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Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date). UNIVERSITY ------OF ------JOHANNESBURG

THE ETHNOBOTANICAL, ANTIMICROBIAL AND PHYTOCHEMICAL SCREENING OF SELECTED MEDICINAL PLANTS FROM GA- MASHASHANE, LIMPOPO PROVINCE, SOUTH AFRICA

BY

LESIBA ABRAM PAPO

Dissertation submitted in fulfilment of the requirements for the degree o f

Masters in Botany In

Botany and Plant Biotechnology

Faculty of Science

University of Johannesburg

Supervisor: Professor A Moteetee Co-supervisor: Professor S van Vuuren

July, 20 17 DECLARATION

I, Lesiba Abram Papo, student number: 200935782, solemnly declare that this dissertation is my own original work. It is being submitted for the degree of Master of

Botany at the University of Johannesburg, South Africa. This work has not been submitted before for any degree or examination and where sources are used in the study, they have been well referenced.

Lesiba Abram Papo Date

i DEDICATIONS

I would like to dedicate this study to my grandfather, Elias Moshemane Papo and my beloved grandmother, Raesetsa Rachel Papo. To my grandfather, thank you for being the mouthpiece and the source of this indigenous information documented in the study.

Your gentle spirit, guidance and the care you bestowed upon the entire family have always been the chief cornerstone to draw our strength to endure. To my grandmother, I have no words, you clothed us with your love, your gentle spirit and compassion and taught us to fix our eyes to the God above, and for that, thank you for shaping me and raising me into the person that I am today. Ke tloga ke leboga ditlou!!

ii ACKNOWLEDGEMENTS

❖ Blessed be the name of God, our Heavenly father for providing me with the

strength, opportunity, and time to start and seal the study.

❖ I would like to thank my supervisor, Professor Annah Moteetee. You have always

being a mother to me and the rest of the Taxeth group. Your ability to see the good

and the potential in us, and also to nurture us with your wisdom. It is an honour to

work under your supervision, for you to grant us opportunities to challenge

ourselves and for you to modify us into successful individuals, thank you for

everything and above all for your Ubuntu.

❖ To my co-supervisor, Professor Sandy Van Vuuren, thank you for the tireless

assistance and guidance with the antimicrobial screening.

❖ To my informants, thank you for your warm-hearted souls and providing me with

your invaluable ethnobotanical information, your co-operation is highly

appreciated.

❖ Malakia Manamela, Inter Letjeku and Johannes Mantjiu, thank you for assisting

me with some plants collection and surveys, your efforts are highly appreciated.

❖ Mutsinda Ramavhunga, thank you for assistance with the study map area.

❖ To the Papo and Makuya family, thank you for your prayers, for the love, care and

believing in me that I can make it.

❖ To my beloved mother, Josephine Papo, thank you for everything and moulding

me into a success. Your love and words of encourangement are highly

appreciated.

in ❖ To my colleagues, Lerato Seleteng-Kose, Phumzile Madondo and the rest of the

Taxeth group, thank you for being part of the academic family and strengthening

each other through tough times.

❖ My beloved sister, Mpho Papo, National Research Foundation and the University

of Johannesburg, thank you for financial assistance to make this study possible.

❖ Moloko Dikgale, Katlego Riet, Lesetja Kgobe, and Karabo Robaya thank you for

being the brothers who kept me focused throughout.

❖ To my beloved lady and friend, Abigail Lethuba, thank you for the love, care,

compassion and your ability to constantly keep me happy and focused. Your

presence in my life is indeed a blessing.

IV CONFERENCE PRESENTATIONS

❖ Papo, L. A., van Vuuren, S.F., and Moteetee A.N., 2016. The ethnobotanical,

antimicrobial and phytochemical screening of selected medicinal plants from Ga-

Mashashane, Limpopo Province, South Africa. Podium presentation at the joint

conference of the 42nd annual SAAB and 12th bi-annual SASSB conferences held

at University of the Free State, 10-13th January, 2016.

❖ Papo, L. A., van Vuuren, S.F., and Moteetee A.N., 2016. The ethnobotanical,

antimicrobial and phytochemical screening of selected medicinal plants from Ga-

Mashashane, Limpopo Province, South Africa. Oral presentation for the 5th

postgraduate centre, Research and innovation cross-faculty symposium held at

University of Johannesburg, 13th October 2015.

❖ Papo, L. A., van Vuuren, S.F., and Moteetee A.N., 2016. The ethnobotanical,

antimicrobial and phytochemical screening of selected medicinal plants from Ga-

Mashashane, Limpopo Province, South Africa. Oral presentation at the

postgraduate Botany Symposium held at University of Johannesburg Lapa, 27th

October, 2015.

v ABSTRACT

Medicinal plants are widely recognised and acknowledged in rural areas and globally they contribute to the healthcare system within various countries. The core indigenous knowledge on the use of these medicinal plants resides in elderly people and it is traditionally passed from one generation to the next through oral transmission, and often not efficiently documented. The Ga-Mashashane area, situated in the Capricorn District of the Limpopo Province is no exception to the risk of losing some of this information through oral transmission. However, ethnobotanical studies have been conducted at

District level within the Province covering Waterberg, Capricorn, Sekhukhune, Mopani and Vhembe. The aim of the current study was to explore and document the indigenous knowledge on the use of medicinal plants by the people in the Ga-Mashashane area and to determine the efficacy of these medicinal plants through antimicrobial and phytochemical screening.

Knowledge on the use of medicinal plants was gathered through structured questionnaires, which considered socio-demographics, plant organs used, dosage, and administration method. Forty-five medicinal plants were recorded to treat various human ailments in the area, and the majority of these plants were noted from Anacardiaceae,

Asteraceae, Fabaceae, and Verbenaceae. Twelve new uses were recorded in the current study with Senecio barbertonicus Klatt (used as a purgative), recorded for the first time.

Priority was given to gastrointestinal ailments treated with 22 medicinal plants, followed by respiratory ailments (18), skin ailments (16), and sexually transmitted infections (eight), while non-infectious ailments are treated with four species. Peltophorum africanum was the most highly utilized medicinal plant to treat all infectious ailments, followed by

Acokanthera oppositifolia, Carissa bispinosa, Carpobrotus edulis, and Eucalyptus

VI camaldulensis used to treat three infections. Oral administration, inhalation, soaking, topical ointment application, and smoke therapy were recorded as the preferred method for administration of the medicinal plants. The plants were further dried and ground for pathogen-specific antimicrobial screening against 16 pathogens of the gastrointestinal, respiratory, skin, and sexually transmitted infections, as well as qualitative phytochemical screening. Plants with outstanding noteworthy actitities were recorded from the organic extracts of Ozoroa sphaerocarpa (MIC range 0.1 - 1.0 mg/ml) and Searsia lancea (MIC range 0.1 - 1.0 mg/ml) against all six gastrointestinal pathogens. The organic extract of

Peltophorum africanum exhibited potent activity against two respiratory pathogens

(Klebsiella pneumoniae and Staphylococcus aureus both at 1.0 mg/ml) while the organic extract of Ozoroa insignis demonstrated excellent activity against Gardnerella vaginalis and Neisseria gonorrhoea at 1.0 mg/ml and 0.1 mg/ml respectively. Interestingly, the organic extracts of eight medicinal plants highlighted to treat STIs exhibited noteworthy activity against N. gonorrhoea at MIC range of 0.1 - 0.8 mg/ml. Antimicrobial screening against skin pathogens resulted in the organic extracts of Asparagus racemosus (MIC range of 0.1 - 1.0 mg/ml), Peltophorum africanum (0.3 - 1.0 mg/ml), and Ximenia caffra

(0.5 -1.0 mg/ml) exhibiting potent activity by inhibiting all four pathogens known to cause skin infections. Noteworthy activity with essentials oils were recorded from Eucalyptus camaldulensis (1.0 mg/ml) and Lippia javanica (1.0 mg/ml) when screened against S. aureus. Phytochemical screening was performed on plants with newly recorded uses and previously deficient phytochemical information. Zanthoxylum humile emerged as the only plant with all screened phytochemicals; alkaloids, coumarins, flavonoids, glycosides, tannins, terpenoids, and saponins. In addition the aqueous extract of Z. humile demonstrated the lowest MIC value of 0.75 mg/ml against K. pneumoniae.

Englerophytum magaliesmontanum and Ozoroa sphaerocarpa also exhibited an array of

Vii phytochemicals (except saponins) detected mostly from their organic extracts. The study has indicated that the use of medicinal plants in Ga-Mashashane area concurs with other cultures in South Africa and furthermore, there is a correlation between findings of antimicrobial screening and qualitative phytochemical screening which together validates the efficacies of the plants.

Viii TABLE OF CONTENTS

DECLARATION...... i DEDICATIONS...... ii ACKNOWLEDGEMENTS...... i ii CONFERENCE PRESENTATIONS...... v ABSTRACT...... vi TABLE OF CONTENTS...... ix LIST OF FIGURES...... xi LIST OF TABLES...... xii LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS...... xiv CHAPTER 1 ...... 1 1. Introduction...... 1 1.1 General introduction...... 1 1.2 Literature Review...... 3 1.2.1 Ethnobotany in South Africa...... 3 1.2.2 Infectious diseases and medicinal plant use...... 7 1.3 Antimicrobial activity...... 12 1.4 Phytochemistry...... 13 1.5 Study area...... 13 1.6 Aims and Objectives...... 15 1. CHAPTER 2 ...... 16 2 . Materials and Methods ...... 16 2.1 Ethnobotanical survey ...... 16 2.2 Collection of plant material ...... 16 2.3 Phytochemical screening ...... 17 2.3.1 Preparation of plant extracts ...... 17 2.3.2 Sample extraction...... 19 2.4 Antimicrobial assay...... 25 2.4.1 Selection of bacteria ...... 25 CHAPTER 3 ...... 28 3. Results and Discussion...... 28 3.1 Ethnobotany ...... 28 3.2 ANTIMICROBIAL SCREENING...... 51

ix 3.2.1 Gastro-intestinal pathogens...... 51 3.2.2 Respiratory pathogens...... 55 3.2.3 Skin pathogens...... 61 3.2.4 STI pathogens...... 65 3.3 PHYTOCHEMISTRY...... 68 3.3.1 Introduction...... 68 CHAPTER 4 ...... 76 4. Summary, general conclusions,and future research...... 76 4.1 Summary...... 76 4.1.1 Ethnobotany...... 76 4.1.2 Antimicrobial screening ...... 77 4.1.3 Phytochemical screening ...... 78 4.2 General conclusions...... 79 4.3 Future work and recommendations...... 80 5. CHAPTER 5 ...... 81 References ...... 81 APPENDIX 1: Ethics clearance certificate...... 100 APPENDIX 2: Research Questionnaire...... 108 APPENDIX 3: Description,distribution and ethnobotanical information of medicinal plants gathered from Ga-Mashashane area...... 112

x LIST OF FIGURES

Figure 1.1 Study area: Ga-Mashashane area situated within the Capricorn district, Limpopo Province...... 14

Figure 3.1.1 Representation of the plant families utilized in the Ga-Mashashane area...... 39

Figure 3.1.4 Diagram representing plant parts of medicinal plants utilized in Ga- Mashashane area...... 40

XI LIST OF TABLES

Table 1. Ethnobotanical studies conducted in various regions of South Africa...... 6

Table 2.1 The calculation of medicinal plant yields used for antimicrobial screening. ...21

Table 2.2 Pathogens used for antimicrobial screening of medicinal plants in the Ga- Mashashane area...... 25

Table 3.1 Ethnobotanical information of medicinal plants used in Ga-Mashashane based on interviews and literature (new uses writen in bold)...... 29

Table 3.2 Antimicrobial activites of plants used in Ga-Mashashane area against gastro­ intestinal pathogens...... 54

Table 3.3 Antimicrobial activites of plants used in Ga-Mashashane area against respiratory pathogens...... 57

Table 3.4 Antimicrobial activites of the essential oils of plants used in Ga-Mashashane area against respiratory pathogens...... 60

Table 3.5 Antimicrobial activites of plants used in Ga-Mashashane area against skin pathogens...... 65

Xii Table 3.6 Antimicrobial activites of plants used in Ga-Mashashane area against STIs pathogens...... 66

Table 3.7 Phytochemical screening of medicinal plants used in Ga-Mashashane for various human ailments...... 69

x iii LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS

°C: Degree Celsius

%: Percent

(Pty) Ltd: Proprietary limited company

Aq: aqueous

ATCC: American type culture collection

CF: Chloroform

CFU: colony forming units

DCM: Dichloromethanol

DME: Dimethyl Ether

DMSO: Dimethyl sulfoxide

EtOH: Ethanol g: Gram h: hour

H2O: Water

INT: p-iodonitrotetrazolium

JRAU: University of Johannesburg herbarium

L: liter

MeOH: Methanol mg/ml: milligram per

MHB: Mueller-Hinton broth

MIC: Minimum inhibitory concentration ml: milliliter

Org: organic

TSB: Tryptone Soya broth ul: microliter

x iv CHAPTER 1

Introduction

1.1 General introduction

Traditional medicine plays a significant role in Africa as majority of people relies on medicinal plants to meet their primary healthcare needs (Rukangira, 2003; Van Andel et al., 2012; Moyo et al., 2015). African Traditional Medicine (ATM) takes a holistic approach, focusing on both the physical and spiritual parts of the human being as opposed to Western medicine (Gurib-Fakim, 2006; Ramaway et al., 2009; Van Vuuren,

2008). Western medicine is perceived to possess side effects if not administered properly and thus to some individuals there is a preference towards traditional medicine (Semenya and Maroyi, 2013). However, the main reasons behind the use of traditional medicines are believed to be poverty, lack of proper medical facilities, and unaffordability of Western pharmaceuticals (Makunga et al., 2008; Semenya et al., 2012a; 2013; Otang et al., 2014;

2015). The use of traditional medicine is evident in most developing countries (Rukangira,

2003). South Africa is no exception as more than half of the population is reported to consult traditional healers for healthcare needs (Jager et al., 1996; McGaw et al., 2005;

Le Roux-Kemp, 2010; Semenya et al., 2012a; York et al., 2012; Nair and van Staden,

2013). Traditional healers fulfil an important function in various South African poverty- stricken communities where there is lack of proper medical facilities and traditional medicine remains the only available, accessible, and affordable preference for healthcare needs (Vandebroek et al., 2004). It is for these reasons that traditional doctors have a respected standing in many South African communities (Le Roux-Kemp, 2010).

1 The ethnobotanical knowledge on the use of medicinal plants to treat ailments does not lie only with traditional doctors but also with elderly people of various rural communities

(Steenkamp, 2003; Philander, 2011) and thus the culture and the traditions of the people are preserved. Ethnobotanical knowledge is transferred orally to younger generations and through practice (Kaido et al., 1997; Light et al., 2005; Le Roux-Kemp, 2010; Sacky and

Kasilo, 2010; Mahwasane et al., 2013; De Wet et al., 2013; Moyo et al., 2015), however, it is lost as a result of urbanisation and cultural influences originating from other regions of the world (Van Wyk et al., 2002). For most of the South African ethnic groups, the ethnobotanical knowledge has been well documented from both semi-urban and mostly rural communities where these medicinal plants are highly harvested and brought into muthi markets across the country (Makunga et al., 2008). Medicinal plants are used by various South African cultures to treat infectious diseases and other ailments such as reproductive ailments (Semenya et al., 2013b), diabetes (Semenya et al., 2012a), and gynaecological and obstetric problems (De Wet and Ngubane, 2014). The importance of medicinal plants in South Africa and Africa at large cannot be underestimated, as they are the backbone of rural healthcare (Light et al., 2005). In addition, Ndhlala et al. (2011) have highlighted that medicinal plants have resulted in a huge herbal industry, creating jobs for people involved in printing, manufacturing, and also distribution particularly to urban areas, and thus they ameliorate poor living conditions. There are 1100 medicinal plants commercialized globally, of which 8% emanate from plants endemic to Africa

(Brendler et al., 2010 and Moyo et al., 2015). Van Wyk (2008a) indicated that 16 South

African indigenous medicinal plants: Agathosma betulina (Berg.) Pillans, Artemisia afra

Jacq. ex Willd, Aspalathus linearis (Burm.f.) R.Dahlgren, Cyclopia species,

Harpagophytum procumbens (Burch.) DC. ex Meisn. subsp. procumbens and subsp. transvaalense Ihlenf. & H.E.K. Hartmann, Hoodia gordonii (Masson) Sweet ex Decne,

2 Hypoxis hemerocallidea Fisch., C.A.Mey. & Ave-Lall., Lippia javanica (Burm.f.) Spreng.,

Pelargonium sidoides DC., Sceletum tortuosum (L.) N.E. Brown, Siphonochilus aethiopicus (Schweif.) B.L. Burt, Lessertia frutescens (L.) R.Br., Warburgia salutaris

(Bertol.f.) Chiov, and Xysmalobium undulatum (L.) Aiton f. var. undulatum, have a potential to enter into the pharmaceutical market.

1.2 Literature Review

1.2.1 Ethnobotany in South Africa

Approximately 3 000 indigenous plant taxa are used in southern Africa for medicinal purposes by people from diverse ethnic groups (Van Wyk et al., 1997; Van Wyk and

Gericke, 2000). Watt and Breyer-Brandwijk (1962) remains one of southern and eastern

Africa’s ethnobotanical pillars of knowledge for reference in which ca.170 species recorded are still utilized in most rural areas for various ailments (Light et al., 2005, Van

Wyk, 2008a). In the South African context, documentation of ethnobotanical knowledge has either been at regional (or provincial) level or based mainly on ethnicity. The ethnobotany of certain ethnic groups has been a focus of numerous studies in the past few years, in particular the Khoi-San (Cape herbal medicine), the Xhosas (Amathole

District), the Zulus (especially the Maputaland-Pondoland-Albany region), the Vhavenda and Bapedi (Limpopo Province). Emphasis is placed on the latter group, since this is the main focus of this study.

The history on the utilization of medicinal plants in South Africa can be traced back to the

Khoi-San culture. Their ethnobotanical knowledge was preserved in the form of rock paintings. For example Aloe species depicted in their rock painting are still currently used

(Van Wyk, 2008b). Their interaction with the early European settlers has resulted in a unique traditional medical system (Cape herbal medicine) still incorporated hitherto for

3 treating various human ailments (Van Wyk, 2008b). The Cape herbal medicine comprises a number of species endemic to one of South Africa’s biodiversity hotspots, the Cape

Floristic Region (Myers et al., 2000; Philander, 2011 and Moyo et al., 2015). In the

Western Cape Province there is also a group of people which utilises a mixture of ethnomedicine from other South African cultures (e.g. Nguni tribes), the Khoi-San, and the Cape herbal medicine, known as the Rastafarians (Philander, 2011).

Another biodiversity hotspot of ethnobotanical interest in southern Africa is the

Maputaland-Pondoland-Albany region (Moyo et al., 2015), with studies undertaken within the Nguni tribes (Zulus and Xhosas). An ethnobotanical study carried out by Hutchings

(1996) on the exploration of medicinal plants used by the Zulu people remains one of the solid foundations in documented literature, whereby 1 032 plants were recorded to be used in the KwaZulu-Natal (KZN) Province for various human ailments. Prior to this publication, Hutchings and Van Staden (1994) documented 96 medicinal plants used for stress related ailments, in particular headaches, by the Sotho, Xhosa, and the Zulu people. More recently, ethnobotanical documentation in the Maputaland region includes studies done by De Wet et al. (2010; 2013), York et al. (2011), Naidoo et al. (2013) and

De Wet and Ngubane (2014).

Ethnobotanical knowledge of the Xhosa people in the Eastern Cape Province, has also been relatively well-documented, although a comprehensive study has not been compiled. In the area formerly known as Transkei, Hutchings (1989) recorded 794 medicinal plants used for the treatment of various illnesses, while Bhat (2013) recorded

35 species for this area. Other studies have focussed more on documenting plants used for specific illnesses in certain regions, e.g. Afolayan et al. (2014) - 106 plants used for skin conditions in the Amatole district, Gail et al. (2015) - 17 plant species used to treat

HIV and related opportunistic infections in the Alfred Nzo municipality, Ndamane et al.

4 (2015) - 17 plants to treat chest and cough related diseases in the OR Tambo District, and Madikizela et al. (2012) - 34 species used to treat diarrhoea in the Bizana area.

Table 1, gives an indication of the ethnobotanical studies performed in South Africa from

2006 to 2015.

In the Limpopo Province, much of the documented ethnobotanical literature is on the

Bapedi and Vhavenda people. This is not surprising since they are the most dominant tribes (with the Afrikaans, Ndebeles, and Tsonga making up the rest of the population)

(Igumbor et al., 2003). The Province is dominated by rural areas whereby the use of traditional medicinal plants to treat diseases is highly practised (Vandebroek, 2013). The documentation of ethnobotanical knowledge in the Limpopo Province can be traced back to 1981, undertaken in the greater Gazankulu area situated in the Mopani District

(Liengme, 1981). Interestingly, the study was based on a broad spectrum of the ethnobotany of the Tsonga people and more than 200 plant species were recorded. Later on, Mabogo (1990) recorded approximately 245 plant species used for medicinal and non-medicinal purposes in the Vhembe District, specifically in the Venda region. This study laid a firm foundation and served as a basis and referral point for other ethnobotanical and antimicrobial studies (Samie et al., 2005, Steenkamp et al., 2007,

Mulaudzi et al., 2011; 2012). Within the Vhembe District, Steenkamp et al. (2007) documented 36 medicinal plants used against infectious diseases by the Vhavenda people. Mahwasane et al. (2013), documented 16 medicinal plants used for various human ailments by the traditional healers situated in the Lwamondo area, also found in the Vhembe district. As far as the documentation of the Bapedi ethnobotany is concerned, no comprehensive review has been published, much emphasis has been on the medicinal plants used for the treatment of various ailments such as diarrhoea, diabetes mellitus,

5 reproductive problems, tuberculosis, etc. The majority of this work has been published by

Semenya and co-workers (Semenya and Maroyi, 2012; 2012b; 2013a).

Table 1.2 Ethnobotanical studies conducted in various regions of South Africa No. of medicinal Ailment treated Geographical References plant species region recorded 21 Gastro-intestinal Limpopo Mathabe et al., disorders (diarrhoea) Province 2006) (Capricorn district) 23 Gastro-intestinal KwaZulu-Natal De Wet et al., 2010 disorder (diarrhoea) (Maputaland) 181 Various ailments Western Cape Philander, 2011 30 Respiratory infections Maputaland York et al., 2011 (KwaZulu-Natal) 34 Gastro-intestinal Eastern Cape Madikizela et al., disorder (diarrhoea) (Pondoland) 2012 18 HIV Eastern Cape Omoruyi et al., (Mpoza) 2012 20 Gastro-intestinal Limpopo Semenya and disorders (diarrhoea) Province Maroyi, 2012 (Capricorn, Sekhukhune and Waterberg district) 47 Skin disorders KwaZulu-Natal De Wet et. al., (Maputaland) 2013 19 Sexually transmitted Maputaland Naidoo et al., 2013 infections (STIs) (KwaZulu-Natal) 21 Respiratory infections Limpopo Semenya and (TB) Province Maroyi, 2013 (Capricorn, Sekhukhune and Waterberg district) 37 STIs Limpopo Semenya et al., Province 2013a (Capricorn, Sekhukhune and Waterberg district) 106 Skin disorders Eastern cape Afolayan et al., (Amathole 2014 District) 17 HIV and opportunistic Eastern Cape Gail et al., 2015 infections (Mpoza village)

6 17 Respiratory infections Eastern Cape Ndamane et al., (OR Tambo 2015 district)

1.2.2 Infectious diseases and medicinal plant use

1.2.2.1 Gastrointestinal disorders

Gastrointestinal disorders are very broad disorders and can originate from the oesophageal, gastroduodenal, bowel, and include abdominal pain (Drossman, 2006).

The World Health Organization (2003) defines gastroenteritis as the inflammation of the digestive tract, involving both the stomach and the small intestines which is characterized by symptoms such as stomach pain, diarrhoea, dysentery, vomiting, fever, inflammatory infections of the colon, and abdominal cramps. Of these, diarrhoea and dysentery have overwhelmed most of the African children under the age of five (Madikizela et al., 2012), and claim over nine million lives annually in the world as a result of poor sanitation and poor hygiene (WHO, 2009; De Wet et al., 2010). This condition is caused by both foodborne and waterborne pathogens (Mathabe et al., 2006). The mortality rate from diarrhoea is higher in rural areas than in urban areas due to the lack of clean water and this is perpetuated by the sharing of water with livestock as noticed in the Bizana area,

Pondoland region (Madikizela et al., 2012). The majority of the people in developing countries use medicinal plants to treat illnesses such as diarrhoea (Lin et al., 2002) and

Limpopo Province is no exception. The very first ethnobotanical study on the Bapedi traditional healers was undertaken by Mathabe et al. (2006), documenting 21 medicinal plants used to treat diarrhoea within the Capricorn district, whereas Semenya and Maroyi

(2012) recorded 20 medicinal plants used in Capricorn, Sekhukhune, and Waterberg districts. Punica granatum L., Grewia bicolor Juss., Dombeya rotundifolia Hochst.,

Commiphora marlothii Engl., and Senegalia Senegal (L.) Britton & P. Wilson are highly

7 preferred by the Bapedi traditional healers to treat diarrhoea (Semenya and Maroyi,

2012a).

Medicinal plant species such as Rapanea melanophloeos (L.) Mez., Ficus craterostoma

Warb. ex Mildbr. & Burret, A. ferox, Ledebouria ovatifolia Roth, Dais cotinifolia L.,

Berkheya bipinnatifida (Harv.) Roessler subsp. bipinnatifida, Alysicarpus rugosus (Willd.)

DC., Cyperus dives L., Senecio serratuloides DC., Maesa lanceolata Forssk., and

Brunsvigia grandiflora Lindl. were recorded for the first time for the treatment of diarrhoea in the South African Materia Medica (Madikizela et al., 2012). The juxtaposing of Mabogo

(1990) with Semenya and Maroyi (2013) revealed that Sanseviera hyacinthoides (L.)

Druce and Ximenia americana L. were the only common plant species utilised by both the Bapedi and Vhavenda traditional healers to treat diarrhoea. The Anacardiaceae and

Asteraceae emerged as the most dominant plant families used for combating diarrhoea in the Limpopo Province (Semenya and Maroyi, 2012a). In the Capricorn district, preference is given to the use of stem bark (Mathabe et al., 2006), and roots to prepare traditional medication against diarrhoea (Semenya and Maroyi, 2012a).

1.2.2.2 Respiratory disorders

According to the WHO (2005), chronic respiratory infections are the leading cause of human suffering globally. Acute respiratory infections claim over four million lives especially in children in the world annually (York et al., 2011). In developing countries, especially South Africa, influenza, pneumonia, and tuberculosis (TB) cause the highest mortality rates (Shann et al., 1999; Statistics South Africa, 2005; York et al., 2011).

The co-existence of TB with HIV/AIDS has become a greater concern with exerted research efforts to find ways to combat TB infection. The TB infection causes symptoms

8 such as coughing, fever, haemoptysis, chest pain, fatigue, and weight loss. Tuberculosis spreads through sneezing and cough and is thus perpetuated by crowded and poor living conditions (Pereira et al., 2005). In the Vhembe district, medicinal plants such as

Combretum molle (Sond.) and Ficus sycomorus (L.) are used for respiratory ailments

(Mabogo, 1990) whereas Combretum hereroense Schinz. and Ficus carica L. are preferred by the Bapedi traditional healers specifically for the treatment of tuberculosis

(Semenya and Maroyi, 2013). When comparing Mabogo (1990) and Semenya and

Maroyi (2013), it is evident that the only medicinal plants used in common by the Bapedi and the Vhavenda traditional healers for respiratory ailments are Myrothamnus flabellifolius (Sond.) and L. javanica.

Researchers such as Van Wyk and Gericke (2000), Semenya and Maroyi (2013), have noted the use of A. afra for the treatment of respiratory ailments including asthma, bronchitis, colds and coughs apart from TB which is ranked the second death causing agent in the Limpopo Province based on mortality profile report of 2013 (Statistics South

Africa, 2015). Preference of both A. afra and L. javanica was highlighted as well as exotics such Cannabis sativa L. used mainly to treat dry cough which can be a symptom of TB

(Hutchings, et al., 1996; Semenya and Maroyi, 2013)

1.2.2.3 Skin infections

Skin is the largest organ of the body and is associated with a number of vital roles which

include protection, thermoregulation, percutaneous absorption, and having both secretory and sensory activities (Njoroge and Bussmann, 2007). The association of skin disease with Human Immunodeficiency Virus, Acquired Immunodefiency Syndrome (HIV/AIDS) has become a major concern as more than 90% of individuals living with HIV/AIDS

9 encounter skin and mucosal complications such as mucosal erosion and ulcers

(Tschachler et al., 1996; De Wet et al., 2013). Medicinal plants are perceived to quicken healing of wounds, stop bleeding, and improve skin condition, and for these reasons they are very popular in rural areas for treating skin infections (Naidoo and Coopoosamy,

2011). More than 100 medicinal plants have been recorded in southern Africa to treat dermatological problems (Mabona and Van Vuuren, 2013; De Wet et al., 2013).

Transmission of skin infections from one person to the other can be perpetuated by overcrowded households situated in hot and humid climate (De Wet et al., 2013). Some studies (Hutchings et al., 1996; De Wet et al., 2013) performed in South Africa have shown a high incidence of overcrowding in coastal regions than inland regions. Bapedi traditional healers utilize only two exotic plant species to treat skin ailments, whereas in the Vhembe district C. marlothii is used to treat skin condition called Pellagra characterised by drying and peeling of the skin (Mabogo, 1990). Rapanea melanophloes

(L.) Mez, Solanum panduraeforme (E.Mey), and Rauvolfia caffra (Sond.) are used in the

Vhembe region for the treatment and dressing of wounds with Diospyros whyteana

(Hiern) F. White used for skin rashes (Mabogo, 1990).

The most common skin infliction amongst many people noticeably in teenagers is acne vulgaris, characterized by the inflamed sebaceous glands and hair follicles in the skin

(Magin et al., 2006). Plant species such as Psidium guajava L. (Guiterrez et al., 2008),

Elephantorrhiza elephantina (Burch.) skeels and Eucalyptus camaldulensis Dehnh. have been reported to combat the acne condition (Mabona, 2013). Skin infections in the

Limpopo Province remain one of the least researched and investigated ailments.

1.2.2.4 Sexually transmitted infections (STIs)

1 0 According to the WHO (2007) report, developing countries are faced with over a million

STI infections annually (Semenya et al., 2013a). Globally, STIs have the highest mortality rate with a large number of death cases occurring in Africa (Tshikalange et al., 2005).

