Screening of Plants Used as Traditional Anticancer Remedies in Mkuranga and Same Districts, Tanzania, Using Brine Shrimp Toxicity Bioassay

Background Inadequate specialized cancer hospitals and high costs are contributing factors that delay cancer patients from accessing health care services in Tanzania. Consequently, majority of patients are first seen by Traditional Health Practitioners (THPs) before they access specialized services. This study presents ethnomedical information and preliminary evaluation of 25 plant species claimed by THPs in Mkuranga and Same districts of Tanzania on use for treatment of cancer. Literature search and laboratory investigation results are presented to support evaluation. Methods. This study was a single disease ethnomedical enquiry focusing on plants being used for cancer treatment Face-to-face interviews and questionnaires were administered to eight (8) THPs in Mkuranga and Same districts on the claimed plants and their use for management of cancer. Plants were selected based on being frequently mentioned and emphasis given by THPs. Literature search and brine shrimp toxicity (BST) of methanol : dichloromethane (1:1) extracts was used as surrogates to evaluate strength of the claims. Results This study reports 25 plant species used by the THPs in two districts of Tanzania. Eight plants (32%) have been reported in the literature to have activity against cancer cells. BST results revealed, 14 (56%) plants exhibited high toxicity against brine shrimps. The most active plants included Croton pseudopulchellus Pax (LC50 4.2 μg/ml), Dalbergia melanoxylon Guill. & Perr. (LC50 6.8 μg/ml), Loranthus micranthus Linn (LC50 4.0 μg/ml), Ochna mossambicensis Klotzsch (LC50 3.3 μg/ml), and Spirostachys africana Sond. (LC50 4.4 μg/ml); their toxicity was comparable to that of Catharanthus roseus (L) G. Don. (LC50 6.7 μg/ml), an established source of anticancer compounds. Nine other plants had LC50 values between (19.8 and 71.6) μg/ml, indicating also potential to yield anticancer. Conclusion Literature search and BST results provide a strong support of the potential of the claimed plants to yield active anticancer compounds.


Introduction
Cancer is a major public health problem affecting both developing and developed nations, but whereas 50% of cancer patients in developed countries die of the disease, in developing countries 80% of cancer victims are diagnosed with late-stage incurable tumors [1]. In the same vain while the incidence of cancer in Africa is about one-third that of North America, the death rate is nearly the same, thus further illustrating the cancer treatment challenges that African countries face and the need to develop solutions for addressing this challenge.
According to the Foundation for Cancer Care, an estimated 30,000 new cancer cases are diagnosed each year, in Tanzania, but there is only one specialized cancer treatment center to serve the entire population of nearly 55 million people, with a capacity to see only 5000 patients in a year. Hence, majority of patients access treatment too late, when 2 Evidence-Based Complementary and Alternative Medicine their cancers have already advanced and are at stages for which the only remaining option is palliative care [2,3]. Lack of cancer education, inadequate screening services, and detection methods contribute to late diagnosis and access to treatment [3,4]. The Ocean Road Cancer Institute, situated on the eastern coast of the country, in Dar es Salaam, is the only specialized cancer treatment hospital for the whole country; hence patients have to incur high transport costs to reach the center, and this, combined with the high costs of anticancer drugs, leaves the majority of patients with no option, but to depend on Traditional Health Practitioners (THPs) to care for them.
Anecdotes abound of successful treatment of cancers by THPs in Tanzania, but only a few of the plants that are used have been evaluated for efficacy using cancer cell lines [5][6][7] and some have only been evaluated using proxy assays such as the brine shrimp lethality test [8][9][10][11][12]. Two studies on Tanzanian plants are particularly inspiring to efforts to screen Tanzanian plants for anticancer activity [13,14]. In one study, aguiaine sesquiterpene, Englerin A, was isolated from a Tanzanian medicinal plant, Phyllanthus engleri Pax (Euphorbiaceae), which has been demonstrated to have selective activity against kidney cancer cells [13]. More recently isoflavone derivatives, rhynchoviscin, genistein, sophoraisoflavone, licoisoflavone A, and 3'O-methylorobol with antiangiogenic activity and hence potential for cancer treatment, were isolated from another Tanzanian plant, Rhynchosia viscosa (Roth) DC that is used in traditional medicine [14].
We report in this study documentation of 25 plant species that are used by THPs in Mkuranga district (Coast Region) and Same district (Kilimanjaro Region) of Tanzania, for treatment of cancer. We also used information from the literature and results of their toxicity profile against brine shrimp larvae (Artemia salina) as surrogates for interrogating their potential as sources of anticancer compounds.

