercially available herbal formulations were studied using in vivo and in vitro methods to assess their claimed antimalarial properties.
Mus musculus -the animal model chosen for this studyprovides an easy to manage, cheap and excellent animal model for human malarial which cell-based studies do not offer at the moment. Also, cell-based study does not offer the physiological environments which animal model offers. Environmental factors can readily be controlled in M. musculus compared to human studies. These obvious factors informed our choice of M. musculus as a model animal for the study.

Materials and Methods
Plasmodium parasites The P. falciparum strain 3D7 used for the in vitro study (chloroquine sensitive parasite) was obtained from Immunology Department, Noguchi Memorial Institute for Medical Research, Legon, Accra, Ghana and chloroquinesensitive strain of P. berghei, maintained in mice, from the National Institute of Pharmaceutical Research and Development, Nigeria. The parasite was subsequently inoculated into fresh mice, which served as donor mice in this study.
Drug extraction/preparation Six herbal formulations were obtained from commercial drug markets in Anambra State, Nigeria. Twenty millilitres (20 mL) of each herbal formulation was measured and dispensed into Eppendorf tube and stored in the freezer at -20 º C. The herbal formulations were removed and placed in a freeze dryer (Labconco FreeZone 6, USA) with reduced pressure of 12 mbar at -40 º C to remove all moisture for complete dryness. The dry extracts were weighed using electronic weighing balance, transferred into Eppendorf tubes and stored for further analysis.
Herbal formulation preparation Stock concentrations of 50 mg/mL of each extract of the 6 herbal preparations (Ruzu Bitters, Deep Root, Chuka Trin Cleanser, Blood Purifier, M&T Capsule and African Iba), as well as chloroquine and artesunate were prepared in 50% ethanol and filter-sterilized through a 0.22 μm membrane (Millipore) filter. The extracts, artesunate and chloroquine stocks were diluted to 2000 μg/mL, 630nM and 1000nM, respectively in complete parasite medium and further diluted three-fold serially into six different concentrations for the assays with extracts, chloroquine and artesunate (final concentrations, 0.41-100 µg/ mL, 0.41-100nM and 0.03-6.3nM, respectively). Both chloroquine and artesunate served as the positive controls.
In vitro culture of Plasmodium falciparum The chloroquine-sensitive P. falciparum strain 3D7 was cultured and maintained as described in earlier studies with slight modification (17,25). The parasites were cultured in a complete media consisting of RPMI 1640 (Sigma) supplemented with 1% L-glutamine, 25mM HEPES, 0.2% sodium bicarbonate, 0.5% Albumax II, 100μM hypoxanthine, and 1% gentamycin and incubated at 37 o C. The parasites were cultured in fresh O + human erythrocytes at 4% hematocrit. Estimation of parasitaemia was done using Giemsa stain and visualization performed under the normal light microscope (X100) using oil immersion. The antiplasmodial activity of the six herbal formulations was assessed against P. falciparum: 3D7 (chloroquinesensitive) strain in vitro using SYBR® Green assay. The activity of the extract was measured over the six concentrations prepared. All experiments were performed in triplicates. At least two independent experiments were performed. The SYBR green I-based fluorescence antiplasmodial assay as described by Leidenberger et al (26) was used for the activity screening of the extracts and drugs. Sorbitol synchronized parasites were incubated with the extracts and drugs (100 μL final volume) under culture condition as described above at 2% hematocrit and 1% parasitaemia. Parasites without drug treatment were used as negative controls while the wells containing chloroquine and artesunate were the positive controls. The plates were covered and shaken slightly to ensure a thorough mixing. The cultures were incubated for 72 hours. After incubation, aliquots of 100 μL of SYBR GREEN lysis buffer containing 20mM Tris-Cl (pH 7.5), 5mM EDTA, 0.008% saponin, 0.08% triton-X 100 and 1X SYBR green I (10 000X in DMSO) were added. The plates were shaken gently and incubated in the dark for 3 hours. The fluorescence in each well was read using a Tecan fluorescence (Tecan Infinite M200, Austria) multiwell plate reader at excitation and emission wavelengths of 485 and 530nm, respectively. The experiments were performed in triplicate and each repeated at least once. The intensities of the fluorescence signals were plotted against the extracts or drug concentrations to obtain a concentration response curve. The curves were analysed to determine 50% inhibitory concentrations (IC 50 ) of the drugs.

