Ethnobotanical survey and toxicity evaluation of medicinal plants used for fungal remedy in the Southern Highlands of Tanzania.

BACKGROUND/AIM: Some of the antifungal drugs used in the current treatments regime are responding to antimicrobial resistance. In rural areas of Southern Tanzania, indigenous people use antifungal drugs alone or together with medicinal plants to curb the effects of antibiotic resistance. This study documented ethnobotanical information of medicinal plants used for managing fungal infections in the Southern Highlands of Tanzania and further assess their safety.
MATERIALS AND METHODS: Ethnobotanical survey was conducted in Makete and Mufindi districts between July 2014 and December 2015 using semi-structured questionnaires followed by two focus group discussions to verify respondents' information. Cytotoxicity study was conducted on extracts of collected plants using brine shrimp lethality test and analyzed by MS Excel 2013 program.
RESULTS: During this survey about 46 plant species belonging to 28 families of angiosperms were reported to be traditionally useful in managing fungal and other health conditions. Among these, Terminalia sericea, Aloe nutii, Aloe lateritia, Zanthoxylum chalybeum, Zanthoxylum deremense, and Kigelia africana were frequently mentioned to be used for managing fungal infections. The preparation of these herbals was mostly by boiling plant parts especially the leaves and roots. Cytotoxicity study revealed that most of the plants tested were nontoxic with LC50 > 100 which implies that most compounds from these plants are safe for therapeutic use. The dichloromethane extract of Croton macrostachyus recorded the highest with LC50 value 12.94 µg/ml. The ethnobotanical survey correlated well with documented literature from elsewhere about the bioactivity of most plants.
CONCLUSIONS: The ethnobotanical survey has revealed that traditional healers are rich of knowledge to build on for therapeutic studies. Most of the plants are safe for use; and thus can be considered for further studies on drug discovery.

INTRODUCTION

The history of mankind has continuously remained interlocked to the surrounding environment. The first civilizations realized that there were plants with healing potential. The value of plants has a long history in saving human beings cutting across different cultures in the world [1]. Utilization of medicinal plants by individuals lies on the knowledge accumulated through the interaction of people with the environment and the diffusion of information, traditionally transmitted orally through subsequent generations [2]. In the contemporary world of conventional medicine, the practice of herbal medicine has attracted more attention and is becoming accepted globally [3]. Traditional medicine is not well documented in most African societies [4]. However, the practices and resources have been orally transferred from one generation to another thus limiting its reliability.

Documenting the indigenous knowledge through ethnobotanical studies is important for sustainable utilization of medicinal plants in drug discovery. Several active compounds have been discovered from plants based on ethnobotanical information, some used directly as therapeutic drugs [3]. Therefore, the focus of the study was to collect and document information on the use of antifungal medicinal plants and their therapeutic practices among the Hehe and Kinga tribe in Southern Highland of Tanzania. The information could further help scientific research in drug development.

MATERIALS AND METHODS

Study Area

The study was conducted in Mufindi District found in Iringa Region and Makete District based in Njombe Region. Makete District is one of the six districts of Iringa Region and is located in the Southern Highlands of Tanzania about 115 km from the regional headquarters (Figure 1). It is situated within 9°15’0” S 34°10’0” E [5]. Mufindi district on the other hand lies between 08°35’40’’S 035°17’20’’E. Both districts are dominated by Hehe, Kinga and Bena ethnic tribes. Furthermore, these districts experience high levels of migration and mobility (61.4%) caused by seasonal workers to numerous plantations in the areas and being a logistical hub for transport infrastructural facilities by road and railway (Tanzania-Zambia route) [6]. These unique dynamics increase the risk for HIV transmission in the communities. Most of the livelihoods are from agriculture which is the major source of subsistence, occupying about 80 % of the households in the districts [5]. Other activities include livestock keeping, timber production, and petty businesses at small scale. Most household members are thus compelled to engage in multiple jobs and activities to make a living [5].

Figure 1

Map of Tanzania showing the study areas (Mufindi and Makete Districts) Ethnobotanical survey

During the ethnobotanical survey that was done between July 2014 and September 2015 semi-structured questionnaire was used as data collection tools to interview traditional health practitioners, elders and selected villagers who have knowledge on medicinal plants. This study employed a purposive sampling, in which selection of respondents do only focus to people who are considered by the community as having exceptional knowledge about the use of plants such as traditional healers, herbalists and elders. The questionnaire aimed to collect and document ethnobotanical information of plants that are used to treat various infections including fungal infections. Documentation of plants, parts used and their preparations whenever possible was done. Focus group discussion was employed to validate information collected using questionnaire method.