Southern Africa is reported to be leading in the world with STI transmission rates (Van

Vuuren and Naidoo, 2010). Sexually transmitted infections and HIV/AIDS are currently ranked the nineth cause of death in the Limpopo Province (Statistics South Africa, 2015).

The Vhavenda people make use of 19 medicinal plant species as reported by Mabogo

(1990) to treat STIs (venereal diseases). Recently Masevhe et al. (2015), focused on medicinal plants used to treat candidiasis in the main areas of Venda (Mashau, Mutale,

Nzhelele and Thohoyandou), and a total of 45 medicinal plants were reported by the traditional healers within those villages. Sexually transmitted infections are treated with

37 indigenous plant species by Bapedi traditional healers (Semenya et al., 2013a). When comparing the Bapedi and the Vhavenda pharmacopeia, specifically on the treatment of

STIs, only E. elephantina is used in common and its use is recorded only by the Bapedi traditional healers of Sekhukhune district (Semenya et al., 2013a). Medicinal plants such

Annona senegalensis Pers. and Terminalia sericea Burch ex. DC are recorded for STIs

(Mabogo’s 1990).

Most plant species utilised by the Bapedi traditional healers for the treatment of STIs emerged from the following plant families; Asteraceae (e.g. Callilepis salicifolia Oliv.),

Fabaceae (e.g. Burkea africana Hook.), Asphodelaceae (e.g. Aloe marlothii A. Burger subsp. marlothii), and Hyacinthaceae (e.g. Drimia elata Jacq.). The underground parts were also preferred by the Bapedi traditional healers for treating STIs (Semenya et al.,

2013a).

11 1.3 Antimicrobial activity

Medicinal plants like all other plants possess secondary metabolites, used in defence against micro-organisms (Yazaki et al., 2008). Some micro-organisms are causative agents of infectious diseases such as gastro-intestinal disorders (e.g. Escherichia coli), respiratory infections (e.g. Mycobacterium tuberculosis), skin infections (e.g

Staphylococcus epidermidis) and also STIs (e.g. Neisseria gonorrhoeae). These micro­ organisms are prone to develop resistance towards pharmaceuticals (Masoko and

Nxumalo, 2013). The efficacies of some of the medicinal plants used for the treatment of the infectious diseases have been validated in numerous antimicrobial studies across

South Africa (Kelmanson et al., 2000; Motsei et al., 2003; Steenkamp et al., 2007; van

Vuuren, 2008; Buwa and Afolayan, 2009; Fawole et al., 2009; Ncube et al., 2012).

In the Limpopo Province, antimicrobial studies were performed on medicinal plants that treat diarrhoea: 21 medicinal plants - Mathabe et al. (2006), 16 medicinal plants - Shai et al. (2013). Five medicinal plants used for the treatment of TB were screened by Masoko and Nxumalo (2013). Tshikalange et al. (2005), investigated the antimicrobial activity of six medicinal plants whereas Steenkamp et al. (2007) screened 32 medicinal plants used by the Vhavenda people to treat STIs. The amount of ethnobotanical studies conducted versus antimicrobial studies reveals that there is still paucity on antimicrobial studies

(specifically pathogen-specific tests) in the Limpopo Province to validate the use of medicinal plants. So far gaps still exist for screening of medicinal plants used for skin infections against the causative pathogens.

1 2 1.4 Phytochemistry

Secondary metabolites are compounds not essential for the normal growth, development or reproduction of the plant but rather may serve for defence against micro-organisms,

insects and protection against UV light from the sun (Yazaki et al., 2008; Vaishnav and

Demain, 2010). Secondary metabolites are present in all plants including medicinal plants and are therefore of great benefit to human beings. They are incorporated into pharmaceuticals, fragrances, and dyes (Yazaki et al., 2008; Ramawat et al., 2009). They are categorised into distinct groups based on their chemical properties: nitrogen and non­ nitrogen containing compounds, and sulphur containing groups (Raghuwashni and Sinha,

2014). The constituents of these groups include alkaloids, flavonoids, lignins, phenolics, sterols, tannins, and terpenes (Ramawat et al., 2009).

Secondary metabolites possess numerous medicinal properties such as antibacterial, anticancer, antifungal, antiparasitic, and act as an immune-suppressants (Raghuwanshi and Sinha, 2014; Vaishnav and Demain, 2010), and are thus the compounds having

importance in the combating of micro-organisms that give rise to infectious diseases.

Tshikalange et al. (2005), has validated the antibacterial activity of the compound Luteolin extracted from Senna petersiana (Bolle) Lock, which was previously investigated in studies conducted by Mori et al., 1987; Basile et al., 1999; Sato et al., 2000. The various medicinal plants mentioned during this ethnobotanical survey (and previously not tested), will be screened for their phytochemical properties to validate traditional use.

1.5 Study area

The study area (Ga-Mashashane) depicted in Figure 1.1 is situated in the Capricorn district (23°53’0” S, 29°7’60”E), Limpopo Province between Polokwane city and

1 3 Mokopane. The area is made up of 22 rural villages occupied by a mixture of the Bapedi,

Ndebeles, and the Tsongas. Patients scattered amongst various villages of Ga-

Mashashane area make use of a single clinic and mobile clinics to attend to their health needs. However, there are patients within the area that make use of traditional medicine by consulting traditional healers, herbalists, and also perform self-medication for various ailments. This study focuses on gathering ethnobotanical information from individuals around Ga-Mashashane area as opposed to most ethnobotanical studies previously conducted, where knowledge was attained from traditional healers. The study area is diverse (three ethnic groups) and thus enhances a good chance to excavate new ethnobotanical knowledge.

Figure 1.1 Study area: Ga-Mashashane area situated within the Capricorn district, Limpopo Province (ArcGIS). 1.6 Aims and Objectives

1.6 AIMS & OBJECTIVES

The aim of the study is to evaluate the ethnobotany of the medicinal plants used in Ga-

Mashashane area in the Limpopo Province. Furthermore the study aims at validating the use of the medicinal plants based on their antimicrobial and phytochemical properties.

The information obtained is then compared with available literature on medicinal plant use in Limpopo in general, in order to identify previously unrecorded plants and new uses.

Specific objects are to:

(a) Conduct an ethnobotanical survey amongst 20 participants scattered within

Ga-mashashane area.

(b) Gather the medicinal plants from the wild, and confirm the identity of these

plants by comparing with voucher specimens at the herbarium of University of

Johannesburg (JRAU).

(c) Perform pathogen-specific antimicrobial screening.

(d) Perform phytochemical screening of unscreened medicinal plants.

1 5 CHAPTER 2

Materials and Methods

1.1 Ethnobotanical survey

A survey was conducted from October 2014 to March 2015 to gather ethnobotanical knowledge from 20 participants residing within the 22 villages of Ga-Mashashane area situated in the Capricorn District. The panel of participants was composed of 12 males

(including our primary informant; Mr Elias Moshemane Papo) and eight females and were taken through ethical clearance (Appendix 1) obtained from the University of

Johannesburg.The study was translated into the local vernacular language of Sepedi.

The survey was conducted in the form of structured interviews using questionnaires

(Appendix 2) to source information such as sociodemographic data (age, gender and educational background), plant information (plant parts used, plant form) and the administration method used.

1.2 Collection of plant material

Plant specimens were collected during the same period when the survey was conducted with the assistance of Mr Elias Moshemane Papo. Plants were mostly collected from their natural habitat and very few were harvested from households. Identification of the plants was done at the University of Johannesburg herbarium (JRAU) where voucher specimens have also been deposited. The plants were then washed, air dried for a period of approximately one week and then ground into fine powder for both antimicrobial and phytochemical screening.

1 6 1.3 Phytochemical screening Phytochemical screening was performed according to the method by Tiwari et al. (2011) and Zohra et al. (2012).

1.3.1 Preparation of plant extracts

Different plant parts were dried at room temperature and ground to a fine powder with an electrical grinder (MRC Lab equipments). Plant extractions were performed using chloroform, dimethyl-ether, ethanol, methanol, and water. Two and half grams of powdered material were mixed with 25 ml of each solvent. The mixtures were left overnight, at room temperature, and were covered with para-film to avoid evaporation.

Thereafter, the extracts were filtered through Whatman No. 1 filter paper, using the

Buchner funnel. The filtrates prepared from various solutions were weighed, recorded and then stored at 4°C for further use. The extracts were then screened for the presence of alkaloids, flavonoids, coumarins, terpenoids, saponins, tannins, and glycosides as described as follows:

1.3.1.1 Alkaloids The filtrate was treated with Wagner’s reagent which composed of 1.27 g of iodine and 2 g of potassium iodide in 100 ml distilled water. The formation of reddish-brown precipitate indicated presence of alkaloids.

1.3.1.2 Flavonoids The filtrate was treated with a few drops of sodium hydroxide. Addition of few drops of dilute hydrochloric acid (HCl) turned the yellow solution to colourless as an indication for the presence of flavonoids

1 7 1.3.1.3 Coumarins Five ml of ethanolic solutions was evaporated and the residue was dissolved in 2 ml of hot water. The solution was then divided into two halves, one half served as a control while 0.5 ml of 10% ammonium hydroxide was added to the other. A few drops of each solution were blotted on a piece of filter paper and put underneath UV light. An intense fluorescence indicated the presence of the coumarins.

1.3.1.4 Terpenoids Presence of the terpenoids was tested through the Salkowki’s test. One ml of chloroform was added to the filtrate followed by a few drops of concentrated sulphuric acid.

Immediate production of reddish brown precipitate indicated the presence of terpenoids.

1.3.1.5 Saponins The filtrate was shaken vigorously to foam and allowed to stand for 20 minutes. The presence of saponins is confirmed by the presence of foam.

1.3.1.6 Tannins The filtrate was treated with a few drops of 1% gelatine solution which contained sodium chloride. Formation of white precipitate indicated the presence of tannins.

1.3.1.7 Glycosides The Modified Borntragers test was incorporated for the detection of glycosides. The filtrates were treated with ferric chloride and immersed in boiling water for a period of 5 minutes. The mixture was then cooled and added to benzene, which was later separated from the mixture and treated with ammonia solution. The presence of the glycosides was detected by the pink colour formation within the ammonical layer.

1 8 1.3.2 Sample extraction

Extraction of samples for antimicrobial screening was performed using (i) distillation technique in the case of essential oils, (ii) distilled water for aqueous extraction, and (iii) organic solution consisting of dichloromethane and methanol at a ratio of 1:1. For the organic extraction, an empty flask was weighed prior to and after addition of plant material. Solution of dichloromethane and methanol was added to the ground material at a ratio of 1:1, sealed with para-film to minimize evaporation and then put in the shaker

incubator at 37 °C for 24 hours. After the 24 hour duration the supernatant was poured

in a beaker. The pellet from the first mixture was then re-mixed with dimethyl ether- methanol solution at a ratio of 1:1 and incubated for another 24 hours. The second supernatant was then added to the first and put underneath a fume hood to enhance dryness within 3 days. For the effective transfer of dry mass solution into a weighed and

labelled vial, acetone was added into the beaker with the residue and then transferred

into the vial with the total mass recorded. Once the acetone has evaporated from the vial, the dry mass was weighed and the yield determined.

Aqueous extracts were prepared by weighing an empty flask and recording the mass, followed by placing of ground powdered material. The total mass of both the flask and the ground material was weighed and recorded followed by the addition of sterile water at an equal volume to the ground powder material and incubated at 37°C for 24 hours. The supernatant was then transferred into a previously weighed and labelled vial, and then frozen at -80°C for 24 hours. To drain all the moisture, the vial was then put into a

lyophilizer (United Scientific (Pty) Ltd.) and the final dry residue weighed and recorded to determine the yield.

1 9 Essential oils were obtained through oil distillation technique using the Clevenger apparatus. Cold water of about 1L was added to cover the plant material and the system was switched on and left to run for about 3 hours to enable enough distillation of the oil.

When the 3 hour period had elapsed, the oil was collected in a weighed vial and the yield was calculated according to Table 2.1

2 0 Table 2.1 The calculation of medicinal plant yields used for antimicrobial screening. Species Name Voucher Parts Extraction Empty Flask + Empty vial Vial + dry Plant Extract (D % yield number Used flask (A) plant (C) mass or material (B - C = F) (F/E x 100) material ess. oil (D) - A = E) (B) Acokanthera oppositifolia LP 29 Lvs Org 102.14 112.63 4.9812 5.2352 10.49 0.2540 2.42 (Lam.) Codd Aq 148.92 159.1 4.9396 5.2349 10.18 0.2953 2.9

Aloe arborescens Mill. LP 04 Lvs Org 99.3 109.56 4.9464 5.0342 10.26 0.0878 0.86 Aq 106.05 116.75 4.8383 5.0340 10.70 0.1957 1.83

Artemisia afra Jacq. ex Willd LP 02 Lvs Org 87.52 97.64 4.8381 5.8749 10.12 1.0368 10.25 Aq 155.71 165.91 4.8606 5.8747 10.20 1.0141 9.94 Ess. oil 84.83 184.93 5.0134 7.1272 100.1 2.1138 2.11 Asparagus racemosus Willd. LP 33 Rts Org 113.14 123.18 4.9305 5.0093 10.04 0.0788 0.78 Aq 112.56 119.62 4.9194 5.0068 7.060 0.0874 1.24

Callilepis laureola DC. LP 18 Blb Org 84.83 94.84 4.9101 5.3563 10.01 0.4462 4.46 Aq 182.91 193.13 4.9275 5.3560 10.22 0.4285 4.19

Carissa bispinosa (L.) Desf. LP 15 Rts Org 95.71 105.84 4.9971 5.1013 10.13 0.1042 1.03 ex Brenan Aq 93.12 103.99 4.8776 5.1010 10.87 0.2234 2.06

Carpobrotus edulis (L.) L. LP 42 Lvs Org 87.8 97.92 4.9353 5.1226 10.12 0.1873 1.85 Bolus Aq 159.21 170.02 4.9150 5.1201 10.81 0.2051 1.9 Clerodendrum glabrum LP 35 Lvs Org 69.78 79.79 4.9344 5.4227 10.01 0.4883 4.88 E.Mey. var. glabrum Aq 172.38 182.58 4.8690 5.4202 10.20 0.5512 5.4 Combretum imberbe LP 11 Brk Org 117.72 127.73 4.9059 4.9540 10.01 0.0481 0.48 Wawra. Aq 168.8 178.82 4.9132 4.9538 10.02 0.0406 0.41

Datura stramonium L. LP 22 Lvs Org 100.49 110.54 4.9268 5.0939 10.05 0.1671 1.66 Aq 98.48 108.58 4.8429 5.0936 10.10 0.2507 2.48

Dicoma anomala Sond. LP 03 Tbr Org 84.56 94.04 4.9825 5.1164 9.480 0.1339 1.41 subsp. anom ala Aq 92.77 102.05 4.8588 5.1162 9.280 0.2574 2.77 21 Species Name Voucher Parts Extraction Empty Flask + Empty vial Vial + dry Plant Extract (D % yield number Used flask (A) plant (C) mass or material (B - C = F) (F/E x 100) material ess. oil (D) - A = E) (B) Dombeya rotundifolia LP 37 Lvs Org 66.71 76.81 4.9349 5.4880 10.10 0.5531 5.48 (Hochst.) Planch. Aq 150.39 160.54 4.8483 5.4855 10.15 0.6372 6.28

Drimia elata Jacq. LP 34 Blbs Org 106.99 118.4 5.0132 5.2376 11.41 0.2244 1.97 Aq 97.52 108.71 4.9223 5.2351 11.19 0.3128 2.8 Elaeodendron transvaalense LP 01 Rts/Brk Org 100.81 111.31 4.9210 6.5952 10.50 1.6742 15.94 (Burtt Davy) R.H. Archer A 100.57 110.82 4.8714 5.7950 10.25 0.9236 9.01

Elephantorrhiza elephantina LP 45 Blb Org 85.56 96.04 4.9815 5.1064 10.48 0.1249 1.19 (Burch.) Skeels Aq 95.72 100.99 4.8776 5.1039 5.270 0.2263 4.29 Eucalyptus camaldulensis LP 12 Lvs Org 108.41 118.76 4.9529 5.6513 10.35 0.6984 6.75 Dehnh. Ess. oil 98.30 194.93 4.9194 7.3826 96.63 2.4632 2.549 Aq 96.32 105.46 4.8689 5.6511 9.140 0.7822 8.56 Euclea crispa (Thunb.) LP 32 Rts Org 84.83 95.68 4.9201 5.4563 10.85 0.5362 4.94 Gurke subsp. crispa Aq 94.29 103.91 4.8894 5.4538 9.620 0.5644 5.87

Grewia occidentalis L. LP 38 Lvs Org 96.79 103.94 4.9978 5.1003 7.150 0.1025 1.43 Aq 113.27 120.37 4.8967 5.0978 7.100 0.2011 2.83 Helichrysum caespititum LP 08 Rts Org 87.12 92.14 4.9157 5.1024 5.020 0.1867 3.72 (DC.) Harv. Aq 95.34 100.46 4.8689 5.1022 5.120 0.2333 4.56

Jatropha zeyheri Sond. LP 07 Tbr Org 95.45 106.16 4.9374 5.1130 10.71 0.1756 1.64 Aq 141.93 152 4.9207 5.1128 10.07 0.1921 1.91

Lannea edulis (Sond.) Engl. LP 36 Lvs Org 86.57 97.4 4.9112 5.0884 10.83 0.1772 1.64 Aq 94.4 104.17 4.8965 5.0859 9.770 0.1894 1.94

Lantana rugosa Thunb. LP 19 Lvs Org 97.93 108.07 4.9328 5.1777 10.14 0.2449 2.42 Aq 94.72 104.09 4.8776 5.1774 9.370 0.2998 3.2 Ess. oil 95.71 176.24 4.8294 5.3194 80.53 0.49 0.61

2 2 Species Name Voucher Parts Extraction Empty Flask + Empty vial Vial + dry Plant Extract (D % yield number Used flask (A) plant (C) mass or material (B - C = F) (F/E x 100) material ess. oil (D) - A = E) (B) Ledebouria revoluta (L. f.) LP 31 Rzm Org 101.49 110.64 4.9268 5.0939 9.150 0.1671 1.83 Jessop Aq 105.29 110.38 4.8726 5.0936 5.090 0.2210 4.34

Lippia javanica (Burm.f.) LP 24 Lvs Org 105.85 115.96 4.9582 5.1254 10.11 0.1672 1.65 Spreng Aq 94.9 99.91 4.8894 5.1251 5.010 0.2357 4.7 Ess. oil 106.05 205.31 4.9112 7.5803 99.26 2.6691 2.69 Ozoroa insignis Delile LP 39 Lvs Org 107.99 118.4 5.0134 5.2366 10.41 0.2232 2.14 Aq 99.48 109.58 4.8429 5.2341 10.10 0.3912 3.87

Ozoroa sphaerocarpa R. & LP 27 Brk Org 97.19 104.94 4.8978 5.1103 7.750 0.2125 2.74 A. Fern. Aq 98.52 108.71 4.9223 5.1100 10.19 0.1877 1.84 Peltophorum africanum LP 40 Brk Org 98.17 108.29 5.0788 5.4985 10.12 0.4197 4.15 Sond. Aq 111.61 121.9 4.9123 5.4960 10.29 0.5837 5.67 Ptaeroxylon obliquum LP 14 Lvs Org 65.71 76.81 4.9339 5.4781 11.10 0.5442 4.9 (Thunb.) Radlk Aq 68.02 79.27 4.9199 5.4778 11.25 0.5579 4.96

Punica granatum L. LP 43 Lvs/Rts Org 94.79 104.94 4.9978 5.1003 10.15 0.1025 1.01 Aq 96.24 105.46 4.8689 5.0978 9.220 0.2289 2.48

Ricinus communis L. var. LP 13 Lvs Org 107.99 118.51 4.9316 5.1208 10.52 0.1892 1.8 co m m un is Aq 97.06 102.1 4.8558 5.1206 5.040 0.2648 5.25

Schotia brachypetala Sond. LP 26 Lvs Org 86.55 96.95 4.8652 5.9987 10.40 1.1335 10.9 Aq 117.87 123.38 4.8690 5.9984 5.510 1.1294 20.5

Searsea lancea (L.f) F.A. LP 10 Frt Org 99.3 109.47 4.9433 5.4868 10.17 0.5435 5.34 Barkley Aq 95.77 101.05 4.8588 5.4866 5.280 0.6278 11.89 Siphonochilus aethiopicus LP 41 Rts Org 84.83 94.84 4.9101 5.3563 10.01 0.4462 4.46 (Schweif.) B.L. Burt Aq 93.89 98.91 4.8894 5.3538 5.020 0.4644 9.25

2 3 Species Name Voucher Parts Extraction Empty Flask + Empty vial Vial + dry Plant Extract (D % yield number Used flask (A) plant (C) mass or material (B - C = F) (F/E x 100) material ess. oil (D) - A = E) (B) Solanum supinum Dun. LP 21 Rts Org 109.41 119.76 4.9629 5.6513 10.35 0.6884 6.65 Aq 113.25 121.37 4.8967 5.6510 8.120 0.7543 9.29

Vangueria infausta Burch. LP 09 Lvs Org 104.95 114.96 4.9682 5.1164 10.01 0.1482 1.48 subsp. infau sta Aq 175.74 185.91 4.8606 5.1162 10.17 0.2556 2.51

Ximenia caffra Sond. var. LP 30 Lvs Org 108.41 119.76 4.9629 5.6513 11.35 0.6884 6.07 caffra Aq 97.64 108.03 4.9024 5.6510 10.39 0.7486 7.21

Zanthoxylum humile (E.A LP 17 Brk Org 99.17 108.29 5.0688 5.4995 9.120 0.4307 4.72 Bruce) P.G. Waterman Aq 103.6 113.11 4.9247 5.4992 9.510 0.5745 6.04 Ziziphus mucronata LP 44 Lvs Org 135.89 146.53 4.9752 5.2346 10.64 0.2594 2.44 Aq 69.02 79.27 4.9199 5.2321 10.25 0.3122 3.05 Plant plants: Brk, bark; Blb, bulb; Frt, fruits; Lvs, leaves; Rzm, rhizome; Rts, roots; Tbr, tuber. Type of extract: Ag, aqueous; Ess. oil, essential oils, Org, organic.

2 4 1.4 Antimicrobial assay

1.4.1 Selection of bacteria

The selection of bacteria was based on the type of the infection to be treated (Table 2.4)

Table 2.2 Pathogens used for antimicrobial screening of medicinal plants in the Ga- Mashashane area. Category of ailments Pathogens and strain number

Gastro-intestinal disorders Bacillus cereus ATCC 11775 Enterococcus faecalis ATCC 29212 Escherichia coli ATCC 8739 Salmonella typhimurium ATTC14028 Shigella sonnei ATCC 9290 Listeria monocytogenes ATCC19111 Respiratory ailments Klebsiella pneumoniae ATCC 13883 Moraxella catarrhalis ATCC 23246 Staphylococcus aureus ATCC 29123 Skin infections Candida albicans ATCC10231 Pseudomonas aeruginosas ATCC9027 Staphylococcus aureus ATCC29213 Staphylococcus epidermidis ATCC12228 STIs Candida albicans ATCC10231 Gardnerella vaginalis ATCC14028 Neisseria gonorrhoeae ATCC19424

1.4.1.1 Antimicrobial activities

For the MIC study, a starting concentration of 32 mg/ml was determined using the extraction yield and the amount of water/essential oils that had to be added. The antibiotic ciprofloxacin (Sigma) was used as the positive control and was made up to a starting concentration of 0.01 mg/ml, with the negative control (acetone prepared with water) prepared to the same concentration to those of plant extracts (32 mg/ml). The culture control was left overnight at room temperature and was used as an indicator for contamination by the presence of murkiness.

2 5 The MIC values were determined using the method described in Eloff (1998). In order to eliminate occurrence of contamination during the assay, ethanol was sprayed and wiped with autoclaved cotton wool to sterilise the laminar flow cupboard. This was followed by the opening of 96-well sterile microplates which were marked and numbered to avoid confusion.

Two types of broths were prepared for the MIC assay; the Tryptone Soya broth (TSB,

Oxoid) and Mueller-Hinton broth (MHB, Oxoid). The former was prepared by weighing 30 g of TSB powder which was added into 1 L Schott bottle and dissolved with 1 L of purified water, and then autoclaved at 121°C for a duration of 15 minutes. The latter was prepared by weighing 21 g of MHB powder, followed by dissolving in 1 L purified water and autoclaved at similar conditions as mentioned above. Fifty ml of sheep blood was added to 1 L of MHB to make a 5% solution concentration.

Depending on the pathogen being screened against, 100 pl of the broth was added into the columns of the microplates then followed by the pipetting of 100 pl of the plant extract or essential oils across the first row. The plant extracts, essential oils, and both positive and negative controls prepared in triplicates were then serially diluted from the first row

(8 mg/ml) of the microplate using a multichannel pipette to the last row (0.075 mg/ml) with the discarding of the last 100 pl withdrawn from the last row.

This was followed by the addition of 100 pl of bacterial culture indicated in Table 2, across the columns of the microplates. The 1X106 colony forming units (CFU) was attained by diluting bacterial cultures at ratio of 1:100, where by 500 pl of bacterial culture was diluted into 50 ml broth. Once the bacterial cultures were added into the wells, the plates were then sealed and incubated overnight. The viability of the bacteria was determined by streaking bacteria on the media plates and incubating overnight along with the broth.

2 6 After 24 hours (except for Neisseria gonorrhoeae and the yeast Candida albicans which both required 48 hours of incubation), the reagent p-Intiodonitrotetrazolium chloride (INT)

(Sigma-Aldrich) was added to the wells. The reagent was prepared by weighing 0.08g

INT and dissolving in 200 ml of pure water on a shaker incubator for duration of 30 minutes. Forty gl of INT was pipetted into wells and after three hours detection of results was performed - the pink colour formation indicated bacterial growth. The microplates were then put on a lightbox and read. The results were then recorded on the template sheet, and the MIC values averaged to determine the actual MIC value. The MIC value less than 1.0 mg/ml was recorded as noteworthy activity for extracts (Gibbons, 2004; Rios and Recio, 2005) and essential oils (Orchard and van Vuuren, 2016), whereas MIC values greater than 8.0 g/ml were recorded as poor activity.

2 7 CHAPTER 3

Results and Discussion

2.1 Ethnobotany

The results of the ethnobotanical knowledge obtained from 20 informants (12 males and eight females) residing in the Ga-Mashashane area is summarised in Table 3.1. The average age was 64.5 years and the oldest and youngest informants were both females aged 90 and 34 years respectively. The oldest male informant was 84 years while the youngest was 39. The informants were selected randomly, however, preference was given to men as they tend to regularly visit the field and were knowledgeable on various localities for collecting the plants. Due to the fact that there were more males than females, men contributed more ethnobotanical information, with the primary informant contributing the highest number of 31 medicinal plants. The ethnobotanical survey has recorded 45 medicinal plants, belonging to 43 genera in 26 plant families used in the Ga-

Mashashane area for treating various infectious ailments including gastro-intestinal disorders, respiratory ailments, skin conditions, sexually transmitted infections (STIs), as well as non-pathogenic ailments.

