Toxicology of medicinal plants and combinations used in rural northern KwaZulu-Natal (South Africa) for the treatment of hypertension

Toxicology of medicinal plants and combinations used in rural northern KwaZulu-Natal (South Africa) for the treatment of hypertension

Accepted Manuscript Title: Toxicology of medicinal plants and combinations used in rural northern KwaZulu-Natal (South Africa) for the treatment of hy...

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Accepted Manuscript Title: Toxicology of medicinal plants and combinations used in rural northern KwaZulu-Natal (South Africa) for the treatment of hypertension Authors: Mmbulaheni Ramulondi, Helene de Wet, Sandy van Vuuren PII: DOI: Reference:

S2210-8033(18)30061-7 https://doi.org/10.1016/j.hermed.2018.12.001 HERMED 251

To appear in: Received date: Revised date: Accepted date:

22 March 2017 11 May 2018 4 December 2018

Please cite this article as: Ramulondi M, de Wet H, van Vuuren S, Toxicology of medicinal plants and combinations used in rural northern KwaZulu-Natal (South Africa) for the treatment of hypertension, Journal of Herbal Medicine (2018), https://doi.org/10.1016/j.hermed.2018.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Toxicology of medicinal plants and combinations used in rural northern KwaZulu-

Mmbulaheni Ramulondi a, Helene de Wet a*, Sandy van Vuuren b a

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Natal (South Africa) for the treatment of hypertension

Department of Botany, University of Zululand, Private Bag 1001, KwaDlangezwa 3886,

b

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South Africa

Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of

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Witwatersrand, 7 York Road, Parktown, 2193, South Africa

The most frequently used anti-hypertension plants in northern KwaZulu-Natal

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Highlights



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were found to be mostly non-toxic.

Five of the medicinal plants (Citrullus lanatus, Cladostemon kirkii, Hyphaene

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coriacea, Pyrenacantha kaurabassana and Strychnos madagascariensis) investigated in the current study have not been previously evaluated for any toxicity

Toxicity was evident for the organic extract of P. kaurabassana (roots) in both

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the BSLA and in the Ames test at all concentrations tested. Results of both assays (BSLA and Ames test) demonstrated that in general

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lower toxicity was observed for the aqueous extracts rather than the organic



Synergistic interactions (never been evaluated previously) were observed

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extracts.

between plant combinations, however, some of the plant combinations resulted in increased toxicity (antagonistic interactions).

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Abstract: Ethnobotanical surveys showed that medicinal plants play a major role in the health-care of people residing in the rural areas of northern KwaZulu-Natal (South Africa). Because of the long-term usage, these medicinal plants are often assumed to be safe. The aim of this study was to evaluate the safety of 26 medicinal plants and 19 plant combinations which are routinely used for the treatment of hypertension in this rural area. Five of the medicinal plants (Citrullus lanatus, Cladostemon kirkii, Hyphaene coriacea, Pyrenacantha kaurabassana and Strychnos

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madagascariensis) investigated in the current study have not been previously

evaluated for any toxicity. Two extracts [organic (dichloromethane:methanol) and

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aqueous] were assessed using the brine shrimp assay (BSLA) and the Ames test.

The results showed that for the organic extracts, 17 plant extracts tested were toxic in the BSLA while six plant extracts were toxic in the Ames test. Extracts tested in various concentrations demonstrated that toxicity was dosage dependent i.e. as the

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concentration increased, mortality percentage increased. Results of both assays

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(BSLA and Ames test) demonstrated that in general lower toxicity was observed for

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the aqueous extracts as only Catharanthus roseus (roots), Citrus limon (peel) and Ozoroa engleri (roots) showed toxicity. Synergistic interactions were observed

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between plant combinations; however, some of the plant combinations resulted in increased toxicity (antagonistic interactions). This study provides some insight into

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the possible toxicity implications of medicinal plants routinely used in rural northern

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Keywords

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KwaZulu-Nat.

Anti-hypertension plants, Medicinal plant combinations, Cytotoxicity, Ames test,

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Brine shrimp assay, KwaZulu-Natal, South Africa.

1. Introduction

According to the World Health Organisation (WHO, 2003), medicinal plant usage, although having beneficial effects, may exhibit undesired harmful effects and knowledge of adverse effects is greatly needed. Even for commercialized herbal products worldwide, less than 10% have standardised active components (Obidike 2

and Salawu, 2013). Although the WHO has emphasized in 2004 that research must focus on the potential therapeutic and toxic effects of herbal medicine, little has been done to validate the safety of medicinal plants. Several studies across the world have acknowledged the importance of herbal remedies and recognised the significance of determining plant safety. These studies have been undertaken in countries such as South Africa (Ndhlala et al., 2013), Nigeria (Usman et al., 2014), Kenya (Kirira et al., 2006), Cameroon (Assob et al., 2011), Mexico (Deciga-Campos

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et al., 2007), Brazil (De sa Ferraira and Ferrao Vargas, 1999), Peru (Bussman et al., 2011), India (Singh and Singh, 2012) and Australia (Kassie et al., 1996).

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Potential toxicity of medicinal plants is not a new concept. It is well known that

some medicinal plants must be used with caution since they might cause different toxicity profiles (acute toxicity, cytotoxicity, cardiotoxicity, genotoxicity, hepatotoxicity among others) (Bnouham et al., 2006). One of the problems of using medicinal

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plants in a traditional and rural settings is that no definite dose is prescribed,

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particularly for medicinal plants which are used by lay people as documented in

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previous studies carried out in northern Maputaland (York et al., 2011; De Wet et al., 2016). This may result in an overdose (Usman et al., 2014). Acute toxicity as a result

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of taking herbal medicine in South Africa is not uncommon. It has been observed that due to inadequate records, mortality rates have been estimated to be

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approximately 80 000 per annum (Meel, 2007). However, the number is probably higher, as not every death due to toxicity is reported. In rural areas few cases of

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human poisoning are documented as patients either do not arrive at the hospital in time (distance from homestead) or the plant is not identified (Botha and Penrith, 2008). It is important to note that the estimated mortality rate does not specify

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whether medicinal plants are used singularly or in combination with conventional drugs. This is not surprising in South Africa as this country is rich in its plant diversity which includes a high variety of medicinal plants usage (3000 plant species) with

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very little data on toxicity. Toxicity of medicinal plants may also depend on various factors such as the quantity consumed, duration of treatment, plant part used, as well as individual body chemistry. Studies in these areas are thus warranted (Mounanga et al., 2015). In Africa and more specifically South Africa, individuals still prefer to use medicinal plants due to their cultural beliefs, ease of availability, and the belief that the use of medicinal plants promote healthy living. The belief that medicinal plants are safe to 3

use together with the high cost of allopathic medicine and concurrent shortage of drugs in available clinics and hospitals further supports the preference for medicinal plant use (Tagwireyi et al., 2002; Lanini et al., 2012; Ekor, 2013; Ngarivhume et al., 2015). Although clinics are provided by the government and are free of charge in South Africa, several studies have demonstrated that medicinal plant use still plays a major role in the treatment of various diseases in rural areas such as northern KwaZulu-Natal (De Wet et al., 2016). Some known South African medicinal plants

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tested positive for various toxicities include Callilepis laureola D.C Asteraceae

(nephrotoxicity) (Wainwright et al., 1977), Catha edulis (Vahl.) Endl. Celastraceae

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(nephrotoxicity) (Al-Mamary et al., 2002), Catharanthus roseus (L.) g. Don

Apocynaceae (mutagenicity) (Elgorashi et al., 2003), Hippobromus pauciflorus (L.F) Sapindaceae (hepatotoxicity) (Pendota et al., 2010) and Senecio latifolius D.C Asteraceace (carcinogenicity) (Steenkamp et al., 2001) to mention a few.