Materials and Methods
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e Study Area. The study was done in Mkuranga district (Coast region) and Same district (Kilimanjaro region) located on the eastern coast and north-eastern parts of Tanzania, respectively. Selection of the two areas was based on information from patients who visited Traditional Health Practitioners in the two districts to seek treatment for cancer as well as other informants and local government leaders. There is no documented evidence of successfully treated patients but there are famous THPs who are frequently being consulted by cancer patients for their remedies in these two areas.
. . Documentation of Ethnomedical Information and Selection of Plants for Testing. Physical visits were made to the two areas and meetings convened with Traditional Health Practitioners who were interviewed orally and, then, requested to fill questionnaires concerning their knowledge on treatment of cancer. Information documented included age, how they became experts in cancer treatment, how they identify cancer patients, the medicinal plants used, and methods of preparation. Traditional healers who seemed knowledgeable and claimed to treat cancer patients were selected for further discussion.
. . Selection and Collection of Plants for Biological Testing. Selection of plants for documentation was based on the number of practitioners who used the same plants for treatment of cancer. Plant materials were collected, labelled, and kept away from direct sunlight to minimize metabolic degradation and the effects of ultra violet light. In all cases, a botanist from the Botany Department, University of Dar es Salaam, assisted in the identification and preparation of voucher specimens for plants of interest. Collected plant materials were dried in open air at room temperature and away from direct sunlight.
. . Information from the Literature. Literature search was done to determine if any of the plants was being used somewhere else for treatment of cancer or had been screened for anticancer activity. The information obtained is reported as information that supports the Traditional Health Practitioners' claims, but where there is no information the interpretation was taken as an indication for need of studies to establish proof having anticancer activity.
. . Brine Shrimp Toxicity Testing Materials. Brine shrimp eggs were obtained from Aquaculture Innovations (Grahams Town, South Africa). Solvents and other chemical reagents used in this study (with their sources) included Dimethyl sulphoxide (DMSO; Sigma: Poole, Dorset, UK); methanol, ethanol, dichloromethane, petroleum ether, and ethyl acetate (CARLO ERBA, Van de Reut, France); sulphuric acid, sodium bicarbonate, sodium hydroxide, hydrogen peroxide, and vanillin (Fisher Scientific Ltd., Leicestershire, UK). Sea salt was prepared by evaporation of water collected from the Indian Ocean along the Dar es Salaam coast.
. . Extraction of Plant Materials. Dried plant materials were ground using a milling machine and then 100g of each powdered plant material was extracted by maceration using methanol: dichloromethane (1:1), for about 24 hours, after which the extract was decanted and filtered. The extracts were concentrated using a rotary evaporator (Heldolph instruments Gmbh, Walpersdorfer, Germany) at 40 ∘ C, under reduced pressure, after which they were dried further with a freeze-drier (Edwards, BOC Ltd., Crawley Sussex, England), to remove any traces of water. The dried crude extracts were kept in vials in a freezer at -20 ∘ C until when needed for biological tests.
. . Evaluation for Brine Shrimp Toxicity. Stock solutions (40 mg/ml) of extracts were prepared in DMSO, diluted into varying concentrations. A solution of DMSO (0.6%) in artificial sea water was used as a negative control, while a methanol: dichloromethane (1:1) leaf extract of Catharanthus roseus (Apocynaceae), a plant with known anticancer activity, was used as a positive control. Ten brine shrimp larvae were introduced into each vial containing 5mls of Evidence-Based Complementary and Alternative Medicine 3 the test solution or control; after 24 hours the nauplii were examined against a lighted background and the number of live larvae counted. The mean percentage mortality was plotted against the logarithm of concentrations using the Fig  P computer program (Biosoft Inc., USA), which also gives regression equations. The regression equations were used to calculate LC 16 , LC 50 , LC 84 , and 95% confidence intervals (95%CI).

Results
. . Ethnomedical Information and Proof of Concept from the Literature. Table 1 presents a list of 25 plant species that are used by Traditional Health Practitioners (THPs), in Mkuranga and Same districts, for the treatment of cancer. Among the listed plant species 22 are used in Same district, two are used in Mkuranga, and one is used in both districts. The plant species belong to 17 plant families, with 1-4 plant species, being from each family. The family Fabaceae was represented by more plant species (4) than the other families, followed by Bignoniaceae, Ebenaceae, and Malvaceae, each of which was represented by 2 plant species and one species for each of the remaining families. Information from the literature [10, indicates that 8 of the listed plants including, Carissa spinarum L. [15,16], Markhamia obtusifolia (Baker) Sprague [17,18], Kigelia africana (Lam) Benth. [19][20][21][22][23], Diospyros zombensis (B.L Burtt) F. White [30], Euclea natalensis A.DC. [31], Acacia nilotica (L) Delile [34,35], Cassia abbreviata Oliv. [5,37], and Ochna mossambicensis [44,45] have confirmed activity against one or more cancer cell lines.

