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How to cite this thesis

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 [Unpublished]: University of Johannesburg.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).

An inventory of the most popular medicinal barks sold on Johannesburg muthi markets and the antimicrobial activity of selected extracts and isolated chemical compounds

Gugulethu Philadelphia Khumalo

Dissertation submitted In fulfilment of the requirements For the degree

MAGISTER SCIENTIAE In

BOTANY In the

FACULTY OF SCIENCE At the

UNIVERSITY OF JOHANNESBURG

SUPERVISOR: PROF. B-E. VAN WYK CO-SUPERVISOR: DR. N.J. SADGROVE CO-SUPERVISOR: PROF. S.F. VAN VUUREN

JULY 2018 Affidavit

I, Gugulethu Philadelphia Khumalo, declare that this dissertation is my own work. It has only been submitted (by myself) for the degree of Master of Science in Botany at the University of Johannesburg. It has never been submitted before for any degree or examination at any other University. I also state that all the sources that I have used herein have been appropriately acknowledged.

Signature (Student) : ______

This 25th day of July 2018

Signature (Supervisor) : ______

This 25th day of July 2018

i Dedication

I dedicate this Master’s dissertation to God whom I praise, for I do not believe that I could have made it possible. I thank Him for my life. I am also dedicating this dissertation to my 11 year old little sister, Samukelisiwe Khumalo. I want you to know that if I managed to achieve this, so can you. This should be like footprints I am leaving you with, to follow a peaceful educational path that will train you to be independent for, I will not always be there throughout your life to guide you. To my parents, even though you did not make it to University but you managed to create a

University graduate. Therefore, you should be proud of yourselves.

Acknowledgements

 I would first like to thank my supervisor, Professor Ben-Erik van Wyk, for

believing in my abilities by offering me an opportunity to work under his

guidance as one of the best and internationally recognised researchers. A

great learning experience indeed that channelled me in the right direction. I

thank you Prof for your financial, mental and academic support – it has greatly

contributed to my personal growth.

 I would also like to thank the experts who allowed for collaboration, with their

commitment and inputs in my research project thus making it a success; my

co-supervisors, Professor Sandy van Vuuren and Dr Nick Sadgrove. I thank

you Prof for welcoming me into your lab and giving me an opportunity to learn

and understand antimicrobial screening. To Dr Nick Sadgrove, I thank you so

much for sharing your chemistry skills with me.

 I would like to thank Dr Ekaterina Kotina, a bark anatomist, for her assistance

with the interpretation of morphological bark characters that can be used for

identification.

 I thank the National Research Fund for financial assistance in pursuing my

Master’s degree and the University of Johannesburg for giving me the

platform to learn.

 I would like to thank the postgraduate students and staff members from the

Department of Botany and Plant Biotechnology for their support.

 I would like to thank the Johannesburg muthi market traders for allowing me

to communicate with them and for sharing their knowledge on the uses of

medicinal barks. Special thanks to Gogo Makhaza, Mkhulu Madondo, Bab’

Nqulunga and Mancube.

iii

 I would like thank my mother, Ntombizodwa Khumalo, brother, Sfiso

Buthelezi, and mamncane Mrs Bonisiwe Ngcenwa for financial support,

particularly during the final stage of this Master’s degree study.

 I would like to thank my brothers and sisters in Christ under The Twelve

Apostles Church in Christ for allowing God to give me spiritual guidance

through them. I thank you for all the support you invested in me.

 I would like to give special thanks to Zamani Ncanana for being an inspiration

to further my studies, for being a true friend, for psychological, emotional and

spiritual support. I thank you so much.

 I would like to thank my family and the community of Diepkloof in Soweto for

being a silent inspiration that has encouraged me to work hard.

List of Contents

Affidavit ...... i Dedication ...... ii Acknowledgements ...... iii List of Contents ...... v List of Figures ...... viii List of Tables ...... xi Summary ...... xiii CHAPTER 1. INTRODUCTION ...... 1

1.1 General introduction ...... 1 1.2 Objectives of this study ...... 9 CHAPTER 2. INVENTORY OF POPULAR MEDICINAL BARKS ...... 11

2.1 Introduction ...... 11 2.2 Materials and methods ...... 14 2.2.1 The Muthi Markets ...... 15 2.2.1.1 The Faraday Muthi Market ...... 15 2.2.1.2 The Kwa Mai-Mai Muthi Market ...... 17 2.2.2 Trader information and data recorded ...... 21 2.3 Results and discussions ...... 22 2.3.1 Traders on the muthi markets...... 22 2.3.2 Gender information on traders at the Johannesburg muthi markets ...... 24 2.3.3 Diversity of bark on the muthi markets ...... 25 2.3.4 Method of preparation and mode of administration ...... 46 2.3.5 Main uses of medicinal barks ...... 49 2.3.6 Identification of medicinal barks ...... 57 2.3.7 Conservation status of bark species ...... 87 2.4 Summary ...... 91 2.5 Conclusions ...... 92 CHAPTER 3. ANTIMICROBIAL ACTIVITY OF SELECTED EXTRACTS ...... 93