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The usage of plant combinations to treat various conditions is not a new concept.

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Plant combinations have been used for centuries (Biavatti, 2009). Benefits include

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improved efficacy, reduced toxicity, reduced adverse effects, increased bioavailability and lower dosage concentrations (Cottarel and Wierzbowski, 2007;

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Inui et al., 2007). It has already been acknowledged in the literature that the lay people and healers do not only depend on a single plant extract but regularly

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combine several plant species for treatment in the belief that the efficacy may be enhanced (Van Vuuren and Viljoen, 2011). Combination therapy, although frequently

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used may also be dangerous when the interaction is antagonistic, since it may result in higher toxicity (Meletiadis et al., 2010). Several studies have been conducted across the world such as in the United States of America (Huang et al., 2011), China

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(Che et al., 2013), India (Parasuraman et al., 2014), Pakistan (Azmi et al., 2010), Tanzania (Haule et al., 2012), Ghana (Koffuor et al., 2011) as well as in South Africa (Zonyane et al., 2013) on the efficacy of plant combinations in other pharmacological

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assays, however little has been done scientifically to determine the safety of plants when used in combination. In a survey carried out by De Wet et al. (2016), 28 plant species and 19 plant combinations were documented for the treatment of hypertension in a rural area of northern KwaZulu-Natal. In brief, 100 people comprising 98 lay people and two traditional health practitioners were interviewed to gain insight into the medicinal plants used for the treatment of hypertension. Structured questionnaires were used 4

to obtain information about the plants which are used to treat hypertension, plant parts used, method(s) of preparation, dosage form and method of administration. On average, one handful of fresh plant material which is equivalent to 10 g was reported to be prepared in one litre of water as either maceration or decoction. A glass of decoction/maceration was then taken three times a day for the management of hypertension. Five of these medicinal plants (Citrullus lanatus, Cladostemon kirkii, Hyphaene coriacea, Pyrenacantha kaurabassana and Strychnos madagascariensis)

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have never been tested for any cytotoxicity. Only one plant combination (Hypoxis

hemerocallidea and Senecio serratuloides) has been evaluated for cytotoxicity and

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was found to be non- toxic (Naidoo et al. 2013). Hypertension requires ongoing

medication and some of the interviewees have been using these medicinal plants for as long as 20 years, which can cause accumulation of some toxicants in their systems. These accumulated toxicants can cause genetic interference with long term

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use and thus requires investigation to confirm safety. The aim of this study was to

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evaluate the potential cytotoxicity of 26 medicinal plants and 19 plant combinations

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used for the treatment of hypertension in rural northern KwaZulu-Natal (South Africa)

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2. Material and methods

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(De Wet et al., 2016).

2.1. Plant material and extraction process

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The collection of plant material occurred during February and March 2014 and details regarding traditional use and preparation of mixtures for traditional use are previously recorded (De Wet et al., 2016). Various plant parts were collected

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including leaves, roots, corm, flowers, fruits and the whole plant, depending on the traditional use. Voucher specimens were prepared and deposited in the herbarium of the Botany Department at the University of Zululand. Botanical identification was

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made by Dr THC Mostert and further confirmation of authenticity was undertaken by Mr Ngwenya from the South African National Biodiversity Institute, KwaZulu-Natal Herbarium. All the plants were collected in and around the homesteads with the permission of the homestead owners. None of the plants collected is on the red data species list as being rare or in danger of extinction (Raimondo et al., 2009). Only enough material was collected for the research project. The plant materials were left to dry at room temperature and thereafter chopped into smaller pieces and ground 5

into a fine powder using a Sciencetec RSA hammer mill. The grinder was rinsed thoroughly with water and alcohol between all samples ground to eliminate cross contamination of the plants. Two types of extracts (aqueous and organic) were prepared. 2.1.1. Organic extracts For the organic extraction, the ground material (10 g) was extracted twice with 200

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ml of a methanol and dichloromethane (1:1) mixture. Preparation of organic extracts was undertaken to ensure the extraction of both polar and non-polar compounds.

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The powdered material was left on a platform shaker for 24 h at 37°C, and thereafter filtered through 90 mm filter paper. The filtrate was left in open bottles in a fume hood for the complete evaporation of the solvent, leaving behind the solid extract.

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After the evaporation, the solid extracts were stored at 4°C until further analysis.

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2.1.2. Aqueous extracts

Aqueous extracts were extracted in a similar manner in which people from northern

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KwaZulu-Natal prepare their traditional remedies (De Wet et al., 2016). Two methods

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were considered, either a decoction where plant material was boiled in water or a maceration where plant material was soaked in water. Ten grams of dried plant

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material is approximately equivalent to one handful of fresh material. For the decoction, 10 g was boiled in 200 ml of distilled water for 30 min. For the maceration,

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10 g was soaked in 200 ml warm water. Thereafter, the extract was then left for 24 h in a platform shaker (Merck), filtered and stored at -80°C before lyophilization (Labcon). The freeze dried materials were then stored at room temperature and

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protected from light until further analysis. 2.2 Cytotoxicity studies

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2.2.1 Brine shrimp lethality assay Artificial sea water was prepared by dissolving 16 g of Tropic Marine® Sea Salt in 500 ml of deionised water. Brine shrimp (Artemia franciscana) eggs (Ocean Nutrition™) (0.5 g) were then added to the salt water. To ensure a high hatch rate, the salt water was aerated with a rotary pump (Kiho) and a constant source of light for warmth (220– 240 V). Brine shrimp eggs were incubated at 25°C for 18–24 h. After the incubation 6

period, the container was tilted for approximately 30 min. This was done so that the brine shrimps moved towards the light which increased the sample size when collecting. After 30 min, 48-well micro-titre plates were prepared by adding 400 μl salt water containing 40–60 live brine shrimps to each well together with 400 μl of the test sample (positive control, solvent control, negative control or plant extract). The negative toxic-free control was prepared by adding 16 g Tropic Marine® Sea salt in 500 ml of deionised water which mimics the natural environment for the brine shrimp

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to support growth and survival. The solvent control was DMSO, which was kept at

10%. The positive control consisted of 1.6 mg/ml potassium dichromate, which is

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known to be a highly toxic compound (Sigma-Aldrich). Dead brine-shrimp were

counted after 24 and 48 h by viewing plates under a light microscope (Olympus) at 40× magnification.

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After counting at 48 h a lethal dose of acetic acid (Saarchem; 100% (v/v); 50 μl) was added to each well, and after 30 min a final count was undertaken such as to calculate

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the percentage mortality (Cock and Kalt, 2010; Cock and Van Vuuren, 2013). A

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mortality percentage of 50% and above was considered to be toxic (Bussmann et al.,

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2011; Hubsch et al., 2014). Plant extracts (aqueous) which were toxic in the BSLA were further analysed by a dose response at the concentrations of 0.031, 0.063, 0.125,

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0.25, 0.5 and 1 mg/ml. The organic extracts were dissolved in dimethylsulfoxide (DMSO) and diluted with sterile water. DMSO is a widely used solvent to dissolve evaporated plant extracts since brine shrimp nauplii show no significant sensitivity to

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this solvent up to 11% concentration (Hadimi et al., 2014). Aqueous extracts were dissolved in sterile water. Samples that were tested singularly were prepared to a

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concentration of 2 mg/ml. Extracts that were tested in combination, were prepared in two concentrations (2 and 4 mg/ml). All results were analysed as the mean of experiments undertaken in triplicate where replicates were tested on separate days.

