Ethnobotanical survey and in vitro antiplasmodial activity of medicinal plants used to treat malaria in Kagera and Lindi regions , Tanzania

Tanzania has over 12,000 plant species, some of which are endemic and have potential to yield useful medicines. This study seeks to document such plants used as traditional medicines for treatment of malaria in Kagera region of northwestern Tanzania and Lindi region in south eastern Tanzania. The study also reports on the antiplasmodial activity against chloroquine-resistant Plasmodium falciparum (Dd2) strain of some of the documented plants using the parasite lactate dehydrogenase method. A total of 108 plant species, among which the families Compositae (14; 12.96%), Fabaceae (12; 11.11%), Euphorbiaceae (8; 7.41%), Melastomataceae (6; 5.56%) and Myrtaceae (4; 3.70%) were documented. Sixteen (16; 44.4%) of 36 extracts from 31 plant species that were tested inhibited malaria parasites growth by more than 50%. Bersema abyssinica stem bark extract was the most active with 86.67% inhibition rate followed by Bridelia micrantha stem bark extract with 71.87% inhibition rate. These results confirm the potential for plants used in traditional medicine to yield active antimalarial compounds. Further in vitro and in vivo screening supported by bioassay-guided isolation of active compounds from plants showing good safety margin is suggested.


INTRODUCTION
Human malaria is caused by five Plasmodium species namely; P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi, but Plasmodium falciparum is the most widespread and virulent species (World Health Organization (WHO), 2013;Cox-sigh and Singh, 2008).Malaria in Tanzania is mainly caused by P. falciparum with Anopheles gambiae complex being the main vector United States Agency for International Development (USAID, 2014).Tanzania has high malaria prevalence and it is among six African countries that have many reported cases of malaria, with an estimated 10 to 12 million cases and 60,000 to 80,000 malaria-related deaths per year (USAID, 2014;WHO, 2012).Although the Tanzania HIV/AIDS and Malaria Indicator Survey (2011/2012) reported a decrease in the prevalence of malaria in Tanzania, the trend remains unchanged.Prevalence is still high in rural areas and the Lake Victoria zone as compared to other parts of the country Tanzania Commission for AIDS (TACAIDS, 2013).
Malaria is a curable disease that is treated by both modern drugs and herbal medicines (Kinung'hi et al., 2010;Gessler et al., 1995).However, the emergence of P. falciparum strains resistant to almost all classes of antimalarial drugs dictates that efforts be increased to develop new antimalarial drug candidates (Dondorp et al., 2009;Wongsrichanalai et al., 2002).Since most antimalarial drugs that are currently being used like quinine and artemisinin derivatives originate from traditionally used medicinal plants (Wells, 2011), this source has a great potential to provide new antimalarial molecules.
Tanzania is estimated to have over 12,000 higher plant species, of which 10% are used for medicinal purposes, and may yield active antimalarial compounds (Mahunnah et al., 2012).Previous reports confirm that some of these plants are used in traditional medicine for treatment of malaria (Gessler et al., 1995), and malaria is leading among diseases that are popularly treated with medicinal plants (Moshi et al., 2009;Mahunnah, 1987).Some of these plants have been reported in previous studies (Moshi et al., 2009;Gessler et al., 1995;Mahunnah, 1987), but many have not been documented and only a few have been tested for antimalarial activity.Therefore, this study reports plant species used for treatment of malaria in Kagera and Lindi regions of Tanzania and results of some of the plants that were screened for in vitro antiplasmodial activity.

Documentation, identification and collection of the medicinal plants used for treatment of malaria
Disease-specific ethnobotanical survey was conducted in six villages in Kagera region (North-west of Tanzania) and one village in Lindi region (South east of Tanzania).In Kagera region the study was conducted in November, 2012 in Buzi Bukombe, Buzi Kishura and Kwamkenge villages in Bukoba rural district, Buleza village in Muleba disctrict, Rwambaizi village in Karagwe district, and Kashozi village in Misenyi district.The study in Lindi region was conducted in July, 2012 in Mchakama village located in Kilwa district.Information was collected from well known and experienced traditional healers and herbalists who were informants in a previous ethnomedical study (Moshi et al., 2009).Before collecting information all the participants were informed about the study objectives and agreed to participate by signing an informed consent form.Vernacular names of the plants, part(s) used, method for preparation, route of administration and possible signs of toxicity were documented.