3.1 Introduction ...... 93 3.1.1 Role of bark for skin ailments ...... 95 3.1.2 Role of bark for respiratory ailments ...... 96 3.1.3 Role of bark for gastrointestinal tract ailments ...... 98 3.2 Materials and methods ...... 100 3.2.1 Plant extraction ...... 100

3.2.2 Minimum inhibitory concentration (MIC) technique ...... 101 3.3 Results and discussions ...... 104 3.3.1 Skin ailments ...... 104 3.3.2 Gastrointestinal tract ailments ...... 108 3.3.3 Respiratory tract ailments ...... 114 3.3.4 The influence of solvent extract on the antimicrobial activity of the barks ...... 118 3.3.5 Overall antimicrobial activity of medicinal barks...... 118 3.4 Summary ...... 121 3.5 Conclusions ...... 122 CHAPTER 4. CYTOTOXICITY OF SELECTED EXTRACTS AND COMPOUNDS 123

4.1 Introduction ...... 123 4.2 Materials and methods ...... 125 4.2.1 Sample preparation for the brine shrimp assay ...... 126 4.2.2 The brine shrimp assay ...... 126 4.3 Results and discussions ...... 127 4.4 Summary ...... 133 4.5 Conclusions ...... 133 CHAPTER 5. ANTIMICROBIAL ACTIVITY OF ISOLATED COMPOUNDS ...... 134

5.1 Introduction ...... 134 5.2 Materials and methods ...... 135 5.2.1 Compound isolation and structural elucidation of Elaeodendron transvaalense 135 5.2.2 Compound isolation and structural elucidation of Erythrina lysistemon ...... 136 5.2.3 Compound isolation and structural elucidation of Warburgia salutaris ...... 137 5.2.4 Antimicrobial screening of isolated compounds ...... 138 5.3 Results and discussion (chemical structure of isolated compounds) ...... 138 5.3.1 Elaeodendron transvaalense ...... 138 5.3.2 Erythrina lysistemon ...... 140 5.3.3 Warburgia salutaris ...... 142 5.4 Results and discussion (Antimicrobial screening of isolated compounds) ...... 144 5.4.1 Antimicrobial activity of compounds from Elaeodendron transvaalense ...... 144 5.4.2 Antimicrobial activity of compounds from Erythrina lysistemon ...... 146 5.4.3 Antimicrobial activity of compounds from Warburgia salutaris ...... 149 5.5 Summary ...... 152 5.6 Conclusions ...... 152 CHAPTER 6. GENERAL SUMMARY AND CONCLUSIONS ...... 153

6.1 Study of bark on the muthi markets ...... 153

6.2 Antimicrobial activity ...... 155 6.3 Cytoxicity ...... 156 6.4 Antimicrobial activity and chemistry of selected bark species ...... 156 6.5 Conclusions ...... 157 6.6 Recommendations for future studies ...... 157 References ...... 159

APPENDICES – SUPPLIED AS SEPARATE PDF FILES:

APPENDIX 1. INVENTORY OF THE MOST POPULAR MEDICINAL BARKS SOLD ON MUTHI MARKETS IN JOHANNESBURG

APPENDIX 2. 13C NMR SPECTRA OF THE COMPOUNDS ISOLATED FROM THE BARK OF ELAEODENDRON TRANSVAALENSE

APPENDIX 3. 1H NMR SPECTRA OF THE COMPOUNDS ISOLATED FROM THE BARK OF ERYTHRINA LYSISTEMON

APPENDIX 4. 13C NMR SPECTRA OF THE COMPOUNDS ISOLATED FROM THE BARK OF ERYTHRINA LYSISTEMON

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List of Figures

Figure 2.1 Examples of bark products as they are sold at the Johannesburg muthi markets. Photograph by E.L. Kotina...... 14 Figure 2.2 Entrance to the Faraday Muthi Market. Photograph by E.L. Kotina...... 15 Figure 2.3 Typical stall at the Faraday Muthi Market, showing bark products. Photograph by E.L. Kotina...... 16 Figure 2.4 Processed bark products on the Faraday Muthi Market. Photograph by E.L. Kotina...... 16 Figure 2.5 Entrance to the Kwa Mai-Mai Muthi Market. Photograph by E.L. Kotina. 18 Figure 2.6 Typical selling stalls at the Kwa Mai-Mai Muthi Market. Photograph by E.L. Kotina...... 18 Figure 2.7 Inside a traditional medicine stall at the Kwa Mai-Mai Muthi Market with the trader, Mr Gift Nzimande (a traditional healer), on the left. Photograph by E.L. Kotina...... 19 Figure 2.8 Inside the same trading stall shown in Figure 2.7, illustrating the way in which processed bark is stored and dispensed. Photograph by E.L. Kotina...... 20 Figure 2.9 Plant families of 70 medicinal bark species purchased at the Faraday and Kwa Mai-Mai Muthi Markets in Johannesburg...... 26 Figure 2.10 Method of preparation (dosage forms) of medicinal barks according to traders on the Johannesburg muthi markets...... 46 Figure 2.11 Mode of administration of medicinal barks as recorded by the muthi market traders...... 47 Figure 2.12 Main uses of the most popular bark samples on the Faraday and Kwa Mai Mai muthi markets in Johannesburg (Table 2.3), compared with literature data (see Appendix 1)...... 54 Figure 2.13 Comparison of the appearance (colour and texture) of powdered bark from the 70 medicinal bark species that were purchased at the Johannesburg muthi markets. The horizontal distance of spread of all samples was 50 mm. Photographs by B.-E. van Wyk...... 61 Figure 2.14 Comparison of the colour of bark extracts from the 70 medicinal bark species that were purchased at the Johannesburg muthi markets (numbered as in Appendix 1). The solvents were a 75:25 mixture of water and methanol

viii

(numbered vials) and pure dichloromethane (unnumbered vials). The way in which colours was coded in Table 2.4 is shown in the bottom row. Photographs by B.-E. van Wyk. The samples are 1, Araucaria heterophylla*; 2, Albizia adianthifolia; 3, Balanites maughamii; 4, Berchemia discolor; 5, Bersama lucens; 6, Bersama tysoniana; 7, Brackenridgea zanguebarica; 8, Bridelia micrantha; 9, Calodendrum capense; 10, Carissa bispinosa; 11, Cassipourea flanaganii; 12, Cinnamomum camphora*; 13, Combretum caffrum; 14, Commiphora africana; 15, Croton sylvaticus; 16, Cryptocarya latifolia; 17, Cunonia capensis; 18, Curtisia dentata; 19, Dichrostachys cinerea; 20, Dombeya rotundifolia; 21, Ekebergia capensis; 22, Elaeodendron croceum; 23, Elaeodendron transvaalense; 24, Erythrina lysistemon; 25, Erythrophleum lasianthum; 26, Eucalyptus sideroxylon*; 27, Euclea natalensis; 28, Ficus glumosa; 29, Garcinia gerrardii; 30, Garcinia livingstonei; 31, Harpephyllum caffrum; 32, Heteromorpha arborescens; 33, Hippobromus pauciflorus; 34, Ilex mitis; 35, Kigelia africana; 36, Macaranga capensis; 37, Maytenus undata; 38, Ochna holstii; 39, Ocotea bullata; 40, Ozoroa paniculosa; 41, Pappea capensis; 42, Peltophorum africanum; 43, Philenoptera violacea; 44, Pittosporum viridiflorum; 45, Pleurostylia capensis; 46, Protorhus longifolia; 47, Prunus africana; 48, Ptaeroxylon obliquum; 49, Pterocarpus angolensis; 50, Pterocelastrus rostratus; 51, Rapanea melanophloeos; 52, Rauvolfia caffra; 53, Rhus chirindensis; 54, Schotia brachypetala; 55, Sclerocarya birrea; 56, Securidaca longepedunculata; 57, Sideroxylon inerme; 58, Spirostachys africana; 59, Strychnos henningsii; 60, Syzygium cordatum; 61, Terminalia sericea; 62, Trichilia emetica; 63, Vachelia xanthophloea; 64, Vachelia karroo; 65, Vachelia robusta; 66, Vitex obovata; 67, Voacanga thouarsii; 68, Warburgia salutaris, 69, Zanthoxylon capense and 70, Ziziphus mucronata...... 62 Figure 2.15 Conservation status of the 70 most popular medicinal bark species sold at the Johannesburg muthi markets...... 88 Figure 2.16 The trunk of a marula tree (Sclerocarya birrea) showing signs of repeated bark harvesting and subsequent wound healing (Rustenburg district). Photographs by B.-E. van Wyk...... 90 Figure 3.1 Medicinal barks (32 species) with noteworthy activity (≤ 1 mg/ml) against the tested bacterial strains...... 120

Figure 5.1 Structures of chemical compounds isolated from the bark of Elaeodendron transvaalense. Chemical structures drawn by N.J. Sadgrove...... 139 Figure 5.2 Structures of chemical compounds isolated from the bark of Erythrina lysistemon. Chemical structures drawn by N.J. Sadgrove...... 140 Figure 5.3 Structures of chemical compounds isolated from the bark of Warburgia salutaris. Chemical structures drawn by N.J. Sadgrove...... 142