2 8 Table 3.1 Ethnobotanical information of medicinal plants used in Ga-Mashashane based on interviews and literature (new uses writen in bold)______Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Acokanthera oppositifolia LP 2 9 Tsebe-dinthla L e a v e s Stomach and respiratory O r a l Stress related ailm ents (Hutchings and

(Lam.) Codd (Apocynaceae) ailm ents, also used as blood Van Staden, 1994), gynaecological p u r i f ie r complaints, (Steenkamp, 2003),

toothache, stomach ache, colds, anthrax,

tapeworm s, spider and snake bites

(Kumar and Singh, 2005)

A lo e arborescens M il l. LP 0 4 A lo e L e a v e s Inflam ed feet or swollen feet Soaking/wash W ounds, burns and various skin problems (Asphodelaceae) (inflam m ation) (Bruce, 1975; Van W yk et al., 2000),

cardiac problems (Amusan et al., 2002),

gastrointestinal complaints, blood

purifier (Philander, 2011), STIs (Semenya

et al., 2013a)

Artemisia afra Jacq. ex W illd LP 0 2 L e n g a n a L e a v e s Respiratory ailm ents such as cold O r a l a n d Stomach-ache, dry dyspepsia, malaria (Asteraceae) a n d flu in h a la t io n (Burits et al., 2001; Van W yk and Gericke,

2000; Vagionas et al., 2007; Semenya and

M aroyi, 2013), infertility (W att and

Breyer-Brandwijk, 1962; Steenkamp,

2 0 0 3 )

Asparagus racemosus W illd . LP 3 3 Morakadimane R o o ts B o d y s o re s O r a l Gonorrhoea, piles, diabetes, rheumatism , (Asparagaceae) cough, diarrhoea, dysentery, gastric

troubles, headache (Sharma and Sharma ,

2 0 1 3 )

Callilepis laureola D C . LP 1 8 P h e la B u lb Lung problem / respiratory O r a l Tape worm infestations, cough (W att and (Asteraceae) p r o b le m s Breyer-Brandwijk, 1962), induces fertility

(Debetto, 1978; Seedat and Hitchcock,

1971; Semenya, 2013h), stomach

problems (Steenkamp et al., 1999),

vomiting, abdominal pain, headache

(W ink and Van W yk, 2008)

2 9 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Carissa bispinosa (L.) Desf. ex LP 1 5 M o r o k o l o R o o ts Chicken-pox, cough and sores O r a l Im potence (Amusan et al., 2002) coughs,

B r e n a n . (Apocynaceae) diarrhoea (M aroyi, 2013)

Carpobrotus edulis (L .) L. LP 4 2 D its h ip s L e a v e s Toothache and digestive Oral, gargle Skin ailments (W att and Breyer-

B o lu s ailments, cough and rubbed on Brandwijk, 1962; Rood, 1994; Van W yk et (Mesembryanthemaceae) s o re s al., 2000; Van der W att and Pretorius,

2001; Van Vuuren and Mabona, 2013),

poultices for sores, burns, scalds,

infections of the m outh and throat,

dysentery, digestive troubles,

tuberculosis, diuretic (M artins et al.,

2005), STIs (Van Vuuren and Naidoo,

2 0 1 0 )

Clerodendrum glabrum LP 3 5 Mothlokothloko L e a v e s Stomach-ache, extract taken as a O r a l M alaria (Clarkson et al., 2004; Tshikalange

E .M e y . var. glabrum la x a tiv e et al., 2008) (Verbenaceae)

Combretum imberbe W a w r a . LP 1 1 M o h w e l e r e B a rk Burnt and sniffed for headache Smoke therapy Chest cough, fever and infections (Combretaceae) a n d f e v e r (Hutchings et al., 1996; McGaw et al.,

2 0 0 8 )

Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Datura stramonium L. LP 2 2 M o k h u r e L e a v e s Inflam ed body parts Soaking/wash Boils and abscesses W att and Breyer- (Solanaceae) Brandwijk , 1962; Bruton 1995; Rabe and

Van Staden , 1997; Van W yk et al., 2000;

Priya et al., 2002; McGaw and Eloff, 2008;

Saadabi & Moglad; 2011 and Mabona and

Van Vuuren, 2013)

Dicoma anomala S o n d . LP 0 3 H lo e n y a T u b e r Intestinal problems, stom ach­ Oral, direct Toothache, hypertension, diabetes, s u b s p . a n o m a la (Asteraceae) ache and wounds application period pains, pneumonia, backache,

wounds and sores (Van W yk and Gericke,

2000; M oteetee and Van Wyk, 2011)

3 0 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Dombeya rotundifolia LP 3 7 M o k g o b a L e a v e s The leaves are mixed along with O r a l Chest complaints (Smith, 1966; McGaw et

(Hochst.) Planch. other plants to quicken healing al., 2008), heart problems, nausea in

(Sterculiaceae) process of variety of ailm ents pregnant wom en, intestinal ulcers,

including stomach ailments headaches, stomach complaints,

haem orrhoids, diarrhoea and dyspepsia

(Reid et al., 2001)

Drimia elata J a c q . LP 3 4 S e k a n a m a B u lb s Blood purifier, also used as a T o p ic a l Used to relieve pain and asthma (Hyacinthaceae) rubbing ointm ent for swollen (Hutchings et al., 1996, van W yk et al., f e e t a n d S TIs. 2009; Ndhlala et al., 2013), arthritis

(Philander, 2011), gonorrhoea, HIV/AIDS

(Semenya et. al., 2013a)

Elaeodendron transvaalense LP 0 1 M o n a m a n e Roots/ Bark High-blood pressure a n d S TIs O r a l Dysmenorrhoea (van Wyk, 1972; (Burtt Davy) R.H. Archer Steenkamp, 2003) herpes ( Mabogo,

(Celastraceae) 1990; Bessong et al., 2005) HIV (Semenya

et al., 2013a) and impotence (Semenya et

al., 2013c)

Elephantorrhiza elephantina LP 4 5 M o s h i t s a n e B u lb Blood purification and stom ach­ O r a l Abdominal pains, perforated peptic

(Burch.) Skeels (F a b a c e a e ) a c h e ulcers, (bloody) diarrhoea, dysentery

(W att and Breyer-Brandwijk, 1962; Jacot

Guillarmod, 1971; Gelfand et al., 1985;

Hutchings, 1989; Pujol, 1990; M athabe et

al., 2006; Appidi et al., 2008; Bisi-Johnson

et al., 2010; Madikizela et al., 2012;

Mpofu et al., 2014)

3 1 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Englerophytum LP 2 8 M o h l a t s w e L e a v e s Leaf extract is taken as blood O r a l Contraceptive (Mabogo, 1990; magalismontanum (S o n d .) p u r ifie r . Steenkamp, 2003), root extract is used to

T . D . P e n n . (Sapotaceae) enhance fertility (Van der M erw e et al.,

2001; McGaw and Eloff, 2008), diabetes

(Semenya et al., 2012a)

Eucalyptus camaldulensis LP 1 2 M o b u l u k o m o L e a v e s Steam -vapour is inhaled for colds, O r a l a n d Diarrhoea, dysentery, haemorrhage,

D e h n h . (Myrtaceae) coughs, flu, stom ach-ache and in h a la t io n laryngalgia, laryngitis, pharyngitis, sore

s o re s throat, spasm, trachalgia, and wounds

(Duke and W ain, 1981; Mouna and Segni,

2011), treatm ent of acne (Hutchings,

1996; Babayi et al., 2004; Ayepola and

Adeniyi, 2008; Musa et al., 2011 and

Mabona, 2013), TB (Semenya and Maroyi,

2 0 1 3 )

Euclea crispa (Thunb.) Gurke LP 3 2 Mokwerekwere R o o t Coughs, STIs and respiratory O r a l Dysmenorrhoea (Steenkamp, 2003; subsp. crispa a il m e n t s M oteetee and Van Wyk, 2011), coughs (Ebenaceae) (M cGaw et al., 2008; Maroyi, 2013), bark

decoction drunk as a purgative (M oteetee

and Van W yk, 2011), HIV/AIDS (Semenya

et al., 2013a)

Eucomis pallidiflora B a k e r LP 2 5 Mathuba-difala R o o ts Used for im potence O r a l Erectile dysfunction, tuberculosis, blood subsp. pole-evansii (N .E .B r .) clotting, cough (Semenya and Potgieter,

Reyneke ex J.C.Manning 2013; Semenya et al., 2013b) (Asparagaceae)

3 2 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Grewia occidentalis L. LP 3 8 M o g w a n e L e a v e s STIs. The leaves are crushed and T o p ic a l The bark is soaked in w ater and used to

(Malvaceae) mixed with Vaseline and applied dress wounds (W att and Breyer-

to sores and wounds Brandwijk, 1962; Hutchings , 1996;

Grierson and Afolayan, 1999; Van

Vuuren, 2008; Mabona and Van Vuuren,

2013), infertility (Steenkamp, 2003), STIs

(M ulaudzi et al., 2011)

Helichrysum caespititum LP 0 8 M a t s a n a R o o ts Used as blood purifier, colds and O r a l Headaches and chest colds (W att and

(DC.) Harv. (Asteraceae) flu Breyer-Brandwijk, 1962; Jacot Guillarmod,

1971; Dekker et al., 1983; Gelfand et al.,

1985; Hutchings and Van Staden , 1994;

Neuwinger, 1996; Swanepoel, 1997;

Pooley, 1998; Mathekga et al., 2000;

Mathekga , 2001; Arnold et al., 2002;

M eyer et al., 2002; Pooley , 2003), STIs

(M oteetee and Van W yk, 2011; Erasmus

et al., 2012; Semenya et al., 2013a)

Hypoxis hemerocallidea LP 0 6 T s h ik a -p o o T u b e r Used as a pain-killer O r a l Bladder problems (Van W yk et al., 2013),

Fisch., C.A.Mey. & Ave- Lall inflam m ation, testicular tum ours, urinary

(Hypoxidaceae) complaints, cancer and HIV/AIDS (W att

and Breyer-Brandwijk, 1962; Crouch et al.,

2006; Ncube et al., 2012)

Hypoxis obtusa L. LP 0 5 Monna-maledu F ru its Used as a pain-killer O r a l Prostrate hypertrophy (Nel, 1914), urinary (Hypoxidaceae) diseases (M arini- Bettolo et al., 1985;

Galeffi et al., 2002), aphrodisiac,

gonorrhoea and infertility in wom en

3 3 (Gelfand et al., 1985; Semenya et al.,

2013b); impotence (Moeng, 2010);

abdominal pains (M aroyi, 2013)

Jatropha zeyheri S o n d . LP 0 7 Sefapabadia T u b e r Inflam ed feet or swollen feet Soaking/Wash M enstrual pains, irregular periods and to (Euphorbiaceae) (inflam m ation) ensure a strong foetus during pregnancy

(Van W yk and Gericke, 1997), STIs and

urinary tract infections (M ongalo et al.,

2013), purify blood, applied externally to

dress wounds and boils (Van W yk et al.,

2 0 1 3 )

Lannea edulis (Sond.) Engl. LP 3 6 Mokgolokgotwane L e a v e s Diarrhoea and stomach O r a l Sore eyes, boils and abscesses (W att and

(Anacardiaceae) complaints and wounds Breyer-Brandwijk, 1962; Hutchings, 1996;

Van W yk, 1997), constipation (Amusan et

al., 2002; Maroyi, 2011)

Lantana rugosa T h u n b . LP 1 9 Sekwebatana L e a v e s Eye-pains/problem s, used as eye Soaking/Wash The leaf paste is used to treat eye (Verbenaceae) purifier and for flu problems (W att and Breyer-Brandwijk,

1962; Mabogo, 1990; McGaw, 2008). The

Vhavenda people use the leaves and stem

o f L. rug osa to make a paste for

troublesome eyes (Mabogo, 1990).

Leaves, stems or ripe fruit paste treats

festering sores (Hutchings, 1996; Suliman,

2010; Mabona and Van Vuuren, 2013).

Ledebouria revoluta (L . f.) LP 3 1 L e h w a m a R h iz o m e Foot inflammation and stomach Soaking/Wash Used as an expectorant, diuretic, irritant

J e s s o p (Hyacinthaceae) a il m e n t s to gasto-intestinal tract (W att and Breyer-

Brandwijk, 1962; Dold and Cocks, 2001;

Abegaz, 2002; M oodley et al., 2006),

sores, wound and skin eruptions (M uleya

et al., 2014)

3 4 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Lippia javanica ( B u r m .f .) LP 2 4 Moshunkwane Leaves/twigs Steam -vapour inhaled for colds, In h a la t io n Paste (Gelfand et al., 1895; Hutchings et

S p r e n g (Verbenaceae) flu treatm ent al., 1996; van W yk et al., 1997; van W yk

and Gericke, 2000; Samie et al., 2005;

Mabona et al., 2013; Semenya and

M aroyi, 2013), skin disorders such as heat

rash, scratches, stings and bites

(M thethwa, 2009)

Mentha longifolia (L.) Huds. LP 2 3 M e n t h o l o L e a v e s Steam -vapour inhaled for colds, In h a la t io n Dressing of wounds (W att and Breyer- s u b s p . p o ly a d e n a (B r iq .) flu and respiratory ailments Brandwijk, 1962; Van W yk et al., 2000;

B riq . (Lamiaceae) Scott et al., 2004, McGaw and Eloff,

2008), headache, indigestion, insomnia

(M oteetee and Van W yk, 2011; Philander

2011; Van W yk, 2011; Mabona et al.,

2 0 1 3 )

Ozoroa insignis D e lile LP 3 9 M o n o k o a n a L e a v e s The leaves are used to treat O r a l Diarrhoea, STIs (M aroyi, 2011; 2013) (Anacardiaceae) digestive ailm ents and STIs

Ozoroa sphaerocarpa R. & A . LP 2 7 M o n o k o B a rk A s th m a , diarrhoea and wounds O r a l Dressing of wounds (Amusan et al., 2001),

F e rn . diarrhoea (Semenya et al., 2012a;

(Anacardiaceae) Sibandze et al., 2010), im potence

(Semenya et al., 2013c)

3 5 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Peltophorum africanum LP 4 0 M o s e h la B a rk The bark piece is kept in m outh O r a l Female infertility, post-partum depression

S o n d . (Leguminosae) like a lozenge to prevent stomach (Semenya et al., 2013b), eye infections,

infiltration in children known as joints and back pains, toothache,

"Makgom e".Bark decoction is diarrhoea, dysentery, infertility, skin

used also for diarrhoea, rash, STIs rashes, blisters, venereal diseases,

a n d flu depression, anthelm intic, coughs, sore

throat, ascites, abdominal disorders

(Mazim ba, 2014)

Ptaeroxylon obliquum LP 1 4 M o l o p e L e a v e s Running stomach O r a l Rheumatism, arthritis and heart disease

(Thunb.) Radlk (R u ta c e a e ) (W att and Breyer-Brandwijk, 1962;

Hutchings, 1996; Van W yk et al., 2009),

used as sneeze inducer (Hutchings et al.,

1996), headaches (Philander, 2011)

Punica granatum L. LP 4 3 M o g a r e n a t e Leaves/roots Stomach-ache and digestive O r a l Diarrhoea (M athabe et al., 2005; (Lythraceae) a il m e n t s Semenya and Maroyi, 2012; Semenya and

M aroyi, 2012), enhances fertility (Ndhlala

et al., 2013)

Ricinus communis L. var. LP 1 3 M o t h o b a L e a v e s Steam -vapour is inhaled for colds, In h a la t io n Applied as poultice in the treatm ent of co m m u n is (Euphorbiaceae) flu, sores and STIs boils (W att and Breyer-Brandwijk, 1962;

Hutchings, 1996; Van W yk et al., 2000;

Luseba et al., 2007; M alik et al.,2011;

Mabona et al.,2013), breast tumours,

m enstrual disorders, vaginal discharges (

De Boer and Cotingting, 2014)

3 6 Schinus molle L. LP 2 0 M o t h o b a L e a v e s Steam -vapour inhaled for colds, In h a la t io n Toothache, rheumatism, (Anacardiaceae) flu and respiratory ailments m enstrual disorders, respiratory and

urinary tract infections (Perez and

Anesini,1994; dos Santos et al., 2009)

Schotia brachypetala S o n d . LP 2 6 M o lo p a L e a v e s Used as an im mune booster O r a l Dysentery (Hutchings, 1996), ulcers (F a b a c e a e ) against various ailm ents (Venter and venter, 1996), Diarrhoea

(M cGaw et al., 2000; M athabe et al.,

2006; Shai et al., 2013)

Searsea lancea (L.f) F.A. LP 1 0 M o s o t lh o F ru its Measles in children and gastro­ O r a l Diarrhoea (Van der M erw e et al., 2001;

B a r k le y (Anacardiaceae) intestinal complaints McGaw and Eloff, 2008)

Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Senecio barbertonicus K la tt. LP 1 6 M a p h o lo L e a v e s Mixed with milk as to vom it and O r a l N o r e c o r d s (Asteraceae) curb heart-burn

Siphonochilus aethiopicus LP 4 1 S e r o k o lo R o o t Colds and flu O r a l Amenorrhoea (Pujol, 1990),

(Schweif.) B.L. Burt dysmenorrhoea (van W yk and Gericke,

(Zingiberaceae) 2000; Steenkamp, 2003 ), colds, cough,

influenza, hysteria, pain, asthm a, anti­

inflam m atory, bronchodilatory (Van W yk,

2 0 1 1 )

Solanum supinum D u n . LP 2 1 Thola-ye-kgolo R o o ts To cure skull problem s in children O r a l Diarrhoea (M athabe et al., 2006) (Solanaceae)

Vangueria infausta B u rc h . LP 0 9 M m i l o L e a v e s High-blood pressure, cancer, O r a l Menstrual pains (W att and Breyer- subsp. infausta (Rubiaceae) diarrhoea and coughs Brandwijk, 1962; SteenKamp, 2003),

infertility (M abogo, 1990), chest

complains, coughs (Pooley 1993; McGaw

and Eloff, 2008), diarrhoea (M aroyi, 2011)

Ximenia caffra S o n d . Var. LP 3 0 M o c h i d i Leaves, roots Purify kidneys and urinary tract, Oral, topical septic sores (Von Koenen 1996; Fabry et c a ffra (Olacaceae) diarrhoea and sores al., 1998; Van W yk et al., 2011; Van

Vuuren and Mabona, 2013), diarrhoea

(M athabe et al., 2006), candidiasis,

headaches, scurvy (M ulaudzi et al., 2014).

3 7 Species name & fam ily V o u c h e r Vernacular plant P a rts u s e d Medicinal uses (interviews) M o d e o f Other reported uses and references

n u m b e r n a m e administration

Zanthoxylum humile (E .A LP 1 7 M o n o k w a n e B a rk Cold and flu, mouth and throat O r a l HIV/AIDS (Semenya et al., 2013a),

Bruce) P.G. W aterm an s o re s , the bark is kept in the im potence (Semenya et al., 2013b) (R u ta c e a e ) m outh like a lozenge.

Ziziphus mucronata W illd . LP 4 4 M o k g a lo L e a v e s Sores, coughs, stomach-ache O r a l Sores (W att and Breyer-Brandwijk, 1962; (Rhamnaceae) Rood, 1994; Hutchings, 1996; Rabe and

van Staden, 1997; Luseba et al., 2007; van

W yk et al., 2000; Van W yk et al. 2011;

Mabona and Van Vuuren, 2013),

m enorrhagia and infertility (Arnold and

Gulumian, 1984; Steenkamp, 2003),

coughs, chest problems, and diarrhoea

(Van W yk and Gericke, 2000; Nyila et al.,

2012), boils (M aroyi), gonorrhoea

(Semenya et al., 2013a)

3 8 The most dominant plant families were Anacardiaceae and Asteraceae each with five species, followed by Fabaceae and Verbenaceae with three species each, as depicted in

Figure 3.1.1. Other represented families (with two species in each) are Apocynaceae,

Asparagaceae, Euphorbiaceae, Hyacinthaceae, Hypoxidaceae, Rutaceae, and

Solanaceae. Asteraceae and Fabaceae have been highlighted in several other ethnobotanical studies (e.g. Semenya and Maroyi, 2012a; Megersa et al., 2013; Afolayan et al., 2014; Williams et al., 2014) as some of the most utilised families, studies of Maroyi

(2013) and Rasethe et al. (2013) identified Anarcadiaceae as well.

6

S 5 u

2 E 1 1 1 111 1111111 1 1 _L 0 ,zr/S ' czr 'S ' rzr /S' fe> /S' .zr /S' .zr /S' rzr/S' ,e /S' ,er /S' fe- /S' ,er /S' ezr /S' .zr /S' czr /S' .zr/S' .zr/S' .zr /S' /S' /S' /S' /S' /S' /S' /S' /S' sjF ^/S1 ^/S /S' /S' /S' .-jF /S' /S' /S' /S' /S' /S' /S' /S'

Plant families

Figure 2.1.1 Representation of the plant families utilized in the Ga-Mashashane area.

Preference on the utilization of plant parts was given to leaves (53%), underground parts

(31%), bark (11%), and fruits (5%) as indicated in Figure 3.1.2. This could be due to the fact that leaves are readily available, easy to harvest and prepare as either a decoction or infusion, whereas other parts may require maceration and grinding. The high preference of leaves and underground parts has also been reported in other studies (Van

Vuuren and Naidoo, 2010; York et al., 2011; Semenya and Maroyi, 2013; Masevhe et al.,

3 9 2015). In particular, underground parts act as reservoirs of various healing agents (Louw et al., 2002; Ncube et al., 2011) and are highlighted to possess great healing efficacy as compared to other plant parts (Kunwar et al., 2006; Maroyi, 2011; Semenya & Maroyi,

2012a). However, there is a great concern on the use of underground parts as this leads to destruction, unsustainability, and overharvesting of most of the indigenous plants

(Street et al., 2008; Semenya et al., 2012b). To obtain the same healing efficacy as displayed by underground parts in some of the plants, enough quantity of leaves should be utilised instead of underground parts as leaves are easy to regrow (Zschocke et al.,

2000; Gail et al., 2015).

Figure 2.1.2 Diagram representing plant parts of medicinal plants utilized in Ga- Mashashane area.

The ethnobotanical study has revealed 12 new medicinal uses recorded from the following species; Acokanthera oppositifolia, Aloe arborescens, Elaeodendron transvaalense, Englerophytum magalismontanum, Hypoxis hemerocallidea., Ledebouria revoluta, Ozoroa sphaerocarpa, Ptaeroxylon obliquum, Ricinus communis var. communis, Vangueria infausta subsp. infausta, and Zanthoxylum humile while the use of

4 0 one plant, Senecio barbertonicus is reported for the first time in the South African Materia

Medica. The majority of these plants are used for the treatment of infectious diseases falling into four categories (gastrointestinal, respiratory, skin conditions, and STIs).

Twenty-two species are utilised to treat gastrointestinal disorders such as diarrhoea, heartburn, and stomach-ache; 19 species for respiratory ailments including asthma, cold, coughs, and flu; 16 for skin ailments, for example sores, burns, and wounds, while eight

(A. oppositifolia, Drimia elata, E. transvaalense, Euclea crispa subsp. crispa, Grewia occidentalis, Ozoroa insignis, Peltophorum africanum, and R. communis var. communis) are utilized to treat STIs. Of the non-pathogenic ailments, most of the plants are used for inflammation (especially of the limbs) and blood purification, whereas conditions such as cancer are rarely treated. It is worth noting that several of these plants are used to treat more than one condition. Morphological descriptions, geographic distributions, and the details of the mode of administration of these plants are presented in Appendix 3.

Peltophorum africanum emerged as the most utilized plant species for the treatment of various ailments. According to the literature, the plant has numerous medicinal uses, for example the Vhavenda women use it to treat herpes (Mabogo, 1990), whereas the Bapedi traditional healers use the plant to induce female fertility and treat post-partum depression

(Semenya et al., 2013b). Mazimba et al. (2014), noted that the plant is also used for the treatment of eye infections, joints and back pains, toothache, diarrhoea, dysentery, infertility, skin rashes, blisters, STIs, depression, coughs, sore throat, ascites, abdominal disorders, and as an anthelmintic, thus justifying its versatility within the study area. Other most utilized plants are Carissa bispinosa, Carpobrotus edulis, Eucalyptus camaldulensis, and Ziziphus mucronata. These plant species are used to treat gastrointestinal, respiratory, and skin ailments. Carissa bispinosa is also used as a root decoction by the people of south-central Zimbabwe to treat coughs and diarrhoea

4 1 (Maroyi, 2013). A root decoction is also reported to treat impotence (Amusan et al., 2002).

The Xhosa people of Amathole District in the Eastern Cape use both C. bispinosa and C. edulis to treat skin disorders (Afolayan et al., 2014). The sap from the leaves of C. edulis is squeezed out and applied topically for sores, wounds, and burns (Buwa and Afolayan,

2009). Van Wyk et al. (1997), highlighted that C. edulis has a wide range of uses which include eczema, TB, vaginal thrush, toothache, and earache. Eucalyptus camaldulensis is well known for the treatment of respiratory ailments such as colds and flu. The Zulu people use a leaf decoction topically to treat acne (Hutchings, 1996; Babayi et al., 2004;

Ayepola and Adeniyi, 2008; Musa et al., 2011; Mabona and Van Vuuren, 2013), while

Bapedi traditional healers utilize the plant to treat TB (Semenya et al. 2012c). The plant has broader use among different cultures with a leaf decoction reported to treat diarrhoea, dysentery, haemorrhage, laryngitis, pharyngitis, sore throat, spasm, trachalgia, and wounds (Duke and Wain, 1981; Mouna and Segni, 2011).

A decoction prepared from the leaves of Ziziphus mucronata is taken orally by South

African women for menorrhagia and infertility (Arnold and Gulumian, 1984; Steenkamp,

2003). The boiled leaves of this plant are applied as a poultice for wounds and boils (Rabe and Van Staden, 1997). In the Nhema communal area of Zimbabwe, the powder from crushed fruits and leaves is used for the treatment of boils and wounds (Maroyi, 2011), while Bapedi traditional healers utilize a decoction against STIs, specifically gonorrhoea

(Semenya et al., 2013a). In addition, Z. mucronata is used for the treatment of tuberculosis (Green et al., 2010), coughs, chest problems, and diarrhoea (Van Wyk and

Gericke, 2000; Nyila et al., 2012). Acokanthera oppositofolia emerged as one of the medicinal plants with a wide range of uses within the study area. It is used for treating gastrointestinal problems, respiratory ailments, and STIs. According to the literature, it is also indicated in a number of conditions ranging from stress-related disorders (Hutchings

4 2 and Staden, 1994) to reproductive ailments in which case, a bark decoction is taken orally to treat menorrhagia by Vhavenda women (Steenkamp, 2003). Similar to the current study, Kumar and Singh (2005), reported that the plant is used for the treatment of stomach ache, tapeworm, colds, toothache, anthrax, spider, and snake bites. Although the plant has multiple uses, its leaves are reported to be extremely dangerous (fatal) if taken in large quantities (Kumar and Singh, 2005).

Ricinus communis L. var. communis also has a number of uses by the people of Ga-

Mashashane area, as it is used for treating respiratory ailments, skin infections, and STIs.

The leaves are applied as poultice for the treatment of boils (Watt and Breyer-Brandwijk,

1962; Hutchings, 1996; Van Wyk et al., 2000; Luseba et al., 2007; Malik et al., 2011;

Mabona et al., 2013). The Bapedi traditional healers prepare root decoctions to treat STIs

(Semenya and Maroyi, 2013a). The same dosage form also treats breast tumours, menstrual disorders, and abnormal vaginal discharges (De Boer and Cotingting, 2014).

The species Callilepis laureola, Schotia brachypetala, and Vangueria infausta subsp. infausta are used for treating only two types of ailments, i.e. gastrointestinal and respiratory conditions. A bulb decoction of C. laureola is also reported as being used to induce fertility (Debetto, 1978; Seedat and Hitchcock, 1971; Semenya, 2013c), for stomach problems (Steenkamp et al. 1999), tape worm infestation, and coughs (Watt and

Breyer-Brandwijk, 1962). However, it is reported that C. laureola could cause vomiting, abdominal pain, headache leading to coma and death (Wink and Van Wyk, 2008). A bark decoction of S. brachypetala is used by the Zulu people to treat dysentery (Hutchings,

1996) and ulcers (Venter and Venter, 1996). In the Limpopo Province, within the

Capricorn District, a bark decoction is used for diarrhoea (Mathabe et al., 2006; Shai et al., 2013). The people of Manzini District in Swaziland use the bark of Sclerocarya birrea and S. brachypetala to prepare a concoction used as an emetic (Amusan et al., 2002). A

4 3 root decoction of V. infausta is reported to treat abnormal menstruation (Watt and Breyer-

Brandwijk, 1962; Steenkamp, 2003), and is used by the Vhavenda people to treat

infertility (Mabogo, 1990). A root and leaf decoction can also be prepared for chest complaints and coughs (Pooley 1993; McGaw and Eloff, 2008). The people of the Nhema communal area in Zimbabwe drink a hot root decoction of V. infausta for the treatment of diarrhoea (Maroyi, 2011).

Dicoma anomala subsp. anomala, Ledebouria revoluta, Ozoroa sphaerocarpa, and

Ximenia caffra var. caffra are all utilised for the treatment of gastrointestinal and skin ailments in the Ga-Mashashane area. Dicoma anomala has many other recorded uses, for example the tuber extract of D. anomala subsp. anomala is used to treat intestinal problems, stomach-ache and infertility (Watt and Breyer-Brandwijk, 1962; Steenkamp,

2003). The Basotho people use a root stock infusion for colic, diarrhoea, and constipation

(Moteetee and Van Wyk, 2011). In addition, the root stock treats toothache, hypertension, diabetes, period pains, pneumonia, backache, wounds and sores (Van Wyk and Gericke,

2000). Ledebouria revoluta is used for respiratory complaints as an expectorant and for gastrointestinal tract conditions (Watt and Breyer-Brandwijk, 1962; Dold and Cocks,

2001; Abegaz, 2002; Moodley et al., 2006). The pounded bulb is also used to dress wounds, sores, and skin eruptions (Muleya et al., 2014). A mixture prepared from the bark of O. sphaerocarpa and Athrixia phylicoides is used to dress wounds (Amusan et al.,

2002). A bark decoction of O. sphaerocarpa also treats diarrhoea, asthma, and impotence

(Semenya and Maroyi, 2012; Semenya et al. 2013c). In addition, the plant is also used in combination with other medicinal plants by the people of Swaziland for diarrhoea

(Sibandze et al., 2010). The stem bark of Ximenia caffra is utilized by the Bapedi people within the Capricorn district to prepare a decoction incorporated into soft porridge for diarrhoea (Mathabe et al., 2006). The Zulu people located in the Maputaland region,

4 4 KwaZulu-Natal, apply root powder in the form of a paste for skin infections and septic sores (Van Vuuren and Mabona, 2013). The Vhavenda people utilize roots and leaf decoction for candidiasis, headaches, and scurvy (Mulaudzi et al., 2011). Previous records also indicate that root powder is used to treat septic sores through topical application (Von Koenen 1996; Fabry et al., 1998).

In the current study, Ozoroa insignis, is recorded to treat both gastrointestinal ailments and STIs. Literature reports indicate that the stem bark of this plant is used in Limpopo,

Capricorn District, as a decoction for diarrhoea (Mathabe et al., 2006) and for a similar purpose in the Nhema communal area in Zimbabwe (Maroyi, 2011; 2013). Euclea crispa subsp. crispa is the only plant species used for the treatment of skin ailments and STIs.

A decoction of E. crispa has a wide range of uses (Steenkamp, 2003), and South African women use a leaf decoction to treat dysmenorrhoea. Moteetee and Van Wyk (2011), highlighted that a bark decoction is used by the Basotho as a purgative. The people of the Nhema communal area in Zimbabwe, prepare a root decoction for coughs (Maroyi,

2011). The Bapedi traditional healers use a root decoction for STIs (Semenya et al.,

2013a).

The remaining plants are utilised to curb one ailment only, although there are other uses recorded elsewhere, for example eight plants (Clerodendrum glabrum var. glabrum,

Dombeya rotundifolia, Englerophytum magalismontanum, Ptaeroxylon obliquum, Punica granatum, Searsea lancea, and S. supinum) were recorded for the treatment of gastrointestinal ailments only. The Vhavenda people prepare leaf decoctions from C. glabrum to treat STIs (Tshikalange et al., 2008), specifically candidiasis (Masevhe et al.,

2015). McGaw et al. (2008), highlighted that leaf decoctions of C. glabrum and D. rotundifolia treat respiratory ailments. A root decoction of D. rotundifolia treats heart problems, nausea in pregnant women, intestinal ulcers, headaches, stomach complaints,

4 5 haemorrhoids, diarrhoea, and dyspepsia (Reid et al., 2001). Bapedi traditional healers use a root decoction for treatment of diarrhoea (Semenya and Maroyi, 2012). A root decoction prepared from E. magaliesmontanum is used by the Vhavenda people as a contraceptive (Mabogo, 1990; Steenkamp, 2003). The Bapedi traditional healers utilize a bark decoction to treat diabetes (Semenya et al., 2012a). Ptaeroxylon obliquum has various uses in three provinces of South Africa. In KwaZulu-Natal, the Zulu people utilize wood powder as a sneeze inducer to treat headaches (Hutchings et al., 1996), as well as the Rastas (commonly known as the Bush doctors) in the Western Cape (Philander,

2011). The Xhosa people of the Eastern Cape Province bath with the bark infusion to remove body odour (Afolayan et al., 2014). The plant is also recorded to treat rheumatism, arthritis, and heart disease (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996; van Wyk et al., 2009). The people residing in the Capricorn District of the Limpopo Province prepare leaf decoction of P. granatum which is incorporated into a soft porridge to treat diarrhoea (Mathabe et al., 2005; Semenya and Maroyi, 2012a). The seeds of P. granatum are reported to enhance fertility (Ndhlala et al., 2013). A decoction is prepared from the

leaves of S. lancea and treats diarrhoea in children (Van der Merwe et al. 2001; McGaw and Eloff, 2008). A root decoction of S. supinum is used to treat diarrhoea as well

(Mathabe et al., 2006).