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2.2.2. Ames test

For mutagenicity, the standard plate incorporation method (Ames test) established by Ames et al. (1973) was used. Bacterial strains TA98 and TA100 were used because they identify the majority of the mutagens. Strain TA98 gives an indication of frame modification mutation, whereas strain TA100 indicates base pair substitution (Ghazali et al., 2011; Mashele and Fuku, 2011; Florinsiah et al., 2013; 7

Yasin and Ahmet, 2014). The aqueous plant extracts were prepared using distilled water. The organic plant extracts were made up in 10% DMSO. Plant extracts tested singularly were prepared at the concentration of 5 mg/ml and plant extracts tested in combinations were prepared at the concentration of 2.5 mg/ml each to make up a total of 5 mg/ml. Salmonella typhimurium bacterial strains TA98 and TA100 were used. Bacterial stocks were incubated in 10 ml of nutrient broth for 24 h at 37°C. For the preparation of minimum glucose plates, 20 ml of sterile 50 X Vogel-Bonner

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medium salts (VB salts) and 50 ml of sterile 40% glucose (prepared by dissolving 40 g of glucose in 100 ml of distilled water) was added to the agar and poured into the

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petri dishes. Vogel-Bonner medium salts (50 X VB salts) was prepared by dissolving

10 g of magnesium sulphate together with 100 g of citric acid and 500 g of potassium phosphate as well as 175 g of sodium ammonium phosphate in 670 ml of distilled water. The salts were added respectively and each salt was allowed to dissolve

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completely before adding another. The mixture was made up to 1000 ml using

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distilled water and sterilized before use.

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A phosphate buffer was prepared by dissolving one tablet (containing sodium

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phosphate, sodium chloride, potassium chloride and potassium phosphate) in 200 ml of distilled water to yield 0.01 M phosphate buffer, 0.027 M potassium chloride and

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0.137 M at the PH of 7.4. A histidine/biotin solution was also prepared. D-biotin (12 mg) was dissolved in 100 ml hot autoclaved water. When the solution had cooled down (50°C), L-histidine (10.5 mg) was also added and the solution was stored at

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4°C. The top agar was prepared by dissolving 6 g of agar and 5 g of sodium chloride. And thereafter 10 ml of histidine/biotin solution was added and kept molten until use.

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For the Ames test, 100 µl of the overnight culture (TA98 and TA100) was added together with 100 µl of the test sample [negative control, positive control (2 μg/ml) or plant extracts (5 mg/ml)] and 500 µl of phosphate buffer. This combination was then

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added to 2 ml of minimal top agar. This was then poured over the surface of minimum glucose agar plates. The plates were inverted and incubated at 37°C for 48 h. After 48 h, the colonies were counted and the reversion rate was compared to the colonies which were on the control plates. For the negative controls, 10% DMSO, and water were used and for the positive control, 4-nitroquinoline N-oxide (4NQO) and sodium azide were used and both were prepared to the concentration of 2 μg/ml. Strain TA98 detects base pair substitution mutagens while strain TA100 8

detects frameshift mutagens. In order for a test constituent (in this case plant extracts) to be classified as a mutagen, the number of revertant colonies on the plate containing the test extract should be double the number of colonies produced by the positive controls (Mashele and Fuku, 2011). All results were analysed as the mean of experiments undertaken in triplicate where replicates were tested on separate

2.2.3 Fractional inhibitory concentration (FIC) assessment

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days.

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The cytotoxicity of the plant combinations (two to four) were based specifically on traditional use and further assessed using the sum of the fractional inhibitory concentration (ΣFIC), which allowed the classification of the type of the cytotoxicity. This method is commonly used for the classification of antimicrobial combinations;

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however, it has been adapted in this study to classify the types of toxicity which

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occurs when different plants are combined. The interaction of cytotoxicity was

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defined as being considerably less toxic (in other words the extracts interact synergistically to produce a less toxic therapeutic outcome) if the ΣFIC index was ≤

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0.5. When the ΣFIC index was between 0.5 and 1.0, it was regarded as having reduced cytotoxicity (additive effects) and when the ΣFIC value was between 1.0

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and 4.0, the combination was regarded as not having any interactive effects (noninteractive) on cytotoxicity. When the ΣFIC value was above 4.0, the combination

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was considered as having increased cytotoxicity; in other words, an antagonistic response. The modified ΣFIC calculation was determined using the methods

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described by Van Vuuren and Viljoen (2011) as follows;

ΣFIC = FIC (i) + FIC (ii).

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FIC(i) =

FIC (ii) =

Cytotoxicity (a) in combination with (b) Cytotoxicity (a) in dependently Cytotoxicity (b) in combination with (a) Cytotoxicity (b) in dependently

Where (a) and (b) represent different plant samples. The equation was adapted to include more combinations where multiple plants were combined.

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3. Results and discussion 3.1.1. The Brine shrimp assay All the plant samples (organic and aqueous) were tested at 2 mg/ml (Table 1). Extracts were considered toxic if the mortality rate was 50% and more (Bussmann et al., 2011). The positive control (potassium dichromate) killed all the brine shrimps after 48 h of exposure. The negative control DMSO only killed 2% of the brine shrimp

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after 48 h while the sea salt water did not kill any brine shrimp. For the aqueous

extracts, two extracts were found to be toxic after 48 h, namely Catharanthus roseus

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and Citrus limon demonstrating mortality percent of 92% and 63% respectively. For the organic extracts, 63% of the extracts were toxic. Some of the extracts

demonstrated non- toxic effects at 24 h while toxicity was noted at 48 h. This could be because, mostly in stage one (up to 24 h), the larvae only consume their yolk as

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the main source of food and they are resistant to toxic compounds as a result of their

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poor developed epithelium of the digestive tract that disables the normal absorption of nutrients and toxic compounds from the external medium (Hamidi et al., 2014).

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Catharanthus roseus and Citrus limon were further analysed in varied concentrations

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and the dose responses are depicted in Fig. 1. The line which is demonstrated at 50% indicate the cut-off of the cytotoxicity mortality i.e. everything above the line is

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considered cytotoxic. These samples demonstrated increased cytotoxicity at concentrations higher than 0.25 mg/ml and cytotoxic at concentrations of 1 mg/ml.