In vivo Assay
Experimental animals Forty albino mice -species: M. musculus (20-26 g, 5-6 weeks old) of both sexes, caged separately, were tested separately with extracts and controls. The animals were inbred by the principal investigator and when fully grown, they were taken to the animal house of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Agulu Nnamdi Azikiwe University in Anambra State for the experimental procedures. They were first housed in pathogen-free metal cages (n=16) for 7 days to acclimatize under standard environmental conditions of temperature: 26 ± 2 o C, relative humidity: 45 ± 2% and 12 hours natural dark-light cycles before the experimentation. Sixteen cages (same sex per cage) were chosen to prevent mating. These standard environmental conditions were maintained throughout the experimental periods. During the acclimatization period and throughout experimental periods, the animals were handled in accordance with established guidelines (27)(28)(29) for care and use of laboratory animals. The study protocols were approved by the Proposal/Ethics Committee for animal studies of the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Awka Nigeria.

Induction of infection
After 7 days acclimatization period, standard inoculums of 1×10 5 P. berghei infected M. musculus erythrocytes were prepared by diluting the infected M. musculus blood with 0.9% normal saline such that 0.2 mL of the blood contained approximately 1×10 5 infected erythrocytes. A 0.2 mL of the diluted blood was injected intraperitoneally into each experimental animal on the first day.

Malarial curative test (schizonticidal activity in established infection)
The evaluation of the curative potential of the herbal formulations on established malarial infection was evaluated using the method described by Fidock et al (30) and modified by Tarkang et al (31). The animals were first placed into 8 groups of each sex with 5 animals per group and after 72 hours post-infection; the treatment began with oral administration of single daily dose and lasted for 3 days (parasitemia was checked before commencement of treatment). Oral route was chosen to mimic the natural means of administration of the herbal formulation in human. All the animals were allowed free access to feed and clean water throughout the experimental period.  Animals' allocation into groups was such that the mean weights of the groups were equal (or near equal) and there were approximately equal number of each sex per group. The administration of the herbal drugs and results recording were all done by the investigator. The animals were allowed free access to their feed and water throughout the period of the study. All treatments were carried out in the animal house in the morning periods. On days 4 and 7 post-treatment, blood samples were collected from the tip of the tail of each lab animal for the preparation of thin smears. The smears were prepared, fixed with methanol, and stained with 10% Giemsa solution at pH 7.2 for 25 minutes and examined under microscope with an oil immersion objective (×100 magnification power).
All the animals used in the study were observed for 30 days for signs of morbidity and for mortality.

Parasitaemia monitoring
Parasitaemia was determined by collecting blood sample from the tail on day 0 (before treatment), day 4 and day 7 post treatment. Parasitaemia was assessed by thin blood films made by collecting blood from the cut tip of the tail, dry blood films were fixed with methanol for 15 seconds and subsequently stained with 10% Giemsa for 25 min. They were then washed with water and allowed to dry. The slides were then microscopically examined using ×100 magnification in oil immersion. Triplicate experiments were performed. The level of parasitaemia was determined by counting the number of parasitized erythrocytes out of 200 erythrocytes in random fields of the microscope. The percentage parasitaemia was determined using the following equation (33): Where 'a' is the mean percentage parasitaemia in the corresponding days (day 4 and 7) of the treatment groups and 'b' is the mean percentage parasitaemia in the basal day (day 0) of the treatment groups.
Fifty percent inhibitory concentrations (IC 50 ) for the in vitro study were obtained from plots of extract concentration versus percentage inhibition.

Statistics and data analysis
The results were presented as the mean ± SEM (standard error of mean) for each group of experiments. The test groups were compared with the negative control group using one-way analysis of variance (ANOVA). All data were analysed at a 95% confidence interval using GraphPad Prism 3 Software, San Diego, California, USA. P values less than 0.05 were considered statistically significant. The IC 50 values were obtained from the log-linear regression analysis of log-dose response curves using the GraphPad Prism 3. Table 1 shows the Latin binomial nomenclature of the constituents of the herbal formulations that were investigated.