List of Tables

Table 2.1 The names of traders on the Faraday (F) and Kwa Mai-Mai (M) muthi markets in Johannesburg who provided information on the traditional uses of medicinal bark. The abbreviations are used in Table 2.2...... 21 Table 2.2 Seventy species of medicinal barks recorded on the Faraday (F) and Kwa Mai-Mai (M) muthi markets in Johannesburg and their ethnobotanical uses as reported by the traders (for abbreviations of the traders, see Table 2.1). Exotic (non-indigenous) species are indicated by an asterisk*...... 28 Table 2.3 Summary of medicinal and magical uses of the most popular medicinal barks (70 species) sold on Johannesburg muthi markets, arranged by main categories of use (see Appendix 1 for details about all uses). M = use- reports from the muthi markets – number of traders (out of 14) who reported that use; Lit = use-reports from the literature (see Appendix 1); M+Lit = number of use categories where muthi market and literature use-reports correspond ...... 50 Table 2.4 Characters and character states that can be used for the identification of important and popular medicinal barks as sold at the Faraday and KwaMai- Mai muthi markets in Johannesburg (see Appendix 1 for colour photographs). Colour codes according to the Munsell Soil Chart; Ts = transverse section; RLS = radial longitudinal section. Odour probably variable, depending on the freshness of the sample. [Colour of extracts:

H2O: MeOH – 3:1, DCM – see Figure 2.14 for the colour codes used in column 3 for extracts] ...... 65 Table 3.1 Bark sample yields and selected pathogens for further study...... 102 Table 3.2 Antimicrobial activities of medicinal barks that were tested against skin pathogens (mean value for three repetitions). Values are given in mg/ml. 105 Table 3.3 Antimicrobial activities of medicinal bark extracts tested against gastrointestinal tract pathogens (mean value for three repetitions). Values are given in mg/ml...... 109 Table 3.4 Medicinal bark extracts tested against respiratory tract pathogens (mean value of three replicates). Values are given in mg/ml...... 115

Table 4.1 Cytotoxicity of 20 medicinal bark extracts and some isolated compounds that were tested in the brine shrimp assay...... 128 Table 5.1 Minimum inhibitory concentrations (mg/ml) of solvent extracts and isolated compounds from Elaeodendron transvaalense bark ...... 145 Table 5.2 Minimum inhibitory concentrations (µg/ml) of solvent extracts and isolated compounds from Erythrina lysistemon bark ...... 147 Table 5.3 Minimum inhibitory concentrations (mg/ml) of solvent extracts, essential oils and isolated compounds from Warburgia salutaris bark...... 150

xii

Summary

There is an urgent need for the development of new and more effective natural products derived from plant species due to the constantly increasing rate of infectious diseases, especially in developing countries, resulting in high mortality rates. The popularity of bark for medicinal, spiritual and magical purposes is evident from the large-scale trade in these materials on the Johannesburg muthi markets.

Bark is frequently used in the preparation of traditional remedies in southern Africa, and is mostly prefered by the muthi market traders. In the present study, the 70 most popular and most frequently traded medicinal barks used in traditional medicine were purchased from the Faraday and Kwa Mai-Mai Muthi Markets in Johannesburg.

Commercial ethnobotanical knowledge associated with medicinal barks was recorded from traders on the Johannesburg muthi markets. The following information was provided: vernacular names, method of preparation, and mode of administration, ailment used for and less often the dosage form. The muthi market traders are very knowledgeable about the bark products they sell, as some information given was similar to the literature data. The majority of traders were females and they were herbalists, either sangomas, traditional healers or ordinary people from rural areas.

An inventory of the 70 most commonly traded medicinal barks is presented. It includes the data referred to above, as well as literature data on the recorded uses, main chemical compounds and antimicrobial activities. Also recorded in the inventory are detailed descriptions of the bark itself, including digital images of bark materials and extracts that can be used for the identification purposes.

Selected extracts of the bark (64 samples from 32 medicinal barks) were subjected to antimicrobial screening against infectious diseases associated with the

xiii skin, gastrointestinal tract and respiratory tract. The following were used as test organisms: Bacillus cereus ATCC 11175; Enterococcus faecalis ATCC 29121;

Escherichia coli ATCC 8739; Klebsiella pneumoniae ATCC 13883; Moraxella catarrhalis ATCC 23246; Pseudomonas aeruginosa ATCC 743971; Salmonella typhimurium ATCC 14028; Shigella sonnei ATCC 9290; Staphylococcus aureus

ATCC 25923 and Staphylococcus epidermidis ATCC 12228. The overall noteworthy antimicrobial activity of the medicinal barks supported their traditional uses. The lowest minimum inhibitory concentration (MIC) values of 0.004 mg/ml against S. epidermidis, 0.06 mg/ml against B. cereus and 0.20 mg/ml against P. aeruginosa were noted for Erythrina lysistemon, Garcinia livingstonei and Pterocelastrus rostratus bark extracts, respectively.