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Twenty-one out of the 26 plants have previously been assessed for toxicity using various assays including in vivo assays. The five plants that have not been tested previously for any toxicity are Citrullus lanatus, Cladostemon kirkii, Hyphaene

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coriacea, Pyrenacantha kaurabassana and Strychnos madagascariensis. A summary of the medicinal plants that have been investigated for their cytotoxicity and are documented in Table 2 together with the plant part used and their known

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toxicity status. Although some of the potential toxicity of these medicinal plants has been reported in previous studies, it is worth noting that the reports are based on different assays, different solvents used for extraction and different plant part tested. Even though most of the plants in the current study have been subjected to some toxicity tests, the majority of the studies focused only on testing the organic extracts (De Wet et al., 2007; Sumathy et al., 2011; Kevin et al., 2012; Suad et al., 2012; Sanon et al., 2003) instead of the aqueous extracts which are more important to test 10

considering that they relate to traditional use. The plant parts previously tested differ mostly from the current plant parts tested. Some of the organic extract species tested in the current study (Acanthospermum hispidium, Altertisia delagoensis, Cannabis sativa, Carpobrotus dimidiatus, Hyphaene coriacea, Hypoxis hemerocallidea, Lippia javanica, Momordica balsamina, Psidium guajava, Ptaeroxylon obliquum Pyrenachantha kaurabassana, Sarcophyte sanguinea Strychnos madagascariensis and Tetradenia riparia) showed to be cytotoxic, yet

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demonstrating non-cytotoxicity with the aqueous extracts. The only two aqueous

extracts which were cytotoxic were Catharanthus roseus (roots) and Citrus limon

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(peel). Several studies have been conducted on the cytotoxicity of Catharanthus

roseus (leaves and twig) (Van de Venter et al., 2008; Kevin et al., 2012; El-Seedi et al., 2013). However, no literature could be found on the toxicity of the aqueous roots extracts of Catharanthus roseus. The ethanol peel extract of Citrus limon has been

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previously investigated for genotoxicity (Ali and Celik, 2007), however, the aqueous

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extracts of the peel have never been tested for any toxicity. The results demonstrate

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that the primary method of using water to prepare plant extracts by lay people is the safer option. Traditional methods of preparation are often aimed at eliminating toxins

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of the plants according to Van Wyk and Wink (2015). The results of the plants tested in different concentrations revealed that the degree of lethality was directly

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proportional to the concentration of the extracts. This effect has also been observed in other previous studies tested on other plant extracts at different concentrations

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(Olowa and Nuneza, 2013; Socorro et al., 2014; Asaduzzaman et al., 2015).

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3.1.2 Ames test

The mutagenicity properties of the plant extracts derived from 26 medicinal plants

are recorded in Table 1. For the aqueous extracts, two extracts (Catharanthus

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roseus and Ozoroa engleri) were found to be mutagenic. For the organic extracts, six plant extracts (Catharanthus roseus, Cannabis sativa, Ozoroa engleri, Pyrenacantha kaurabassana, Sarcophyte sanguinea and Strychnos madagascariensis) were found to be mutagenic towards two strains tested (TA98 and TA100). Thirteen of the 26 medicinal plants (50%) have been previously assessed for mutagenicity using various Salmonella strains. The strains include TA98, TA100, TA1535, TA1537 and TM677. The 13 plants that have not been investigated for mutagenicity are Albertisia 11

delagoensis, Citrullus lanatus, Citrus limon, Cladostemon kirkii, Hyphaene coriacea, Lippia javanica, Musa acuminata, Ozoroa engleri, Ptaeroxylon obliquum, Pyrenacantha kaurabassana, Sarcophyte sanguinea, Strychnos madagascariensis and Vangueria infausta. Among the plants that have never been subjected to mutagenicity testing, four plant species were found to be mutagenic. These results further emphasize the importance of screening all the medicinal plants that has not been subjected to mutagenicity testing especially the newly documented plants. In

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general, the frequency of the plants mostly taken for treating hypertension correlated with their non-mutagenic profile (Aloe marlothii, Hypoxis hemerocallidea, Momordica

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balsamina, Musa acuminata and Senecio serratuloides). The only two aqueous extracts which were mutagenic were Catharanthus roseus (roots) and Ozoroa

engleri (roots). Dichloromethane and methanol extracts of the leaf of Catharanthus roseus were reported to be mutagenic against strain TA98 and non-mutagenic

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against strain TA100 (Elgorashi et al., 2003). However, no data has previously been

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reported regarding the cytotoxicity of the aqueous roots extracts of Catharanthus

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roseus. Furthermore, to the best of our knowledge no evidence could be found in the literature regarding the mutagenicity of Ozoroa engleri. Thus it is important to

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continue screening the aqueous extracts of the plants since it is the common form in

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which lay people use traditional remedies.

Table 1

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Cytotoxicity of the individual plants expressed in percentage death of the brine shrimps as well as the mutagenicity results tested using Salmonella typhimurium bacterial strains, namely TA98 and TA100.

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Plant names and parts used

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Acanthospermum hispidium (roots)

Albertisia delagoensis (roots)

Aloe marlothii (leaves)

Extracts

BSLA (percentage Ames test mortality) (Strains CFU/ml) 24 h

48 h

TA98

TA100

Organic

67*

92

100

100

Aqueous

7

10

28

18

Organic

91

100

16

24

Aqueous

22

35

87

71

Organic

2

10

105

100

Aqueous

6

6

87

78 12

Citrus limon (peel)

Cladostemon kirkii (roots)

1000

1000

Aqueous

4

6

97

102

Organic

14

65

39

44

Aqueous

9

11

100

100

Organic

14

90

1000

1000

Aqueous

6

92

Organic

7

16

Aqueous

5

Organic

72

Aqueous

60

Organic

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Lippia javanica (leaves)

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Momordica balsamina (leaves)

Musa acuminata (flower bracts)

Ozoroa engleri (roots)

1000

36

41

27

13

100

52

63

63

35

44

0

30

200

200

4

4

200

200

Organic

54

67

200

200

Aqueous

4

44

200

200

Organic

29

54

102

117

Aqueous

4

5

100

105

Organic

46

52

100

200

Aqueous

6

8

200

200

Organic

34

50

109

99

Aqueous

10

39

78

71

Organic

0

4

35

44

Aqueous

5

6

98

84

Organic

28

58

1000

1000

Aqueous

1

7

1000

1000

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Hypoxis hemerocallidea (corm)

1000

6

Aqueous Hyphaene coriacea (roots)

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Citrullus lanatus (fruit)

100

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Catharanthus roseus (roots)

96

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Carpobrotus dimidiatus (leaves)

Organic

N

Cannabis sativa (leaves)

13

Pyrenacantha kaurabassana (roots)

Ricinus communis (leaves)

Sarcophyte sanguinea (stem)

7

72

84

Aqueous

6

11

59

67

Organic

13

59

91

99

Aqueous

4

20

88

101

Organic

100

100

200

200

Aqueous

4

5

100

100

Organic

90

100

Aqueous

4

4

Organic

100

Aqueous

2

Organic

Tetradenia riparia (leaves)

A

Trichilia emetica (leaves)

Vangueria infausta (leaves)

DMSO (negative control)

200

200 200

3

100

91

0

1

1000

1000

2

11

200

200

Organic

2

16

200

200

Aqueous

7

8

100

100

Organic

1

36

109

100

Aqueous

5

6

71

88

Organic

7

68

1000

1000

Aqueous

3

3

70

62

Organic

82

100

74

79

Aqueous

3

4

69

61

Organic

38

40

88

76

Aqueous

2

4

100

109

Organic

5

26

200

200

Aqueous

1

5

200

200

2

2

NA

NA

A

N

CC E

PT

Strychnos madagascariensis (seed)

1000

200

M

ED

Senecio serratuloides (whole plant)

1000

100

Aqueous Sarcostemma viminale (whole plant)

IP T

Ptaeroxylon obliquum (roots)

4

SC R

Psidium guajava (roots)

Organic

U

Psidium guajava (leaves)

14

Sea water (negative control)

0

0

NA

NA

Potassium dichromate (positive control)

100

100

NA

NA

Positive control (4NQO)

NA

NA

500

500

Positive control (Sodium azide)

NA

NA

500

500

Negative control (10% DMSO)

NA

NA

0

0

Negative control (water)

NA

NA

0

0

*Results in bold are toxic.