Collection of Plant Materials

Identification of plant species was done by the botanist from the Department of Botany, University of Dar es Salaam, Tanzania, and all voucher specimens were deposited at the Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences. Collection of the identified plants was aided by the traditional health practitioners and elders. Decision on which plant and/or part of plant to be collected for further studies was mainly influenced by the information given by respondents in the field validated first by focus group discussion and by literature.

Reagents

Absolute ethanol, dichloromethane, and petroleum ether were purchased from Fluka Chemie GmbH (Sigma-Aldrich®, Zwijndrecht, Netherlands), dimethyl sulfoxide (DMSO) was purchased from Sigma® (Poole, Dorset, UK) while sea salt was prepared locally by evaporating water collected from the Indian Ocean, along the Dar es Salaam Coast.

Extraction and Concentration

Plant materials from the field were cut into small pieces, air-dried and ground using a machine grinder consequently soaked, sequentially using petroleum ether, dichloromethane, and ethanol for 48 h for each solvent. The method of percolation was employed during extraction process. The crude extracts were obtained by concentrating the filtrate in vacuo using a rotary evaporator with the bath temperature maintained at 40°C. The crude extract obtained was placed in the refrigerator for few hours and then subjected to freeze drier to remove solvent that could have remained.

Brine Shrimp Lethality Test

The brine shrimp lethality assay was used as an indication for bioactivity of different tested plant extracts as well as investigation for toxicity [7,8]. Artificial seawater was prepared by dissolving 3.8 g of sea salt in 1 L of distilled water. Brine shrimp eggs (2 g) were added and left for 24 h to hatch in light condition. Stock solutions (40 mg/mL) of all extracts were dissolved in DMSO. Different levels of concentrations (240, 120, 80, 40, 24, 8, 4.5, 3, 1.5 and 1 µg/ml) were prepared by drawing different volumes from the stock solutions and then added into vials, each containing ten brine shrimps larvae. The volume was adjusted with the prepared artificial seawater. Each level of concentration was tested in duplicate. The negative control contained brine shrimp, artificial seawater and DMSO (0.6%) only. The vials were incubated under light for 24 h. The dead larvae were counted and mean percentage mortality calculated.

Data Analysis

The mean percentage mortality was plotted against the logarithm of concentrations and the concentration killing 50% of the larvae (LC50) were determined from the graph using Microsoft Excel 2013 computer software. Regression equation obtained enabled calculation of lethal concentrations, i.e., LC50, LC16, and LC84. The 95% confidence interval was then calculated using method reported by Litchfield and Wilcoxon [9]. The results were used to document safety and cytotoxicity activity of plant extracts.

RESULTS

Ethnobotanical Survey

During the ethnobotanical survey, a total of 40 respondents (traditional healers, herbalists, and elders) were interviewed from the selected regions. 5 different villages in Njombe and Iringa regions were visited for the survey including three villages; Tambalang’ombe, Mayale, Kingege, and Ifwagi from Mufindi, Iringa region as well as Lupalilo and Maliwa villages of Makete district in Njombe region. These villages were chosen based on the information of registered or known traditional health practitioners obtained from the District Medical offices.

A total of 46 plant species used by the Hehe, Bena and Kinga tribe for the treatment of various microbial related ailments were documented [Table 1]. The plants represent about 28 families with the most prominent families being Euphorbiaceae (6 species), Combretaceae, and Rubiaceae (4 species each) and followed by Rutaceae, and Fabaceae (each with 3 species). Most of the ethnobotanical information were related to fungal infections since the study focused on documenting plants that were used in managing fungal infections among these ethnic groups. Out of 46 reported plant species, 14 (32%) had similar cited antifungal activity while 8 (18%) of plant species traditionally used for managing other nonfungal infections in Mufindi and Makete districts were reported by the literature to have antifungal activity [Table 1].

Table 1

List of medicinal plants reported for managing various diseases in Iringa and Njombe regions

Brine Shrimp Lethality Assay

The brine shrimp test is used as a preliminary test for testing toxicity of a plant and anticancer activity after a single dose administration. In this study, the LC50 values were clustered per Moshi et al., [95]. The LC50 of <1.0 µg/ml is considered highly toxic; LC50 1.0-10.0 µg/ml is toxic; LC50 10.0-30.0 µg/ml - moderately toxic; LC50 > 30 < 100 µg/ml - mildly toxic and LC50 > 100 µg/ml as nontoxic. Studies done by Moshi et al., [96,97] provided the evidence that plant extract with the LC50 <20 µg/ml could be a source for anticancer compounds. The results from this study revealed that most (77.1%) of the plants tested were nontoxic with LC50 value <100 [Table 2]. The present findings imply that most compounds from these plants were safe for therapeutic use. Among the tested plant extracts dichloromethane extract of Croton macrostachyus had moderate toxicity with LC50 value 12.94 µg/ml.