The results of the antimicrobial screening of selected bark extracts influenced further evaluation for cytotoxicity studies, using the brine shrimp lethality assay.

Cytotoxicity screening of 20 medicinal barks selected based on their antimicrobial activity was undertaken. The tested bark extracts, as well as isolated compounds from Erythrina lysistemon bark, were found to be non-toxic since the mortality after

48 hours was below 50% for all species. Barks from Ziziphus mucronata,

Sclerocarya birrea, Harpephyllum caffrum and Dombeya rotundifolia showed less than 15% mortality after 48-hour period. The methanol extract of Garcinia livingstoneidisplayed the maximum levels of toxicity compared to other barks, with a

46.85% mortality after 48 hours. Therefore, overall brine shrimp lethality activity of the medicinal bark extracts are regarded as non-toxic.

For medicinal barks with noteworthy chemical patterns or antimicrobial activity, detailed studies of their main chemical compounds were undertaken.

Seventeen individual chemical compounds were isolated using column

xiv chromatography and identified by Dr Sadgrove using Nuclear Magnetic Resonance spectroscopy (NMR). The stem bark of Elaeodendron transvaalense yielded three major lupeol triterpenes and a polyphenol, namely lup-20(30)-ene-3a,29-diol (1), 6β- hydroxylup-20(29)-ene-3-one (2), 30-hydroxylup-20(29)-ene-3-one (3) and 4’-O- methyl epigallocatechin (4). The stem bark of Erythrina lysistemon yielded seven prenylated flavonoid derivatives, namely erybraedin A (1), phaseollidin (2), abyssinone V-4’ methyl ether (3), eryzerin C (4), alpumisoflavone (5), lysisteisoflavone (6) and cristacarpin (7). The stem bark of Warburgia salutaris yielded six sesquiterpenes, as follows: drimenol (1), E-nerolidol (2), 12 -acetal- polygodial (3), polygodial (4), ugandensidial (5) and warburganal (6). Compound 4 from the bark of Erythrina lysistemon and compound 1, 2, 3 and 5 from the bark of

Warburgia salutaris are reported for the first time in the bark of these two plant species.

CHAPTER 1. INTRODUCTION

1.1 General introduction

Tree bark provides protection for the plant from various destructive abiotic and biotic factors. It includes all the tissues outside the vascular cambium, which is an actively dividing layer of cells that is responsible for the production of xylem and phloem conducting tissues. The rhytidome is the outermost layer of the bark and functions as a mechanical support and physical barrier to protect the tree against solar radiation, dehydration, fire and plant pathogens. The phloem is a living tissue and forms the inner part of the bark, functioning mainly as a critical structure that transports nutrients throughout the plant (Evert, 2006; Delvaux et al., 2010; Senkoro et al.,

2014; Angyalossy et al., 2016).

In southern Africa, bark has been used extensively in traditional medicine, accounting for almost one third of plant material used in South African traditional medicine (Williams, 1996; Grace et al., 2002). An ethnobotanical survey by Mathabe et al. (2006) conducted in the Limpopo Province also revealed that trees are widely used or preferred by traditional healers, and that bark was the most frequently used plant part in preparing the remedies. A high demand for traditional medicine, especially those derived from woodland and forest plants, has become a key concern for sustainable use of plant products. According to a survey carried out in

Durban, KwaZulu-Natal, a reported 84% of the black population is dependent on both western and traditional medicine (Mander, 2004). Thus, there is an estimated

28 million consumers of traditional medicinal plant products in South Africa. An estimated 75% of plant species traded on muthi markets are collected from

1 woodlands and forests, which accounts for over 60 million plants that are traded annually in order to meet the health care needs of a large number of muthi markets customers (Mander, 2004). The lack of effective management measures has resulted in a continuous decline in the supply of many woodland and forest species into the traditional medicine markets (Mander, 2004). Afromontane forest is the smallest and probably the most vulnerable biome in South Africa, comprising only

0.56% (717 770 ha) of the total land surface area (Cocks et al., 2004). According to

Williams (2004), conservational strategies for plant species should involve rural communities and muthi market traders.