Similar to the foregoing discussion, eight plants are recorded for treating respiratory ailments only; Artemisia afra, Combretum imberbe, Helichrysum caespititum, Lippia javanica Lantana rugosa, Schinus molle, Siphonochilus aethiopicus and Zanthoxylum humile (E.A Bruce) P.G. Waterman. The ethnobotanical knowledge on the use of A. afra has been extensively documented globally. A leaf decoction treats fever, coughs, and influenza (Dykman, 1891; Watt & Breyer-Brandwijk, 1962). Additionally, the decoction is administered to treat stomach-ache, dry dyspepsia, and malaria (Burits et al. 2001; Van

4 6 Wyk and Gericke, 2000; Vagionas et al. 2007; Semenya and Maroyi, 2013). A leaf decoction of C. imberbe is used to curb a chest cough and fever (Hutchings et al., 1996;

McGaw et al., 2008). Helichrysum caespititium is smoked to relieve headaches, chest colds, and also dresses open wounds (Mathekga et al., 2000). Similarly, in Lesotho, the plant is crushed, burnt and snuffed for chest and head colds (Moteetee and Van Wyk,

2011). Lippia javanica has been extensively studied globally. Its uses include treatment of asthma, chest ailments, colds, cough, influenza, bronchial complaints, fever, and headaches (Watt and Breyer-Brandwijk, 1962; Gelfand et al., 1985; Mabogo, 1990;

Roberts, 1990, Hutchings et al., 1996; Van Wyk and Gericke, 2000; Green et al., 2010).

A leaf decoction is also used for TB treatment (Green et al., 2010), while the essential oil

is inhaled and effective for treating colds and influenza (Makunga et al., 2008; York et al.,

2011). The Vhavenda people use leaves and stems of L. rugosa to make a paste for troublesome eyes (Mabogo, 1990). The paste prepared from the leaves, stems, or ripe fruit treats festering sores (Hutchings, 1996; Suliman, 2010; Mabona and van Vuuren,

2013). A root infusion of L. rugosa is also used to treat bronchitis, tuberculosis, colds, and flu (Mabogo, 1990; McGaw et al., 2008; Mahwasane et al., 2013). A leaf infusion of S. molle is inhaled or taken orally to treat colds and influenza (Hutchings, 1992). A topical application of fruit paste of this plant is used by the Xhosa people to treat eczema

(Afolayan et al., 2014). A bulb decoction of S. aethiopicus treats amenorrhoea (Pujol,

1990) and dysmenorrhoea by South African women (van Wyk and Gericke, 2000;

Steenkamp, 2003). Furthermore, the species is recorded as a treatment for colds, coughs, influenza, hysteria, pain, asthma, inflammation, and as a bronchodilator (van

Wyk, 2011). The Bapedi traditional healers macerate the bark of Z. humile in preparing a decoction for impotence (Semenya et al. 2013c) and HIV/AIDS (Semenya et al. 2013a).

4 7 Five medicinal plants were recorded to treat skin ailments only; Aloe arborescens,

Asparagus racemosus, Datura stramonium, Jatropha zeyheri, and Lannea edulis. The sap from the leaves of A. arborescens treats wounds, burns, and various other skin problems (Bruce, 1975; Van Wyk et al. 2000), while a leaf decoction taken orally assists with cardiac problems (Amusan et al., 2002), gastrointestinal complaints, blood purification or cleansing (Philander, 2011), and STIs (Semenya et al., 2013a). Asparagus racemosus treats nervous disorders, dyspepsia, tumours, inflammation, and is purported to increase fertility and vitality in women (Garde and Vagbhat, 1970; Alok et al., 2013;

Sharma and Sharma, 2013). The species is extensively used in the Indian pharmacopoeia on the Asian continent. The leaves of D. stramonium are placed onto affected area to treat boils and abscesses (Watt and Breyer-Brandwijk, 1962; Bruneton

1995; Rabe and Van Staden, 1997; Van Wyk et al., 2000; Priya et al., 2002; McGaw and

Eloff, 2008; Saadabi & Moglad 2011; Mabona and Van Vuuren, 2013). Van Wyk and

Gericke (1997), reported that the root stock decoction of J. zeyheri treats menstrual pains, irregular period pains, and ensures a strong foetus during pregnancy. The decoction is also beneficial against STIs and urinary tract infections (Mongalo et al., 2013). The oral intake of the decoction also purifies the blood and is applied externally to dress wounds and boils (Van Wyk et al., 2013). Crushing the bark of L. edulis with topical application also treats boils and abscesses (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996; Van

Wyk et al., 1997). The people of the Manzini region in Swaziland use the root bark decoction for constipation (Amusan et al., 2002). In the Zimbabwean Nhema communal area, root decoction treats diarrhoea (Maroyi, 2011).

Drimia elata Jacq. and Elaeodendron transvaalense (Burtt Davy) R.H. Archer emerged as the only two medicinal plants used for treating STIs only in the Ga-Mashashane area.

The former plant has been recorded to relief pain and asthma by the Zulu people

4 8 (Hutchings et al., 1996, Van Wyk et al., 2009; Ndhlala et al., 2013). In addition, the Zulu people prepare an infusion from the pounded bulbs and leaves to make an enema for fever. Philander (2011), recorded that bulb decoction to treat arthritis, while the Bapedi traditional healers use it for gonorrhoea and HIV/AIDS (Semenya et. al., 2013a). The bark decoction of E. transvaalense treats dysmenorrhoea (Van Wyk, 1972; Steenkamp, 2003), while a root decoction is used by Vhavenda traditional healers to treat herpes (Mabogo,

1990) and candidiasis (Bessong et al. 2005; Masevhe et al., 2015). A bark and root decoction is beneficial for fungal infections, stomach disorders, ulcers, and STIs (Samie et al., 2005). The root/bark is mixed with the root of Peltophorum africanum and the decoction is taken to treat STIs (Semenya et al. 2013a) and female infertility (Semenya et al. 2013b).

Four medicinal plants recorded to treat non-pathogenic ailments are Eucomis pallidiflora,

Hypoxis hemerocallidea, H. obtusa, and Senecio barbertonicus. A bulb decoction of E. pallidiflora is used to treat impotence in the Ga-Mashashane area. In the literature, it is recorded for the treatment of Chlamydia, erectile dysfunction, tuberculosis, blood clotting, coughs as well as tuberculosis by the Bapedi traditional healers (Semenya and Potgieter,

2013; Semenya and Maroyi, 2011; Semenya et al., 2013b). Both H. hemerocallidea and

H. obtusa are recorded for use as pain-killers in the current study, however, they have a wide range of uses in different cultures in southern Africa. A tuber decoction of H. hemerocallidea treats several ailments such as inflammation, testicular tumours, urinary complaints, cancer, and HIV/AIDS (Watt and Breyer-Brandwijk, 1962; Crouch et al., 2006;

Ncube et al., 2012). Van Wyk (2008), highlighted that the tuber decoction is a remedy for bladder problems. Hypoxis obtusa tuber decoction treats prostrate hypertrophy (Nel,

1914), urinary disorders (Marini-Bettolo et al., 1985; Galeffi et al., 2002), gonorrhoea and infertility in women and is also used as an aphrodisiac (Gelfand et al., 1985). Semenya

4 9 et al. (2013b) and Moeng (2010), highlighted that the Bapedi traditional healers prepare a bulb decoction for the treatment of infertility and impotence, whereas the people of

Zimbabwe use the plant species for abdominal pains (Maroyi, 2013). The use of Senecio barbertonicus to treat ailments is being recorded for the first time for the South African pharmacopeia. The plant is utilized as a purgative prepared from boiling the leaves

(cladophylls) and as an emetic where it is mixed with milk and taken orally. The practice

is commonly done when it is suspected that a person has been given a food substance known as ‘Sejeso’ with the intention to kill or to hypnotize (Papo E., 2014, pers. comm.).

Several Senecio species are documented in the South African pharmacopeia, for example S. concolor (Smith 1895; Mabona et al., 2013), S. coronatus (Dold and Cocks,

2002; Street et al., 2008), S. deltoideus (Watt and Breyer-Brandwijk, 1962; York et al.,

2011), S. latifolius (Smith 1895; Mabona et al., 2013), and S. serrulatoides (Hutchings,

1996; Lall and Meyer, 1999; York et al., 2011; Madikizela et al., 2012; De Wet et al.,

2013). Interestingly, the overall utilization of medicinal plants by the people of Ga-

Mashashane area correlates with other cultures in South Africa and southern Africa at large.

50 2.2 ANTIMICROBIAL SCREENING

2.2.1 Gastro-intestinal pathogens

Twenty-two plant species were screened against six gastro-intestinal pathogens; Bacillus cereus (ATCC 11775), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC

8739), Listeria monocytogenes (ATCC 19111), Salmonella typhimurium (ATCC 14028), and Shigella sonnei (ATCC 9290). The results showing activity of plants against these causative agents is presented in Table 3.2. Fifteen plants exhibited noteworthy antimicrobial activity against at least one pathogen (with activities ranging between 0.1 mg/ml-1.0 mg/ml), the most inhibitive being O. sphaerocarpa and S. lancea as they inhibited all the six pathogens, showing broad-spectrum activity. This was followed by C. glabrum, E. elephantina, and P. africanum, which were active against four pathogens each. On the other hand, six species (C. laureola, C. bispinosa, E. camaldulensis, L. revoluta, S. supinum and Z. mucronata) exhibited insignificant activity against all the tested pathogens. The plant extracts displaying the highest activity (all with MIC values of 0.1 mg/ml) were: i) were the organic extracts of C. glabrum , O. sphaerocarpa, and S. lancea against B. cereus; ii) organic extracts of O. sphaerocarpa against E. coli, L. monocytogenes, and S. sonnei; and iii) aqueous extract of X. caffra against B. cereus.

The most susceptible pathogens were S. typhimurium, inhibited by ten organic and six aqueous extracts (with the highest activity shown by aqueous extracts of P. africanum at

0.3 mg/ml and organic extracts of D. rotundifolia, P. africanum, and S. lancea, all at 0.5 mg/ml). Enterococcus faecalis, was inhibited by ten organic and one aqueous extracts, and B. cereus, inhibited by seven organic and four aqueous extracts (with X. caffra

51 displaying the highest activity among all aqueous extracts at 0.1 mg/ml). The most resistant pathogen was L. monocytogenes as it was inhibited only by organic extracts of

O. sphaerocarpa with the rest showing no significant activity.

Organic extracts performed better compared to aqueous extracts with higher susceptibility recorded from B. cereus, E. faecalis, and L. monocytogenes. The three pathogens are classified as the Gram-positive bacteria due to the lack of the outer membranes which acts as a barrier that inhibits interaction of the plant extracts with the pathogen. Although the organic extracts of O. sphaerocarpa demonstrated broad- spectrum inhibition, the aqueous extract showed no significant activity against all the

Gram-positive bacteria, except S. typhimurium. Conversely, a moderate antimicrobial activity of O. sphaerocarpa (1.2 mg/ml) screened against E. coli was highlighted by

Sibandze et al. (2010) in a study investigating its anti-diarrhoeal properties. The organic extracts of S. lancea were inhibitive to all the tested bacterial species. The organic extracts of P. africanum demonstrated noteworthy activity against four pathogens, with E. faecalis inhibited by both organic (0.5 mg/ml) and aqueous extracts (1.0 mg/ml). The organic extracts of E. elephantina showed noteworthy activity against four pathogens, namely; -B. cereus (0.8 mg/ml) and E. faecalis (0.7 mg/ml), S. typhimirium (0.7 mg/ml), and S. sonnei (1.0 mg/ml), with the aqueous extract showing better activity against B. cereus (0.5 mg/ml). Previously, Mpofu et al. (2014), demonstrated noteworthy activity of

E. elephantina against B. cereus with an MIC value of 0.25 mg/ml using dichloromethanol

(organic) and 0.5 mg/ml using aqueous extracts which is in correlation with the current findings. Bacillus cereus, was also susceptible to the aqueous extract of Xaminia caffra

(0.1 mg/ml). The use of X. caffra to treat diarrhoea has been reported and validated by

Mathabe et al. (2006), although B. cereus was excluded from their antimicrobial screening study. Listeria monocytogenes showed the highest resistance amongst the Gram-positive

52 bacteria and was only susceptible to the organic extract of C. glabrum (0.5 mg/ml), O. sphaerocarpa (0.1 mg/ml) and S. lancea (0.4 mg/ml).

Escherichia coli, Salmonella typhimirium, and Shigella sonnei were susceptible to the organic extracts of O. sphaerocarpa, P. africanum, and Searsia lancea. The organic extract of Clerondendrum glabrum demonstrated noteworthy activity by inhibiting the growth of E. coli at an MIC value of 0.3 mg/ml and S. sonnei at the MIC value of 0.5 mg/ml. Masevhe (2013), obtained MIC values of 0.16 - 0.47 mg/ml from hexane, acetone, and methanol extracts respectively, of C. glabrum against E. coli. Both the organic and aqueous extracts of O. sphaerocarpa at MIC values of 0.7 mg/ml and 0.1 mg/ml, P. africanum at MIC values of 0.5 mg/ml and 0.3 mg/ml, D. rotundifolia at MIC values of 0.5 mg/ml and 0.5 mg/ml, and E. elephantina at MIC values of 0.7 mg/ml and 0.5 mg/ml demonstrated noteworthy activity when tested against S. typhimirium. Interestingly, aqueous extracts of three species, O. sphaerocarpa, P. africanum, and E. elephantina performed better compared to their organic extracts. The antimicrobial activity of P. africanum against S. sonnei was highlighted by Obi et al. (2003), with an inhibition zone of 12 mm.

53 Table 3.2 Antimicrobial activites of plants used in Ga-Mashashane area against gastro-intestinal pathogens Antim icrobial activity (MIC values in m g/m l) P l a n t V o u c h e r Plant species p a r t B c E f L m E c S t S s n u m b e r a s s e s s e d (ATCC 11775) (ATCC 29212) (ATCC 19111) (ATCC 8739) (ATCC 14028) (ATCC 9290) Org Aq Org Aq Org Aq Org Aq Org Aq Org Aq A. oppositifolia LP 29 Leaves 2.0 >8.0 1.0 4.0 3.3 8.0 6.7 8.0 1.5 0.8 5.3 8.0 C. laureola LP 18 Bulb 1.3 4.0 2.7 >8.0 2.7 4.0 6.7 8.0 8.0 6.0 4.0 >8.0 C. bispinosa LP 15 Roots 2.0 4.0 2.0 >8.0 3.3 4.0 8.0 >8.0 1.7 8.0 3.3 >8.0 C. edulis LP 42 Leaves 1.7 >8.0 1.3 8.0 2.0 4.0 4.0 8.0 1.0 2.0 3.3 4.0 C. glabrum LP 35 Leaves 0.1 >8.0 1.3 >8.0 0.5 4.0 0.3 >8.0 1.7 >8.0 0.5 4.0 D. anomala LP 3 Tuber 1.0 4.0 2.0 8.0 2.0 >8.0 2.7 >8.0 2.0 4.0 2.0 >8.0 D. rotundifolia LP 37 Leaves 2.0 0.4 2.5 3.0 7.0 2.0 4.0 8.0 0.5 0.5 5.3 4.0 E. elephantina LP 45 Bulb 0.8 0.5 0.7 2.0 1.7 8.0 2.0 2.0 0.7 0.5 1.0 >8.0 E. magaliesmontanum LP 28 Leaves 2.7 8.0 1.0 2.0 2.0 4.0 4.0 8.0 1.0 1.5 3.3 8.0 E. camaldulensis LP 12 Leaves 1.7 2.0 2.0 8.0 3.3 >8.0 2.7 >8.0 1.8 8.0 2.0 >8.0 L. revoluta LP 31 Rhizome >8.0 4.0 2.0 >8.0 >8.0 >8.0 >8.0 >8.0 2.3 >8.0 >8.0 >8.0 O. insignis LP 39 Leaves 1.3 >8.0 0.8 4.0 3.0 4.0 2.0 >8.0 0.7 >8.0 1.7 4.0 O. sphaerocarpa LP 27 Leaves 0.1 >8.0 1.0 2.0 0.1 >8.0 0.1 8.0 0.7 1.0 0.1 >8.0 P. africanum LP 4 0 Leaves 1.7 4.0 0.5 1.0 3.0 >8.0 0.8 >8.0 0.5 0.3 1.0 >8.0 P. obliquum LP 14 Leaves 1.3 2.0 1.3 >8.0 2.0 >8.0 8.0 4.0 0.8 2.0 2.0 >8.0 P. granatum LP 43 Leaves 2.0 0.3 1.3 2.0 3.3 8.0 4.0 4.0 0.8 2.0 3.3 >8.0 S. brachypetala LP 26 Leaves 1.7 4.0 1.0 >8.0 >8.0 >8.0 2.0 >8.0 5.3 >8.0 6.7 >8.0 S. lancea LP 10 Leaves 0.1 8.0 1.0 3.0 0.4 >8.0 0.3 8.0 0.5 4.0 0.3 >8.0 S. supinum LP 21 Roots 1.7 >8.0 1.7 >8.0 >8.0 2.0 2.0 >8.0 >8.0 >8.0 >8.0 2.0 V. infausta LP 9 Leaves 0.3 >8.0 1.0 4.0 4.0 >8.0 1.7 >8.0 1.7 4.0 2.7 >8.0 X. caffra LP 30 Leaves 1.0 0.1 1.0 1.5 1.7 3.0 2.0 2.0 1.7 0.8 3.3 4.0 Z. mucronata LP 4 4 Leaves 1.7 3.0 1.3 4.0 2.7 2.0 2.0 6.0 2.7 4.0 1.7 2.0 Ciproflaxicin +C 0.078 0.31 0.039 0.31 0.31 0.31 0.17 0.18 0.31 0.31 0.17 3 Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml Acetone -C >8.0 Type of extract: Ag, aqueous; Org, organic. Pathogens: Bc, Bacillus cereus , Ef, Enterococcus faecalis ; Lm, Listeria monocytogenes , Ec, Escherichia coli; St, Salmonella typhimurium ; Ss, Shigella sonnei ; MIC, minimum inhibition concentration values expressed in mg/ml Noteworthy activities given in bold

54 2.2.2 Respiratory pathogens

Eighteen medicinal plants were screened against three respiratory pathogens; Klebsiella pneumoniae (ATCC 13883), Moraxella catarrhalis (ATCC 23246), and Staphylococcus aureus (ATCC 29213) as indicated in Table 3.3. Eleven species exhibited noteworthy antimicrobial activity against at least one of the pathogens with an MIC range of 0.1-1.0 mg/ml. Peltophorum africanum emerged as the most inhibitive plant as it inhibited two pathogens, while the rest of the plants inhibited only one pathogen each, with the exception of C. bispinosa, C. edulis, E. camaldulensis, L. rugosa, R. communis, S. brachypetala, and Z. mucronata which exhibited no significant activity. Plant extracts displaying highest activity were organic extracts of H. caespititium against M. catarrhalis with MIC value of 0.1 mg/ml, followed by aqueous extract of A. afra against M. catarrhalis and S. aethiopicus against S. aureus (both at 0.5 mg/ml), and aqueous extracts of Z. humile (0.75 mg/ml) against K. pneumoniae. Other noteworthy activities can be summarised as follows (all with MIC values of 1.0 mg/ml): i) organic extracts of A. oppositifolia, C. laureola, E. crispa, P. africanum, and Z. humile against K. pneumoniae; ii) aqueous extract of L. javanica against K. pneumoniae; iii) organic extracts of C. imberbe, P. africanum, S. aethiopicus, and V. infausta against S. aureus; iv) the aqueous extract of C. imberbe against M. catarrhalis and S. aureus. The most susceptible pathogens were K. pneumoniae inhibited by five organic and two aqueous extracts (with highest activity shown by aqueous extract of Z. humile at 0.75 mg/ml), followed by S. aureus inhibited by four organic and one aqueous extract (with highest activity shown by organic extract of S. aethiopicus at 0.5 mg/ml and aqueous extract of C. imberbe at 1.0 mg/ml). Moraxella catarrhalis was the most resistant amongst all the pathogens.

55 The screening of P. africanum against S. aureus has been carried out in several other studies. Interestingly, none of them demonstrated noteworthy activity, with much higher

MIC values obtained when screening against S. aureus, e.g. 3.0 mg/ml (Samie et al.,

2005), 2.0 mg/ml (Steenkamp et al., 2007), and 3.125 mg/ml (Mulaudzi et al., 2011) as opposed to the current study. The differences can be attributed to geographical and seasonal variations due to temperature, light and water availability of the collected species (Romero et al., 2005; Celiktas et al., 2007). The recorded MIC value of V. infausta in the current study is in correlation with prior noteworthy activity of 0.08 mg/ml recorded by Shai et al. (2013). Staphylococcus aureus was only susceptible to the aqueous extracts of C. imberbe (1.0 mg/ml). Katerere et al. (2003), indicated that four pentacyclic triterpenoids isolated from C. imberbe have antimicrobial activity with an MIC range of

6.25 - 0.025 mg/ml. The DCM extract of the root of Z. humile was recorded with an inhibition zone of 8.3 mm when screened against S. aureus and indicated the presence of antimicrobial activity (Dzomba and Gwizangwe, 2013).

In the current study the organic extract of P. africanum screened againt S. aureus showed better results when compared to a previous study (MIC value of 1.56 mg/ml) noted by

Mulaudzi et al. (2011). The screening of the extracts of A. oppositifolia, C. laureola, E. crispa, and Z. humile against K. pneumoniae are investigated here for the first time. The use of Z. humile to treat respiratory disorders with validity through antimicrobial screening was performed by Dzomba and Gwizangwe (2013). In their study, the DCM extracts of Z. humile was screened against S. aureus - the only respiratory pathogen. Interestingly, aqueous extracts of Z. humile and L. javanica against K. pneumoniae as well as A. afra against M. catarrhalis showed much better results when compared to their organic extracts. This indicated that the most bioactive compounds are hydrophilic compounds

(Suleiman et al., 2010). The lower MIC value of the aqueous extract of L. javanica as

56 compared to its organic extract concurs with the finding by York et al. (2012). The current study revealed the antimicrobial activity of the crude extracts of A. afra against M. catarrhalis. Buwa and Afolayan (2009) indicated that the DCM extract prepared from leaves of A. afra (0.390 mg/ml) has noteworthy activity when screened against K. pneumoniae.

Table 3.3 Antimicrobial activites of plants used in Ga-Mashashane area against respiratory pathogens.______P lant species V o u c h e r P l a n t p a r t Antim icrobial activity (MIC values in mg / m l ) n u m b e r a s s e s s e d K p M c S a (ATCC 13883) (ATCC 23246) (ATCC 29213) Org Aq Org Aq Org Aq A. a fra LP 2 L e a v e s 3 .3 6 .0 4 . 0 0 . 5 2 .0 6 .0

A. oppositifolia LP 2 9 L e a v e s 1 .0 8 .0 > 8 .0 8 .0 2 .0 > 8 .0

C. lau re o la LP 1 8 B u lb 1 .0 > 8 .0 4 . 0 8 .0 1 .7 > 8 .0

C. bisp ino sa LP 1 5 R o o ts 4 . 0 > 8 .0 > 8 .0 > 8 .0 > 8 .0 > 8 .0

C. edulis LP 4 2 L e a v e s 1 .7 2 .0 4 . 7 > 8 .0 2 .0 3 .0

C. im b erb e LP 1 1 B a r k 1 .1 2 .0 3 .3 1 .0 1 .0 1 .0

E. ca m a ld u le n sis LP 1 2 L e a v e s 2 .0 8 .0 2 .0 > 8 .0 1 .3 > 8 .0

E. crispa LP 3 2 L e a v e s 1 .0 2 .0 4 . 0 8 .0 2 .0 4 . 0

H. caespititium LP 8 R o o ts > 8 .0 4 . 0 0 . 1 8 .0 > 8 4 . 0

L. ja v a n ic a LP 2 4 L e a v e s 2 .0 1 .0 2 .7 8 .0 2 .0 8 .0

L. rug osa LP 1 9 L e a v e s 6 .0 > 8 .0 4 . 0 > 8 .0 > 8 > 8 .0

P. a frica nu m LP 4 0 L e a v e s 1 .0 2 .0 2 .0 4 . 0 1 .0 8 .0

R. communis v a r. LP 1 3 L e a v e s 2 .0 2 .0 2 .7 2 .0 2 .0 2 .0

S . brachypetala LP 2 6 L e a v e s 4 . 0 8 .0 2 .0 > 8 .0 > 8 > 8 .0

S. a e th io p icu s LP 4 1 R o o t 2 .0 > 8 .0 1 .3 > 8 .0 0 . 5 > 8 .0

V. infausta LP 9 L e a v e s 1 .3 > 8 .0 2 .0 > 8 .0 1 .0 > 8 .0

Z. hum ile LP 1 7 B a r k 1 .0 0 . 7 5 4 . 0 4 . 0 1 .7 2 .0

Z. mucronata LP 4 4 L e a v e s 2 .0 6 .0 3 .3 > 8 .0 2 .0 > 8 .0

Ciprofloxacin + C 0 . 0 3 9 0 . 6 3 p g 0 . 0 6 3 p g 1 .3 0 . 0 3 9 0 . 6 3 p g /

P g /m l / m l / m l P g /m l P g /m l m l A c e t o n e -C > 8 . 0 Type of extract: Ag, aqueous; Org, organic. Pathogens: Kp, Klepsiella pneumoniae , M c , M oraxella catarrhalis ; S a , Staphylococcus aureus; MIC, minimum inhibition concentration values expressed in mg/ml.

Values in bold considered noteworthy

Moraxella catarrhalis demonstrated resistance towards all plant extracts with the exception of organic extract of H. caespititium (0.1 mg/ml) and aqueous extracts of A. afra (0.5 mg/ml) and C. imberbe (1.0 mg/ml). Several publications (Watt and Breyer-

Brandwijk, 1962; Jacot Guillarmod, 1971; Dekker et al., 1983; Gelfand et al., 1985;

57 Hutchings and Van Staden, 1994; Neuwinger, 1996; Swanepoel, 1997; Pooley, 1998;

Mathekga et al., 2000; Mathekga, 2001; Arnold et al., 2002; Meyer et al., 2002; Pooley,

2003; Moteetee and Van Wyk, 2011) have reported on the use of H. caespititium in treating headaches, chest complaints, and colds. The low MIC value of 0.1 mg/ml obtained from the screening of the organic extract against M. catarrhalis in the current study validates its traditional use to some extent.

2.2.2.1 Essential oils

Essential oils distilled from four medicinal plants were screened against three respiratory pathogens (Klebsiella pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus), and the results obtained gave MIC ranges of 1.0 - 4.0 mg/ml. Van Vuuren (2008), recommended MIC values of <2.00 mg/ml for essential oils as noteworthy, while a most recent study (Orchard and Van Vuuren, 2017) recommends values of <1.00 mg/ml. The latter reference is followed in interpreting the results in the current study. Essential oils displayed less antimicrobial activity when compared to the crude extracts. The most inhibitive essential oils were displayed by E. camaldulensis and L. javanica against S. aureus both having an MIC value of 1.0 mg/ml. Klebsiella pneumoniae and Moraxella catarrhalis demonstrated resistance to all screened essential oils.

Essential oils are recognized as having several therapeutic applications to treat respiratory disorders through inhalation therapy and skin disorders (Van Vuuren and

Viljoen, 2006). These oils possess an array of properties, ranging from antifungal, bactericidal (Gundindza; 1993; Van Vuuren and Viljoen, 2006), insecticidal (Cheng et al.

2004; Traboulsi et al. 2005; Yang et al. 2005 Kishore et al., 2013), anti-inflammatory, and also having sedative effects (Lovkova et al., 2001). Essential oils are produced by plants and stored in secretory cells, cavities, canals, epidemic cells or glandular trichomes (York

58 et al., 2011). They play a major role in the plant’s defence mechanism and they are complex volatile groups of compounds consisting of monoterpenes, sesquiterpenes, and aromatic compounds along with their derivatives (Lovkova et al., 2001; Mouna and segni,

2011). Due to their volatile nature essential oils have better diffusability through pathogens’ cell membranes, and have been screened as potential remedies for treatment of various diseases (Dhanavade et al., 2011).

Respiratory ailments specifically cold, coughs and flu are treated with four aromatic plants

in the Ga-Mashashane area; A. afra, E. camaldulensis, L. javanica, and L. rugosa. Their common mode of administration is through inhalation therapy of leaf infusion/decoction and also through oral intake. Crushing of leaves in the hands and inhalation for immediate symptom relieve can still be effective in terms of L. javanica and E. camaldulensis due to their strong aromatic compounds found in the leaves (Pujol, 1990; York et al. 2011). They also have a reported wide use, e.g. treatment of infertility with A. afra (Watt and Breyer-

Brandwijk, 1962; Steenkamp, 2003), and wounds, scratches, and stings with L. javanica

(Gelfand et al., 1985; Hutchings et al., 1996; Van Wyk et al.,1997; Van Wyk and Gericke,

2000; Samie et al., 2005; Mabona et al., 2013; Semenya and Maroyi, 2013). Eucalyptus camaldulensis is used for acne (Hutchings, 1996; Babayi et al., 2004; Ayepola and

Adeniyi, 2008; Musa et al., 2011; Mabona, 2013) and L. rugosa used to treat eye problems (Watt and Breyer-Brandwijk, 1962; Mabogo; 1990; McGaw, 2008).

Several antimicrobial studies on the essential oils of the medicinal plants screened in the current study against S. aureus have been reported. Van Vuuren and Viljoen (2006) previously recorded an MIC value of 4.5 mg/ml for A. afra which is similar to the value of

2.0 mg/ml obtained by Hubsch et al. (2014), who is in support of the moderate activity recorded in the current study. Previously, Van Vuuren and Viljoen (2006), recorded an

59 MIC value of 16.0 mg/ml for the screening of L. javanica essential oils against S. aureus while York et al. (2012) and Hubsch et al. (2014) reported values of 5.33 mg/ml and 1.5 mg/ml respectively, indicating weak to moderate activity respectively. However difference with essential oil composition is affected by geographical distribution as well as the seasonal variation (Amoo et al., 2013). An MIC value of 0.5 mg/ml was recorded by Akin et al. (2010) when screening E. camaldulensis against S. aureus. The susceptibility of S. aureus towards L. rugosa is revealed for the first time in the current study.

