Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3631

Full Length Research Paper

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

Ramadhani S.O. Nondo*
  • Ramadhani S.O. Nondo*
  • Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences P.O.Box 65001, Dar es Salaam, Tanzania.
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Denis Zofou
  • Denis Zofou
  • Biotechnology Unit, University of Buea, P.O.Box 63 Buea, South West Region, Cameroon.
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Mainen J. Moshi
  • Mainen J. Moshi
  • Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences P.O.Box 65001, Dar es Salaam, Tanzania.
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Paul Erasto
  • Paul Erasto
  • National Institute for Medical Research P.O.Box 9653, Dar es Salaam, Tanzania.
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Samuel Wanji
  • Samuel Wanji
  • Research Foundation in Tropical Diseases and Environment P.O.Box 474, Buea, South West Region-Cameroon.
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Moses N. Ngemenya
  • Moses N. Ngemenya
  • Biotechnology Unit, University of Buea, P.O.Box 63 Buea, South West Region, Cameroon.
  • Google Scholar
Vincent P.K. Titanji
  • Vincent P.K. Titanji
  • Biotechnology Unit, University of Buea, P.O.Box 63 Buea, South West Region, Cameroon.
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Abdul W. Kidukuli
  • Abdul W. Kidukuli
  • Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences P.O.Box 65001, Dar es Salaam, Tanzania.
  • Google Scholar
Pax J. Masimba
  • Pax J. Masimba
  • Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences P.O.Box 65001, Dar es Salaam, Tanzania.
  • Google Scholar


  •  Received: 10 November 2014
  •  Accepted: 05 February 2015
  •  Published: 10 February 2015

 ABSTRACT

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.

 

Key words: Ethnobotanical survey, medicinal plants, malaria, treatment, in vitro antiplasmodial, Tanzania. 


 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.


 MATERIALS AND METHODS

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. 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/01st August 2011) from the Muhimbili University of Health and Allied Sciences Institutional Review Board.

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 antiplasmodial screening

Malaria parasites

Blood stage chloroquine-resistant P. falciparum Dd2 strains (Pf Dd2; MRA-156 deposited by TE Wellems, Lot# 59443398) were used. The parasites were donated to the University of Buea in Cameroon by BEI-resources (MR4/ATCC® Manassas, VA, USA).

Malaria culture medium

RPMI-1640 (Lot# RNBC 8874) culture medium with L-glutamine and 20 mM HEPES (Sigma®, Steinheim, Germany) was used.

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 NaHCO3, 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% CO2, 5% O2, 90% N2 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). Non-synchronized 1% parasitized red blood cells (pRBCs) at 2% 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% CO2, 5% O2, 90% N2. 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.

 


 RESULTS

Documentation and identification of medicinal plants used for treatment of malaria in Kagera and Lindi regions, Tanzania

A total of 108 plants species distributed into 41 plant families were documented and identified in six villages in Tanzania (Table 1). Fourteen plant species (12.96%) belonged to the family Compositae, 12 plant spp (11.11%) belonged to Fabaceae, 8 plant spp (7.41%) belonged to Euphorbiaceae, 6 plant spp (5.56%) belonged to Melastomataceae and 4 plant spp (3.70%) belonged to Myrtaceae. The families Anacardiaceae, Graminae, Labiatae, Meliaceae and Rutaceae each had 3 plant spp (2.78%) while other families were represented by 2 or 1 plant spp (Figure 1). The reported medicinal plants were identified as trees (37%), herbaceous plants (34%), shrubs (20%), climbers (5%), grass (2%) and wood climbers (2%) as shown in Figure 2. In addition, all the reported medicinal plants are administered orally, mostly as decoctions. Boiling was the most common method of preparation (Table 1). Leaves were the most used part of the plants, representing 46% of all plant parts reported followed by stem bark (19%), aerial parts (15%), roots (8%), whole plants (4%),  seeds  (4%),  fruits (3%) and whole stem (1%) as shown in Figure 3.

 

 

 

 

 

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 chloroquine- resistant Dd2 strains (Table 2).

 


 DISCUSSION

The results of the current study support results of previous ethnobotanical studies done in Tanzania and outside Tanzania. In previous studies Abrus precatorius, Adansonia     digitata,      Azadirachta     indica,    Cassia didymobotrya, Dombeya shupangae, Ethrina sacleuxii, Lantana camara, Mangifera indica, Maytenus senegalensis, Momordica foetida, Parinari excelsa, Pseudospondias microcarpa, Psidium guajava, Syzygium cordatum, Todalia asiatica, Vangueria infausta, Vernonia amygdalina, and Zanthoxylum chalybeum were reported to be used in the treatment of malaria in Tanzania and some of them have shown good in vitro antimalarial activity against multi-drug resistant P. falciparum K1 malaria parasites (Amri et al., 2012; Augustino et al., 2011; Gessler et al., 1994; Weenen et al., 1990). Similarly, Erythrina abyssinica, Markhamia lutea, Teclea nobilis, Adansonia digitata, Lantana camara, Azadirachta indica, Zynthoxylum chalybeum, Maytenus senegalensis, Vernonia amygdalina, Momordica foetida, Mangifera indica, Moringa oleifera, Leonotis nepetifolia, Maesa lanceolata, Psidium guajava, Funtumia africana, Canna indica, Cymbopogon citratus and Pycnanthus angolensis are used in traditional medicine for malaria treatment in Kenya, Uganda, Cameroon and Nigeria (Lacroix et al., 2011; Nguta et al., 2010; Tabuti et al., 2008; Titanji et al., 2008; Katuura et al., 2007; Odugbemi et al., 2007).

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 IC50 = 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 IC50 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.


 CONFLICT OF INTEREST

The authors declare that they have no competing interests


 ACKNOWLEDGEMENTS

This work was financed by Sida through MUHAS capacity building grants. Authors are very grateful to Sida for the financial support. We are grateful to Mr. Mohamedi Ngalanga, Mr. Didas Ngemera, Mr. Dominic Mushwahili, Mr. Buchadi Tibikunda and Mr. Papianus Rwechungura for showing us the medicinal plants traditionally  used  for 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.



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