Table 2

SC R

IP T

NA - Not applicable

Medicinal plants tested and their documented evidence of cytotoxicity. Plant part used

Known cytotoxicity

Correlation with current study

References

Acanthospermum hispidium DC.

Seed and whole plant

Showed cytotoxicity in BSLA.

*Not tested

Suad, 2012

Asteraceae

Stem and leaf

Showed weak cytotoxicity against the following three cell lines, monocytes (THPl), normal melanocytes (MCDB) and malignant melanoma cells (HTB66).

Not tested

Sanon et al., 2003

Leaf and twigs

Showed low cytotoxicity when evaluated using the MTT assay.

Not tested

Bero et al., 2009

Whole plant

The in vivo acute (2000 mg/kg) and sub-acute (1000 mg/kg) toxicity studies did not show any toxicity in mice.

Not tested

Ganfon et al., 2012

Aerial part

Non-mutagenic towards strain TM677 at the concentrations of 0.31, 0.62, 1.25, 2.5 and 5.0 mg/ml.

Not tested

Hussain et al., 1990

A

CC E

PT

ED

M

A

N

U

Plant species (scientific names, author names and family)

15

Showed low cytotoxicity at 200 μg/ml against Graham cell line.

Cytotoxic for rhizome

De Wet et al., 2007

Leaf and gel

The cell viability indicated no cytotoxicity against human hepatocellular cells (HepG2), human neuroblastoma cells (SH-SY5Y) and human adenocarcinoma epithelial cells (HeLa).

Noncytotoxic in BSLA

Du Plessis and Hamman, 2013

Leaf

Found to be noncytotoxic at 100 µg/ml against the human kidney epithelial cell line.

Noncytotoxic in BSLA

Leaf

Found to be nonmutagenic when tested against TA98 at the concentrations of 50, 500 and 5000 μg/ml.

Menispermaceae Aloe marlothii A.Berger

Non-

U

Asphodelaceae

IP T

Leaf and rhizome

Naidoo et al., 2013

SC R

Albertisia delagoensis N.E.Br

Luseba et al., 2007

Was mutagenic in the presence of metabolic activation at the concentration of 1 mg/ml

Mutagenic without metabolic activation

Busch et al., 1979

Found to be noncytotoxic at concentrations of 1, 10, 100, 1000 μg/ml towards BSLA.

Cytotoxic in leaf

Hurinantha et al., 2009

Leaf and flower

Reported to be nonmutagenic toward TA98 and TA100 strains at the concentrations of 5, 10, 20, 100 and 1000 μg/ml.

Nonmutagenic in leaf

Hurinantha et al., 2009

Leaf

Shown cytotoxicity against the human lymphoma U-937 GTB cell line.

Not tested

El-Seedi et al., 2013

Leaf and twigs

Showed cytotoxicity in the Chang liver and 3T3-L1 cell lines at

Not tested

Van de Venter et al., 2008

Leaf

M

Cannabis sativa L.

A

N

cytotoxic

Carpobrotus dimidiatus L.

Leaf and flower

A

CC E

PT

Mesembryanthemaceae

ED

Cannabaceae

Catharanthus Roseus (L.) G.Don Apocynaceae

16

concentration of 12.5 µg/ml. Not tested

Kevin et al., 2012

Mutagenic against strain TA98 while being nonmutagenic towards strain TA100 at the concentration of 0.05, 0.5 and 5.0 mg/ml.

Mutagenic in roots

Elgorashi et al., 2003

No toxicity tests done.

Noncytotoxic in BSLA

Cucurbitaceae Shown to have genotoxic effect on Allium cepa.

Peel

Shown to have cytotoxic effect on human colorectal carcinoma cell line at the concentration of 0.5-48 μg/ml.

Ali and Celik, 2007

Cytotoxic in BSLA

Jomaa et al., 2012

No toxicity tests were evident.

Noncytotoxic in BSLA

No toxicity tests were evident.

Non – cytotoxic and nonmutagenic in roots

Corm

Showed cytotoxicity on BSLA at the concentrations of 10, 100 and 1000 µg/ml.

Noncytotoxic in BSLA

Jooste et al., 2012

Corm

Non-cytotoxic at 100 µg/ml against human kidney epithelial cell line.

Not tested

Naidoo et al., 2013

PT

Cladostemon kirkii (Oliv.) Pax & Gilg

ED

M

A

Rutaceae

Nonmutagenic in AMES

U

Peel

N

Citrus limon (L.) Osbeck

IP T

Citrullus lanatus (Thunb.) Matsum. & Nakai

Reported to have subacute toxicity on Sprague Dawley rats at the concentrations of 0.1, 0.5 and 1 g/kg.

SC R

Leaf

CC E

Hyphaene coriacea Gaertn. Arecaceae

A

Hypoxis hemerocallidea Fisch & Ave-Lall Hypoxidaceae

17

Not tested

Corm and leaf

Non-mutagenic at the concentrations of 0.05, 0.5 and 5.0 mg/ml against the strains TA98 and TA100.

Nonmutagenic in corm

Leaf

Displayed minimal inhibitory effect (LC50 of 0.51 ± 0.03 mg/ml).

Roots

Shown to have weak cytotoxicity in BSLA at different concentrations of 50, 100, 200, 400, 500, 1000 and 2000 μg/ml.

Not tested

Ayuko et al., 2009.

Non-cytotoxic towards BSLA at the concentration of 2 mg/ml.

Cytotoxic in BSLA

Cock and van Vuuren, 2014

Acute toxicity study done on mice showed that the mice were lethargic and had mortality of 37.5%.

Cytotoxic in BSLA

Madzimure et al., 2011

Leaf

Shown to be cytotoxic against C2C12 muscle cells and hepatocytes cell lines when tested at the concentration of 12.5 µg/ml.

Cytotoxic in BSLA

Van de Venter et al., 2008

Stem bark

Reported to have acute toxic at very high dosages (0.56 mg/100 g) on tissues and organs of rats.

Not tested

Geidam et al., 2007

Reid et al., 2006; Elgorashi et al., 2003

SC R Cytotoxic in BSLA

Hubsch et al., 2014

ED

Leaf

M

A

N

Verbenaceae

Steenkamp and Gouws, 2006

IP T

Reported to be noncytotoxic against three human cancer cell lines; prostate cancer cells (Du-145), breast cancer cells (MDA-MB-231 and MCF-7) and nonmalignant breast cell line (MCF-12A) at the concentration of 50 μg/ml.

U

Lippia javanica (Burm.F.) Spreng.

Corm

CC E

PT

Leaf

Momordica balsamina L.

A

Cucurbitaceae

18

Leaf

Non-mutagenic toward S. typhimurium strain TA98 at the concentrations of 50, 500 and 5000 μg/ml.

Non mutagenic in AMES

Ndhlala et al., 2011

Red flower bracts

Showed to be noncytotoxic against brine shrimps with the LC50 value of 9.97 mg/ml.

Noncytotoxic in BSLA

Sumathy et al., 2011

Roots

The plant was found to be non-cytotoxic at 100 µg/ml against human kidney epithelial cell line.

Not tested

Stem bark

Cytotoxic with the ID50 at 35 µg/ml against vervet monkey kidney cell line.

Leaf

Non-cytotoxic at 100 µg/ml against the human kidney epithelial cell line.

Not tested

Naidoo et al., 2013

Non-cytotoxic in BSLA with the LC50 value of 707,2 µg/ml.