Essential oils demonstrated moderate activity on the two Gram-negative bacteria,

Klebsiella pneumoniae and Moraxella catarrhalis. Poor antimicrobial activity of essential oils of A. afra screened against K. pneumoniae was recorded by Sulieman et al. (2010) at MIC 9.3 mg/ml and Hubsch et al. (2014) at MIC >8.0 mg/ml. Furthermore, Sulieman et al. (2010) noted that A. afra inhibits M. catarrhalis at 8.0 mg/ml. The presence of poor antimicrobial activity of L. javanica (6.67 mg/ml) was recorded by York et al. (2012) when screening against K. pneumoniae and at MIC 5.33 mg/ml against M. catarrhalis. However

Hubsch et al. (2014) recorded MIC 3.0 mg/ml which is much closer to the MIC value (2.0 mg/ml) in the current study. The moderate activity of E. camaldulensis against M. catarrhalis and L. rugosa against K. pneumoniae at MIC 2.0 mg/ml are revealed for the first time through the current study. The use of the essential oils of E. camaldulensis and

L. javanica to treat respiratory ailments were partially validated in the current study.

Table 3.4 Antimicrobial activites of the essential oils of plants used in Ga-Mashashane area against respiratory pathogens______P lant species V o u c h e r P l a n t Antim icrobial activity (MIC values in m g/m l) n u m b e r p a r t a s s e s s e d K p M c S a (ATTC 13883) (ATTC 23246) (ATTC 29213)

A. afra LP 2 L e a v e s 2 .0 2 .0 1.3

60 E. ca m a ld u le n sis LP 1 2 L e a v e s 2 .0 2 .0 1 .0

L. ja v a n ic a LP 2 4 L e a v e s 2 .0 4 . 0 1 .0

L. rug osa L P 1 9 L e a v e s 2 .0 4 . 0 2 .0

Ciproflaxicin + C 6 . 3 1 . 3 6 . 3 1 . 3 6 . 3 1 . 3

P g /m l p g /m l P g /m l A c e t o n e -C > 8 Type of extract: Ag, aqueous; Org, organic. Pathogens: Kp, Klepsiella pneumoniae , M c , M oraxella catarrhalis ; S a , Staphylococcus aureus; MIC, minimum inhibition concentration values expressed in mg/ml

2.2.3 Skin pathogens

Sixteen plant species were screened against four skin pathogens; Candida albicans

(ATCC 10231), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC

29213), and Staphylococcus epidermidis (ATCC 12228), as indicated in Table 3.5.

Thirteen species exhibited noteworthy antimicrobial activity against at least one of the pathogens with an MIC range of 0.1-1.0 mg/ml. The most inhibitive extracts were recorded from A. racemosus, P. africanum, and X. caffra which inhibited all four pathogens, followed by L. edulis inhibiting three, G. occidentalis and O. sphaerocarpa inhibiting two pathogens each, and the rest inhibiting one pathogen with the exception of

C. bispinosa, D. anomala, and L. revoluta which showed insignificant activity. The highest noteworthy activity (0.1 mg/ml) were recorded from i) organic extract of A. racemosus against S. aureus and S. epidermidis; ii) organic extract of O. sphaerocarpa against S. epidermidis (0.2 mg/ml); iii) organic extracts of A. racemosus against P. aerigunosa, as well as L. edulis and P. africanum against S. epidermidis (with MIC of 0.3 mg/ml); iv) organic extracts of O. sphaerocarpa against P. aerigunosa (with MIC of 0.5 mg/ml); v) organic extracts of A. racemosus, E. camaldulensis, J. zeyheri, and X. caffra against S. epidermidis (with MIC of 0.5 mg/ml); vi) aqueous extract of X. caffra against P. aeruginosa

(0.75 mg/ml); vii) organic extracts (MIC of 0.8 mg/ml) of R. communis and Z. mucronata against S. epidermidis; and viii) aqueous extracts of A. racemosus and O. sphaerocarpa

61 against S. epidermidis both with MIC of 0.8 mg/ml. Other noteworthy activities (all MIC of

1.0 mg/ml) can be summarized as follows: i) organic extracts of A. racemosus, L. edulis,

P. africanum and X. caffra against C. albicans; ii) organic extract of P. africanum against

P. aeruginosa; iii) aqueous extracts of O. sphaerocarpa and R. communis against P. aeruginosa; iv) organic extract of L. edulis and P. africanum against S. aureus; v) aqueous extracts of G. occidentalis and X. caffra against S. aureus; vi) organic extracts of C. edulis,

D. stramonium and G. occidentalis against S. epidermidis; and vii) aqueous extracts of

A. arborescens and G. occidentalis against S. epidermidis. Aqueous extracts with highest activity were recorded from X. caffra (0.75 mg/ml) against P. aeruginosa whereas the aqueous extracts of A. racemosus (0.8 mg/ml) and O. sphaerocarpa (0.8 mg/ml) showed significant activity against S. epidermidis. The most susceptible pathogen was S. epidermidis inhibited by 13 organic and four aqueous extracts (with the highest activity noted from organic extract of A. racemosus at 0.1 mg/ml and aqueous extract of A. racemosus and O. sphaerocarpa at 0.8 mg/ml each), followed by P. aeruginosa inhibited by three organic and three aqueous extracts (with the highest activity shown by organic extract of A. racemosus at 0.3 mg/ml and aqueous extract of X. caffra at 0.75 mg/ml), and

S. aureus inhibited by three organic and two aqueous extracts (with the highest activity shown by the organic extract of A. racemosus at 0.1 mg/ml and aqueous extract of G. occidentalis and X. caffra at 1.0 mg/ml each).

The traditional use of three medicinal plants, A. racemosus, L. edulis, and P. africanum were validated by inhibiting all the Gram-positive bacteria and the fungus C. albicans.

Candida albicans has been noted in several studies (Heisey and Gorham, 1992; Buwa and Van Staden, 2006; Ncube et al., 2011; 2012) as the most resistant pathogen towards most plant extracts. In the current study the pathogen was susceptible to organic extracts of A. racemosus (1.0 mg/ml), L. edulis (1.0 mg/ml), P. africanum (1.0 mg/ml), and X. caffra

62 (1.0 mg/ml). None of the aqueous extracts demonstrated significant activity against C. albicans. Mulaudzi et al. (2014) showed that the ethanol and aqueous extracts of X. caffra both possess noteworthy activity against C. albicans at the MIC values of 0.78 mg/ml. In their study, the DCM extract showed no significant activity which is contrary with the findings in the current findings (1.0 mg/ml). Generally, aqueous extracts perform poorly as they only extract hydrophilic compounds for interaction with the pathogen excluding other compounds. Their poor performance is evident in studies such as Muleya et al.

(2014), in which focus was only given to the organic extracts when screening against C. albicans.

Staphylococcus aureus was susceptible to three organic extracts of A. racemosus (0.1 mg/ml), L. edulis (1.0 mg/ml) and P. africanum (1.0 g/ml) and aqueous extracts of G. occidentalis and X. caffra both at MIC value of 1.0 mg/ml. The organic extract of A. racemosus was previously reported to possess noteworthy activity and inhibited the growth of S. aureus at MIC value of 0.15 mg/ml (Mandal et al., 2000; Singh & Geetanjali,

2016). Mulaudzi et al. (2011), indicated noteworthy activity using the ethanol extract of the root of G. occidentalis (0.78 mg/ml). The difference could be attributed to different organs used, as underground parts were indicated to possess higher concentration of bioactive compounds (Maroyi, 2011; Ndivhaleni et al, 2015) contrary to the current study in which leaves were used. Aloe arborescens is one of the most frequently used medicinal plants to treat wounds, burns and various skin problems (Bruce, 1975; Van Wyk et al.,

2000; Mabona et al., 2013)., McGaw and Eloff (2008), Di Luccia et al. (2013), and Amoo et al. (2013), all showed that the organic extracts of A. arborescens lack noteworthy activity and only the homogenized leaf sap has antimicrobial activity (Jia et al., 2008). As previously mentioned, Staphylococcus epidermidis was inhibited by the majority of the plant extracts as follows: organic extracts of A. racemosus (0.1 mg/ml), O. sphaerocarpa

63 (0.2 mg/ml), L. edulis and P. africanum both at MIC 0.3 mg/ml. Additionally the organic extracts of A. arborescens, E. camaldulensis, J. zeyheri and X. caffra at MIC of 0.5 mg/ml followed by R. communis (0.8 mg/ml) with three organic extracts of C. edulis, D. stramonium, and G. occidentalis all at MIC of 1.0 mg/ml. Aqueous extracts with noteworthy activity against S. epidermidis were noted from A. racemosus and O. sphaerocarpa both at MIC value of 0.8 mg/ml followed by A. arborescens and G. occidentalis both at 1.0 mg/ml.

Previous studies have also confirmed antimicrobial activity of P. africanum (0.5 mg/ml) against S. epidermidis (Steenkamp et al., 2007), as well as the DCM organic extract of

A. arborescens (1.0 mg/ml), D. anomala (0.5 mg/ml) and E. camaldulensis (0.5 mg/ml) all screened against S. epidermidis (Mabona et al., 2013). The current findings are contradictory to the findings of Mongalo et al. (2013) which indicated that organic extracts of J. zeyheri displayed no significant activity against S. epidermidis. However, the study did not screen DCM extracts but methanol, acetone, and ethyl acetate. Steenkamp et al.

(2007), further noted that X. caffra (1.42 mg/ml) possess moderate activity, Similarly

Mabona et al. (2013), indicated that the DCM extract of Z. mucronata (2.0 mg//ml) possessed moderate activity.

Pseudomonas aeruginosa was susceptible to three organic extracts of A. racemosus (0.3 mg/ml), O. sphaerocarpa (0.5 mg/ml), and P. africanum (1.0 mg/ml). The methanol extracts of the bark and root of P. africanum has previously been shown to have moderate activity at an MIC value of 1.5 mg/ml and 3.0 mg/ml respectively against this pathogen

(Samie et al., 2005). The current study has demonstrated lower MIC values, indicating that extraction with methanol and dichloromethane yields more bioactive compounds as previously noticed in study by Komape et al. (2017). Interestingly, P. aeruginosa was also susceptible to the aqueous extracts of O. sphaerocarpa (1.0 mg/ml), R. communis (1.0

64 mg/ml), and X. caffra (0.75 mg/ml). This indicates that a simple preparation

infusion/decoction as used in traditional medicine has the potential to yield very different

efficacies.

Table 3.5 Antimicrobial activites of plants used in Ga-Mashashane area against skin pathogens______Plant species V o u c h e r P l a n t p a r t Antim icrobial activity (M IC values in m g/m l) n u m b e r a s s e s s e d C a P a S a S e (ATCC 10231) (ATCC 9027) (ATCC 29213) (ATCC 12228) Org Aq Org Aq Org Aq Org Aq

A. arborescens LP 4 L e a v e s 2 .7 > 8 .0 3 .3 2 .0 > 8 .0 > 8 .0 0 . 5 1 .0

A. racemosus LP 3 3 L e a v e s 1 .0 4 . 0 0 . 3 8 .0 0 . 1 4 . 0 0 . 1 0 . 8

C. b isp ino sa LP 1 5 R o o ts 1 .7 > 8 .0 > 8 .0 > 8 .0 > 8 .0 > 8 .0 4 . 0 > 8 .0

C. e du lis LP 4 2 L e a v e s > 8 .0 > 8 .0 4 . 0 > 8 .0 2 .0 3 .0 1 .0 4 . 0

D. stramonium LP 2 2 L e a v e s 1 .7 > 8 .0 1 .3 > 8 .0 1 .7 8 .0 1 .0 4 . 0

D. ano m a la LP 3 T u b e r > 8 .0 > 8 .0 > 8 .0 > 8 .0 2 .7 > 8 .0 1 .7 > 8 .0

E. camaldulensis LP 1 2 L e a v e s 2 .7 8 .0 2 .0 > 8 .0 1 .3 > 8 .0 0 . 5 > 8 .0

G. o ccid en ta lis LP 3 8 L e a v e s 2 .0 4 . 0 2 .7 1 .5 1 .3 1 .0 1 .0 1 .0

J. z e y h e ri LP 7 R o o ts 2 .7 > 8 .0 1 .3 3 .0 2 .0 4 . 0 0 . 5 1 .5

L. e du lis LP 3 6 L e a v e s 1 .0 8 .0 5 .3 > 8 .0 1 .0 2 .0 0 . 3 2 .0

L. re vo lu ta LP 3 1 R h iz o m e 2 .0 > 8 .0 4 . 0 > 8 .0 > 8 .0 > 8 .0 > 8 .0 > 8 .0

O.sphaerocarpa LP 2 7 L e a v e s 2 .0 > 8 .0 0 . 5 1 .0 1 .3 8 .0 0 . 2 0 . 8

P. africanum LP 4 0 L e a v e s 1 .0 8 .0 1 .0 2 .0 1 .0 > 8 .0 0 . 3 3 .0

R. communis LP 1 3 L e a v e s > 8 .0 2 .0 2 .0 1 .0 2 .0 2 .0 0 . 8 2 .0

X . caffra LP 3 0 L e a v e s 1 .0 8 .0 2 .0 0 . 7 5 1 .7 1 .0 0 . 5 1 .5

Z. mucronata LP 4 4 L e a v e s 2 .0 8 .0 4 . 0 8 .0 2 .0 > 8 .0 0 . 8 2 .0

Ciprofloxacin + C 0 . 0 3 9 1 .3 0 . 3 1 0 .6 3 0 . 4 8 0 . 6 4 0 . 0 7 8 1 .3

P g /m l P g /m l P g /m l P g /m l P g /m l P g /m l P g /m l P g /m l A c e t o n e -C > 8 Type of extract: Ag, aqueous; Org, organic. Pathogens: Ca, Candida albicans ; P a , Pseudomonas

aeruginosa; S a , Staphylococcus aureus; S e , Staphylococcus epidermidis; MIC, minimum inhibition

concentration values expressed in mg/ml

2.2.4 STI pathogens

Eight plant species were screened against three pathogens; Candida albicans (ATCC

9290), Gardnerella vaginalis (ATCC 14028), and Neisseria gonorrhoea (ATCC 19424)

responsible for STIs as shown in Table 3.6. Interestingly, all the plant species used

traditionally to treat STIs were active against N. gonorrhoea with very good MIC values

ranging from 0.1 - 0.8 mg/ml. The most inhibitive plant was O. insignis since it inhibited

65 two pathogens. The highest activity was noted from the organic extracts of i) O. insignis

(0.1 mg/ml), ii) D. elata and E. transvaalense (0.2 mg/ml), and iii) A. oppositifolia and R.

communis var. communis (0.3 mg/ml) all against N. gonorrhoea. Similarly with the skin

pathogens, C. albicans displayed the most resistance to all the screened extracts.

Mulaudzi et al. (2011) recorded an MIC value of 6.25 mg/ml for G. occidentalis against C.

albicans and thus indicated the absence of antimicrobial activity, which is also evident in

the current study. Gardnerella vaginalis is an opportunistic pathogen, occurring as a result

of disruptions in the normal vaginal microflora, causing infections such as endometriosis,

pelvic inflammatory diseases, and vaginal cellulites (Van Vuuren and Naidoo, 2010). The

pathogen is susceptible only to the organic extract of O. insignis (1.0 mg/ml) and least

susceptible to all the screened aqueous extracts. Little attention has been given to this

pathogen with regards to antimicrobial activity of medicinal plants used for treating STIs.

Neisseria gonorrhoea has a characteristic infection on the cervix, urinary tract, mouth or

rectum (Murray and Pizzorno, 1999: Van Vuuren and Naidoo, 2013) and causes infertility

in women amongst other conditions (Mulaudzi et al., 2011). Eight medicinal plants utilized

to treat STIs in the Ga-Mashashane area were able to inhibit the growth of this pathogen.

The susceptibility of N. gonorrhoea towards the organic extract of P. africanum (0.5

mg/ml) was highlighted in a study by Naidoo et al. (2013) and concurs with the current

findings. The pathogen was further susceptible to the aqueous extract of E. transvaalense

and E. cripsa both at MIC 1.0 mg/ml.

Table 3.6 Antimicrobial activites of plants used in Ga-Mashashane area against STIs pathogens______P lant species V o u c h e r P l a n t Antim icrobial activity (MIC values in m g/m l) n u m b e r p a r t a s s e s s C a G v N g e d (ATCC 10231) (ATCC 14028) (ATCC 19424) Org Aq Org Aq Org Aq

A. oppositifolia LP 2 9 L e a v e s 8 .0 4 . 0 4 . 0 > 8 .0 0 . 3 8 .0

D. elata LP 3 4 B u lb 1 .3 > 8 .0 4 . 0 > 8 .0 0 . 2 > 8 .0

66 E. transvaalense R o o ts / LP 1 4 . 0 2 .0 4 . 0 4 . 0 0 . 2 1 .0 B a r k

E. crispa s u b s p . crispa LP 3 2 L e a v e s 3 .3 4 . 0 4 . 0 2 .0 0 . 7 1 .0

G. o ccid e n ta lis LP 3 8 L e a v e s 1 .3 4 . 0 5 .0 8 .0 0 . 8 8 .0

O. in sig n is LP 3 9 L e a v e s 1 .7 > 8 .0 1 .0 4 . 0 0 . 1 > 8 .0

P. a frica n u m LP 4 0 L e a v e s 1 .7 8 .0 4 . 0 8 .0 0 . 3 8 .0 R. communis L. v a r. L e a v e s co m m un is LP 1 3 2 .7 2 .0 4 . 0 4 . 0 0 . 3 2 .0

Amphotericin B / + C 0 . 0 3 9 1 .3 1 .3 1 .3 0 . 0 3 9 0 . 1 6

Ciproflaxicin P g /m l P g /m l P g /m l P g /m l P g /m l P g /m l A c e t o n e -C > 8 . 0 Type of extract: Ag, aqueous; Org, organic. Pathogens: Ca, Candida albicans ; G v , Gardnerella vaginalis;

N g , Neisseria gonorrhoeae; MIC, minimum inhibition concentration values expressed in mg/ml

67 2.3 PHYTOCHEMISTRY

2.3.1 Introduction

Phytochemistry is the study of chemicals derived from plants, these can be primary or secondary metabolites. Secondary metabolites are used by plants as defence mechanisms against insects and herbivores (Wink, 2013). Plants are used for medicinal purposes due to the presence of these phytochemicals, particularly the secondary compounds (Wadood et al., 2013). Plants produce a great diversity of secondary metabolites, these include alkaloids, saponins, phenolic compounds, terpenoids, essential oils, and glycosides (Berdy 2005). These compounds exhibit numerous pharmacological activities, for example terpenoids have anti-inflammatory, anti-cancer, anti-viral, and anti-bacterial activities (Berdy 2005, Wadood, 2013), and alkaloids have anti-diabetic, anti-malarial, and anaesthetic properties, which assist in the curbing of both human and animal ailments. Phytochemical screening of traditional medicinal plants in the Limpopo Province has been given attention in recent years but studies have so far been limited to a few plants, e.g. plants used for treatment of tuberculosis (Masoko, et al.,

2013). The current study therefore focuses on the qualitative phytochemical evaluation of some of the medicinal plants (Table 3.7) obtained from the Ga-Mashashane area that were not previously screened and also with newly recorded uses.

68 Table 3.7 Phytochemical screening of medicinal plants used in Ga-Mashashane for various human ailments Parts Alkaloids Saponins Flavonoids Terpenoids Tannins Glycosides Coumarins Species used H2 O CF DME MeOH H2 O MeOH H2 O CF DME MeOH H2 O CF DME MeOH H2 O CF DME EtOH Callilepis laureola Root + + + + Drimia elata Bulb + + + Elaeodendron Leaf + + + + + + + transvaalense — — — — — — — — — — — Englerophytum Bark + + + + + + + + + + magaliesmontanum _ — — — — — — — Grewia occidentalis Leaf + + + + — — + — + — — — — — — — — Ledebouria Bulb + + + + + revoluta — — — — — — — — — — — — — Ozoroa Leaf + + + + + + + + + + + + + sphaerocarpa — — — — — Ptaeroxylon Leaf + + + + + obliquum — — — — — — — — — — — — — Senecio Leaf + + + barbertonicus _ — — — — — — — — — — — — — — Solanum supinum Root — — — — + — — — — — — — — — — — — Zanthoxylum Bark + + + + + + + + + humile — — — — — — — — — Solvents: H2O - Water, CF - Chloroform, MeOH - Methanol, DME - Dimethyl Ether and EtOH - E hanol, + presence, - absence

69 Table 3.7 indicates that the alkaloids were detected in all plant species excluding C. laureola, D. elata, S. barbertonicus, and S. supinum. Alkaloids were detected in the water extracts of E. transvaalense, L. revoluta, O. sphaerocarpa, and P. obliquum. The non­ polar solvents, chloroform and dimethyl ether, had a trend of similar results by manifesting the presence of alkaloids in E. magaliesmontanum, G. occidentalis, and O. sphaerocarpa with alkaloids of P. obliquum only detected in the DME solvent. Alkaloids were detected better in methanol compared to all solvents, from the following five plant species; E. traansvalense, E. magaliesmontanum, G. occidentalis, O. sphaerocarpa, and P. obliquum.

The presence of flavonoids was detected in all species with E. transvaalense, S. barbertonicus, and P. obliquum as exceptions. Flavonoids dissolved better in polar solvents and were thus detected using only water and methanol. Their presence was better detected in plant species E. magaliesmontanum, G. occidentalis, O. sphaerocarpa,

Z. humile, and C. laureola prepared in water solvents. Flavonoids were also detected in the methanol extracts, however, only from a few plant species (C. laureola, D. elata, L. revoluta and S. supinum.

Ledebouria revoluta and S. supinum emerged as the only species that lacked terpenoids.

The presence of terpenoids was detected in chloroform and methanol extracts of E. magaliesmontanum, G. occidentalis, and Z. humile, as well as in the methanol extracts of D. elata, S. barbertonicus, and C. laureola. Terpenoids were poorly detected in all water extracts with an exception of C. laureola and E. transvaalense.

Tannins were better detected in the water extracts of E. transvaalense, E. magaliesmontanum, L. revoluta, O. sphaerocarpa, S. barbertonicus and Z. humile compared to extracts prepared from non-polar solvents. In the non-polar solvents, tannins

70 were only detected from the extracts of L. revoluta using chloroform, and E. magaliesmontanum and S. barbertonicus using DME. Tannins were only detected in methanol extracts of two plant species; E. transvaalense and O. sphaerocarpa.

The foam test was incorporated to detect the presence of saponins by shaking the extracts vigorously in water and those that had persistent foam were regarded to contain saponins. Saponins were detected from four plant species: D. elata, E. transvaalense, L. revoluta, and Z. humile.

Glycosides dissolved effectively in non-polar solvents; chloroform and dimethyl-ether.

Incorporating the two solvents, glycosides were detected in both extracts of O. sphaerocarpa, Z. humile, and E. transvaalense of which could not be detected in the dimethyl ether extract. Coumarins were detected in ethanol and their presence was revealed in E. transvaalense, E. magaliesmontanum, O. sphaerocarpa, and Z. humile.

Callilepis laureola exhibited the presence of flavonoids and terpenoids. The plant is used for lung and respiratory problems in the Ga-Mashashane area. Flavonoids have been proven to exhibit various physiological activities in the human body such as anti-ulcer, antibacterial, anti-inflammatory, antihyperlipidemia, and analgesic activities (Zafra-Stone et al., 2007; Yin et al., 2015). Terpenoids are derivatives of terpenes, which are reported to exhibit biological activities which include anticancer, antimicrobial and anti­ inflammatory (Liu et al., 2000). However, C. laureola has a toxic diterpenoid glycoside, atractyloside reported to cause liver and kidney necrosis (Obatomi and Bach, 1998;

Tamokou and Kuete, 2014). A decoction of root stock of C. laureola was suggested to be the major cause of death in women residing in the KwaZulu-Natal Province, where the plant is incorporated into a mixture known as ‘Isihlambezo’ used as a labour-inducer

(Varga and Veale, 1997).

71 Drimia elata displayed presence of flavonoids, terpenoids, and saponins. It is taken orally as a blood purifier, for the treatment of STIs, and used in the preparation of a topical ointment for swollen feet or inflammation. Bozorgi et al. (2017), reviewed the phytochemistry of the various species of the genus Drimia and reported the following biological activities; antibacterial, antifungal, antiviral, anti-oxidant, anti-inflammatory, and anti-insecticidal. Saponins have been reported to have a wide array of biological activities as they are also derivatives of terpenes. Their biological activity includes anticancer, anti­ inflammatory, antifungal, antiviral and also anti HIV-1 (Luyt et al., 1999; Yang et al., 1999;

Liu et al., 2000; Parsaeimehr et al., 2011). Okem et al. (2015), highlighted that D. elata also produces a cardiac glycoside of the bufadienolide-type, the derivative of proscillaridin

A, reported to be effective against cardiac conditions - heart failure, and hypertension

(Komajda et al., 2003). Drimia elata exhibits anti-inflammatory activity (Bozorgi et al.,

2017) and its use for the treatment of swollen feet/inflammation in the Ga-Mashashane area is thus some what validated.

Elaeodendron transvaalense displayed the presence of alkaloids, terpenoids, tannins, saponins, and coumarins and the root/bark decoction is taken orally to treat high blood pressure and STIs. The ethyl acetate extract of the stem bark of the plant was proven to exhibit anti-HIV activity by inhibiting the activity of the transcriptase protein a- glycohydrolase and viral proteins (NF-kB and Tat) which play a vital role in the HIV virus lifecycle (Tshikalange et al., 2008). The presence of triterpenoids was highlighted by

Tshikalange (2007), with a suggestion of antidiabetic activity contributed by phenolic compounds; elaeocyanidin, gallotannins, and ouratea proanthocyanidin A (Drewes and

Mashimbye, 1993).

Englerophytum magalismontanum displayed the presence of alkaloids, flavonoids, terpenoids, tannins, glycosides, and coumarins and the leaf extract is taken orally as

72 blood purifier and for stomach problems. The plant was previously shown to exhibit anti­

inflammatory, anticholinesterase and anti-oxidant activity (Dzoyem and Eloff, 2015).

Alkaloids and phenolic compounds are the main constituents in plants with anticholinesterase activity (Adewusi et al., 2012, Aderogba et al., 2013; Dzoyem and

Eloff, 2015).

Grewia occidentalis exhibited the presence of alkaloids, flavonoids, and terpenoids. Leaf decoction of the plant is used for the treatment of STIs, in addition, the leaves are crushed and mixed with Vaseline and topically applied to sores and wounds. Aqueous and methanol extracts of the bark of G. occidentalis was previously proven to exhibit anti­ oxidant activity (Steenkamp et al., 2005). Plant extracts with anti-oxidant activity play a major role as they interact with reactive oxygen species (ROS), which are known to cause state of oxidative stress resulting in numerous human diseases by damaging lipids, proteins, and DNA (Gutteridge, 1993; Steenkamp et al., 2005; Ahmed et al., 2012).

Ledebouria revoluta displayed the presence of alkaloids, flavonoids, tannins, and saponins, and the bulb extract is taken orally for feet inflammation and stomach ailments.

Muleya et al. (2014), reported that L. revoluta exhibits antibacterial, antifungal, and anti­

inflammatory activity. The detection of numerous phytochemicals from L. revoluta with anti-inflammatory activity validates its use for the treatment of swollen feet or

inflammation. The presence of phenolic compounds such as homoisoflavanones was previously isolated from the bulb of L. revoluta in a study conducted by Moodley et al.

(2006). Homoisoflavanones are suggested to have broader biological activities apart from anti-inflammatory activity (Moodley et al., 2006).

Ozoroa sphaerocarpa showed the presence of all the screened classes of compounds

(i.e. alkaloids, flavonoids, terpenoids, tannins, glycosides, and coumarins), with only

73 saponins not detected. The bark extract of this plant is taken orally to treat asthma, diarrhoea, and wounds. Sibandze et al. (2016), isolated alkyl phenols from bark extract of O. sphaerocarpa and were proven to exhibit both antibacterial and antifungal activity.

Tannins are effective in wound treatment as they are capable to arrest bleeding (Dzomba and Gwizangwe, 2013), thus validate the use of the plant for wound healing.

Ptaeroxylon obliquum displayed the presence of alkaloids, terpenoids, and coumarins and the leaf extract is taken orally to treat gastrointestinal disorders. Agostinho et al.

(2013), showed the presence of terpenoids and isolated ptaerobliquol which has antiparasitic activity specifically against Toxoplasma gondii. Furthermore, their study isolated chromones and coumarins such as a scopoletin and prenyletin. Scopoletin is a sesquiterpene coumarin which has been reported to exhibit numerous biological activities such as antiviral, antibacterial, antileishmanial, anti-inflammatory, and antitumor activity

(Gliszczynska and Brodelius, 2012). The diterpenoid Cneorubin X was isolated from wood bark of P. obliquum (Mulholland and Mahomed, 2000). However, its activity remains unknown.

Senecio barbertonicus displayed the presence of terpenoids and tannins and the leaves

(cladophylls) are boiled in milk and the mixture is taken orally as a purgative. The mode of action to justify the use of S. barbertonicus as purgative is unclear, however, tannins have antibacterial activity believed to be attained through the complexing with bacterial proteins (Dzomba and Gwizangwe, 2013). Therefore, it is suggested that the interaction of terpenoids and tannins with milk bacteria could justify its use as a purgative.

Solanum supinum displayed the presence of flavonoids and it is incorporated into soft porridge for the treatment of diarrhoea in babies. The plant contains the least number of phytochemicals compared to all the screened plants, with the assumption of exhibiting

74 anti-oxidant activity due to the presence of flavonoids. The current study has confirmed that S. supinum is neither antibacterial nor antifungal as previously highlighted by

Mathebe et al. (2006).

Zanthoxylum humile displayed the presence of alkaloids, flavonoids, terpenoids, tannins, saponins, glycosides, and coumarins. The bark of Z. humile is used for cold and flu, mouth and throat sores, the bark is kept in the mouth like a lozenge. The phytochemical profile of Z. humile was previously reported by Dzomba and Gwizangwe (2013), in which all the phytochemicals in the present study were detected with the exception of coumarins.

Furthermore, the study indicated that Z. humile exhibit several biological activities such as anti-oxidant, antibacterial, and anti-inflammatory, and thus validating its utilization for cold and flu related illnesses.