Exploitation of non-timber forest products has increased because of extensive use in traditional medicinal plant trade. Large populations from developing countries throughout the world are dependent on non-timber forest products for their survival needs. Harvesting of these products can create one of the main demands on indigenous forest resources (Cocks et al., 2004). Tree bark is an important component of non-timber forest products and is not only used as a source of raw material in traditional medicine but also in various applications including dyes, tanning of leather, as a detergent, in pottery and construction. An ethnobotanical survey in Mozambique (Senkoro et al., 2014), revealed that the bark of seven species are used for non-medical purposes, of which four are used as detergents

[Albizia adianthifolia (Schum.) W.Wight], Albizia versicolor (Oliv.), Sclerocarya birrea

[(A.Rich.) Hochst.], and Terminalia sericea (Burch. ex DC.). Other applications of bark included the making of ropes and strings (Dialium schlechteri Harms and

Strychnos sp.), pottery (Trichilia emetica Vahl), and house construction (Strychnos sp.). The population structure and reproduction of individual tree species are frequently altered by over harvesting of non-timber forest products (Delvaux et al.,

2010). A survey in KwaZulu-Natal revealed that bark from tree trunks and roots from mature trees are the most important and popular medicinal plant materials sold at the muthi markets and accounts for 54% of the plant products traded (Mander et al.,

2007). Another study by Moyo and co-workers (2011), also reported a high preference of 32% for plant material harvested from mature trees, with 83% of the market traders preferring the use of mature bark.

Extensive harvesting of bark causes internal stress for the tree, which could result in progressive deterioration of health and ultimately death. In some species, severe stress may be associated with bark recovery (to close a wound). Protection against external harmful effects is drastically reduced when a tree is debarked

(Geldenhuys, 2004; Delvaux et al., 2010; Senkoro et al., 2014). A few plant species that have been observed to survive ring-barking include Quercus suber L.,

Eucommia ulmoides Oliv., Prunus africana (Hook.f.) Kalkman, Warburgia salutaris

(G.Bertol.) Chiov., Ocotea bullata (Burch.) E. Mey., Ficus natalensis (Hochst.) and

Carapa procera DC. Species observed with poor bark recovery rates after being harvested include Cryptocarya myrtifolia Stapf, Rapanea melanophloeos (L.) Mez,

Elaeodendron transvaalense (Burtt Davy) R.H.Archer and Curtisia dentata (Burm.f.)

C.A.Sm. Anatomical differences in the structure of wood and bark between individual plant species may explain variances in the reaction to wound closure (Geldenhuys,

2004; Twine, 2004; Delvaux et al., 2010).

Traditional medicine is made up of various health practices, approaches, knowledge and beliefs (WHO, 2005). The healing power of medicine is influenced by the incorporation of material from plant, animal, and/or mineral sources. Spiritual therapies, manual techniques and exercises are also used singularly or in combination to treat, diagnose, or prevent illness in order to maintain the human

3 well-being. This is in contrast to allopathic medicine that is based on Western culture whereby practitioners emphasise a scientific approach that is not characterised by cultural values (WHO, 2005). In addition, allopathic medicine is largely centred on the philosophies of science, technology and clinical analysis and is typically linked with diseases that affect only the physical body (Richter, 2003).

In African traditional medicine, the incorporation of animal parts to traditional remedies is frequently considered as an addition of magical power to a potion, although these animal products are taken in much smaller doses than the plant material. The use of mammalian parts is more popular compared to birds, reptiles and invertebrates (White et al., 2004). Williams and Whiting (2016) mention that animal derivatives are also vital medicinal sources that promote curative, protective and preventative effects against a wide variety of ailments and are not restricted to magical charms.

African traditional medicine has been reported to be one of the oldest healing systems since Homo sapiens are known to have originated in Africa. Although it is amongst the most diverse medicinal systems, it is also regarded as the least systematised and poorly recognised (Van Wyk and Wink, 2015). Cultural diversity has led to the development of many medicinal healing systems that are unique to particular regions throughout sub-Saharan Africa. African traditional medicine is holistic, i.e., the mind and body are both treated simultaneously as a unit (Mander et al., 2007; Van Wyk and Wink, 2015).

Natural products have a long history of use in various medical applications in the form of traditional medicines, remedies, potions and oils; hence, many biologically active natural products are still being identified (Dias et al., 2012). Plant

4 species hold a unique chemical composition and these bioactive compounds may be present in a number of species of a genus, in several related genera, or in different families (McGaw et al., 2002).

Proteins, fats, nucleic acids and carbohydrates are vital components to all living organisms and their biosynthesis and breakdown in plants is known as primary metabolism (Pengelly, 2004). The biosynthesis of secondary metabolites (natural products) originates from the key processes of photosynthesis, glycolysis and the

Krebs cycle. The mechanism by which these natural products are produced is known as secondary metabolism, and their biosynthesis is frequently considered unique to an organism or is an expression of the distinctiveness of a species (Pengelly, 2004).

