Brine shrimp cytotoxicity and antimalarial activity of plants traditionally used in treatment of malaria in Msambweni district

Abstract

Context: In Kenya, most people use traditional medicine and medicinal plants to treat many diseases including malaria. To manage malaria, new knowledge and products are needed. Traditional herbal medicine has constituted a good basis for antimalarial lead discovery and drug development.

Objectives: To determine in vivo antimalarial activity and brine shrimp toxicity of five medicinal plants traditionally used to treat malaria in Msambweni district, Kenya.

Materials and methods: A 0.2 ml saline solution of 100 mg/kg aqueous crude extracts from five different plant parts were administered orally once a day and evaluated for their in vivo chemosuppressive effect using Plasmodium berghei berghei-infected Swiss mice for four consecutive days. Their safety was also determined using Brine shrimp lethality test: Grewia trichocarpa Hochst ex A. Rich (Tiliaceae) root, Dicrostachys cinerea (L) Wight et Am (Mimosaceae) root, Tamarindus indica L. (Caesalpiniaceae) stem bark, Azadirachta indica (L) Burn. (Meliaceae) root bark, and Acacia seyal Del. (Mimosaceae) root.

Results: Parasitaemia was as follows: A. indica, 3.1%; D. cinerea, 6.3%; T. indica, 25.1%; A. seyal, 27.8%; and G. trichocarpa, 35.8%. In terms of toxicity, A. indica root bark extract had an LC₅₀ of 285.8 µg/ml and was considered moderately toxic. T. indica stem bark extract and G. trichocarpa root extract had an LC₅₀ of 516.4 and 545.8 µg/ml, respectively, and were considered to be weakly toxic while A. seyal and D. cinerea root extracts had a LC₅₀ >1000 µg/ml and were, therefore, considered to be non-toxic.

Discussion and conclusion: All extracts had antimalarial activity that was not significant compared to chloroquine (p ≥ 0.05). No extract was toxic to the arthropod invertebrate, Artemia salina L. (Artemiidae) larvae, justifying the continued use of the plant parts to treat malaria.

• Aqueous plant extracts
• chemosuppression
• Plasmodium berghei
• Plasmodium falciparum
• Swiss albino mice

Introduction

Approximately 40% of the world’s population, mostly in the world’s poorest countries, is at risk of contracting malaria (WHO, 2002). In 2010, there were 216 million cases of malaria in 2010 resulting in 655 000 deaths equivalent to about 2000 deaths daily (Nadim & Behrens, 2012; WHO, 2011). It is estimated that about 80% of all malaria deaths in the world occur in sub-Saharan Africa with the majority (65%) occurring in children under 15 years old and pregnant women (Olver, 2012; WHO, 2003). In Kenya, of the 22 million people at risk of malaria attacks, 70% of them live in rural areas while about 34 000 Kenyan children under five die every year from malaria compared with a total estimate of 42 000 deaths arising from all diseases combined (KMSI, 2011; WHO, 2002). Poverty can also increase the risk of malaria because of lack of financial capacity to prevent or treat the disease. Malaria has also been estimated to cost Africa $12 billion USD every year. The economic impact includes costs of health care, working days lost due to sickness, days lost in education, decreased productivity due to brain damage from cerebral malaria, and loss of investment and tourism (Greenwood et al., 2005).

Resistance to antimalarial drugs is proving to be a challenge in malaria control in most parts of the world (Greenwood et al., 2005). Virtually all drugs for malaria such as chloroquine, quinine, sulphadoxine–pyrimethamine, and artemisinin combined therapies (ACTs) have developed resistance against Plasmodium parasites (Jambou et al., 2005). Therefore, to effectively manage malaria, new knowledge, products, and tools are urgently needed and particularly new drugs (Omulokoli et al., 1997). An effective malaria vaccine would be the most powerful and most cost-effective measure with a potential long-term impact including the possibility of eradicating the disease. While substantial progress has been made, a real breakthrough towards a malaria vaccine is still missing.

Natural products could provide a starting point in drug discovery. A typical example is quinine, which was the first antimalarial drug of plant origin isolated from the bark of Cinchona succirubra Pav. ex Klotsch (Rubiaceae) tree in 1820 (Bruce, 1988). The vastly unexplored flora of East Africa (90% of total species) could provide other new leads and drugs for chemotherapy, for the isolation of certain natural products in large amounts, total synthesis by chemical approaches, or limited scope for chemical modification (David et al., 2004). For decades, traditional herbal medicine has constituted a good basis for antimalarial lead discovery and drug development (David et al., 2004). In Africa, more than 80% of people use traditional medicines and most families have recourse to this medicine based on plant extracts for the curative treatment of malaria (Wright & Phillipson, 1990). In fact, the traditional medicine of this continent constitutes an important source for ethnopharmacological investigation. Collaborative research between academics and traditional healers have resulted in identification of herbal remedies for different ailments including malaria, broadening horizons of knowledge in the areas of natural products chemistry as well as benefiting people immensely by lowering the cost of treatment (Bayer et al., 1998).