Noncytotoxic in BSLA

Ajaiyeoba et al., 2006

Non-cytotoxic against 3T3 (mouse embryonic fibroblast cell) and 4T1 (mouse breast cancer cell) at 100 µg/ml.

Noncytotoxic in BSLA

Ling et al., 2010

Leaf

No acute toxicity was found on rats which were administered 1050 mg/100g.

Noncytotoxic in BSLA

Etuk and Francis, 2003

Roots and leaf

Found to be cytotoxic against Chang liver and 3T3-L1 fat cell lines at the concentration of 12.5 µg/ml.

Noncytotoxic in BSLA

Van de Venter et al., 2008

Leaf

ED

Psidium guajava L. Myrtaceae

CC E

PT

Leaf

A

Naidoo et al., 2013

SC R Not tested

U

Anacardiaceae

IP T

Karumi et al., 2006

N

Ozoroa engleri R.Ferm & A.Ferm

Cytotoxic in BSLA

A

Musaceae

Acute toxicity study done did not show any signs of toxicity.

M

Musa acuminata Colla

Leaf

Prozesky et al., 2001

19

Noncytotoxic in BSLA

Joseph and Priya, 2010.

Leaf

Caused a decrease in cell viability and growth on human gingival fibroblast.

Noncytotoxic in BSLA

Fernandes et al., 2010

Roots

Found to have acute and sub-chronic toxicity.

Noncytotoxic in BSLA

Onyekwe et al., 2011

Stem bark

Toxic at the LC50 value of 1.0009 µg/ml in BSLA.

Noncytotoxic in BSLA

Leaf

Non-cytotoxic in BSLA.

Pyrenacantha kaurabassana Baill

N

No cytotoxicity tests were evident.

Ricinus communis L. var. communis

Leaf

Euphorbiaceae

Leaf

A

Icacinaceae

Cock and van Vuuren, 2014

Roots cytotoxic in BSLA McGaw et al., 2007

No acute toxicity found on mice with a dosage of 2, 4, 6, 8 and 10 g/kg.

Cytotoxic in BSLA

Sadashiv, 2011

Resulted in damage to the kidney and liver.

Not tested

Kumar et al., 2003

Seed

Acute toxicity study on birds showed dizziness, diarrhoea and mortality after 48 h.

Not tested

Ukachukwa et al., 2011

Root

Non-toxic in Wistar rats at the dosages of 200 and 1000 mg/kg.

Not tested

Ilavarasan et al., 2011

Leaf

Reported to be nonmutagenic against strain TA98 at concentrations of 50, 500 and 5000 μg/ml.

Nonmutagenic in AMES

Luseba et al., 2007

Root

Non-mutagenic at the concentrations of 0.05, 0.5 and 5.0 mg/ml

Not tested

Elgorashi et al., 2003

ED

M

Cytotoxic in BSLA

PT CC E

Not Tested

Cytotoxic in BSLA at 0.1, 1.2 and 5 mg/ml.

Seed

A

Fasola et al., 2011

U

Rutaceae

IP T

Inhibited pronounced cytotoxic effects at the concentration of 10 µg/ml on HeLa cell lines.

SC R

Ptaeroxylon obliquum (Thunb.) Radlk

Leaf

20

Sarcophyte sanguinea Sparm. subsp. Sanguinea

Stem

Non-cytotoxic in monkey kidney cell line with the LD50 of 50 µg/ml.

Noncytotoxic in BSLA

Naidoo et al., 2013

Leaves

Poisonous to fish.

Not tested

Neuwinger, 2004

Leaves

Toxic to animals.

Not tested

Whole plant

Found to be nonmutagenic when tested against TA98 at the concentrations of 50, 500 and 5000 μg/ml.

Cytotoxic in

IP T

against strains TA98 and TA100.

Balanophoraceae Sarcostemma viminale (L.) B.r. Saps Apocynaceae

Luseba et al., 2007

SC R

BSLA and

Van Wyk et al., 2002

Non-

mutagenic

Senecio serratuloides DC.

Non-cytotoxic at 100 µg/ml against the human kidney epithelial cell line.

Noncytotoxic in BSLA and Nonmutagenic in AMES

Naidoo et al., 2013

Found to be nonmutagenic toward strains TA98 and TA100 at the concentrations of 0.05, 0.5 and 5.0 mg/ml.

Not tested

Elgorashi et al., 2003

Stem

Poisonous to fish.

Not tested

Neuwinger, 2004

Tetradenia riparia (Hochst.) Codd

Leaf

High cytotoxicity in MTT assay.

Cytotoxic in BSLA

Gazim et al., 2014

Lamiaceae

Leaf

Non-mutagenic towards TA98 at the concentrations of 0.005, 0.5 and 5 mg/ml and 50, 500 and 5000 μg/ml.

Nonmutagenic in AMES

Elgorashi et al., 2003; Ndhlala et al., 2011

Stem bark

Non-cytotoxic in monkey kidney cell line with the LD50 of 50 µg/ml.

Not tested

Prozesky et al., 2001

N

Whole plant

U

in AMES

A

CC E

Strychnaceae

PT

Strychnos madagascariensis Poir.

ED

Leaf

Trichilia emetica Vahl Meliaceae

M

A

Asteraceae

21

Not tested

Germano et al., 2005

Leaf

Low cytotoxicity on human normal fibroblast at 20 mg/ml.

Noncytotoxic in BSLA

Bero et al., 2009

Leaf

Poisonous to fish.

Not applicable

Neuwinger, 2004

Leaf

Found to be nonmutagenic toward strains TA98 and TA100 at the concentrations of 0.05, 0.5 and 5.0 mg/ml.

Nonmutagenic in AMES

Elgorashi et al., 2003

Leaf and roots

Non-cytotoxic in MTT.

Leaf nonmutagenic in AMES

Noncytotoxic in BSLA

Munodawafa et al., 2012

A

* Current study evaluated different plant part.

Mthethwa et al., 2014

N

Moderately safe in BSLA.

U

Rubiaceae

IP T

Non-cytotoxic in BSLA (LC50 > 1000 g/ml).

SC R

Vangueria infausta Burch. subsp. infausta

Roots

M

3.1.3. Plant combinations

3.1.3.1 Plant combinations tested in the BSLA

ED

The results of the combinations traditionally used for the treatment of hypertension (De Wet et al., 2016) which were tested for preliminary cytotoxicity using the BSLA

PT

are depicted in Table 3 along with the FIC indices. For the organic extracts, 13 plant combinations were found to be toxic either at 2 mg/ml or 4 mg/ml or both. For the aqueous extracts, three plant combinations were toxic at 2 mg/ml while six plant

CC E

combinations were toxic at 4 mg/ml. The results of the two concentrations which were tested demonstrated that the toxicity of plant combinations was dosage dependant i.e. as the concentration increases, toxicity levels also increased. Some

A

of the combinations were toxic at 4 mg/ml while non-toxic at 2 mg/ml. Such combinations include the combination between Albertisia delagoensis and Senecio serratuloides (organic extract), Catharanthus roseus and Aloe marlothii (organic extract) and Aloe marlothii, Hypoxis hemerocallidea and Momordica balsamina (organic extract), Catharanthus roseus and Musa acuminata (organic extract), Trichilia emetica, Aloe marlothii and Hyphaene coriacea (organic and aqueous