Secondary metabolites are usually not critical for the growth, development or reproduction of an organism. Their production is largely influenced by the development of survival traits or strategies in order to adapt to harsh environmental conditions, or are produced to act as a possible defence mechanism against predators (Dias et al., 2012).

Natural products (secondary metabolites) are considered as potential drug leads and have been used since ancient times in traditional medicine for the treatment of many diseases (Eldeen et al., 2005; Mahesh and Satish, 2008; Dias et al., 2012; Tchinda et al., 2016). Early records of plant species displaying the use of natural products are found in oils from Cupressus sempervirens L. and Commiphora species, which are still used today to treat coughs, colds and inflammation. These were described on clay tablets in cuneiform from Mesopotamia since 2600 B.C.

(Dias et al., 2012). The development of antimicrobial, antioxidant and anti- inflammatory screening techniques have led to many discoveries of useful and effective medicinal plants. Among the estimated 250 000 to 500 000 plant species

5 used medicinally, only a small portion has undergone phytochemical investigation and biological or pharmacological screening (Mahesh and Satish, 2008).

Bark has historically been an important source of medicine, and there are famous examples of barks that were initially used without any scientific understanding of their efficacy. Quinine for example, is an alkaloid that occurs naturally in Cinchona tree bark. Lay people (Peruvian Indians) used this medicinal bark since ancient times as a remedy for treatment of fever. Cinchona bark was later discovered by scientists to have the ability to cure malaria. It was only in the 1640’s when Cinchona bark was first introduced to Europe (from Peru), after the discovery of its antimalarial properties. Hence, Cinchona bark has been universally recognised as an antimalarial substance since 1681. However, it was only in the 17th century that quinine was first isolated after many unsuccessful attempts by various scientists.

The German chemist Friedrich Vilhem reported that morphine forms salts in the presence of acids, which then led him to the successful isolation of ‘quinine’, the active principle from the bark (Misra et al., 2008; Nguta and Mbaria et al., 2013).

A more recent example was based on scientific methodology and not on traditional knowledge. Paclitaxel (Taxol®), isolated from the bark of Taxus brevifolia

Nutt. (Pacific yew), is the most widely used cancer drug (Donehower, 1996;

Chowdhury et al., 2017). Another well-known example is the synthesis of the anti- inflammatory agent, acetylsalicyclic acid (aspirin), derived from the natural product, salicin. It is generally considered to have been isolated from the bark of the willow tree, Salix alba L. (Dias et al., 2012). However, the real source was actually

Filipendula ulmaria (L.) Maxim., previously known as Spiraea ulmaria L. The name aspirin (AspirinTM) was derived from this plant name (Van Wyk and Wink, 2017). In southern Africa, the best known active compounds discovered in medicinal barks

6 include warburganal (Kubo et al., 1977), mukaadial, polygodial and isopolygodial

(Mashimbye et al., 1999), isolated from Warburgia salutaris, acyclic triterpenoids,

2,3,22,23-tetrahydroxy- 2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene and 2-hydroxymethyl-2,3,22,23-tetrahydroxy-6,10,15, 19,23-pentamethyl-6,10,14,18- tetracosatetraene (Nishiyama et al., 1996) isolated from Ekebergia capensis

Sparrm., betulinic acid, (+) – catechin and bergenin (Theo et al., 2009) isolated from

Peltophorum africanum Sond., lupeol and aurantiamide acetate (Tamokou et al.,

2012) isolated from Albizia adianthifolia and triterpenes such as 3β-hydroxylanosta-

9, 24-dien-21-oic acid and methyl-3β-hydroxylanosta-9,24-dien-21-oate (Mosa et al.,

2014) isolated from Protorhus longifolia (Bernh.) Engl. As will be seen in Chapter 5, new and highly active compounds can still be discovered in well-known and seemingly well-studied medicinal barks (e.g. Elaeodendron transvaalense, Erythrina lysistemon and Warburgia salutaris).

The antimicrobial activity of medicinal plants is still highly relevant because of resistance to existing antimicrobial agents. There have been regular reports in the literature of the last 10 years that there is an increase of resistance to Gram-negative bacteria. Hence, there is a worldwide search for new active compounds and it is likely that some will be discovered in traditional medicines that have not yet been properly evaluated. More than 50% of all the drugs used in clinical applications are claimed to be natural products and their derivatives, with a quarter originating from higher plants (Eldeen et al., 2005; Mahesh and Satish, 2008; Tchinda et al., 2016).

Therefore, due to the ability of plants to produce antimicrobial chemical compounds, they are considered worthwhile for further exploration as a source for antibiotic leads

(Eldeen et al., 2005). Less than 10% of the world’s biodiversity has been subjected to evaluations of biological activity, so that there must be numerous plant species

7 with unknown chemical profiles that have potential as sources of new drug leads

(Dias et al., 2012).