In Kenya, traditional medicine is a vital and popular part of health care as the conventional system provides for only 30% of the population, implying that more than two-thirds of Kenyans depend on traditional medicine for their primary health care needs (WHO, 2003). The Digo community, for example, use traditional herbal remedies to treat malaria which is a health challenge affecting mostly children, pregnant mothers and the elderly among Kenyan coastal communities. Validation of traditional remedies can be problematic because of the lack of sufficient information, documentation and standardization of extracts (Corson & Crews, 2007). Therefore, Digo community of Msambweni district was chosen in order to assess their efficacy of their commonly used antimalarial plants with the aim of adding to the database of scientifically approved standard antimalarial plant extracts. The toxic effects of the remedies should also be tested in order to ascertain safety during treatment. The objective of this study was therefore to determine the in vivo antimalarial effect of the aqueous extracts of five commonly used plants in this community of Kenya using Plasmodium berghei-infected Swiss mice and to evaluate their cytotoxicity using Brine shrimp lethality test.

Materials and methods

Collection of plant materials and preparation of crude extracts

The five plant samples used in this study were collected in December 2010 from Msambweni district of Kenya based on ethnopharmacological use through interviews with local communities and traditional health practitioners (Nguta et al., 2010). The plants were identified by Mr. Kimeu Musembi, a taxonomist at the University of Nairobi Herbarium, Nairobi, where voucher specimens were deposited. The plants voucher numbers (in parenthesis) are as follows: Grewia trichocarpa Hochst ex A. Rich (Tiliaceae) root (JN022); Dichrostachys cinerea (L) Wight et Am (Mimosaceae) root (JN016); Tamarindus indica L. (Caesalpiniaceae) stem bark (JN038); Azadirachta indica (L) Burn. (Meliaceae) root bark (JN09), and Acacia seyal Del. (Mimosaceae) root, JN01 (Table 1). The plant parts were chopped into small pieces; air dried at room temperature (25 ± 5 °C) under shade and pulverized using a laboratory mill (Christy & Norris Ltd., England, UK). An aqueous hot infusion of each plant part was prepared (50 g of powdered material in 500 ml of distilled water) in a water bath at 60 °C for 1 h. The extracts obtained were filtered through muslin gauze and the filtrate was kept in a deep freezer for 24 h. The frozen filtrate was then lyophilized, collected in stoppered sample vials, weighed, and kept at −20 °C until use.

Table 1. Parasitaemia, chemosuppression, and survival time of Plasmodium berghei-infected mice treated orally with five aqueous crude plant extracts at a dose of 100 mg/kg body weight, once a day for 4d.

Plant species	Voucher specimen number	Plant part	Parasitaemia(%)	Chemo-suppression (%)	Survival on day 7 (%)
A. indica	JN09	Root bark	67.9 ± 0.27^1	3.1 d	20.0
D. cinerea	JN016	Root	65.7 ± 1.08	6.3 d	40.0
T. indica	JN038	Stem bark	52.4 ± 6.17	25.1 c	20.0
A. seyal	JN01	Root	50.5 ± 3.12	27.8 c	40.0
G. trichocarpa	JN022	Root	45.0 ± 2.51	35.8 b	40.0
Chloroquine^2	N/D	–	0.0 ± 0.00	100.0 a	100.0
Distilled water^3	N/D	–	70.0 ± 3.59	N/D	0.0

N/D, not determined.

¹Mean ± SD

²Positive control.

³Negative control.

^a–dSignificantly different or not.