75 CHAPTER 4

Summary, general conclusions, and future research

3.1 Summary The ethnobotanical, antimicrobial, and phytochemical properties of selected medicinal plants of the Ga-mashashane area, Limpopo, were investigated. Twenty informants in the age group of 34 - 90 were interviewed on the use of medicinal plants to treat various ailments in the form of structured interviews. The findings are summarized as follows

3.1.1 Ethnobotany

Twenty informants in the ratio of 12:8 male to female were interviewed from October 2014 to March, 2015 to gather ethnobotanical knowledge. Forty-five medicinal plants were noted to treat various ailments within the area; 22 were recorded for the treatment gastrointestinal ailments, 18 for respiratory ailments, 16 for skin ailments, eight used for

STIs, and four for non-infectious ailments. For infectious conditions, the most utilized medicinal plants were Peltophorum africanum which is used to treat gastro-intestinal, respiratory, skin, and sexually transmitted infections. Carpobrotus edulis, Eucalyptus camaldulensis, Acokanthera oppositifolia, and Ziziphus mucronata were used for treating three infections each. The dominant plant families were Anacardiaceae and Asteraceae both with five species each, followed by Fabaceae and Verbenaceae both at three species each. The dominance of the Anacardiaceae family supports prior findings by

Maroyi (2013) and Rasethe (2013). Preference of plant parts was given to the leaves

(53%), underground parts (31%), bark (11%) and fruits (5%), with this trend highlighted in previous studies (Van Vuuren & Naidoo, 2010); York et al.2011; Semenya &

Maroyi,2013; Masevhe et al.,2015). Oral administration of the medicinal plants was the

76 most preferred mode of administration followed by inhalation/steaming, soaking/wash, topical application of ointment, and smoke therapy. The current study has resulted with one new medicinal plant, Senecio barbertonicus and 12 new uses of the medicinal plants;

Acokanthera oppositifolia, Aloe arborescens, Elaeodendron transvaalense,

Englerophytum magalismontanum, Hypoxis hemerocallidea, Ledebouria revoluta,

Ozoroa sphaerocarpa, Ptaeroxylon obliquum, Ricinus communis var. communis,

Vangueria infausta subsp. infausta, and Zanthoxylum humile. Furthermore the recorded uses of the medicinal plants in Ga-mashashane area are in correlation with other cultures in South Africa.

3.1.2 Antimicrobial screening

Antimicrobial screening was performed with 38 medicinal plants screened against 16 plants associated with gastrointestinal infections (six pathogens), respiratory infections

(three pathogens), skin infections (four pathogens), and STIs (three pathogens). The screening was performed with an objective to validate the use of the plants based on the antimicrobial activity of the plant extracts. The use of 15 plants from 22 plants highlighted to treat gastrointestinal infections was validated as all of these plants exhibited noteworthy antimicrobial activity against at least one gastrointestinal pathogen. The most inhibitive plants were O. sphaerocarpa and S. lancea having inhibited all the six pathogens. Other inhibitive plants were C. glabrum, E. elephantine, and P. africanum as they inhibited four pathogens. The fact that these plants have demonstrated a wide spectrum of activity against gastrointestinal bacteria does validate their traditional use for the treatment of gastrointestinal infections. The antimicrobial screening against respiratory pathogens has shown Peltophorum africanum as the most inhibitive plant as it inhibited two pathogens,

Klebsiella pneumoniae and Staphylococcus aureus. The highest antimicrobial activity

77 was noted from the organic extract of H. caespititium (0.1 mg/ml) screened against

Moraxella catarrhalis. The highest susceptibility was noted from K. pneumoniae which was inhibited by five organic and two aqueous extracts, followed by S. aureus inhibited by four organic and one aqueous extract. Asparagus racemosus, Peltophorum africanum, and Ximenia caffra emerged as the most inhibitive plant species against skin pathogens as they inhibited all the four skin pathogens, followed by Lannea edulis which inhibited three pathogens, and G. occidentalis and O. sphaerocarpa (MIC range 0.2 - 1.0 mg/ml) inhibiting two pathogens each. Staphylococcus epidermidis had the highest susceptibility towards plant extracts and was inhibited by 13 organic extracts and four aqueous extracts. Eight medicinal plants indicated in the treatment of STIs exhibited noteworthy activity against Neisseria gonorrhoea with MIC values ranging from 0.1 - 0.8 mg/ml. Plant extracts with the highest noteworthy activity was recorded for O. insignis in which the organic extracts inhibited two pathogens, Gardnerella vaginalis at 1.0 mg/ml and N. gonorrhoea at the lowest MIC value of 0.1 mg/ml. Essential oils of Eucalyptus camaldulensis and Lippia javanica exhibited noteworthy activity against S. aureus and since the pathogen is a common cause of respiratory and skin infections, the use of the above two plant species is validated to treat this ailment.

3.1.3 Phytochemical screening

The qualitative phytochemical evaluation of 11 medicinal plants used traditionally in the

Ga-Mashashane area previously not screened and with newly recorded uses was performed. Zanthoxylum humile emerged as the only plant showing the presence of all the screened phytochemicals, followed by Englerophytum magaliesmontanum and

Ozoroa sphaerocarpa which also exhibited an array of all the chemicals, but lacking only saponins. Interestingly, the aqueous extract of Z. humile had the lowest MIC value against

78 K. pneumoniae with its organic extract showing noteworthy activity against the same pathogen. The organic extract of E. magaliesmontanum demonstrated noteworthy activity against two pathogens of the gastrointestinal infections. The phytochemical screening of this plant has indicated that the alkaloids, terpenoids, and coumarins are only detected in organic solvents and not with the aqueous extract. This suggests that the bio-active molecules of E. magaliesmontanum may be related to non-polar compounds. Ozoroa sphaerocarpa has exhibited great antimicrobial activity against six pathogens of the gastrointestinal infections with lowest values recorded from the organic extract.

Furthermore, the inhibition of the pathogens by the plant extracts was only attained when using organic extracts, from which the presence of alkaloids, terpenoids, glycosides, and coumarins were detected. Alkaloids were also detected in the aqueous extract of O. sphaerocarpa with a noteworthy activity recorded against S. typhimirium. Solanum supinum exhibited the least amount of phytochemicals, with a record of only flavonoids.

The plant species also demonstrated poor antimicrobial activity. Callilepis laureola

(flavonoids and terpenoids) and Senecio barbertonicus (terpenoids and tannins) exhibited two types of phytochemicals each. Interestingly, the two plant species exhibited no significant antimicrobial activity with the former exhibiting activity only against K. pneumoniae. Thus it can be concluded that although only qualitative screening has been done on some of the medicinal plants, results indicate that there is correlation between antimicrobial activity and the various phytochemical groups of the plants (Akhtar, 2015).

3.2 General conclusions

The current study has highlighted that the people residing in Ga-Mashashane area are knowledgeable on the use of medicinal plants and the knowledge concurs with other cultures in South Africa. Furthermore, the medicinal use of Senecio barbertonicus and 12

79 new uses from 11 medicinal plants were added into the South African pharmacopeia. The study also noted six plants with the high antimicrobial activity; Ozoroa sphaerocapa and

Searsea lancea for gastro-intestinal pathogens, Asparagus racemosus, Peltophorum africanum, and Ximenia caffra against skin pathogens, P. africanum against respiratory infections, and O. insignis against STIs. Some of the the six species could be targeted as potential sources for isolation of bioactive compounds. Further it can be concluded that there is correlation between antimicrobial activity and phytochemical composition of the medicinal plants.

3.3 Future work and recommendations

The current study has provided a lead for further studies and the following recommendations are proposed;

• Isolation of bioactive compounds from the six most antimicrobially active species,

namely; Asparagus racemosus, Ozoroa sphaerocarpa, Ozoroa insignis, Searsia

lancea, Peltophorum africanum, and Ximenia caffra.

• Further in vitro and in vivo studies should be performed on these six species to

determine their toxicity level.

80 CHAPTER 5

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9 9 APPENDIX 1 Ethical Clearance

FACULTY OF SCIENCE

APPLICATION FOR APPROVAL TO INVOLVE HUMANS IN RESEARCH RELEVANT DEPARTMENT:

A: PRINCIPAL INVESTIGATOR /RESEARCHER ( C o r r e s p o n d i n g a u t h o r ) Y E S / N O ______Initials and Surname: L. A. Papo Student number: 200935782 Address: 335 Plane Street Alveda Ext. no 2. Kibler Park, 2091______Or Room 50A6 Student town Phase 2, University of Johannesburg, Kingsway Road, Auckland Park 2092______E-mail address: [email protected] or papo.lesiba4 @yahoo.com Telephone/Cell number: + 27 737944217 or +27 842319345 Current qualification registered for e.g. MSc Botany: MSc Botany

Appropriate experience in human research: My two years’ experience in tutoring and student assistant has equipped me with skills to work with and do research on human beings.______

B : PROJECT TITLE: ______T he ethnobotanical, antim icrobial and phytochem ical screening of selected m edicinal plants from Ga-Mashashane, Limpopo Province, South Africa

C : S U P E R V I S O R / C O R R E S P O N D IN G A U T H O R ( W H E R E A P P L I C A B L E ) ______Initials and Surname: A. Moteetee Personnel number: 720003521 Address: Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland Park, 2006______E-mail address: [email protected] Telephone/Cell number: 082383209 Qualification: PhD

Appropriate experience in human research: As a researcher I have previously conducted numerous interviews with human beings for similar purposes and also at a professional level______

D: CO-W ORKERS (Directly involved) CO-SUPERVISOR/€QLLABORATOR Initials and Surname: S. Van Vuuren Address: Medical School, University of Witwatersrand, 7 York Road, Parktown 2193

E-mail address: [email protected]

1 0 0 Telephone/Cell number: (011) 7172157 Institution currently affiliated with: University of the Witwatersrand

Appropriate experience in human research: N/A

E : F U N D I N G ______Is the above mentioned project fully funded? Yes/No Funding of project depend on the approval of the Ethics Committee? ______Yes/No______Does approval of the project by Research Institutions depend on the approval of the Faculty Ethics Committee? Yes/No

F: TYPE OF RESEARCH Academic Contract For degree purposes X Degree: MSc

G: SIGNATURES PRINCIPLE INVESTIGATOR/STUDENT DATE

SUPERVISOR DATE

HEAD OF DEPARTMENT DATE

H: PROJECT:

1 . C O M M E N C E M E N T O F R E S E A R C H ______Expected starting date: 01 September 2014 Expected completion date: 01 July 2015______I declare that the project has not commenced without approval (signature):

2. BRIEF JUSTIFICATION (Provide a brief introductory statement NOT EXCEEDING 500 WORDS and s u pp o rted b y relevant scientific literature that explains what problems, questions, needs or scientific or clinical observations or new ideas have led to the planning of the experiment.) (Please type)______Limpopo Province is one of the poorest provinces in the country, and most people still rely much on natural resources for shelter, food and well-being (Rasethe et al., 2013). Like most other provinces in South Africa, the majority of the population consult traditional healers and herbalists. This is due to affordability, the close proximity at which traditional healers are found and the familiarity with the local environment (Cunningham, 1988; Muthu et al., 2006; Semenya et al., 2013a). Limpopo is divided into five districts (Waterberg, Capricorn, Sekhukhune, Mopani and Vhembe) and Ga-Mashashane is found in the Capricorn district. The area is dominated by the Bapedi, Ndebele and to a lesser extend the Tsonga tribes. Semenya and co-authors (e.g. Semenya et al., 2012; Semenya et al., 2013a, 2013b) have published several articles on plant use in the province, including the Aganang municipality in which the study area falls. However,

1 0 1 preliminary studies have shown that a number of gaps still exist in the documentation of the ethnobotany of the area. These publications have focussed mainly on the use of plants for medicinal purposes and specifically medicinal plants used for illnesses such as sexually transmitted diseases, (Semenya et al., 2013a), diabetes (Semenya, et al., 2012), tuberculosis (Semenya & Maroyi, 2013), reproductive problems (Semenya et al., 2013b) and diarrhoea (Semenya & Maroyi, 2012). Despite the importance and dependence on herbal and traditional medicines, a majority of the medicinal plants are not well researched, poorly regulated, may contain adulterated products hence produce adverse effects (Mills et al. 2005). Moreover, a majority of the studies have focused mainly on documentation of their indigenous uses and less on confirming their antimicrobial properties. It is therefore important to screen plants used for traditional medicine in Ga-Mashashane, hence gather scientific proof that validates their medicinal potential.______The proposed study is aimed at screening commonly used medicinal plants of Ga- Mashashane for antimicrobial and phytochemical properties. The commonly used plants will be identified through information provided by elders of the community (grandparents) who have ethnobotanical knowledge. Consequently such resource persons will be interviewed to illicit the information.

3. WHERE WILL THE RESEARCH BE CARRIED OUT? (Please furnish name of department)______Ga-Mashashane, Capricorn district, Limpopo

4. WILL CLINICAL TRAILS BE DONE? Y E S N O If yes supply com plete the following Investigator-initiated clinical trials - good clinical practice (gcp) training: date and name of gcp course attended (dd/mm/year) for all investigators (investigators’ meetings do not qualify as gcp training).

F U L L N A M E : ______

G C P C O U R S E N A M E : ______

DATE OF GCP COURSE: DAY/MONTH/YEAR: ______

H OSPITAL/INSTITUTION W HERE EMPLOYED (IF APPLICABLE)

F U L L - T I M E O R P A R T - T I M E E M P L O Y E E : ______

H P C S A N O :

5. REQUIREMENTS FOR THE STUDY If radiation or isotopes are to be used, written approval must be obtained from relevant authority N/A If drugs are to be used, written approval must be obtained from relevant authority N/A

6. STUDY PARTICIPANTS a) State where and how participants will be selected:______Participants will be elders in the community, selected randomly, particularly my grandfather who has a vast knowledge of medicinal plants b) OR will participants be invited to volunteer or will they be selected?______Selected c) Are the participants subordinate to the person doing the recruiting? -Yes No d) If yes, justify the selection______of subordinate participants

1 0 2 e) Will control participants be used? Yes No f) If yes, explain who they are and how they will be recruited g) Participant records: State what records will be used, how they will be selected, whether the study is retrospective or prospective and the approximate range of dates of records.

j) If participants are minors, is an informed consent document provided? Yes ______No______N/A______k) Sex: Male x Female x l) Number of participants and controls?______2______m) Will the research benefit the participants in any direct way? Yes No n) If yes, explain how? Participants will be given feedback on the efficacy of the plants used to treat ailments. o) Will participants receive any remuneration? Yes No

q) Will participation, non-participation or withdrawal from the study disadvantage persons in any way?

Yes No r) If yes, explain in what why:______

s) Explain the steps taken to ensure educated informed consent (make sure that the participants fully understand what they are agreeing to)______Briefing sessions were carried out before interviews commenced. t) Is the following participant information sheets attached? (For written and verbal consent) u) Informed consent form for written consent. v) If informed consent will be verbal - explain why? Participants are old aged people and they were thoroughly explained the relevant information regarding the study in their vernacular language.______w) If informed consent is not considered necessary - explain why not x) If a questionnaire or interview is to be used in the research, it must be attached. If not, the application cannot be considered.______The questionnaire used for interviews is attached. y) How will confidentiality be maintained so that participants are not identifiable to persons not involved in the research? Please answer the questions below:

Will data be confidential?

1 0 3 Yes ii. Will identifiable data be coded and the ‘links’ kept separate?______The filled questionnaires that have individual information and names of resource persons will not be made available to the public______iii. Who will have access to data?______Research supervisors iv. To whom will results be made available?______University of Johannesburg.

7. HYPOTHESIS

Problem statement or hypothesis and results expected (Please type)

The knowledge of ethnobotany in Ga-Mashashane area has not yet been recorded and is fading at an alarming rate, as this great knowledge resides with the elders whose lifespan is almost over, and it is not being effectively passed on to the younger generation (done mostly verbally). However, if the transfer of knowledge could be maintained by appropriate documentation, indigenous knowledge would strengthen. Furthermore validation of in vitro activity may provide credibility to the traditional use.

EXPECTED RESULTS

The expected results are as follows: a) Ethnobotany: An emergence of previously undocumented plant species and uses. b) Antimicrobial activity: New records of antimicrobial activity. c) Plants used in combination may be synergistic in nature. d) Phytochemistry: Screen for bio-active compounds.

8. AIMS/ OBJECTIVES OF THE PROPOSED STUDY (State these briefly and succinctly, please list, please type, please do not attach documents, the space below may be enlarged if required).______Specific objectives are therefore to: (a) Identify hitherto unrecorded plants used in the Ga- Mashashane village for medicinal purposes. (b) Screen the previously unrecorded and unstudied medicinal plants for antimicrobial activity, specifically focusing on pathogens associated with disease related to plant use. (c) Assess these newly recorded medicinal plants for their phytochemical properties. (d) Identify what plants are used in combination and determine if a synergistic effect is observed when tested for antimicrobial activity.______

9. POTENTIAL BENEFITS OF THE RESEARCH FINDINGS What are the benefits and how will the benefits be conveyed to the participants? (These are required to aid the reviewing committee in performing a harm/benefit assessment.) (Please type)______The research findings will provide scientific proof that will help verify claims of medicinal potential for plants used in Ga-Mashashane. The results will be conveyed to the participants through word of mouth.

10. JUSTIFICATION FOR THE USE OF HUMANS If medical or invasive procedures are involved, (briefly justify the use of humans and the number needed. If large numbers are to be used, provide additional rationale for the numbers. State also what non-sentient model/s were considered and on what grounds they were rejected.) (Please type) N/A

11. EXPERIMENTAL DESIGN (W hat will be done?) (Explain the reasoning behind the study design and experimental planning, with particular reference to determination of sample size and statistical analysis. Describe how the subjects will be allocated to experimental and control groups and where applicable, how the experimental treatments will be assigned to each group. The use of flow charts is recommended. The information should be presented in an easily accessible manner.) (Please type)

1 0 4 Elders in rural communities usually possess rich ethnobotanical knowledge. Therefore they were interviewed to provide information on Ga-Mashashane’s traditional medicine. Questionnaires covered aspects such as; common infections encounted, medicinal plants they use for curing such ailment, period of practice on using such medicinal plants.

12. EXPERIMENTAL PROCEDURES (How will it be done?) (Describe briefly in short annotated sentences IN SEQUENCE, all the steps that will be performed in conducting the proposed data survey or experiment. These include: duration of data survey procedure /experiment, and if relevant the collection of samples (if body fluids give routes of collection and volumes), Is/are procedure/(s) routine for: diagnosis/management or specific to this research?) (Please type)______No experimental procedure was/will be performed on humans. Their contribution was only verbal through interviews guided by questionnaires. Each participant was interviewed for less than an hour. Data collected from interviews will help identify common ailments and plants of medicinal importance in Ga-Mashashane used for curing such ailments. The plants will be screened for antimicrobial and phytochemical properties.______

13. SEVERITY OF EFFECTS (RISKS) OF THE EXPERIMENTAL PROCEDURES ON THE SUBJECTS Will the procedure cause physical or psychological discomfort or deprivation? YES NO In the case of data surveys please justify the duration of the procedure (what steps have been taken to minimize the time taken for the survey and to avoid discomfort to the participants?) List the procedures that may cause deprivation, fear, distress and pain.______N/A______Describe what sensations the person may feel.______

Categorise these as minimal, intermediate or high.

* Give their likely duration in time.

Describe what specific steps will be taken to alleviate these conditions through the use of ataractics, dissociative agents, analgesics, anaesthetics or other methods. Estimate how effective these are likely to be.______

Explain if no risks are anticipated) (Please type)

14. STATISTICAL ANALYSIS Is the data descriptive only or will it be statistically analysed? (Describe briefly how the data obtained from the study will be analysed statistically, explain this decision and state by whom the analyses will be performed.) (Please type)______The data is descriptive______

15. REFINEMENT (Describe the specific steps that have been taken to refine the data survey method or experimental procedures to make them as humane as possible i.e. minimising the impact of the proposed procedures on the participants’ wellbeing.) (Please type)______The survey method was structured questionnaires which were used to conduct interviews. The questionnaires were approved by research supervisors.______

16. FEEDBACK: Will feedback of the final outcome of the study be provided to the participants? YES NO If YES give details of how and when the feedback will be provided.______Upon completion of the research, including laboratory assessment, participants will be informed of which plants are effective and have the healing properties.

1 0 5 If NO explain why feedback is not considered necessary or appropriate

17. PERMISSION OF RELEVANT AUTHORITY/IES TO DO THE STUDY (Permit, Letters of approval. State name of authority/ies where applicable)

18. OTHER ETHICS COMMITTEES. (What is the status of the application if submitted?)

PLEASE NOTE

1. Please indicate clearly a correspondence address and contact details. Failure to do so could cause delays.

2. Full details of the study have to be provided with the application. Sufficient information will assist the Committee to understand the protocol detailing the background to the research project, the design of the study and all procedures.

3. Please contact Ms Karien van den Berg, Faculty Officer, Postgraduate Studies, Faculty of Science, C-Ring 202 APK when any uncertainty or questions arise. Tel: 011 559 3718, Email [email protected]

4. Please note that letters of approval will be available 7 days after a Committee meeting. Minutes must be checked, clearance letters drafted and signed by the Committee Chair before dispatching it to applicants.

5. Whether written or verbal consent is to be obtained, the Faculty Ethics Committee requires a Participant Information Sheet written in friendly language understandable to lay persons explaining what is required from a potential participant.

This should include the following: a) Invite participant to take part in the study.

b) Participation is voluntary, and refusal to participate will involve no penalty or loss of benefits to which the participant is otherwise entitled.

c) The participant may discontinue participation at any time without penalty or loss of benefits.

d) Provide a brief description of the research, its duration, procedures and what the participant may expect and/or be expected to do.

1 0 6 e) Explain any foreseeable risks, discomforts, side effects or benefits, including those for placebo.

f) Disclose alternatives available to the participant. (If risks are involved).

g) Explain where and how the data, photographs, videos and other materials will be used and published.

h) Explain how and when feedback will be provided on the final outcome of the study.

6. In the case of m edical and/or invasive procedures, the following are also required:

a) A professional contact name and 24 hour telephone number.

b) Explanation whether medical treatment will be provided in the case of a complication developing.

c) If required - compensation for clinical trial related injuries will be in accordance with the ABPI guidelines.

d) A separate Patient Information and Informed Consent sheet for blood / tissue samples taken for future testing

e) The Participant Information Sheet may be incorporated into the consent form, or the consent. Form may be submitted separately.

1 0 7 APPENDIX 2

Questionnaire used in this study

Research Questionnaire

Date:______Questionnaire no:

Name of the interviewee:______

Particulars of area:

GPS reading:______Name of area:______Name of Sub-location/Sub-area:, Name of Village (exact place):_

Sociodemographic data:

Age: Gender: Educational background:

Plant information: Plant number:

Pedi name:___ English name:_ Botanical name:, Source: Collected from wild Cultivated at home Both Where does the plant naturally prefer to grow (e.g. wetland/ grassland)?

Is there any specific time of the year when you prefer to harvest this plant?

Underground parts Leaves Bark Fruit Stem Whole plant

Part(s) used:

Other:

1 0 8 Preparation method:

a) Method(s) of

administration:

b) Amount of plant

material harvested for

1x usage:

c) Quantify this amount

(e.g. 1 Handful=1 Cup)

d) Is material used dry or

fresh?

e) If dry material is used,

is everything used, or

only some? (Specify

exact amount):

f) Preparation before

taken (e.g. Add 1 cup

of boiling water to

handful of plant

material)

Dosage:

How much of the medicine (as mentioned at (e)) will be taken every time?

How many times a day will this amount be taken?______

1 0 9 If/when this medicine is given to babies or small children, is there a difference in dosage? If yes, describe the difference(s)?

Symptoms:

Any extra information about the disease and/or its symptoms:______

Extra information:

Are there any other uses for the plant?

Where do you get your knowledge from about this plant?

Why do you choose to use traditional medicine, rather than going to a clinic or a hospital?

Is this plant used in combination with any other plant(s)?

Name the plant(s), and describe how this combination is prepared:______

1 1 0 Any other information:

1 1 1 APPENDIX 3

Description, distribution and ethnobotanical information of medicinal plants gathered from Ga-Mashashane area.

1. Acokanthera oppositifolia (Lam.) Codd (Apocynaceae)

Bushman’s poison (E), Tsebe-dintlha (S)

Figure 1: Acokanthera oppositifolia (Photo: G. Nicholis)

Botanical description

Shrub of 2-5 m height, with deeply fissured bark at maturity stage. Leaves opposite, ovate-elliptic to oblong with a pointed tip, glossy, leathery and dark green above, with the lower surface green and a tinge of purple. Flowers white, clustered at the leaf axils, giving rise to ovoid fleshy red to purplish-black fruits (Schmidt et al., 2009).

Geographical distribution

1 1 2 The species is found in South Africa (Limpopo, North-West, Gauteng, Mpumalanga, Western Cape, and Eastern Cape Provinces) and also Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Acokanthera oppositifolia is used by the Zulu people to treat stress related ailments (Hutchings and Staden, 1994). The Vhavenda women use root/bark decoction to treat menorrhagia (Steenkamp, 2003). Kumar and Singh (2005) reported that plant is used for treatment of toothache, stomach-ache, cold, anthrax, tapeworms, and spider and snake bites. The leaves of A. oppositifolia are extremely dangerous (fatal) if taken in large quantities (Kumar and Singh, 2005).

Ethnobotanical information from interviewees

An infusion of Acokanthera oppositifolia is prepared from very little quantity of leaves and is taken orally to treat stomach complaints, respiratory ailments and also taken as a blood purifier. In case of respiratory ailments the infusion can be taken thrice a day (Papo E., 2014, pers. comm.).

1 1 3 2. Aloe arborescens Mill. (Asphodelaceae)

Krantz aloe (E), Alu (S)

Figure 2: Aloe arborescens (Photo: L. Papo)

Botanical description

Aloe arborescens is a succulent shrub that grows up to 2 m in height, consisting of rosette­ bearing branched stems. Leaf margins highly toothed, soft at margins and curved downwards. Flowers are conically clustered and non-branched with a reddish colour (van Wyk and Smith, 2004).

Geographical distribution

Aloe arborescens is distributed in Botswana, South Africa (Limpopo, North-West, Gauteng, Mpumalanga, KwaZulu-Natal, Western Cape, Eastern Cape Provinces), and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

1 1 4 The sap from the leaves is used to treat wounds, burns and various skin problems (Bruce, 1975; Van Wyk et al. 2000). Decoction taken orally can be used for cardiac problems (Amusan et al. 2002), gastrointestinal complaints and as blood purifier (Philander, 2011; Semenya et al. 2013a).

Ethnobotanical information from interviewees

The leaves of Aloe arborescens are used to prepare an infusion for the treatment of swollen feet. The leaves are macerated into a washing container. The infusion should not be too hot but allow swollen feet to submerge into the washing container, and treatment is done until inflammation subsides (Papo E., 2014, pers. comm.).

1 1 5 3. Artemisia afra Jacq. ex Willd (Asteraceae)

Wormwood (E), Lengana (S)

Figure 3: Artemisia afra (Photo: L. Papo)

Botanical description

Multi-stemmed shrub, grows up to 2 m tall. The leaves are feathery, with dark green on the upper surface and silver characteristic colour on the lower surface (Van Wyk et al., 1997).

Geographical distribution

The shrub spreads its distribution from southern Africa (Lesotho, Namibia, South Africa, and Zimbabwe), towards East Africa (mountainous regions of Kenya, Tanzania and Uganda) and as far north Ethiopia (Lui et al., 2008). In South Africa the plant has a very wide distribution and occurs in all the nine provinces (Germishuizen and Meyer, 2003).

Recorded medicinal use

The leaves are boiled to prepare a decoction used to treat fever, coughs and influenza (Dykman, 1981; Watt & Breyer-Brandwijk, 1962). The decoction can also be administered to treat stomach-ache, dry dyspepsia, small-pox and malaria (Burits et al. 2001; Van Wyk and Gericke, 2000; Vagionas et al. 2007; Semenya and Maroyi, 2013).

1 1 6 Ethnobotanical information from interviewees

An infusion is prepared by adding boiling water to a handful of Artemisia afra leaves in a cup and taken orally thrice daily to treat cold and flu. The leaves can also be chewed raw to treat stomach-ache. Large quantity of leaves can also be added in a washing bowl, followed by addition of boiling water and the steam vapour inhaled to treat flu. A blanket is used to cover both the patient and the washing bowl (Papo E., 2014, pers. comm.; Bonoko F., 2015, pers. comm.; Ledwaba D., 2015, pers. comm.).

1 1 7 4. Asparagus racemosus Wild. (Asparagaceae)

Asparagus (E), Morakadimane (S)

Figure 4: Asparagus racemosus (Photo: L. Papo)

Botanical description

Asparagus racemosus is a herbaceous plant of 0.5-4 m in height, with characteristic twining stems. The plant is woody, with branches lacking spines and resembling stems. Leaves modified into numerous cladodes, having spines that turn brown to grey when matured. The plant produces perianth which gives rise to a globose berry with a slightly fissured pericarp (Jessop, 1966).

Geographical distribution

Asparagus racemosus is distributed in Botswana, Namibia, Swaziland, and South Africa where it is found in the Limpopo, Eastern Cape, KwaZulu-Natal, and Mpumalanga Provinces (Germishuizen and Meyer, 2003).

Recorded medicinal use

Asparagus racemosus is reported to treat nervous disorders, dyspepsia, tumours, and inflammation, and to increase fertility and vitality in women (Garde and Vagbhat, 1970; Sharma et al., 2003; Alok et al., 2013).

1 1 8 Ethnobotanical information from interviewees

The leaves and twigs of A. racemosus are used to prepare an infusion or decoction applied topically to treat chicken-pox and sores. The infusion can also be used to prepare soft porridge for children suffering from stomach-ache (Papo E., 2014, Pers. Comm.)

1 1 9 5. Callilepis laureola DC. (Asteraceae)

Ox-eye Daisy (E), Phela (S)

Figure 5: Callilepis laureola (Photo: G. Nicholis)

Botanical description

Callilepis laureola is a herb of 0.8 m height. The plant has a characteristic huge woody rootstock from which erect multiple glabrous or hairy stems develop. Leaves simple, lanceolate to elliptic with young leaves developing from the top of the branches. The main stem produces a capitulum with a ray of white florets encircling the disc-shaped purple florets. Callilepis laureola has a characteristic unpleasant smell (Walker, 1996).

Geographical distribution

The plant species is mostly found in the Eastern Cape, Free State, and Mpumalanga Provinces, as well as in Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Bulb decoction is reported to induce fertility (Debetto, 1978; Seedat and Hitchcock, 1971; Semenya et al, 2013b). In addition the bulb decoction is recorded for treatment of

1 2 0 stomach problems (Steenkamp et al. 1999), tape worm infestations, and cough (Watt and Breyer-Brandwijk, 1962). However it is reported that C. laureola could cause vomiting, abdominal pain, headache leading into coma and death (Wink and Van Wyk, 2008).