Preparation of extracts
Dry powdered plant materials were macerated in 80% ethanol, at room temperature, for 24 h and then filtered through cotton wool to separate the liquid crude extracts from the solid materials.The solid plant materials were macerated again in the same solvent for another 24 h and the extracts obtained from the first and the second extractions were mixed before drying.The liquid crude extracts were concentrated under vacuo by using Heldolph ® rotary evaporator (Heldolph instruments GmbH, Schwabach, Germany) to obtain viscous extracts which were further dried by using a freeze drier (Edwards High Vacuum International, Crawley Sussex, England).
In vitro culture of malaria parasites P. falciparum Dd2 laboratory strains were maintained in culture according to the method of Trager and Jensen (Trager and Jensen, 1976) with modifications (Zofou et al., 2013).The parasites were grown in O + red blood cells (RBCs) maintained in complete malaria culture medium composed of RPMI-1640 medium supplemented with 2 mg/ml NaHCO 3 , 10 µg/ml hypoxanthine, 2 mg/ml glucose, 1% albumax II as source of proteins, lipids and 10 µg/ml gentamicin.The cultures were incubated at 5% CO 2 , 5% O 2 , 90% N 2 in a humidified incubator set at 37°C (SHEL LAB ® Sheldon Mfg Inc, OR, USA).All materials were purchased from SIGMA (Sigma ® , Steinheim, Germany) except Albumax II (GIBCO ™ , Invitrogen) and gentamicin (ROTEX MEDICA, Trittau -Germany) which were supplied locally in Cameroon.

Preparation of plant extracts and standard drug
Stock solutions of 400 µg/ml for each crude extract was prepared by first dissolving 4 mg of crude extract into 100 µl dimethyl sulfoxide (Sigma ® ) followed by addition of RPMI-1640 medium to 10 ml.Artemether (Sigma ® ) was first dissolved in dimethyl sulfoxide and then diluted with RPMI-1640 medium to 5 µg/ml.All solutions were sterilized by using 0.22 µm syringe-adapted filters (Corning ® , NY, USA) and stored at 4°C until use.

In vitro antiplasmodial activity assay
In vitro antiplasmodial activity was assessed using the parasite lactate dehydrogenase (pLDH) assay (Makler et al., 1993).Nonsynchronized 1% parasitized red blood cells (pRBCs) at 2% Nondo et al 181 haematocrit (hct) in 96 well cell culture plates (Costar ® , Corning, NY, USA) were incubated in triplicates with 100 µg/ml crude extracts or with 1.25 µg/ml artemether.Wells with parasitized cells but without extract or drug served as negative controls (100% parasite growth) and wells without parasitized cells but with red blood cells only at 2% hct served as blank controls.The plates were incubated for 48 h at 37°C in a humidified incubator set at 5% CO 2 , 5% O 2 , 90% N 2 .After incubation for 48 h parasite growth was confirmed by the aid of a light/UV fluorescence microscope (TENSION ® , China) with Acridine orange filter (λmax Abs = 490 nm; λmax Em = 525 nm) and a counting device (Lennox Grain Analysis NG NG21, SPI ® , USA) before the plates were frozen overnight at -20°C.In the pLDH assay the plates were thawed at room temperature to hemolyse red blood cells, and the 10 µL of malaria culture were incubated with 50 µL Malstat solutions and 12.5 µL nitroblue tetrazolium/phenazine ethosulfate for 1 h in darkness.
Parasite growth was determined by measuring the activity of pLDH enzymes at 650 nm using a microplate reader (Emax-Molecular Devices Corporation, California, USA) and the optical density (OD) values obtained were used to calculate antiplasmodial activity.The average OD value of the blank control (2%hct RBC only) was subtracted from all OD values.The antiplasmodial activity was expressed as percentage inhibition rate of parasites growth.