Determination of in vivo antimalarial activity and cytotoxicity of the plant extracts

Male and female Swiss albino mice (18–22 g), bred in Kenya Medical Research Institute Laboratory, were used. The animals were housed in the animal house at KEMRI (Kenya Medical Research Institute) and the institute’s Animal care and Use Committee gave approval for the study. In vivo antimalarial testing in mice was done using chloroquine (CQ)-sensitive strain of P. berghei (D6) clones. Each test mouse was injected intraperitoneally with 0.2 ml of the suspension (1.0 × 10⁷ parasitized red blood cells). The assay protocol was based on the 4-d suppressive test (Peters & Robinson, 1975). Thirty-five mice were randomly selected and assigned into seven treatment groups with each group comprising of five mice. Two groups were selected randomly; a positive control group and a negative control group. Each mouse in the negative control group was given distilled water at a dosage of 0.2 ml/d while each mouse in the positive control group was given chloroquine at a dosage of 10 mg/kg in a saline suspension of 0.2 ml. Mice in each of the remaining five groups were given aqueous plant extract at a dosage of 100 mg/kg in a saline suspension 0.2 ml. Plant extracts, distilled water, and chloroquine were administered orally once daily for four consecutive days (day zero to day three). A stained blood smear was made from each mouse (Duguid et al., 1978) on the fifth day using blood squeezed from tail cut. The total number of parasitized erythrocytes was carefully counted as well as the total number of erythrocytes. Counting was done four times in different fields on the same thin blood smear. Investigation and handling of animals were done in accordance to the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Percentage parasitaemia and chemosuppression were obtained by the following formula:

Acute toxicity was determined according to the method described by Meyer et al. (1982). Ten Artemia salina L. (Artemiidae) nauplii were transferred into each sample vial (five replicates for three serial dilutions of different aqueous plant extracts 5, 50, and 500 µl and the control). Surviving nauplii were counted after 24 h, and the percentage of deaths at the three dose levels and control determined. In cases where control deaths occurred, the data were corrected using the formula by Abbott (1925):

The lethal concentration fifty (LC₅₀) at 95% confidence interval and slope were determined from the 24 h counts using the Finney (1971) computer program.

Data analysis

Data from the antimalarial work were entered and analyzed using the SPSS 16.0, software (SPSS Inc., Chicago, IL). The differences among groups were analyzed by the one-way analysis of variance (ANOVA) and inter group comparisons were done using Duncan’s multiple range test (DMRT). Toxicity data were analyzed by Finney’s probit analysis to estimate the LC₅₀. Values of p < 0.05 were considered significant.

Results

Parasitaemia ranged from 45.0 to 67.9% while chemosuppression ranged between 3.1 and 35.8% among plant extract-treated infected mice (Table 1). There were significant (p ≤ 0.05) differences in percentage parasitaemia between treatment with aqueous plant extracts and chloroquine. The highest parasitaemia was recorded when model mice were treated with A. indica root extract. There was no significant (p ≥ 0.05) difference in parasitaemia among sterile distilled water, A. indica and D. cinerea treated mice. Similarly, parasitaemia between A. seyal and T. indica was not significantly different.

Mice treated with G. trichocarpa root extracts showed the lowest mean parasitaemia of 45.0%. There was significant (p ≤ 0.05) difference between chemosuppression produced by chloroquine to that of the five aqueous plant extracts. Grewia trichocarpa extract had a chemosuppression that was significantly different (p ≤ 0.05) from that of the other four plant extracts. On day seven post infection, when there was 100% mortality of mice treated with distilled water, extracts from A. seyal, D. cinerea, and G. trichocarpa maintained a survival rate of 40% while extracts from A. indica and T. indica had a survival rate of 20%. There was significant difference (p ≤ 0.05) chemosuppression in all treatments to the positive control (chloroquine) which cleared all parasites.

There was no 100% mortality rate achieved at the three concentrations tested. A. indica had an LC₅₀ of 285.8 µg/ml and was considered moderately toxic. Two plant extracts, T. indica and G. trichocarpa, had an LC₅₀ of 516.4 and 545.8 µg/ml, respectively, and were considered weakly toxic while the crude plant extracts of A. seyal and D. cinerea had acute toxicity of 1000 and above and were therefore non-toxic (Table 2).

Table 2. Acute toxicity of five aqueous crude plant extracts at different concentrations against Artemia salina larvae.

		% Death after 24 h
Family	Plant species	10 µg/ml	100 µg/ml	1000 µg/ml	LC50
Mimosaceae	A. seyal	10	4	12	5915.6
Mimosaceae	D. cinerea	2	6	17	8298.5
Tiliaceae	G. trichocarpa	2	14	38	545.8
Caesalpiniaceae	T. indica	8	10	36	516.4
Meliaceae	A. indica	26	0	34	285.8
–	Distilled water	0	0	0	80 723 503.0

Discussion

Extracts with a high chemosuppression for Plasmodium parasites offer a potential source for new antimalarial drugs. The mean percentage parasitaemia in the negative control was 70.0% which was the highest witnessed in all the groups treated with the various plant extracts. The mean parasitaemia for each of the five crude aqueous extracts were below this value. This indicated that all the analyzed crude extracts had in vivo antimalarial activity and did suppress the multiplication of P. berghei parasites in mice. Chloroquine, the drug of choice for the treatment of malaria, cleared all the parasites, while parasitaemia in the negative control increased with time, culminating in the death of all mice by day seven post infection.