Ethnobotanical information from interviewees

A decoction prepared from the bulb of C. laureola is used to treat lung and respiratory problems. The bulb is macerated into a pot, boiled, sieved and the mixture is taken orally thrice a day until the condition subsides (Sibela M., 2015, pers. comm.)

1 2 1 6. Carissa bispinosa (L.) Desf. ex Brenan (Apocynaceae)

Num-num (E), Morokolo (S)

Figure 6: Carissa bispinosa (Photo: L. Papo)

Botanical description

Carissa bispinosa is an evergreen multi-stemmed shrub reaching heights of 3 m, with conspicuous Y-shaped thorns. The plant has a greyish smooth bark that wrinkles at maturity, with opposite, ovate leaves. The leaves are glossy, dark green on the upper surface and pale on the under surface. The flowers are pink to whitish, clustered at the tips of the branches and producing sweet scent. The plant produces a red glossy and fleshy edible fruit (Schmidt et al., 2002).

Geographical distribution

Carissa bispinosa is distributed over the entire southern Africa (Germishuizen and Meyer, 2003).

Recorded medicinal use

The plant has been reported to treat impotence (Amusan et al., 2002). The people of south-central Zimbabwe prepare a root decoction of C. bispinosa to treat cough and diarrhoea (Maroyi, 2013).

1 2 2 Ethnobotanical information from interviewees

Root of the plant is macerated into a pot and 1L of water is added to prepare a decoction. The decoction is taken thrice a day to treat chicken-pox and sores until healed (Papo R, 2015, Pers. Comm.).

1 2 3 7. Carpobrotus edulis (L.) L. Bolus (Mesembryanthemaceae)

Sour fig or Cape fig or Hottentots fig (E), Ditships (S)

Figure 7: Carpobrotus edulis (Photo: L. Papo)

Botanical description

Carpobrotus edulis is a perennial succulent plant usually reaching heights of 0.15 m, with branches trailing along the ground. Leaves spread out, triangular, lightly green, occurring in two forms, straight or curved towards the outside to the apex. Flowers occur in 2-4 leaf pairs, solitary, giving rise to a yellowish smooth fleshy edible fruit (Wisura and Glen, 1993).

Geographical distribution

Carpobrotus edulis is distributed in the Eastern, Northern and Western Cape Provinces (Germishuizen and Meyer, 2003). The plant is also cultivated as an ornamental plant.

Recorded medicinal use

Carpobrotus edulis sap has been reported to treat skin ailments (Watt and Breyer- Brandwijk, 1962; Rood, 1994; Van Wyk et al., 2000; Van der Watt and Pretorius, 2001; Van Vuuren and Mabona, 2013). In addition the sap is utilized as poultices for sores, burns, and scalds, gargled to treat infections of the mouth and throat, dysentery, digestive troubles, tuberculosis, and as a diuretic (Martins et al., 2005). Van Vuuren and Naidoo

1 2 4 (2010), reported that the extracted sap is a remedy against STIs in particular, herpes and candidiasis.

Ethnobotanical information from interviewees

The leaves are harvested and the sap is squeezed out and administered orally for the treatment of toothache. The sap is also squeezed out, applied and rubbed on sores. The decoction of the leaves is also used to treat digestive ailments and cough. For cough the decoction can be administered thrice daily (Mahlong S., 2015, pers. comm.).

1 2 5 8. Clerodendrum glabrum E.Mey. var. glabrum (Verbenaceae)

Tinderwood (E), Motlhokotlhoko (S)

Figure 8: Clerodendrum glabrum (Photo: L. Papo)

Botanical description

Clerodendrum glabrum is a medium-sized shrub reaching heights of 10 m, with a single or several thin straight stems. The stems are covered with a pale yellowish-brown bark with noticeable white lenticels. Leaves are deeply sunken, leathery, and slightly pubescent. The plant produces clustered pink to purplish flowers which produce round fleshy purple fruits when ripe (Schmidt et al., 2002).

Geographical distribution

Clerodendrum glabrum is distributed in Botswana, Namibia, and entirely across South Africa with Northern Cape and Western Cape Provinces being the only exceptions (Germishuizen and Meyer, 2003).

Recorded medicinal use

The leaves of Cleronderum glabrum are reported to treat malaria (Clarkson et al., 2004; Tshikalange et al., 2008).

Ethnobotanical information from interviewees

1 2 6 A handful leaves of Clerodendrum glabrum is boiled in 1L of water. The decoction is taken to treat running stomach or stomach cramps. A small quantity of the leaves can also be chewed or an infusion be prepared for the same purpose (Papo E., 2014, pers. comm.).

1 2 7 9. Combretum imberbe Wawra. (Combretaceae)

Leadwood (E), Mohwelere

Figure 9: Combretum imberbe (Photo: L. Papo)

Botanical description

Combretum imberbe is a tree reaching heights of 7-20 m, with a cracked pale grey bark that is rough and deeply fissured, making irregular blocks. Leaves opposite, obovate to oval, appearing grey-greenish on the upper surface and pale green on the under surface. The plant produces creamy-yellow small flowers that produce small 4-winged yellowish- green fruits (Schmidt et al., 2002).

Geographical distribution

Combretum imberbe is distributed in Botswana, Namibia, South Africa (Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North-West Provinces), and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Combretum imberbe is reported to treat chest cough and fever (Hutchings et al., 1996; McGaw et al., 2008).

Ethnobotanical information from interviewees

1 2 8 The dry bark of Combretum imberbe is harvested exposed to fire, burnt and sniffed several times to treat headache and fever. In case of fever, the process can be repeated until the condition subsides (Papo R, 2015, Pers. Comm.)

1 2 9 10. Datura stramonium L. (Solanaceae)

Jimson weed or Devil’s snare (E), Mokhure (S)

Figure 10: Datura stramonium (Photo: L. Papo)

Botanical description

Datura stramonium is an annual herb that grows up to 2 m tall, having diamond to elliptic shaped leaves with strongly toothed margins. The flowers are pale purple or white, and give rise to a spiny capsule fruit (Gongalves, 2005).

Geographical distribution

Having originated in Mexico Datura stramonium has become naturalised in warmer regions across the world and it is widely distributed in South Africa and other southern African countries including Botswana, Lesotho, Mozambique, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

The leaves of Datura stramonium are placed onto infected area to treat boils and abscesses (Watt and Breyer-Brandwijk, 1962; Bruton 1995; Rabe and Van Staden, 1997; Van Wyk et al., 2000; Priya et al., 2002; McGaw and Eloff, 2008; Saadabi & Moglad 2011; Mabona and Van Vuuren, 2013).

1 3 0 Ethnobotanical information from interviewees

The leaves of Datura stramonium are used to prepare an infusion which is applied topically to swollen body parts to treat inflammation. In case of inflamed feet, the patient immerses the feet inside the washing container, treatment is done until inflammation subsides (Papo E., 2014, pers. comm.). The plant should however be used with caution as it is known to be highly poisonous to livestock and humans, especially the seeds, all parts contain toxic tropane alkaloids.

1 3 1 11. Senecio barbertonicus Klatt (Asteraceae)

Succulent bush senecio, Barberton senecio, Lemon bean bush, Finger-leaved senecio (E), Mapholo (S)

Figure 11: Senecio barbertonicus (Photo: L. Papo)

Botanical description

Senecio barbertonicus is a multi-stemmed succulent shrub growing up to 4 m in height. The plant produces cylindrical branches and leaves. The leaves are whorled, borne on very short petioles, and green with pointy axes. Inflorescence is composite flower head which develops terminally on the branches, with tubular orange flowers and a characteristic sweet scent. The flowers produce a cylindrical pubescent fruit (Pooley, 1998).

Geographical distribution

Senecio barbertonicus is distributed in the Limpopo, Free State, Gauteng, and Kwazulu- Natal, Provinces. The distribution extends to Swaziland as well.

Recorded medicinal use

No recorded medicinal use.

1 3 2 Ethnobotanical information from interviewees

The leaves of Senecio barbertonicus are used as a purgative to get rid of ingested poisonous food. A handful of leaves is harvested, washed and boiled in a litre of milk to prepare a decoction. The patient takes the entire decoction and will likely purge to release the ingested poison.

1 3 3 12. Dicoma anomala Sond. Subsp. anomala (Asteraceae)

Fever bush or stomach bush (E), Hloenya (S)

Figure 12: Dicoma anomala (Photo: G. Nicholis)

Botanical description

Dicoma anomala subsp. anomala is a perennial herb characterized by numerous spreading branches of 5-60 cm long, arising from single miniature stem. The herb has linear to elliptic-linear leaves, dark green on the upper surface greyish-green on the lower surface and with a very short petiole. Leaves have two conspicuous veins arising from the base. Flowers solitary, borne on the axis of stems and branches into a capitulum with 1-30 flowers, with florets ranging from 90 to over 200 in shades of purple, pink to white. The florets produce a very small achene fruit (Pope, 1992).

Geographical distribution

Dicoma anomala subsp. anomala is distributed widely in South Africa with exceptions being the Northern and Western Cape Provinces. The plant species also occurs across the borders of South Africa to Lesotho and Swaziland (Germishuizen and Meyer, 2003).

1 3 4 Recorded medicinal use

The tuber extract is used to treat intestinal problems, stomach-ache, and i nferti lity(Watt and Breyer-Brandwijk; 1962; Steenkamp; 2003).The Basotho people use the rootstock to prepare an infusion used to treat colic, diarrhoea, and constipation (Moteetee and Van Wyk, 2011). In addition the rootstock is reported to treat toothache, hypertension, diabetes, period pains, pneumonia, backache, wounds and sores (Van Wyk and Gericke, 2000).

Ethnobotanical information from interviewees

The root-stock of Dicoma anomala is used to prepare an infusion used to treat colds and flu. The root-stock is macerated into a jug and hot water is added and taken orally thrice a day. The root-stock is also burnt, ground, and mixed with fat or Vaseline for dressing wounds (Papo E., 2014, Pers. Comm.)

1 3 5 13. Dombeya rotundifolia (Hochst.) Planch. (Sterculiaceae)

Wild Pear (E), Mokgoba (S)

Figure 13: Dombeya rotundifolia (Photo: L. Papo)

Botanical description

Dombeya rotundifolia is a tree reaching heights of 10 m, with the main stem composed of branchlets characterised by a pale line of lenticels. The branchlets are covered by a roughly fissured dark brown bark. Leaves alternate, simple, round to broadly oblong, dark green on the upper surface compared to the pale green lower surface. Flowers produced in clusters from the leaf axils, white to pink. The flowers give rise to round hairy capsule fruits (Schmidt et al., 2002).

Geographical distribution

Dombeya rotundifolia is confined to Botswana, South Africa (Limpopo, Gauteng, Mpumalanga, KwaZulu-Natal Provinces), and Swaziland within the southern African region (Germishuizen and Meyer, 2003).

Recorded medicinal use

1 3 6 Dombeya rotundifolia is reported to treat chest complaints (Smith, 1966; McGaw et al., 2008), heart problems, nausea in pregnant women, intestinal ulcers, headaches, stomach complaints, haemorrhoids, diarrhoea and dyspepsia (Reid et al., 2001)

Ethnobotanical information from interviewees

The leaves are harvested and a decoction is prepared with other medicinal plants and is used to quicken the healing process for a variety of ailments including stomach problems (Papo E., 2014, pers. comm.).

1 3 7 14. Drimia elata Jacq. (Hyacinthaceae)

Brandui (E), Sekanama (S)

Figure 14: Drimia elata (Photo: L. Papo)

Botanical description

Drimia elata is a herb of 1 m height characterised by a long inflorescence with leaves growing at the base of the plant. The bulb is covered by red, loosely packed dull red scales. The plant produces a raceme of silver green-grey flowers with a tinge of purple- brown on the petals. (Walker, 1996)

Geographical distribution

Drimia elata is distributed widely in South Africa and extends to neighbouring countries such as Botswana and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

The plant has been recorded to relief pain and asthma by the Zulu people (Hutchings et al., 1996, Van Wyk et al., 2009; Ndhlala et al., 2013). In addition the Zulu people prepare an infusion from the pounded bulbs and leaves to make an enema for fever. Philander (2011), recorded the bulb of the plant as used to treat arthritis. The Bapedi traditional

1 3 8 healers use the decoction prepared from the bulb to treat gonorrhoea and HIV/AIDS (Semenya et. al., 2013a).

Ethnobotanical information from interviewees

The bulb of the plant is harvested, pounded and infusion is prepared and sieved for oral intake once or twice a day for blood purification. The bulb is also used to prepare an infusion for treatment of swollen feet whereby the patient immerses the feet in a container with bearable hot water. The pounded bulb can also be mixed with an ointment and rubbed to enhance healing of feet inflammation (Papo E., 2014, pers. comm.; Baloyi G, 2015, pers. comm.; Gwangwa M., 2015, pers. comm.; Malebana R., 2015, pers. comm.)

1 3 9 15. Elaeodendron transvaalense (Burtt Davy) R.H. Archer (Celestraceae)

Bushveld saffron (E), Monamane (S)

Figure 15: Elaeodendron transvaalense (Photo: L. Papo)

Botanical description

Elaeodendron transvaalense is a medium-sized tree reaching heights of 4-7 m, with a grey stem during juvenile stages, and bark fissured longitudinally at maturity. Leaves simple, alternate or opposite, glossy upper surface of the leaf is greener as compared to the pale lower surface. Flowers produced in flower heads, white to green. The flowers give rise to an edible round yellow fruit (Schmidt et al., 2002).

Geographical distribution

The distribution of Elaeodendron transvaalense in South Africa ranges from the North­ West towards the Limpopo Province spreading across Mpumalanga, and KwaZulu-Natal. It also occurs in other southern African countries; Botswana, Mozambique, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Bark decoction is used to treat dysmenorrhoea (Van Wyk, 1972; Steenkamp, 2003). The Vhavenda traditional healers use root decoction to treat herpes (Mabogo, 1990) and candidiasis (Bessong et al. 2005; Masevhe et al., 2015). Samie et al. (2005) has reported that the plant is used to treat fungal infections, stomach disorders, ulcers, and venereal

1 4 0 diseases. The root/bark is mixed with the root of Peltophorum africanum and the decoction is taken to treat STIs (Semenya et al. 2013a) and female infertility (Semenya et al. 2013b).

Ethnobotanical information from interviewees

The root or bark of Elaeodendron transvaalense is macerated into a pot to prepare a decoction. The decoction is sieved and taken to treat high blood pressure, until the condition subsides (Papo E., 2014, Pers. Comm.).

1 4 1 16. Elephantorrhiza elephantina (Burch.) Skeels (Fabaceae)

Eland's bean or Eland's wattle or Elephant's root (E), Moshitsane (S)

Figure 16: Elephantorrhiza elephantina (Photo: L. Papo)

Botanical description

Elephantorrhiza elephantina is a small shrub of 1 m tall, with a single stem arising from a huge underground root-stock. The plant produces bipinnately-compound leaves, with small creamy-white raceme flowers. The fruit is a flat woody pod (Schmidt et al., 2009).

Geographical distribution

Elephantorrhiza elephantina is distributed among the grasslands of South Africa and occurs in all the provinces with Western Cape as an exception. The plant species also occurs in Botswana, Lesotho, Namibia and Zimbabwe (Germishuizen and Meyer, 2003).

Recorded medicinal use

A decoction prepared from the rootstock is used for treatment of abdominal pains, perforated peptic ulcers, bloody diarrhoea and dysentery (Watt and Breyer-Brandwijk, 1962; Jacot-Guillarmod, 1971; Gelfand et al., 1985; Hutchings, 1989; Pujol, 1990; Mathabe et al., 2006; Appidi et al., 2008; Bisi-Johnson et al., 2010; Mpofu et al., 2014). The Xhosa people of Eastern Cape use the decoction prepared from the rootstock to treat

1 4 2 diarrhoea (Madikizela et al., 2012). The root powder of E. elephantina is used to treat wounds (Mabona and Van Vuuren, 2014).

Ethnobotanical information from interviewees

The root-stock of Elephantorrhiza elephantina is macerated into a pot, sieved and a decoction is used to prepare soft porridge to treat diarrhoea in children and it is also administered for blood purification. Administration for blood purification is usually a once- off whereas it is taken thrice a day for diarrhoea (Papo R., 2015, pers. comm.).

1 4 3 17. Englerophytum magalismontanum (Sond.) T.D. Penn. (Sapotaceae)

Transvaal milkplum (E), Mohlatswe (S)

Figure 17: Englerophytum magaliesmontanum (Photo: L. Papo)

Botanical description

Englerophytum magaliesmontanum is a medium-sized tree reaching heights of 2-4 m, commonly having a short and twisted stem. The stem is covered by grey smooth bark that flakes during maturity, with branches conspicuously having remnants of fallen fruits. Leaves alternate, clustered at the terminals of branches, oblong to obovate, leathery with upper surface green and glossy and lower surface brownish. The plant produces small brownish to reddish strongly scented flowers along the branches of leaf axils, that give rise to ovoid edible fruits that turn red during maturity (Schmidt et al., 2009).

Geographical distribution

Englerophytum magaliesmontanum is distributed in the northern parts of South Africa, ranging from North West to Limpopo Province across Gauteng, towards Mpumalanga and KwaZulu-Natal. It also occurs in Botswana and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

1 4 4 The root decoction prepared from E. magaliesmontanum is reported to be used by the Vhavenda people as a contraceptive (Mabogo, 1990; Steenkamp, 2003). The Bapedi traditional healers utilize the bark to prepare a decoction to treat diabetes (Semenya et al., 2012a).

Ethnobotanical information from interviewees

The dried bark of Englerophytum magaliesmontanum is exposed to fire and the smoke from burning bark is sniffed to treat headache (Papo E., 2014, pers. comm.).

1 4 5 18. Eucalyptus camaldulensis Dehnh. (Myrtaceae)

Eucalyptus (E), Mobilikomo (S)

Figure 18: Eucalyptus camaldulensis (Photo: L. Papo)

Botanical description

Eucalyptus camaldulensis is a tree reaching heights of 30 m, with a characteristic smooth bark, erect stem, long, alternate, lanceolate leaves with parallel venation. The plant produces a cluster of small cream-yellow flowers at the terminally on branches and produce capsule fruits with a woody hypanthium (Pooley, 1998).

Geographical distribution

Eucalyptus camaldulensis like other Eucalyptus species are found in most regions of South Africa and are not indigenous to the country (Pooley, 1998).

Recorded medicinal use

The leaves are reported to treat diarrhoea, dysentery, haemorrhage, laryngalgia, laryngitis, pharyngitis, sore throat, spasm, trachalgia, and wounds (Duke and Wayne 1981; Mouna and Segni, 2011). The Zulu people use the leaf decoction topically to treat acne (Hutchings, 1996; Babayi et al.2004; Ayepola and Adeniyi, 2008; Musa et al. 2011

1 4 6 & Mabona, 2013). The Bapedi traditional healers utilize leaf decoction to treat TB (Semenya et al. 2012b).

Ethnobotanical information from interviewees

The leaves are harvested, boiled and steam-vapour is inhaled to treat cold, coughs and flu. During the inhalation process a blanket is used to cover the patient and the bowl in which the decoction is poured. A decoction of the leaves taken thrice daily, can also be administered orally to treat cold/flu (Bonoko F., 2015, pers. comm.; Letseku S., 2015, pers. comm.).

1 4 7 19. Euclea crispa (Thunb.) Gurke subsp. crispa (Ebenaceae)

Blue guarri (E), Mokwerekwere (S)

Figure 19: Euclea crispa (Photo: L. Papo)

Botanical description

Euclea crispa is multi-stemmed tree that reaches heights of 4 m, with characteristic twisted stems and many branches. The plant has pale to grey bark that cracks during maturity. Leaves are slightly opposite and elliptic, lanceolate, ovate to occasionally oblanceolate, slightly leathery, green-blue on the upper surface, and pale greenish on the lower surface. Flowers are tiny and occur in clusters, greenish to cream-whitish/yellowish and yield tiny round pubescent reddish to black fruits during maturity (Schmidt et al., 2002)

Geographical distribution

Euclea crispa is distributed widely in the nine provinces of South Africa, and the distribution extends to neighbouring countries such as Lesotho and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

1 4 8 Steenkamp (2003) reported that most of South African women use a decoction prepared from the leaves of Euclea crispa to treat dysmenorrhoea. Moteetee and Van Wyk (2011), highlighted that decoction prepared from the bark of the plant is used by the Basotho as a purgative. The people of the Nhema communal area in Zimbabwe, prepare root decoction for coughs (Maroyi, 2011). The Bapedi traditional healers make use of the root to prepare a decoction for HIV/AIDS (Semenya et al., 2013a).

Ethnobotanical information from interviewees

The root is macerated into a jug and a decoction or an infusion is prepared, sieved and taken orally to treat respiratory ailments such as coughs and STIs. The medicine is taken thrice a day until the condition subsides (Papo E., 2014, Pers. Comm.).

1 4 9 20. Eucomis pallidiflora Baker subsp. pole-evansii (N.E.Br.) Reyneke ex J.C. Manning (Hyacinthaceae)

Giant pineapple lily (E), Mathuba-difala (S)

Figure 20: Eucomis pallidiflora (Photo: L. Papo)

Botanical description

Eucomis pallidiflora ssp. pole-evansii is an evergreen geophyte with bulb partially emerging from the ground. The bulb is globose covered with papery membranous sheaths. Leaves are few, green, oblong-lanceolate, and emerging from same point at ground level. Flowers are many, green to whitish, forming a raceme attached to a long erect peduncle. Fruit is a globose papery capsule (Manning et al., 2003).

Geographical distribution

Eucomis pallidiflora ssp. pole evansii is distributed in the Mpumalanga, KwaZulu-Natal and the Eastern Cape Provinces in South Africa. It also occurs in neighbouring countries such as Lesotho and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

1 5 0 The Bapedi traditional healers reported the plant to be used for the treatment of chlamydia, erectile dysfunction, tuberculosis, blood clotting, and coughs (Semenya and Potgieter, 2013; Semenya et al., 2013b). In addition Semenya and Maroyi (2011), highlighted that the Bapedi traditional healers utilize the decoction for tuberculosis.

Ethnobotanical information from interviewees

The bulb of Eucomis pallidiflora is macerated to prepare a decoction for the treatment of STIs. The decoction is sieved, taken thrice a day until the condition subsides (Papo E., 2014, pers. comm.).

1 5 1 21. Grewia occidentalis L. (Malvaceae)

Cross-berry or Four-corner (E), Mogwane (S)

Figure 21: Grewia occidentalis (Photo: G. Nicholis)

Botanical description

Grewia occidentalis is a multi-stemmed shrub reaching heights of 4 m with characteristic purple-greenish branchlets and conspicuous white lenticels. Leaves alternate, simple, elliptic to ovate or slightly ovate, shiny and glossy. Flowers borne in clusters of three or less, purple of pink. The flowers give rise to a four-lobed cross-berry fruit that turns from green to reddish brown (Schmidt et al., 2002), from which its common name derives.

Geographical distribution

Grewia occidentalis occurs in all provinces of South Africa except in the Northern Cape. Its distribution extends to Lesotho and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

The bark is soaked in water and used to dress wounds (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996; Grierson and Afolayan, 1999; Van Vuuren, 2008; Mabona and Van Vuuren, 2013). Decoction prepared from the root of G. occidentalis is used in combination

1 5 2 with several other medicinal plants to treat infertility (Steenkamp, 2003). Root decoction is also reported to treat venereal diseases or STIs (Mulaudzi et.al. 2011).

Ethnobotanical information from interviewees

A handful of leaves is boiled in half litre of water and the decoction is taken thrice a day orally to treat STIs. The leaves can also be crushed and mixed with Vaseline and applied topically to dress sores and wounds (Papo E., 2014, Pers. Comm.).

1 5 3 22. Helichrysum caespititum (DC.)Harv (Asteraceae)

Helichrysum (E), Matsana (S)

Figure 22: Helichrysum caespititium (Photo: A. Hankey)

Botanical description

Densely tufted, low prostrate perennial herb with minute leaves of 6-10 mm long growing along stems. The leaves are linear, grasping by the base of stems with a characteristic woolly-feel. Capitula borne in terminal clusters, with diameter less than 1 cm composed of linear leaf-like outer-bracts and narrowly oblong inner bracts. The plant produces small yellow, pink-tipped florets (Pooley, 1998).

Geographical distribution

Helichrysum caespititium is widely distributed in South Africa with the Western Cape Province as an exception. The distribution extends to Lesotho, Swaziland, and Zimbabwe (Germishuizen and Meyer, 2003).

Recorded medicinal use

The plant is collected, crushed, burnt, and sniffed for headaches, chest complains and colds (Watt and Breyer-Brandwijk, 1962; Jacot Guillarmod, 1971; Dekker et al., 1983; Gelfand et al., 1985; Hutchings and Van Staden, 1994; Neuwinger, 1996; Swanepoel,

1 5 4 1997; Pooley, 1998; Mathekga et al., 2000; Mathekga , 2001; Arnold et al., 2002; Meyer et al., 2002; Pooley, 2003; Moteetee and Van Wyk, 2011). A decoction made from the whole plant is used to treat diabetes (Semenya et al., 2012a).

Ethnobotanical information from interviewees

The plant is harvested, dried and crushed burnt and sniffed to relieve headaches and cold. An infusion of the plant is taken orally as blood purifier (Papo E., 2014, Pers. Comm.).

1 5 5 23. Hypoxis hemerocallidea Fisch., C.A.Mey. & Ave- Lall (Hypoxidaceae)

Yellow star (E), Tshika-poo (S)

Figure 23: Hypoxis hemerocallidea (Photo: L. Papo)

Botanical description

Hypoxis hemerocallidea is tuberous perennial with long and slender leaves that meet by the middle of the comb. The underground comb is brown or black, and fleshy and yellow inside. It produces a characteristic star-shaped yellow flower (Baker, 1878).

Geographical distribution

Hypoxis hemerocallidea is distributed in the seven provinces of South Africa Eastern Cape, Free State, Gauteng, Limpopo Province, Mpumalanga, North-West and KwaZulu- Natal. The distribution extends to neighbouring countries; Botswana, Lesotho and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

A decoction prepared from the tuber of Hypoxis hemerocallidea is used to treat several ailments such as inflammation, testicular tumours, urinary complaints, cancer and HIV/AIDS (Watt and Breyer-Brandwijk, 1962; Crouch et al., 2006; Ncube et al., 2012). Van Wyk et al. (2013), highlighted that the decoction is a remedy for bladder problems.

Ethnobotanical information from interviewees

1 5 6 A decoction prepared from the tuber of Hypoxis hemerocallidea is taken orally to ease and relief body pains, used more as a pain-killer. Decoction is taken preferable before bedtime (Papo E., 2014, Pers. Comm.).

1 5 7 24. Hypoxis obtusa L. (Hypoxidaceae)

Yellow stars or (E), Monna-maledu (S)

Figure 24: Hypoxis obtusa (Photo: A. Hankey)

Botanical description

Hypoxis obtusa is a perennial herb reaching heights of 16-50 cm, with long and slander leaves that arise from the miniature sheath stem. The leaves are shiny, erect and becoming irregularly curved ending with pointy apex. Both surfaces of the leaves are smooth or scarcely pubescent. The plant produces a raceme of characteristic yellow star- flowers, usually 2-13, that give rise to an oval black and shiny fruit (Baker, 1878).

Geographical distribution

Hypoxis obtusa is distributed widely in South Africa with exceptions in the Western Cape and Northern Cape Provinces. The plant species has a wide distribution in southern African (Germishuizen and Meyer, 2003).

Recorded medicinal use

Hypoxis obtusa was reported to treat prostrate hypertrophy (Nel, 1914) and urinary diseases (Marini-Bettolo et al., 1985; Galeffi et al., 2002). Gelfand et al. (1985), reported

1 5 8 the extract prepared from the tuber of the plant to be used as an aphrodisiac, in addition to treat gonorrhoea and infertility in women. Semenya et al. (2013b) and Moeng (2010), highlighted that the Bapedi traditional healers prepare a decoction for the treatment of infertility and impotence whereas the people of Zimbabwe use it for treatment of abdominal pains (Maroyi, 2013).

Ethnobotanical information from interviewees

The tuber of H. obtusa is cut into pieces boiled in a litre of water. The decoction is prepared and taken orally to ease and relief body pains (Papo E., 2014, pers. comm.)

1 5 9 25. Jatropha zeyheri Sond. (Euphorbiaceae)

Ugodide (E), Sefapabadia (S)

Figure 25: Jatropha zeyheri (Photo: L. Papo)

Botanical description

Jatropha zeyheri is a perennial herb growing up to 30 cm tall, with a single miniature stem borne from a huge root-stock. Branches brownish, supporting distinctive leaves that are divided halfway to the petiole. Leaves are uniformly green with a rigid and conspicuous midrib. Male and female flowers are borne separately on different plants. The plant produces 3-lobed fruits, smooth or with very small hairs (Radcliffe-Smith, 1996).

Geographical distribution

Jatropha zeyheri has a limited distribution occurring only in Limpopo Province, North­ West, Gauteng Province, east towards Mpumalanga and south towards KwaZulu-Natal Province (Germishuizen and Meyer, 2003).

Recorded medicinal use

Van Wyk and Gericke (2007), reported that the decoction prepared from the rootstock of the plant is used to treat menstrual pains, irregular periods, and to ensure a strong foetus

1 6 0 during pregnancy. In addition the decoction is used for STIs and urinary tract infections (Mongalo et al., 2013). The oral intake of the decoction can also be used to purify blood and applied externally to dress wounds and boils (Van Wyk et al., 2013).

Ethnobotanical information from interviewees

The rootstock of Jatropha zeyheri is macerated and used to treat inflamed feet. Hot water is added to the macerated plant material in a washing bowl and allowed to cool, the patient then submerges the feet into the washing bowl. This treatment is done twice in a day, usually in the morning and the evening. (Papo E., 2014, Pers. Comm.)

1 6 1 26. Lannea edulis (Sond.) Engl. (Anacardiaceae)

Wild grape (E), Mokgolokgotwane (S)

Figure 26: Lannea edulis (Photo: L. Papo)

Botanical description

Lannea edulis is a deciduous suffrutex reaching heights of 30 cm. The plant has short branches which give rise to imparipinnate compound leaves composed of ovate to oblong leaflets. The leaves are glabrous, shiny and light green. The plant produces a raceme of small yellowish to cream flowers which give rise to a drupe (Schmidt et al., 2002).