Table 2

Brine shrimp toxicity results of medicinal plants used in Southern Highland regions

DISCUSSION

Plant-based traditional medicine system continues to play an essential role in primary health care for the wider communities irrespective of the locality. This work has revealed the potential herbal medicines used in managing fungal infection in Njombe and Iringa Regions which are leading in spread of HIV infection in Tanzania with about 14.8% and 9.1% HIV prevalence, respectively [6,94]. Association of opportunistic fungal infections and HIV have been reported from the early days of the HIV/AIDS pandemic in Tanzania and worldwide [98]. The majority of the people living with HIV/AIDS are susceptible to fungal and bacterial opportunistic infections due to immunity suppression [37]. Availability of fungal herbal medicines may subsidize the effect of antifungal drugs resistance and availability to patients due to recurring fungal infections. The findings showed that remedies used in these communities consisted of one or a combination of two or more plant species. According to the traditional health practitioners, combinations of different plant species increases the efficiency of medicine and improves the cure’s power which could be due synergistic effects in treatment of various diseases. Most of plant species collected have been documented to be used in different African communities for the treatment of skin diseases [12]. Furthermore, the study noted that there was a wide use of the leaf part which could be considered as a good sign for the conservation of the environment and ensures sustainable utilization of plants.

Among the frequently mentioned plants, included Terminalia sericea, Aloe nutii, Aloe lateritia, Zanthoxylum chalybeum, Zanthoxylum deremense, and Kigelia africana. The claims on these plants have a special merit as they are also recorded in the literature to be useful in managing various microbial infections. Pharmacological studies by several authors have demonstrated the potency of the mentioned plants in terms of antifungal activity [12,16,21,27,30,81,99,100]. However, the proportion of claims made by traditional health practitioners in Makete and Mufindi districts concerning some of the plants documented in this study and which are supported by literature evidence of proven biological activity or similar ethnobotanical uses elsewhere is remarkable. The results also confirmed the supportive role of traditional health practitioners in offering health-care services to local communities in addition to available conventional medical cares.

Apart from efficacy, safety of herbal medicines is of paramount importance as little is documented about many plants that are used in traditional medicine. Findings from various studies have recommended brine shrimp assay as one of the methods for preliminary investigations of toxicity. This assay is also used in screening bioactive compounds from medicinal plants popularly used for several purposes and for monitoring the isolation of such biologically active compounds [101-103]. This work present few results from plant extracts that were tested for toxicity against brine shrimps. However, not all collected plant samples were screened for toxicity since during extraction yield was very little or none for some samples to be used for the testing. Findings obtained in this study showed that 77.1% of plant tested to be nontoxic supporting the popular use of medicinal plants by communities since they are regarded as safe therapeutic agents. Unlike other plants, C. macrostachyus exhibited high toxicity level that suggests its potential for anticancer agents. The LC50 of C. macrostachyus (12.94 µg/ml) is not statistically different to the standard anticancer drug cyclophosphamide (16.3 µg/ml). Other similar study undertaken on stem barks of this plant to evaluate cytotoxicity and acute toxicity in mice demonstrated the toxicity of the plant resulting in mortality of tested organisms [104]. The genus Croton has been reported to demonstrate moderate to high toxicities with proven the anticancer activity [51]. This knowledge triggers the use of plant products as complementary and alternative therapies both as direct and adjuvant remedy. A growing body of literature suggests the cancer preventive and therapeutic potential of phytochemicals and a lot of research has focused on the cellular mechanisms by which these phytochemicals interfere with the carcinogenic process. With the ability to target a variety of signaling pathways, phytochemicals are considered to be promising therapeutic agents against tumors with limited toxicity to normal cells.

CONCLUSION

The ethnobotanical survey has revealed that traditional health practitioners are rich in knowledge of fungal medicinal plants in these areas. These plants though have received little attention from modern biomedical research could be a promising source of knowledge for the discovery of useful remedies if this wealth is preserved through proper documentation and research. Most of the plants collected were ascertained to be safe for use and hence could be considered for further scientific studies. The reported species may be used for the development of new, affordable, and effective herbal formulations for antifungal health-care management or used in drug discovery.