Mice treated with G. trichocarpa root extract showed a chemosuppression of 35.8%. These findings were in agreement with the literature data of other Grewia species. Ma et al. (2009) demonstrated that some triterpenoids, e.g. 3α,20-lupandiol, grewin, nitidanin, and 2α,3β-dihydroxy-olean-12-en-28-oic acid isolated from Grewia bilamellata are responsible for the antimalarial effect and showed varying degrees of in vitro activity against P. falciparum. This may partially validate ethnomedical use of this herb in the management of malaria.

Root extract of A. seyal had chemosuppression of 27.8%. Similar results were obtained by Garavito et al. (2006) while investigating the in vivo antimalarial activity of ethanolic bark extract of a related species Acacia fernesiana that exhibited activity against P. berghei with parasitaemia inhibition of 32%. According to Doughari (2006), A. seyal aqueous extract of the fruits has shown tannins, saponins, sesquiterpenes, alkaloids, and phlobatannins that have been implicated to have antiplasmodial activity. However, the use of root extract of A. seyal has been documented for the first time. Other studies, however, have shown that other Acacia species, for example, Acacia tortilis root bark possess antimalarial activity of IC₅₀ of >10.0 µg/ml (Koch et al., 2005) and would not be considered for follow up as an antimalarial candidate. Chemosuppression produced by a plant extract is associated with its inherent ability to clear parasite in infected cells. Inability of a plant extract to completely clear parasites in infected cells may be due to increased biotransformation of the crude plant extract.

Stem-bark extract of T. indica showed chemosuppression of 25.1%. Similar results for the methanol extract of the T. indica stem bark showed moderate activity with an IC₅₀ value of 10 mg/ml on 3D7 P. berghei strain (Ahmed et al., 1999). This plant is widely used in folk medicine to treat many infectious diseases including malaria. This might be by stimulation of the immune system leading to a reduction in the level of parasitaemia so that natural immunity can cope with these infections (Ahmed et al., 1999).

Root bark extract of A. indica and root extract of D. cineraea showed 3.1 and 6.3% chemosuppression, respectively. A. indica is the third commonly used herbal medicine to treat malaria in Kenya after Ajuga remota and Caesalpinia volkensii (Kuria et al., 2001). Efficacy of the extract is attributed to azadirachtin, a limonoid of highly oxygenated terpenoids (Roy & Saraf, 2006). Many other communities around the world use these plants as traditional antimalarials. However, activity depends on many factors such as the season in which the plant is collected, the age of the plants, intraspecies variation, the environmental conditions, and the plant part collected among others (Muregi et al., 2004). Azadirachtin (a tetranortriterpenoid) has been detected in all aerial parts of neem plant with concentration decreasing in the older mature seed kernel, leaves, bark, roots, and stem (Sundaram, 1996). The aqueous root extract of D. cinerea had no significant effect in treatment of symptomatic malaria and would not make as a good follow up for discovery of a malarial herbal drug.

In vivo antiplasmodial activity of plant extract is a better screening method compared with an in vitro method because some drugs act as febrifuges, prodrugs, or immuno-modulators (Muregi et al., 2004). It can be predicted that the screened plant species can produce a better recovery of symptomatic malaria following combination with other antimalarial herbal remedies that exert synergy. Indeed, the screened antimalarial herbal remedies are combined with other plant species amongst Digo community traditional healers (Nguta et al., 2010). Most in vivo studies in mice with P. berghei have been uniformly disappointing (Obih & Makinde, 1985). Many reasons could explain these poor in vivo activities. It may be that mice do not metabolise plant extracts in the same way as humans. Murine Plasmodium parasites have different properties and sensitivities compared with human P. falciparum and the 4-d suppressive test may not be sufficient to evaluate plant extracts (Willcox & Bodeker, 2004). If administration of drug is not continued before the host has recovered from infection, the parasite may regain virulence to a level to overcome host’s immune system leading to death.

Extracts of A. seyal, D. cinerea, and G. trichocarpa produced the highest survival of 40%. Lower survival of mice treated with A. indica could partly be contributed by traces of toxins. T. indica gave a lower survival of 20% probably due to the fact that it was weakly toxic. Interestingly, A. seyal gave a high survival of 40%, it was non-toxic. More so, it had one mouse surviving on day 11 of post infection when all plant extract-treated mice had died. Brine shrimp toxicity is a general bioassay that detects a broad range of biological activities and a diversity of chemical structures. One basic premise here is that toxicology is simply pharmacology at a higher dose, thus if we find toxic compounds, a lower, non-toxic, dose might elicit a useful, pharmacological, perturbation on a physiologic system (Jerry, 2008).