22

extract) as well as the combination between Catharanthus roseus and Hypoxis hemerocallidea (aqueous extract). Some of the aqueous extracts combinations tested resulted in increased cytotoxicity and this has raised a concern for the use of these combinations. Such combinations include the combinations between Catharanthus roseus and Aloe marlothii, Hypoxis hemerocallidea and Catharanthus roseus, Musa acuminata and Catharanthus roseus, Trichilia emetic, Aloe marlothii and Hyphaene coriacea as well as the

IP T

combination between Albertisia delagoensis and Senecio serratuloides. moreover, some of the combinations were considered somewhat less cytotoxic (synergistic)

SC R

when combined. For instance, where a plant was cytotoxic singularly it showed

decreased cytotoxicity when combined with another plant. Such instances include the following combinations; Psidium guajava and Carpobrotus dimidiatus (organic extract); Hypoxis hemerocallidea and Aloe marlothii (organic extract) as well as

U

Hypoxis hemerocallidea and Senecio serratuloides (organic extract). One of the

N

combination which is worth highlighting is the combination between Hypoxis

A

hemerocallidea and Catharanthus roseus (organic extract). When tested singularly, both of these plants were found to be cytotoxic, however, their combination resulted

M

in a synergistic effect. The synergistic interactions observed in the current study could justify why lay people use two or more plants in a combination for the

ED

treatment of hypertension. Synergistic interactions are often considered beneficial as it results in reduced cytotoxicity, allowing for dose reduction and fewer side effects.

PT

The only combination relevant to our study which could be found in the literature was the combination between Hypoxis hemerocallidea and Senecio serratuloides which was found to be non-cytotoxic in monkey kidney cell line (Naidoo et al., 2013) and

CC E

these results correlate with the current study Table 3

A

Plant combinations which were tested with the BSLA along with their ΣFIC index and their respective interpretations. Plant combinations

Extracts

Mortality % 2 mg/ml

4 mg/ml

ΣFIC and interpretation† 2 mg/ml

4 mg/ml

A. delagoensis +

Organic

*64

100

1.21 (Non-interactive)

1.89 (Non-interactive)

S. serratuloides

Aqueous

45

57

4.35 (Antagonistic)

5.56 (Antagonistic)

23

Organic

45

93

2.50 (Non-interactive)

5.17 (Antagonistic)

A. marlothii

Aqueous

76

98

6.71 (Antagonistic)

8.70 (Antagonistic)

A. marlothii + H. hemerocallidea

Organic

2

2

0.06 (Synergistic)

0.12 (Synergistic)

Aqueous

2

2

0.37 (Synergistic)

0.37 (Synergistic)

A. marlothii + H. hemerocallidea + M. balsamina

Organic

33

56

1.52 (Non-interactive)

2.60 (Non-interactive)

Aqueous

9

11

1.18 (Non-interactive)

1.45 (Non-interactive)

M. balsamina + A. marlothii

Organic

57

78

3.24 (Non-interactive)

4.68 (Antagonistic)

Aqueous

5

11

0.48 (Synergistic)

1.06 (Non-interactive)

C. sativa +

Organic

62

100

3.41 (Non-interactive)

5.50 (Antagonistic)

A. marlothii

Aqueous

4

11

0.66 (Additive)

1.84 (Non-interactive)

C. kirkii +

Organic

21

33

S. sanguinea +

Aqueous

4

6

M. balsamina + C. sativa

Organic

70

Aqueous

H. hemerocallidea + C. roseus

N

U

SC R

IP T

C. roseus +

12.47 (Antagonistic)

0.67 (Additive)

1.01 (Non-interactive)

91

1.05 (Non-interactive)

1.37 (Non-interactive)

8

15

0.76 (Additive)

1.44 (Non-interactive)

Organic

17

31

0.25 (Synergistic)

0.56 (Additive)

Aqueous

44

66

4.64 (Antagonistic)

6.96 (Antagonistic)

Organic

44

64

5.74 (Antagonistic)

8.36 (Antagonistic)

Aqueous

66

89

5.85 (Antagonistic)

7.98 (Antagonistic)

P. guajava +

Organic

3

4

0.23 (Synergistic)

0.32 (Synergistic)

C. dimidiatus

Aqueous

1

2

0.09 (Synergistic)

0.18 (Synergistic)

H. hemerocallidea +

Organic

7

15

0.15 (Synergistic)

0.36 (Synergistic)

Aqueous

2

5

0.37 (Synergistic)

0.92 (Additive)

CC E

PT

A

M. acuminata + C. roseus

A

M

ED

A. marlothii

7.93 (Antagonistic)

S. serratuloides

24

Organic

66

90

1.29 (Non-interactive)

1.77 (Non-interactive)

M. balsamina

Aqueous

19

29

1.43 (Non-interactive)

2.17 (Non-interactive)

M. balsamina + M. acuminata

Organic

51

90

6.89 (Antagonistic)

2.12 (Non-interactive )

Aqueous

13

21

1.25 (Non-interactive)

2.02 (Non-interactive)

M. balsamina + C. roseus

Organic

67

100

1.04 (Non-interactive)

1.56 (Non-interactive)

Aqueous

73

99

1.30 (Non-interactive)

1.81 (Non-interactive)

M. balsamina + S. serratuloides

Organic

69

96

1.65 (Non-interactive)

2.29 (Non-interactive)

Aqueous

14

24

1.35 (Non-interactive)

2.31 (Non-interactive)

M. acuminata + C. limon

Organic

57

100

7.42 (Antagonistic)

13.0 (Antagonistic)

Aqueous

30

40

4.65 (Antagonistic)

3.62 (Non-interactive)

S. serratuloides

Organic

8

28

0.80 (Additive)

+ M. acuminata + A. marlothii +

Aqueous

4

5

0.85 (Additive)

0.17 (Additive)

2.77 (Non-interactive)

T. emetica +

Organic

46

72

2.14 (Non-interactive)

3.36 (Non-interactive)

A. marlothii +

Aqueous

44

50

7.36 (Antagonistic)

7.34 (Antagonistic)

M

PT

ED

H. hemerocallidea

H. coriacea

A

N

U

SC R

IP T

L. javanica +

CC E

*Results in bold are toxic; ΣFIC index = ≤ 0.5 (interact synergistically to produce a less toxic therapeutic outcome); ΣFIC index 0.5-1.0 (reduced toxicity: additive effects); ΣFIC index 1.0 - 4.0 (non-interactive); ΣFIC > 4.0 (increased toxicity; in other words, an antagonistic response).

3.1.3.2 Plant combinations tested in the Ames test The results of the combinations which were assessed for mutagenicity in the

A

Ames test are depicted in Table 4. For the organic extracts, four plant combinations (Cannabis sativa and Aloe marlothii, Momordica balsamina and Cannabis sativa, Hypoxis hemerocallidea and Catharanthus roseus as well as Musa acuminata and Catharanthus roseus) were found to have an increased mutagenic (antagonistic) effect towards both Salmonella TA strains. For the aqueous extracts, only two combinations (Momordica balsamina and Catharanthus roseus as well as Hypoxis 25

hemerocallidea and Catharanthus roseus) demonstrated some increased mutagenicity. A number of combinations decreased mutagenicity providing a synergistic effect. None of the combinations tested in the current study have been investigated for mutagenicity previously. Table 4 Plant combinations which were tested in the Ames test with two types of Salmonella

interpretations.