Discussion
The purpose of this study was to seek for proof of concept supporting claims by Traditional Health Practitioners (THPs) in Mkuranga and Same districts of Coast and Kilimanjaro regions of Tanzania, respectively, for the treatment of cancer. Medicinal plants such as Cordia africana, Croton pseudopulchellus, Mystroxylon aethiopicum, Spirostachys africana, Trichodesma zeylanicum, and Zanthoxylum chalybeum were mentioned to be used for a given type of cancer by more than one practitioner, despite their different locations. Information from the literature and the brine shrimp lethality test were used as the basis for this enquiry. Information from the literature shows that eight out of the 25 plants that are used by Traditional Health Practitioners (THPs) in Mkuranga and Same districts, for treatment of cancer are substantiated to have activity against various cancer cell lines [15-23, 27-32, 35, 38, 43, 45]. These include cytotoxic or antiproliferative activity against the leukaemia HL-60 cell line [15], breast and lung cancer cell lines [16], and hepatocellular carcinoma [38]. The other substantive evidence that corroborates the THPs claims is the isolation of compounds with cytotoxic activity, such as (-)-carinol, (-)-carissanol, and (-)-nortrachelogenin from Carissa spinarum [16], 7-methyljuglone from Diospyros zombensis [30] and Euclea natalensis [31], and two cytotoxic bioflavonoids from Ochna mossambicensis [45]. Cytotoxic compounds have also been isolated from Kigelia africana of which furanonaphthoquinones were the most active against human breast cancer cell lines [19]. Some of the plants have exhibited antioxidant activity such as Blighia unijugata [48], Securidaca longipedunculata [46], and Loranthus micranthus [40]. Loranthus micranthus contains polyphenols which exhibited antioxidant [40] and immunomodulatory activities which may play a role in the treatment of cancer [41]. Reports from the literature also suggest that some of the plants being used are potentially toxic, such as Senecio deltoideus, belonging to the genus Senecio that is known to contain the hepatotoxic pyrrolizidine alkaloids [8]. In another scenario, while the claims on Mystroxylon aethiopicum are supported by a confirmation that used alone, the plant exhibited low cytotoxic activity against three cancer cell lines [7]; the Traditional Health Practitioners mix it with Senecio deltoideus, thus making this combination potentially toxic due to the presence of pyrrolizidine alkaloids [27]. Another school of thought may purport that such mixing is intended to enhance activity and minimize toxicity, but certainly this remains a subject for further studies.
In our previous studies we have shown that brine shrimp toxicity results frequently predict presence of cytotoxic activity against cancer cell lines [9,10,12]. Thus, from the brine shrimp results Catharanthus roseus, which is a known plant with anticancer activity [49], gave a LC 50 of 6.7 g/ml in support of these previous observations [9,10,12]. Therefore the brine shrimp results of other plants which are likely to have anticancer activity are those with LC 50 values below 100 g/ml. These include extracts of Baphia kirkii (LC 50 71.6 g/ml), Boswellia neglecta (LC 50 27.8 g/ml), Cordia africana (LC 50 19.8 g/ml), Dalbergia melanoxylon (LC 50 6.8 g/ml), Diospyros zombensis (LC 50 67.2 g/ml), Euclea  [15]. In another study the lignans (-)-carinol, (-)-carissanol and (-) nortrachelogenin isolated from the stem exhibited cytotoxic activity against breast (MCF7) and lung (A549) cancer cells [16].   (1) R, L Decoction from boiled root bark is drunk; a tea cup is taken once daily. Trichodesma zeylanicum was reported to be non-cytotoxic to cancer cell lines [24] but in another study it was shown to be a potential source of antioxidants [25].   T B r e a s t c a n c e r ( 1 ) S B , R Paste of grounded dry stem is applied externally on a swelling.

Bignoniaceae/Markhamia
Fresh leaves of Dalbergia melanoxylon are used topically by the Nyamwezi of Tabora, Tanzania, for treatment of cancer [10]. An ethanol extract of the leaves exhibited cytotoxic activity to brine shrimps with an IC 50 of 8.64 g/ml [36]. 10 Evidence-Based Complementary and Alternative Medicine   [33], Euclea natalensis [31], and Ochna mossambicensis [44]. For the other plants with LC 50 values above 100 g/ml, if at all they have anticancer activity, this may probably be ascribed to other mechanisms such as arrest of the cell cycle and induction of apoptosis as reported for Acacia nilotica [34] or inhibition of angiogenesis. In the absence of more evidence the other plants remain to be subject for further research to determine their efficacy.

Conclusion
Reports from the literature and brine shrimp lethality test have provided preliminary information for evaluation of claims from some Traditional Health Practitioners of Mkuranga and Same districts in Tanzania, for use of some medicinal plants in the management and treatment of cancer.
14 Evidence-Based Complementary and Alternative Medicine These results make a strong case for continuing research on most of the plants that are used by THPs in these two districts to explore possibility of isolating novel anticancer compounds.

Data Availability
Materials

Consent
All eight ( ) traditional practitioners interviewed in this study provided informed consent to take part by reading, understanding, and signing a consent form prepared by the Institute of Traditional Medicine of the Muhimbili University of Health and Allied Sciences (ITM-MUHAS).

Disclosure
All participants were conversant with the Kiswahili language and hence Kiswahili version of the consent form was used.

Conflicts of Interest
The authors declare that they have no conflicts of interest.