In general, the smaller the LC₅₀ value, the more toxic the chemical is. The opposite is also true: the larger the LC₅₀ value, the lower the toxicity. A plant extract with a LC₅₀ over 1000 µg/ml is considered to be non-toxic, whereas that with LC₅₀ ranging between 500 and 1000 µg/ml is said to be weakly toxic, that with LC₅₀ ranging between 100 and 500 µg/ml is said to be moderately toxic while that with an LC₅₀ less than 100 µg/ml is said to be highly toxic (Nguta et al., 2011). The least toxic extract against chloroquine (CQ)-sensitive P. berghei was that of D. cinerea root extract which gave LC₅₀ of 8298.5 µg/ml. A. seyal with an LC₅₀ of 5915.6 µg/ml scored second among non-toxic plant extract. Therefore, D. cinerea and A. seyal pose no threat as an antimalarial drug to the community that uses them. The chemosuppression observed can be attributed to having potential to inhibit growth of Plasmodium parasites which is not related with the inherent toxicity of the plant extract. Screening aqueous extracts of T. indica and G. trichocarpa showed LC₅₀ of 516.4 and 545.8 µg/ml, respectively, and were considered to be weakly toxic. It can be argued out that chemosuppression observed was partly due to traces of toxins in the extract but largely due to the extract’s ability to elicit antiplasmodial activity. The extract of A. indica produced LC₅₀ of 285.8 µg/ml and was considered moderately toxic. Toxicity of the crude plant extracts is attributed to the phytoconstituents present in them. More so, the presence and concentration of bioactive ingredients usually vary with the age of the plant, intraspecies variation and time of the season, locality, extraction method used, and length of storage time (Muregi et al., 2004).

All the five plant species reported in the current study exhibited low chemosuppression ranging between 3.1 and 35.8%. Among the investigated plants, G. trichocarpa root extract, A. seyal root extract, and T. indica stem bark extract had high chemosuppression while A. indica root bark extract and D. cinerea had low chemosuppression. Despite the plant extracts exhibiting varying degrees of chemosuppression, none of them exhibited a chemosuppression significantly similar to the positive control (chloroquine) chemosuppression. This can be attributed to the fact that only aqueous extracts were used in the study and the active antimalarial phytoconstituents might not have been present in the aqueous extracts or were present in minute quantities. It may also be argued that mice do not metabolize extracts in the same way as humans and this could have led to the witnessed low chemosuppression of the extracts. In terms of toxicity, A. seyal and D. cinerea were non-toxic while T. indica, G. tricocarpa, and A. indica were moderately toxic to A. salina larvae. It is evident that none of the screened plant extracts is toxic to the arthropod invertebrate, A. salina larvae, justifying the continued use of the plant parts to treat malaria particularly D. cinerea and A. seyal by the Digo community. Results observed in this study suggest that the monotherapy of malaria with aqueous extracts of plant parts under investigation was non-effective. In addition, the results observed in this study confirm antimalarial activities of A. seyal, G. trichocarpa, and T. indica reported by other researchers. Anecdotal efficacy reported by the study community could be related to synergism of phytoconstituents since the assayed plant parts are used in combination with others to treat malaria. Considering the low toxicity on brine shrimp larvae of A. seyal and G. trichocarpa which inhibited parasitaemia better compared with the other aqueous plant extracts, further studies on the antimalarial activity of these plants can be done which will involve the use of other plant parts other than the roots and even the use of organic extracts. The current study reports in vivo antimalarial activity and non-toxicity on brine shrimp A. Salina of A. seyal, G. trichocarpa, and T. indica for the first time.

Acknowledgements

The authors are indebted to the community of Msambweni district for sharing their knowledge and time with them; to Mr. Mutundu Gabriel of Centre for Traditional Medicine and Drug Research laboratories-Kenya Medical Research Institute, Nairobi and Mr. Joseph Gichuki Nderitu of Department of Public Health, Pharmacology and Toxicology, University of Nairobi for their technical assistance and to Mr. Kimeu Musembi, a taxonomist at the University of Nairobi Herbarium, Nairobi, who identified the study plants.

Declaration of interest

The authors declare no conflicts of interest. The authors are grateful to Regional Initiative in Science and Education-African Natural Product Network (RISE-AFNNET) for supporting this study.