Results
The results of the in vitro antiplasmodial assay ( Table 2) showed that the six herbal formulations had IC 50 values of > 100 µg/mL on 3D7 strain of P. falciparum. The products were considered less potent and had low percentage inhibition of parasitaemia when compared with the controls, chloroquine and artesunate, with IC 50 values = 6.92nM and 0.75nM, respectively.
Effect of the herbal medicines on uninfected human red blood cells (RBCs) is also shown on Table 2. None of the extracts could inhibit the viability of untreated RBCs, suggesting that they are not harmful to the cells.

Results of in vivo curative activity using mice infected with Plasmodium berghei
Prior to treatment, the animals were in good health status as evidenced by their agility and good feeding habit and their mean age was 24 ± 0.63 days. Mean parasitaemia of the M. musculus was of the range 9.05 % ± 0.37 to 15.90 % ± 1.08 for day 0 (Basal parasitaemia), 1.60 % ± 0.44 to 8.25 % ± 0.77 for day 4 post treatment and 0.55 % ± 0.18 to 6.11 % ± 0.76 for day 7 post treatment ( Table 3). The mean parasitaemia in Artemether and Lumefantrine (AL) group (positive controls) were 10.65 %±0.9, 1.00 % ± 0.14 and 0.4 % ± 0.20 for days 0, 4 and 7 post treatment, respectively showing good curative effect of the drug while the mean parasitaemia of untreated (negative) control were 16.45 % On days 4 and 7 post treatment, the % change in parasitaemia was higher in Deep root than all the other herbal formulations (Table 4). This change was comparable to the positive control. On the other hand, M&T capsule recorded the lowest change in parasitaemia on both days. Generally, the percentage inhibition was observed to increase with increase in days of administration of the herbal formulation. It was observed that the animals administered M&T capsule and those given African Iba developed sluggish movement on the 29 th and 30 th day respectively.

Discussion
This study utilised both in vitro and in vivo methods to assess the effectiveness of six herbal products in controlling malarial infection. A combination of in vitro and in vivo studies may help show if the herbal products and/or their metabolites are responsible for the observed antimalarial actions. Also, instead of taking the blood samples of the animals immediately, the study refined the method by first anaesthetising the animals with isoflurane cotton balls to minimize pain and distress.
The six herbal formulations did not exhibit inhibitory activity against chloroquine sensitive P. falciparum 3D7 strain (IC 50 > 100 µg/mL), according to the criteria described by Kamaraj et al. (34). Results of earlier in vitro and in vivo investigations into anti-plasmodial activities Table 2. The Effect of the herbal medicines on Plasmodium falciparum 3D7 strain and on uninfected red blood cells