IP T

typhimurium strains used along with their ΣFIC index and their respective

ΣFIC and interpretation

TA98

TA100

TA98

Organic

20

29

0.72 (Additive)

0.75 (Additive)

S. serratuloides

Aqueous

34

27

0.43 (Synergistic)

0.34 (Synergistic)

C. roseus +

Organic

85

90

0.44 (Synergistic)

A. marlothii

Aqueous

200

200

1.25 (Non-interactive)

A. marlothii + H. hemerocallidea

Organic

21

24

Aqueous

29

A. marlothii + H. hemerocallidea + M. balsamina

Organic

U

N

A

A. delagoensis +

TA100

0.49 (Synergistic) 1.38 (Non-interactive)

1.07 (Non-interactive

40

1.23 (Non-interactive)

1.27 (Non-interactive)

37

33

0.35 (Synergistic)

0.31 (Synergistic)

Aqueous

58

49

0.66 (Additive)

0.60 (Additive)

Organic

21

23

0.19 (Synergistic)

0.24 (Synergistic)

Aqueous

35

31

0.42 (Synergistic)

0.42 (Synergistic)

ED

CC E

M. balsamina + A. marlothii

M

1.05 (Non-interactive)

PT

Extracts

SC R

Strains CFU/ml

Plant combinations

Organic

*1000

1000

5.26 (Antagonistic)

5.50 (Antagonistic)

A. marlothii

Aqueous

150

200

1.63 (Non-interactive)

2.26 (Non-interactive)

C. kirkii +

Organic

200

200

1.04 (Non-interactive)

1.07 (Non-interactive)

S. sanguinea +

Aqueous

200

200

1.43 (Non-interactive)

1.52 (Non-interactive)

Organic

1000

1000

5.09 (Antagonistic)

5.55 (Antagonistic)

Aqueous

200

200

2.31 (Non-interactive)

2.39 (Non-interactive)

A

C. sativa +

A. marlothii

M. balsamina + C. sativa

26

Organic

1000

1000

5.40 (Antagonistic)

4.77 (Antagonistic)

Aqueous

1000

1000

1.0 (Non-interactive)

5.26 (Antagonistic)

M. acuminata + C. roseus

Organic

1000

1000

14.79 (Antagonistic)

11.86 (Antagonistic)

Aqueous

100

100

0.56 (Additive)

0.64 (Additive)

P. guajava +

Organic

27

39

0.5 (Synergistic)

0.63 (Additive)

C. dimidiatus

Aqueous

200

200

2.14 (Non-interactive)

1.99 (Non-interactive)

H. hemerocallidea +

Organic

23

19

0.22 (Synergistic)

0.17 (Synergistic)

Aqueous

40

45

0.48 (Synergistic)

0.46 (Synergistic)

L. javanica +

Organic

200

200

1.92 (Non-interactive)

1.51 (Non-interactive)

M. balsamina

Aqueous

100

100

0.89 (Additive)

0.95 (Additive)

M. balsamina + M. acuminata

Organic

19

27

Aqueous

29

M. balsamina + C. roseus

Organic

Aqueous

SC R

IP T

H. hemerocallidea + C. roseus

A

N

U

S. serratuloides

0.45 (Synergistic)

34

0.34 (Synergistic)

0.44 (Synergistic)

200

200

9.27 (Antagonistic)

1.11 (Non-interactive)

1000

1000

6.91 (Antagonistic)

7.54 (Antagonistic)

Organic

33

39

0.44 (Synergistic)

0.31 (Synergistic)

Aqueous

92

100

1.54 (Non-interactive)

1.64 (Non-interactive)

Organic

25

21

0.60 (Additive)

1.41 (Non-interactive)

Aqueous

100

100

1.94 (Non-interactive)

1.73 (Non-interactive)

S. serratuloides

Organic

105

111

1.5 (Non-interactive)

1.43 (Non-interactive)

+ M. acuminata + A. marlothii +

Aqueous

30

25

0.36 (Synergistic)

0.29 (Synergistic)

Organic

100

100

0.87 (Additive)

0.94 (Additive)

ED

PT

CC E

M. balsamina + S. serratuloides

A

M. acuminata + C. limon

M

0.36 (Synergistic)

H. hemerocallidea T. emetica +

27

A. marlothii +

Aqueous

200

200

1.77 (Non-interactive)

1.80 (Non-interactive)

H. coriacea *Results

in bold are mutagenic

. 4. Conclusion

Apart from Catharanthus roseus and Citrus limon (cytotoxic in BSLA) and

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Catharanthus roseus and Ozoroa engleria (mutagenic in Ames test), all the aqueous extracts of the plants taken for treating hypertension were non-toxic. The results

SC R

demonstrated that in general lower cytotoxicity was observed for aqueous extracts than organic extracts. This is encouraging for the safety of lay people in northern

KwaZulu-Natal, (South Africa) utilizing these medicinal plants. It would be interesting to consider future comparisons with traditional healers to determine if the selection of

U

plant material is congruent. It should also be noted that the plant extracts tested for

N

mutagenicity were only evaluated with two strains (TA98 and TA100), thus other strains should also be evaluated before concluding the safety of these extracts as

A

different strains detect different types of mutations. The results of the aqueous

M

extracts which were further analysed at different concentrations showed that the toxicity of medicinal plants was dosage dependent, thus as the concentration

ED

increases, the toxicity also increases. Any substance may be toxic if taken in large quantities, thus it is the dosage that may be the most important factor to be

PT

considered. Lay people were reported to be using one handful of plant material which is equivalent to approximately 10 g. Approximately 250 ml of the remedy was taken three times a day. Thus more attention has to be drawn to the quantity of plant

CC E

material used and to standardize the dosage. When analysing the toxicity profile of the most frequently used combinations; the combination between Catharanthus roseus and Momordica balsamina (aqueous) was found to be toxic in both assays

A

and therefore the use of this combination should be discouraged. The other most frequently used plant combinations (Aloe marlothii and Hypoxis hemerocallidea, Aloe marlothii and Momordica balsamina as well as the combination between Hypoxis hemerocallidea and Senecio serratuloides) were non-toxic in both assays, providing evidence for safety. Although it is believed that the use of plant combinations results in less toxicity (synergistic effect), some of the plant combinations tested in the

28

current study resulted in increased toxicity (antagonistic interactions). Thus the use of these combinations should be avoided.

Conflict of interest

The authors declare no conflict of interest

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Acknowledgements

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Prof Lyndy McGaw, University of Pretoria, for donating the two Salmonella strains. Financial support was provided by the Research Committee of the University of Zululand, University of Witwatersrand (Wits) and the National Research Foundation. We are extremely grateful to the interviewees in northern Maputaland who shared

N

U

their hospitality, time and their valuable information with us.

A

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cell carcinoma of the head and neck. Cancer Epidemiol. Biomark. Prev. 8, 1071-

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1078.

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Zonyane, S., Van Vuuren, S.F., Makunga, N.P., 2013. Antimicrobial interactions of Khoi-San poly-herbal remedies with emphasis on the combination; Agathosma

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crenulata, Dodonaea viscosa and Eucalyptus globulus. J. Ethnopharmacol. 148,

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144-151.

Fig. 1. Dose response curves of the two medicinal plants which were further analysed at different concentrations (BSLA). The line at 50% indicate the cut off of the cytotoxicity mortality i.e. everything above the line is considered toxic. 39

100

C. limon organic extract

90

C. limon aqueous extract C. roseus organic extract

80

C. roseus aqueous extract

60

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Mortality %

70

50 40

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30 20 10

0.031 0.063 0.125

0.25

0.5

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0 1

A

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PT

ED

M

A

N

Concentration of the plant extracts (mg/ml)

40