Geographical distribution

Lannea edulis inhabits mostly rocky grassland and bushveld and has a limited distribution in South Africa (Limpopo, Gauteng, Mpumalanga, and KwaZulu-Natal Provinces). The plant species also occurs in Botswana, Namibia, Swaziland, and Zimbabwe (Germishuizen and Meyer, 2003).

Recorded medicinal use

The crushed bark of Lannea edulis is applied topically for the treatment of boils and abscesses (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996; Van Wyk, 1997). In the Manzini region in Swaziland, the root bark is ground and added in water to make

1 6 2 decoction which is taken orally to treat constipation (Amusan et al., 2002). People of the Nhema community in Zimbabwe prepare root decoction for the treatment of diarrhoea (Maroyi, 2011).

Ethnobotanical information from interviewees

The leaves of Lannea edulis are harvested, boiled and used to prepare a decoction taken orally to treat stomach-ache, the extract is taken as a laxative and thus induces excretion (Papo E., 2014, pers. comm.).

1 6 3 27. Lantana rugosa Thunb. (Verbenaceae)

Sekwebatana (S)

Figure 27: Lantana rugosa (Photo: L. Papo)

Botanical description

Lantana rugosa is a woody perennial, growing up to 2 m in height, with square-shaped stem. Leaves opposite, ovate to lanceolate, upper surface highly fissured giving the plant a characteristic venation. Inflorescence borne on short peduncles of the leaf axils, forming spike of reddish-purple flowers which give rise to small green round fruits that turn purple at maturity (Fernandes, 2005).

Geographical distribution

Lantana rugosa occurs widely in southern Africa including Botswana, Lesotho, Namibia, South Africa (where it occurs in all provinces), and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

The Vhavenda people use the leaves and stem to prepare a paste which is applied to treat troublesome eyes (Mabogo, 1990). A paste prepared from the leaves, stems or ripe

1 6 4 fruit is also used to treat festering sores (Hutchings, 1996; Suliman, 2010; Mabona and Van Vuuren, 2013). An infusion of L. rugosa prepared from the roots is used for the treatment of respiratory ailments such as bronchitis, tuberculosis, cold and flu (Mabogo, 1990; McGaw et al., 2008; Mahwasane et al., 2013).

Ethnobotanical information from interviewees

Leaves are used to make a cold infusion for the purification of the eyes. Cold water is added to the leaves placed in a washing container, and then used to wash off the face. The practice is usually done by elders to better their fading vision. The plant is also used to make an infusion of hot water to treat flu (Papo E., 2014, pers. comm.).

1 6 5 28. Ledebouria revoluta (L. f.) Jessop (Hyacinthaceae)

Common squill (E), Lehwama (S)

Figure 28: Ledebouria revoluta (Photo: G. Nicholis)

Botanical description

Ledebouria revoluta is a bulbous plant 15-20 cm tall with scales, characterised by fully developed leaves during anthesis, in addition the leaves are maculated and have threads when torn. The plant produces multiple raceme inflorescences in the range of 4-10, with a total flower count above 30 per the inflorescence. Flowers have pale yellow anthers, with 6-lobed ovary (Gibson, 1975).

Geographical distribution

Ledebouria revoluta is distributed widely in South Africa with an exception in the Northern Cape Province. The distribution extends to neighbouring countries such as Botswana, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Ledebouria revoluta is reportedly used as an expectorant, diuretic and used to treat gastro-intestinal tract (Watt and Breyer-Brandwijk, 1962; Dold and Cocks, 2001; Abegaz, 2002; Moodley et al., 2006). The pounded bulb is also used to dress wounds, sores, and skin eruptions (Muleya et al., 2014).

1 6 6 Ethnobotanical information from interviewees

Ledebouria revoluta is used for two purposes, to treat feet inflammation and stomach ailments. For inflammation, hot water (bearable) is added to the macerated bulb in a washing container to prepare an infusion into which patient dips the feet, this is repeated until the condition subsides. The bulb is also pounded, an infusion prepared, sieved, and given to the patient to treat stomach ailments (Papo E., 2014, pers. comm.).

1 6 7 29. Lippia javanica (Burm.f.) Spreng (Verbenaceae)

Fever tea or Lemon bush (E), Moshunkwane (S)

Figure 29: Lippia javanica (Photo: L. Papo)

Botanical description

Woody shrub up to 2m in height with highly aromatic leaves that are easily recognised by their deep veins. Yellowish-white flowers are produced in rounded heads (Van Wyk et al., 2013).

Geographical distribution

Lippia javanica is distributed in South Africa however it does not occur in the Northern Cape and Western Cape Provinces. Its distribution extends further into Botswana, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use:

The leaves are used as paste to treat skin disorders such as heat rash, scratches (Gelfand et al. 1895; Hutchings et al. 1996; Van Wyk et al. 1997; Van Wyk & Gericke, 2000; Samie et al. 2005; Mabona et al. 2013 Semenya et al. 2013d). Paste also treats stings and bites (Mthethwa, 2009).

Ethnobotanical information from interviewees

1 6 8 Boiled water is added to the leaves placed in a washing bowl and the patient performs steam inhalation by covering with a blanket, to treat cold and flu. This process is usually done before bedtime. The leaves of L. javanica can also be chewed for stomach-ache (Papo E., 2014, pers. comm.; Bonoko F., 2015, pers. comm.; Gwangwa L., 2015, pers. comm.).

1 6 9 30. Mentha longifolia (L.) Huds. subsp. polyadena (Briq.) Briq. (Lamiaceae)

Wild mint (E), Mentholo (S)

Figure 30: Mentha longifolia (Photo: G. Nicholis)

Botanical description

Perennial shrub of 0.8 m in height. Leaves are produced in pairs opposite to each other along the stem. Flowers arise in clusters directly from the main stem, small, white or purple (van Wyk et al., 2013).

Geographical distribution

Mentha longifolia (L.) Huds. subsp. polyadena (Briq.) Briq. is distributed widely in South Africa with exceptions in the Northern Cape and the North-West Provinces. The plant species also occurs in Lesotho and Swaziland. Its related subspecies Mentha longifolia (L.) Huds. subsp. capensis (Thunb.) Briq. occurs in the Northern Cape, Western Cape, and Eastern Cape Provinces extending its distribution into Lesotho (Germishuizen and Meyer, 2003).

Recorded medicinal use

The leaves of Mentha longifolia are used to dress wounds (Watt and Breyer-Brandwijk, 1962; Van Wyk et al., 2000; Scott et al., 2004; McGaw and Eloff, 2008). Van Wyk (2011), reported that leaves can also be crushed and sniffed for headache and insomnia. An

1 7 0 infusion is taken orally for indigestion (Moteetee and Van Wyk, 2011; Philander, 2011; Mabona et al. 2013). Leaves mashed into pulp and applied topically on skin for shingles and herpes (Gail et al., 2015).

Ethnobotanical information from interviewees

The fresh leaves of Mentha longifolia are put in a washing container and hot water is poured to cover leaves and the resulting steam vapour is inhaled to treat cold and flu. Fresh leaves can also be crushed in the hands of a sick person, inhaled to relieve blocked nose. Infusion prepared from fresh leaves can be also be used for indigestion (Papo E., 2014, pers. comm.).

1 7 1 31. Ozoroa insignis Delile (Anacardiaceae)

Currant Resin Tree (E), Monoko (S)

Figure 31: Image of Ozoroa insignis (Photo: L. Papo) Botanical description

Ozoroa insignis is a medium-sized tree reaching heights of 10-15 m tall with several branches covered in miniature hairs. The trunk of the plant has a characteristic twist/spin and is covered in a rough, deeply fissured grey bark. Leaves simple, alternate, oblong- elliptical or oblong to lanceolate with the upper surface slightly pubescent and the lower surface densely pubescent. Flowers in terminal panicles, unisexual, cream white, borne in a greenish bract giving rise to an ellipsoid black shinny drupe fruit at maturity (Fernandes and Fernandes, 1966).

Geographical distribution

The distribution of O. insignis is restricted to Limpopo Province in South Africa (Schmidt et al., 2002). The distribution extends to Botswana, Namibia, and Zimbabwe (Germishuizen and Meyer, 2003).

Recorded medicinal use

Mathabe et al. (2006) reported that the people of Limpopo Province residing in the Capricorn district prepare a decoction using the stem bark for treatment of diarrhoea. The

1 7 2 use of O. insignis decoction for diarrhoea is also evident in the Nhema communal area in Zimbabwe (Maroyi, 2011; 2013).

Ethnobotanical information from interviewees

Decoction prepared from the leaves is administered orally for treatment of diarrhoea and STIs. For these conditions, the decoction is orally taken thrice a day (Papo E., 2014, Pers. Comm.).

1 7 3 32. Ozoroa sphaerocarpa R. & A. Fern. (Anacardiaceae)

Currant resin tree (E), Monoko (S)

Figure 32: Ozoroa sphaerocarpa (Photo: L. Papo)

Botanical description

Ozoroa sphaerocarpa is a small tree reaching heights of 7.5 m tall. The plant has a grey bark with hairy reddish branchlets composed of lenticels during the juvenile stage of growth. The leaves alternate, elliptic to narrowly oblong, velvety on the upper surface and arranged in groups of 3. Flowers in terminal heads, cream, producing round, fleshy, purple to black drupes (Schmidt et al., 2002).

Geographical distribution

Ozoroa sphaerocarpa grows well in bushveld biome and is distributed in Limpopo, Mpumalanga, KwaZulu-Natal, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

A mixture prepared from the bark of O. sphaerocarpa and Athrixia phylicoides is used to dress wounds (Amusan et al., 2002). Bark decoction is used to treat asthma and impotence by the Bapedi traditional healers (Semenya et al. 2013b). Sibandze et al. (2013), reported that the plant is used in combination with other medicinal plants by the

1 7 4 people of Swaziland to treat diarrhoea, similarly the Bapedi traditional healers use the plant, but not in combination (Semenya and Maroyi, 2012).

Ethnobotanical information from interviewees

Similar to O. insignis, a decoction prepared from the macerated bark is sieved and administered orally for treatment of asthma, diarrhoea, and applied topically for treatment of wounds. For oral administration, decoction is taken thrice a day (Papo E., 2014, pers. comm.).

1 7 5 33. Peltophorum africanum Sond. (Fabaceae)

African wattle or Weeping wattle (E), Mosetlha (S)

Figure 33: Peltophorum africanum (Photo: L. Papo)

Botanical description

Peltophorum africanum is a medium-sized tree in the height range of 4-14 m, with a characteristic branched stem from the base. The tree has a brown and fissured bark, and has branches covered in brown hairs. Leaves double-compound, olive green on the upper surface and pale green on the lower surface, covered with brown hairs. Flowers are yellow, clustered at the end of branches, giving rise to a flat winged pod (Schmidt et al., 2002).

Geographical distribution

Peltophorum africanum grows widely distributed in Africa south of the equator, in South Africa it occurs in the Limpopo, North-West, Mpumalanga, and KwaZulu-Natal Provinces (Germishuizen and Meyer, 2003).

Recorded medicinal use

The Vhavenda women use the plant to treat herpes (Mabogo, 1990). The Bapedi traditional healers use the plant to treat female infertility and post-partum (Semenya et

1 7 6 al., 2013b). Mazimba (2014), recorded the plant for the treatment of eye infections, joints and back pains, toothache, diarrhoea, dysentery, infertility, skin rashes, blisters, venereal diseases, depression, anthelmintic, coughs, sore throat, ascites, and abdominal disorders.

Ethnobotanical information from interviewees

The bark harvested from the branches of Peltophorum africanum is given to children as a lozenge when the elders are from funeral services to prevent stomach infiltration known as Makgome (Mmatli L., 2015, pers. comm.). An infusion prepared from the leaves can also be made for children to take once on the day of the funeral service for the same purpose. A decoction of the leaves is also taken orally, three times a day to prevent diarrhoea. Bark decoction is also used to treat cold/flu and sexually transmitted infections, in which the prepared medication is taken three times daily (Bonoko F., 2015, pers. comm.; Ledwaba M., 2015, pers. comm; Mmatli L., 2015, pers. comm.).

1 7 7 34. Ptaeroxylon obliquum (Thunb.) Radlk (Rutaceae)

Sneezewood (E), Molope (S)

Figure 34: Ptaeroxylum obliquum (Photo: L. Papo)

Botanical description

Ptaeroxylon obliquum is a tree reaching heights of 15-30 m with pale greyish-brown to dark brown bark. Leaves compound, with eight pairs of leaflets (Palmer and Pietman, 1972). Flowers pale yellow, male and female flowers occur in clusters on different trees, and produce an oblong capsule fruit (Van Wyk et al., 2013).

Geographical distribution

In South Africa Ptaeroxylon obliquum is distributed from the Western Cape, through the Eastern Cape, KwaZulu-Natal, Mpumalanga, and Limpopo Provinces. The plant also occurs in Botswana, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

The Zulu people use the wood powder of Ptaeroxylon obliquum as sneeze inducer for headaches (Hutchings et al. 1996). The Rastas commonly known as the Bush doctors of the Western Cape utilize the wood powder to treat headaches (Philander, 2011). The plant is used to treat rheumatism, arthritis, and heart disease (Watt and Breyer-Brandwijk,

1 7 8 1962; Hutchings, 1996; Van Wyk et al. 2009). The Xhosa people use bark infusion to bath as to remove body odour (Afolayan et al., 2014).

Ethnobotanical information from interviewees

Decoction prepared from the leaves is taken orally to treat diarrhoea. The leaves are boiled in a pot containing 1L water and the decoction is taken three times a day for running stomach treatment (Papo R, 2015, Pers. Comm.).

1 7 9 35. Punica granatum L. (Lythraceae)

Pomegranate tree (E), Mogarenate (S)

Figure 35: Punica granatum (Photo: L. Papo)

Botanical description

Punica granatum is a small shrub 5-10 m tall, with multiple woody and spiny stems. The plant has a smooth and dark grey bark with opposite, simple, oblong to obovate leaves. The leaves are glossy on the upper surface but not on the under surface. The plant produces orange-red solitary flowers that give rise to a round berry fruit. Fruit leathery and has characteristic edible red seeds packaged into compartments (Orwa et al., 2009).

Geographical distribution

Punica granatum is native to Afghanistan, Iran, Libyan Arab Jamahiriya, and Tunisia and cultivated in other regions of the world including South Africa (Orwa et al., 2009).

Recorded medicinal use

The people residing in the Capricorn district of Limpopo Province prepare a decoction which is incorporated into soft porridge to treat diarrhoea (Mathebe et al., 2005; Semenya

1 8 0 and Maroyi, 2012; Semenya et al., 2012a). The seeds of P. granatum enhance fertility (Ndhlala et al., 2013).

Ethnobotanical information from interviewees

A handful of leaves of Punica granatum are boiled to prepare a decoction taken thrice a day to treat diarrhoea (Manyashi A., 2015, pers. comm.).

1 8 1 36. Searsia lancea (L.f.) F.A. Barkley (Anacardiaceae)

Karee (E), Mosotlho (S)

Figure 36: Searsia lancea (Photo: L. Papo) Botanical description

Searsia lancea is a semi-evergreen tree reaching heights of 6-14 m, although commonly found at 6-8 m. The plant has a characteristic twisted stem covered with a rough dark brown bark, and reddish branchlets. Leaves characteristically trifoliolate compound, linear to lanceolate, side leaflets sickle-shaped, upper surface dark-olive green, lower surface pale yellow. Flowers yellow, produced at the end of the branches and give rise to a shiny drupe which turns brown at maturity (Schmidt et al., 2002).

Geographical distribution

Searsia lancea is found mostly in highland areas and is distributed widely in South Africa with the exception of KwaZulu-Natal Province. Distribution extends to Botswana, Lesotho, and Namibia (Germishuizen and Meyer, 2003).

Recorded medicinal use

A decoction prepared from the leaves is recorded to treat diarrhoea in children (Van der Merwe et al. 2001; McGaw and Eloff, 2000). Leaf infusion is reported to treat dizziness

1 8 2 as result of anaemia, diabetes, and cardiac problems (Maliehe, 1997), as well as asthma (Moteetee and Van Wyk, 2011).

Ethnobotanical information from interviewees

A decoction is prepared using the fruits and the extract is taken orally thrice a day to treat gastrointestinal complains (Papo R, 2015, Pers. Comm.).

1 8 3 37. Ricinus communis L. var. communis (Euphorbiaceae)

Castorbean or castor-oil-plant (E), Mothoba (S)

Figure 37: Ricinus communis (Photo: L. Papo)

Botanical description

Ricinus communis is a multi-stemmed shrub reaching heights of 7 m. The stem and branches of the plant contain sap. The leaves are alternate and paltate, dark green on the upper surface and pale green on the lower surface. The flowers develop at the end of the branches, female flowers characteristically develop above male flowers (Schmidt et al., 2002).

Geographical distribution

Ricinus communis is naturalised and widely distributed in South Africa with the Free State Province as an exception (Germishuizen and Meyer, 2003). The distribution extends throughout tropical Africa (Schmidt et al., 2002).

Recorded medicinal use

The leaves are applied as poultice in the treatment of boils (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996; Van Wyk et al. 2000; Luseba et al. 2007; Malik et al.2011; Mabona et al.2013). The Bapedi traditional healers use decoction prepared from the roots

1 8 4 to treat STIs. Ricinus communis is also recorded for the treatment of breast tumours, menstrual disorders and abnormal vaginal discharges (De Boer and Cotingting, 2014).

Ethnobotanical information from interviewees

The leaves are harvested and boiled, the steam-vapour is inhaled to treat cold, coughs and flu (Papo E., 2014, pers. comm.; Bonoko F., 2015, pers. comm.; Papo R., 2015, pers. comm.).

1 8 5 38. Schinus molle L. (Anacardiaceae)

False pepper or Peruvian Pepper tree (E), Thoba (S)

Figure 38: Schinus molle (Photo: L. Papo) Botanical description

Schinus molle is a large round tree reaching heights of 15 m with a characteristic short trunk, covered by a deeply fissured dark brown bark, from which sticky latex exudes when damaged. The plant produces several thick branches which are support structures for thin reddish branchlets. Leaves compound imparipinnate, uniformly evergreen, long, resulting in a drooping appearance. Inflorescence panicle, flowers clustered, cream-white and give rise to small, green shinny round fruits. The fruits turn from green to red during maturity (Orwa et al., 2009).

Geographical distribution

Schinus Molle is native to Argentina, Bolivia and Peru, and cultivated in other countries such as Australia, Brazil, Dominican Republic, Eritrea, Ethiopia, Greece, Haiti, India, Kenya, Mexico, Paraguay, South Africa, Spain, Sudan, Tanzania, Turkey, Uganda, and the United States of America (Orwa et al., 2009).

Recorded medicinal use

1 8 6 Leaf infusion of S. molle is inhaled or taken orally to treat cold and influenza (Hutchings, 1992). Topical application of fruit paste of S. molle is used by the Xhosa people to treat eczema (Afolayan et al., 2014).

Ethnobotanical information from interviewees

Hot water (usually more than 1L) is added to the leaves of Schinus molle in a washing bowl, the patient then inhales the steam while covered in a blanket to induce sweat as treatment for colds and flu. This is preferably done before bedtime, (Papo E., 2014, pers. comm.; Letseku S., 2015, pers. comm.; Magongwa L., 2015, pers. comm.).

1 8 7 39. Schotia brachypetala Sond. (Fabaceae)

Weeping boer-bean or African walnut (E), Molopa (S)

Figure 39: Schotia brachypetala (Photo: L. Papo)

Botanical description

Schotia brachypetala is a tree reaching heights of 16 m, with a characteristic round crown and a greyish-brown bark. Branches slightly pubescent when young. Leaves alternate and slightly opposite, compound of 4-8 pairs. Flowers occur in clusters terminally on old wood, deeply red with filamentous petals at maturity. These flowers produce flat smooth woody pods that split open to expose seeds characterized by a yellowish aril (Schmidt et al., 2002).

Geographical distribution

Schotia brachypetala is distributed from the Limpopo Province towards North-West Province,, Mpumalanga, spreading to KwaZulu-Natal and the Eastern Cape Provinces. It also occurs in Botswana, Mozambique, Swaziland, and Zimbabwe (Schmidt et al., 2002).

Recorded medicinal use

The Zulu people use bark decoction of S. brachypetala to treat dysentery (Hutchings, 1996), ulcers (Venter and venter, 1996), and diarrhoea (McGaw et al., 2000; Mathabe et

1 8 8 al., 2006; Shai et al., 2013). The people of Manzini district in Swaziland use the bark of Sclerocarya birrea and S. brachypetala to prepare a concoction used as an emetic (Sibandze et al., 2009).

Ethnobotanical information from interviewees

The leaves of Schotia brachypetala are used to prepare a decoction for treatment of diarrhoea. The leaves can also be used in combination with other medicinal plants such as Ximenia caffra for the same purpose, taken thrice daily (Papo E., 2014, pers. comm.; Ledwaba M., 2015, pers. comm.).

1 8 9 40. Siphonochilus aethiopicus (Schweif.) B.L. Burt (Zingiberaceae)

Wild ginger (E), Serokolo (S)

Figure 40: Siphonochilus aethiopicus (Photo: G. Nicholis)

Botanical description

Siphonochilus aethiopicus is a herbaceous plant characterised by flowers borne at ground level. The leaves are long, linear to broadly lanceolate with a sheath that attaches leaf to the stem-like base. Male and female organs occur on different plants, distinguished by size, female plants shorter compared to male plants. The flowers are tubular, petaloid, whitish to purple-pink by the edge of the petal. Flowers give rise to a small-berry like fruit borne at ground level of flowers (Gordon-Gray et al., 1989).

Geographical distribution

Siphonochilus aethiopicus is distributed and limited to the northern parts towards the eastern parts of South Africa. It occurs in the Limpopo Province, Mpumalanga, Kwa-Zulu Natal and towards the Eastern Cape Province. It also occurs in Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

1 9 0 Pujol (1990), reported that the bulb of S. aethiopicus is used for treatment of amenorrhoea. South African women use the plant to treat dysmenorrhoea (Van Wyk and Gericke, 2000; Steenkamp, 2003). In addition Van Wyk (2011), highlighted the use of S. aethiopicus for the treatment of colds, cough, influenza, hysteria, pain, asthma, and as an anti-inflammatory and bronchodilatory.

Ethnobotanical information from interviewees

The root bulb of S. aethiopicus is macerated into a jug and an infusion is prepared using hot water. The infusion is then sieved and taken orally thrice a day to treat cold and flu (Mahlong S., 2014, pers. comm.; Bonoko F., 2015, pers. comm.; Gwangwa M., 2015, pers. comm.; Letseku S., 2015, pers. comm.).

1 9 1 41. Solanum supinum Dun. (Solanaceae)

Bitter apple or snake berry (E), Mothola (S)

Figure 41: Solanum supinum (Photo: L. Papo)

Botanical description

Solanum supinum is a herb reaching heights of 40 cm, with small pubescent, green- greyish branches borne from a short woody rootstock. Leaves simple, solitary, slightly pubescent and paler on the upper surface with lower surface more pubescent. Flowers solitary, corolla usually found in three shades of white, mauve, blue, and purple, producing a round glossy green fruits that turn completely yellow when ripe (Goncalves et al., 2005).

Geographical distribution

The distribution of S. supinum extends from Limpopo to the North-West Province, Gauteng, and the Free-State Province, towards the Northern and Eastern Cape Provinces. The plant can also be found in Botswana, Lesotho, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Decoction made from S. supinum is used to treat diarrhoea (Mathebe et al., 2006).

Ethnobotanical information from interviewees

1 9 2 The fruit of Solanum supinum is pinched, inserted into the mouth and squeezed onto an aching tooth to cure the pain. In addition, the root decoction is mixed with soft porridge and given to babies to treat depressed fontanelle as it regarded fatal if left untreated (Papo E., 2014, pers. comm.).

1 9 3 42. Vangueria infausta Burch. subsp. Infausta (Rubiaceae)

Wild Medlar (E), Mmilo (S)

Figure 42: Vangueria infausta subsp. infausta (Photo: L. Papo) Botanical description

Vangueria infausta ssp. infausta is a tree reaching heights of 2-8 m, with a single thick stem and pale beige bark and hairy young branches. Leaves opposite, simple, round to ovate, with green upper surface and pale lower surface. Flowers green to yellowish, produced in clusters situated below the leaves and give rise to green smooth fruits that turn brown when ripe. The fruits are edible (Schimdt et al., 2002).

Geographical distribution

Vangueria infausta ssp. infausta occurs widely in South Africa but does not occur in the Western Cape Province. Its distribution extends to Botswana, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

Root decoction of V. infausta is reported to treat abnormal menstruation (Watt and Breyer- Brandwijk, 1962; Steenkamp, 2003), and is used by the Vhavenda people to treat infertility (Mabogo, 1990). The root and leaf decoction can also be prepared for the treatment of chest complaints and coughs (Pooley 1993; McGaw and Eloff, 2008). The people of the Nhema communal area in Zimbabwe drink a hot decoction of V. infausta for the treatment of diarrhoea (Maroyi, 2011).

1 9 4 Ethnobotanical information from interviewees

The leaves of V. infausta are harvested and a decoction is prepared and taken thrice a day for the treatment of high-blood pressure, diarrhoea and cough. A decoction prepared from the dried fruits is believed to lower the chances of catching cancer (Papo E., 2014, pers. comm.; Mahlong S., 2015, pers. comm.; Papo R, 2015, Pers. Comm.).

1 9 5 43. Ximenia caffra Sond. var. caffra (Olacaceae)

Large sourplum (E), Motshidi (S)

Figure 43: Ximenia caffra var. caffra (Photo: L. Papo)

Botanical description

Ximenia caffra var. caffra is a medium to large shrub reaching heights of 1-7 m. The plant is multi-stemmed with a dark greyish stem, characterized by branches which develop into thorns. Leaves alternate or clustered, elliptic and bending upwards at the margins, dark green. Ximenia caffra is dioecious, with small, scented flowers which give rise to large red fruits (Schmidt et al., 2002).

Geographical distribution

Ximenia caffra is distributed in wooded grassland and bushveld biome and is found in Limpopo, Gauteng, and Mpumalanga Provinces. It also occurs in Botswana, Namibia, and Swaziland (Germishuizen and Meyer, 2003), with its distribution extending towards central Africa (Schmidt et al., 2002).

Recorded medicinal use

Root powder is applied topically to treat septic sores (Von Koenen 1996; Fabry et al., 1998; Van Wyk et al., 2011). The Bapedi people in the Capricorn district make use of the stem bark to prepare a decoction incorporated in soft porridge for treatment of diarrhoea

1 9 6 (Mathabe et al., 2006). The Zulu people in the Maputaland region, KwaZulu-Natal, apply the root powder for skin infections and septic sores (Van Vuuren and Mabona, 2013). The Vhavenda people utilize the roots and leaves to prepare a decoction for candidiasis, headache and scurvy (Mulaudzi et al., 2014).

Ethnobotanical information from interviewees

The leaves are boiled in a pot containing 1L of water and the decoction is taken orally thrice a day for purification of kidneys and urinary tract. The decoction is also taken for the treatment of diarrhoea and also applied topically for sores. For stronger decoction, roots can also be used (Papo E., 2014, pers. comm.).

1 9 7 44. Zanthoxylum humile (E.A Bruce) P.G. Waterman (Rutaceae)

Prickly ash (E), Monokwane (S)

Figure 44: Zanthoxylum humile (Photo: L. Papo) Botanical description

Zanthoxylum humile is a small shrub of 3 m in height, with a greyish and hairy stem which has conspicuous dark brown to black curved thorns. Leaves alternate, elliptic to oblong, glabrous, glossy, and dark green on the upper surface and pale green on the lower surface. Zanthoxylum humile is dioecious, with greenish flower heads producing small fruits of ± 5 mm (Schmidt et al., 2002).

Geographical distribution

Zanthoxylum humile is indigenous to Limpopo and Mpumalanga Provinces (Germishuizen and Meyer, 2003), extending towards the south-eastern parts of Zimbabwe (Schmidt et al., 2002).

Recorded medicinal use

The bark is macerated in a pot, boiled and extract taken to treat impotence (Semenya et al. 2013b). The Bapedi traditional healers use the bark decoction to treat HIV/AIDS (Semenya and Maroyi, 2013).

1 9 8 Ethnobotanical information from interviewees

The pieces of bark are harvested off the tree, washed to remove dirt and licked as a lozenge to treat mouth and throat sores (Papo E., 2014, pers. comm.; Ledwaba D., 2015, pers. comm.; Letseku S., 2015, pers. comm.; Mothapo K., 2015, pers. comm.; Shika L., 2015, pers. comm.).

1 9 9 45. Ziziphus mucronata Willd. (Rhamnaceae)

Buffalo thorn (E), Mokgalo (S)

Figure 45: Ziziphus mucronata (Photo: L. Papo) Botanical description

Ziziphus mucronata is a tree reaching heights of 12 m, with a characteristic green and pubescent stem noticeably during the juvenile stages. The plant has reddish-brown or greyish bark with presence of both hooked and straight spines. Leaves alternate, simple, ovate to roundish, dark green, glossy on the upper surface and pale green in the lower surface. Flowers green to yellow, occurring in clusters at the leaf base and giving rise to an edible glossy reddish-brown drupe. (Schmidt et al., 2002).

Geographical distribution

Ziziphus mucronata is distributed widely in South Africa with the Western Cape Province as an exception. It is also found in Botswana, Lesotho, Namibia, and Swaziland (Germishuizen and Meyer, 2003).

Recorded medicinal use

The extract from the leaves has been reported to treat sores (Watt and Breyer-Brandwijk, 1962; Rood, 1994; Hutchings, 1996; Rabe and Van Staden, 1997; Van Wyk et al., 2000; Luseba et al., 2007; Van Wyk et al. 2011; Mabona and van Vuuren, 2013). Leaf decoction

2 0 0 is taken orally by South African women for menorrhagia and infertility (Arnold and Gulumian, 1984; Steenkamp, 2003). The boiled leaves are applied as poultice for wounds and boils (Rabe et al., 1997). In the Nhema communal area of Zimbabwe, powder from crushed fruits and leaves is used for treatment of boils and wounds (Maroyi, 2011). In addition Z. mucronata is used for tuberculosis (Green et al., 2010), coughs, chest problems, and diarrhoea (Van Wyk and Gericke, 2000; Nyila et al., 2012). The traditional healers of Limpopo Province utilize the decoction against gonorrhoea (Semenya and Maroyi, 2013).

Ethnobotanical information from interviewees

The leaves of Z. mucronata are used to prepare a decoction taken orally thrice daily for treatment of coughs, stomach-ache, and can also be applied topically for sores and wounds (Mahlong S., 2014, pers. comm.; Baloyi M, 2015, pers. Comm.).

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