M&T Capsule
African Iba Chloroquine Artesunate Inhibitory effects on P. falciparum 3D7 strain IC 50 (µg/mL) >100 >100 >100 >100 >100 >100 6.92 0.075 Effects on uninfected red blood cells CC 50 (µg/mL) >100 >100 >100 >100 >100 >100 >100 ND ND: Not determined; CC 50 : Concentration of extracts /drugs which causes 50% cytotoxic effect.  of some other medicinal plant extracts showed varying comparisons between the IC 50 values for chloroquine -sensitive 3D7 strain of P. falciparum (23,35,36). Some of the constituents of the test herbal formulations have been reported to show in vitro antimalarial activity individually (37,38). The undetected inhibitory activity in this study could be due to the extracts acting as pro-drugs or the constituent of the mixtures of the medicines diminishing the effects of other active compounds. It is therefore advisable to investigate the individual components and the interactions between two herbal extracts before combining them into a co-formulation. Further studies should also be performed to determine possible pro-drug action of the medicines since in vivo assays showed that they possessed varying antiplasmodial activity against P. berghei. None of the extracts inhibited viability of untreated RBCs. This suggests that they are not cytotoxic to the cells. It has been documented that herbal medicines with CC 50 (µg/mL) values > 100 are non-toxic to RBC (34,39).
The in vivo studies, however, revealed that the herbal formulations were active as % parasitaemia was reduced at least by over 50% in most of the products on days 4 and 7. Deep root formulation exhibited the best reduction in parasitaemia, i.e. 84.91% and 94.81% on days 4 and 7, respectively. This activity is comparable with that the positive control, artemether and lumefantrine, which exhibited 90.61 % and 95.87 % reduction on days 4 and 7, respectively. This could be attributed to the combination of the extracts from plants with known antiplasmodial activity. Although, there has been no previous study on the antimalarial activities of these herbal formulations, many of the medicinal plants that constitute the herbal formulations have been individually reported to exhibit diverse pharmacological actions (35,40,41). M. indica, C. citratus, P. guajava, have all been reported to possess good antimalarial properties in vivo (24,35,42,43).
Similarly, a good in vivo antiplasmodial activity was observed with Ruzu bitters formulation, with percentage parasitaemia reduction of 77.36% and 90.25% on day 4 and 7, respectively. This could be linked to the presence of Carica papaya, Uvaria chamae and Morinda lucida which have all been reported to exhibit good antiplasmodial activity in vivo (18,35,43,44). A similar result has been reported by Ihekwereme et al (33) that indicated high % parasitaemia reduction by fruit pulp of Chrysophyllum albidum (Sapotaceae) against P. berghei in vivo using mouse model. The results obtained are indicative of the curative potential of these herbal formulations. The lowest antiplasmodial activity was observed in M&T formulation with % parasitaemia reduction of 26.34%, and 45.54% on days 4 and 7 post treatment, respectively. These values are below the 50%, thus regarded as demonstration of poor parasitaemia reduction. This could be as a result of the fewer antiplasmodial constituents of the herbal formulation. Chuka Trin Cleanser, with M. oleifera and water as the sole constituents and having % parasitaemia reduction of 50.5%, and 67.3% on days 4 and 7 post treatment, may show that M. oleifera has appreciable antiplasmodial property. Other researchers (19,22,45) have also proved the antimalarial property of the plant. This investigation has demonstrated that the Deep root and Ruzu bitters herbal formulations have very strong inhibitory effect against P. berghei in M. musculus.
Our results lend support to the claims of the traditional medicine practitioners that these herbal formulations, which are used in traditional medicine practice against malaria, possess significant anti-malarial potential and justify their use in traditional medicine. The in vitro assays showed that all herbal formulations had no inhibitory effect on chloroquine sensitive P. falciparum strain 3D7. The difference in activity between the in vivo and in vitro assays may suggest that these herbal formulations act as pro-drugs. In this case, these precursors of the active compounds have to be metabolized in vivo into active antimalarial drugs.

Conclusion
The results of this study confirmed that the six herbal formulations possess antimalarial properties with good curative activity against P. berghei (in vivo assay), which is the animal model of the human P. falciparum. Percentage inhibitions of parasitaemia of the formulations were all above 50% except M&T capsule which had low percentage inhibition of parasitaemia.

Limitations
First, our study did not include the identification of the active principles in these herbal formulations. Secondly, neither mechanistic nor molecular bases of the observed antiplasmodial effects were investigated. Also, comprehensive safety profile in an animal model at the therapeutic dosage was not carried out. These limitations not-withstanding, this study offers scientific information on the usefulness of the formulations in treating human malarial infection.

Conflict of interests
There is no competing interest to declare. The funders were not involved in the writing, editing, approval or decision to publish this manuscript.

Ethics considerations
The study protocols were approved by the Proposal/ Ethics Committee for animal studies of the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Awka Nigeria. Approval Number: FPhS/AEC/Vol.1.002

Funding/Support
The in vitro part of this work was made possible due to the Travel Grant graciously given to CJI by West African research Association (WARC Travel Grant Spring 2016). The in vivo study did not receive sponsorship from external agent but rather was self-sponsored. Funder has no influence whatsoever in the design and execution of the study, analysis and interpretation of data, in the writing of the manuscript nor in the decision to publish the work.