In vitro antiplasmodial activity of the extracts
The results reported in Table 2 show that 16 (44.4%)out of the 36 extracts of 31 plant species that were tested inhibited the growth of the chloroquine-resistant Dd2 malaria parasite strains by more than 50%.The extract of Bersema abyssinica stem barks was the most active with 86.67% inhibition rate followed by the extract of Bridelia micrantha stem barks which inhibited parasite growth by 71.87%.The ethanol extracts of Anthocleista grandiflora stem barks, Funtumia Africana stem bark and leaves, and extracts from leaves of Vernonia glabra, Ipomoea rubens, Pycnanthus angolensis, Eriobotrya japonica and Oxyanthus speciosus were the least active with growth inhibition rate of less than 30% against the chloroquineresistant Dd2 strains (Table 2).
It is notable that some of the reported plants belong to the families Compositae (13%), Euphorbiaceae (7.4%), Fabaceae (11.1%), and Rubiaceae (9.2%) which are known to contain chemical compounds with good antimalarial properties (Ntie-Kang et al., 2014;Batista et al., 2009).The study has provided useful information that supports traditional healers' claims for antimalarial activity and earlier observations that plants used in traditional medicine are a potential source of new antimalarial lead compounds (Onguéné et al., 2013;Bero et al., 2009).
All the extracts tested for in vitro antiplasmodial activity at 100 µg/ml inhibited the growth of malaria parasites to different percentages.Bersama abyssinica, Bridelia micrantha, Canarium schweinfurthii and Antiaris toxicaria stem bark extracts; Aspilia natalensis, Aspilia mossambicensis and Desmodium salicifolium aerial part extracts; Maesa lanceolata and Rhytignia obscura leaf extracts; Pycnanthus angolensis fruit and Hallea rubrostipulata root extracts inhibited parasite growth by more than 60%.The ethanol extract of B. abyssinica was the most active with 86.67% inhibition rate against Dd2.In a previous study Kassa et al. (1996) reported that the ethanol extract of B. abyssinica stem bark exhibited good in vitro antimalarial activity against P. falciparum tine-FAC-2/ Ethiopia with IC 50 = 11 µg/ml.Similarly, a study done in Cameroon by Ngemenya et al. (2005) showed that the methanol extract of B. abyssinica leaves exhibited good in vitro antiplasmodial activity with an IC 50 of 2.7 µg/ml.Meanwhile, the chloroform extract of M. lanceolata was reported to exhibit very good antiplasmodial    activity with IC50 = 1.6 µg/ml against P. falciparum clinical isolates (Katuura et al., 2007).Most plants tested in this study showed low parasite growth inhibition rate.It is not easy to identify the specific reasons for low activity but factors such as the solvent used for extraction, the method of preparation, storage conditions, and variation in the active constituents due to seasonal or geographical and model of testing may also reduce the efficacy of the extract (Weeneen et al., 1990).Furthermore, Chhabra et al. (1993) reported that preparations of medicinal plants can be used orally, rubbed into scarification, inhaled as fumes, splashed on the eyes, poured into the wound or sniffed.In this study we found that all preparations were administered orally in the form of decoction (boiled water extracts), infusion (hot water extract), juice or taken as raw fruits.In the oral route, the bioactive molecules are exposed to various barriers and enzyme systems before reaching the systemic circulation.This causes some bioactive molecules to be modified by metabolism thus, either enhance or reduce their antiplasmodial activity suggesting that the antiplasmodial activity of metabolically activated compounds may not be evident in in vitro assays.

Conclusion
This study reported 108 medicinal plants that are used in the traditional medicine for treatment of malaria and fevers in Kagera and Lindi regions of Tanzania.In vitro assays revealed substantial antiplasmodial activities of 15 plants out of 31 plant species tested.Although questionnaire based evidence suggested that decoctions from these plants were not acutely toxic, further toxicity testing will be required to establish their safety profile.Meanwhile these findings support the use of these plants for the traditional treatment of malaria.Further in vitro and in vivo screening supported by bioassay-guided isolation of active compounds of plants showing good safety margin are suggested.
treatment of malaria in their areas.We would like to extend our gratitude to Mr. Haji.O.Selemani (Botanist) for identification of the documented medicinal plants.Extraction of the plant extracts was done at the Institute of Traditional Medicine in Tanzania whereas in vitro antimalarial study was done at the Research Foundation in Tropical Diseases and Environment, and at the Biotechnology Unit, University of Buea in Cameroon.We are grateful to MR4/BEI-resources (Manassas, VA, USA) for donating malaria parasites used for this work.

Figure 1 .Figure 2 .
Figure 1.Distribution of plant species into different families
Plant part: R = root, ST = Stem, SB = Stem bark, AP = Aerial parts, L = Leaves, F = Fruits, S = Seeds, WP = Whole plant.Nature of the plant: SH = Shrub, H = Herb, T = Tree, CL = Climber, WC = Wood climber, G = Grass Preliminary identification was done by a Botanist, Mr. Haji.O. Selemani, in the field and further authenticated in the Herbarium.Voucher specimens are deposited in the Herbaria at Muhimbili University of Health and Allied Sciences and at the Botany Department, University of Dar es Salaam.The selection of the plants to be tested for antimalarial activity was based on absence in the literature of previous antimalarial testing, reported antimalarial use in other countries or emphasis made by the traditional healers regarding efficacy for malaria treatment.This study received ethical clearance (Ref.No. MU/DRP/AEC/Vol.XIII/01 st August 2011) from the Muhimbili University of Health and Allied Sciences Institutional Review Board.

Table 1 .
Medicinal plants used traditionally in the treatment of malaria in Kagera and Lindi regions, Tanzania