Antimicrobial studies reported in the literature over the period 1978 to 1988 were often not accurate and reliable because of poor techniques or variation within the techniques employed by different authors. As a result, there are discrepancies between the results obtained by different authors for the same plant species and for material that was tested against the same bacteria (Van Vuuren, 2008). Rios and

Reico (2005) argue that even though experimental procedures have been standardised, there are still experimental errors, inconsistencies and a lack of uniformity that may result in inaccurate results. According to Rios and Reico (2005), in most cases “either the selection of microorganism is not well established, the assayed doses are extremely high, the positive control is not clearly defined or the methods are inadequate”. In addition, a review by Van Vuuren (2008) further suggests that lack of uniformity leads to results that are not always clear and predictable. Antimicrobial results produced from natural products should be comparable to other studies in order to ensure reliability and accuracy (Van Vuuren and Holl, 2017).

In recent years, numerous antimicrobial studies using traditional medicinal plants have been carried out, exhibiting noteworthy activity against a diverse range of infectious diseases. Several African traditional medicinal plants demonstrated noteworthy antimicrobial activity against fungi, Gram-negative multi-drug resistant strains and other pathogens. These include, among others, Combretum kraussii

Hochst. (McGaw et al., 2008), Syzygium cordatum Hochst. ex Krauss (York et al.,

2012), Vachellia (Acacia) karroo (Nielsen et al., 2012), Terminalia sericea Burch. ex

DC. (Van Vuuren et al., 2015) and Sclerocarya birrea (Van Vuuren and Holl, 2017),

8 as well as naturalised exotics that have become part of the local medicine system, such as Acacia mearnsii De Wild. (Olajuyigbe and Afolayan, 2012). See Chapter 3 and Appendix 1 for a more detailed review of antimicrobial studies on southern

African barks.

The overall aim of the study was to provide a review and analysis of all commercially important barks sold on Johannesburg muthi markets and to explore morphological and other characters that can be used for identification. A secondary aim was to screen bark extracts for their antimicrobial activity and then to study the chemical compounds and specific antimicrobial activities of three carefully selected barks.

1.2 Objectives of this study

 An inventory of the most important and most popular medicinal barks found in

the Johannesburg muthi markets that are used in traditional medicine. The

aim is to generate data (vernacular names, photographs and descriptions of

the morphology and structure of the bark) for use in the identification of

commonly traded medicinal barks.

 Recording of commercial ethnobotanical knowledge associated with popular

medicinal barks amongst traders on the Johannesburg muthi markets.

 Studying the antimicrobial activity of selected bark extracts against infectious

diseases associated with the skin, the gastrointestinal tract and the

respiratory system.

 Study the cytotoxicity of selected medicinal bark extracts that were subjected

to antimicrobial screening.

 Detailed studies of the main chemical compounds in the bark of a few

carefully selected species (e.g. those with noteworthy chemical patterns or

antimicrobial activity).

CHAPTER 2. INVENTORY OF POPULAR MEDICINAL BARKS

2.1 Introduction

Indigenous knowledge refers to valuable information and skills that have been passed on from earlier generations that is unique to a particular group of people in a community or village and sharing the same values and beliefs (Stafford, 2004).

South Africa holds enormous traditional knowledge that has developed over times and which rural people in particular, engage in throughout the country on a daily basis (Stafford, 2004). Traditional medicinal knowledge may be organized, controlled, taught openly, and practised widely and systematically, and benefit from thousands of years of experience. Alternatively, it may be highly secretive, supernatural and very localised, with knowledge often passed on orally from one person to the next, or from one generation to the next (WHO, 2005).

The use of traditional medicine is strongly based on cultural and spiritual beliefs and is not limited to the rural population, as these plant products are also found in urban areas and widely purchased or used by educated individuals (Ndhlala et al., 2011; Van Vuuren et al., 2014). A survey that was carried out in 2001 at the

Faraday Muthi Market revealed that each year, approximately 12 – 15 million consumers from urban settlements depend on traditional medicine derived from woodlands and forests (Williams, 2004). Furthermore, Van Vuuren and co-workers

(2014) reported the presence of more than 200 vendors in the same market.

Muthi markets can be defined as an informal place of commercial trade in medicinal plants and animal products that are almost invariably harvested from the wild. Plant material may be harvested from communal land, private farms, protected areas, forests and forestry estates (White et al., 2004). According to a report from

11 the Department of Water and Forestry (2005), plant material is usually harvested in bulk from the wild and supplied as raw products with little or no processing to the urban informal street markets. Approximately 2062 indigenous plant species are utilised in traditional medicine in South Africa and 32% of these that used in ethnomedicines have been recorded in muthi markets in the KwaZulu-Natal,

Gauteng, Eastern Cape, Mpumalanga and Limpopo Provinces (Williams et al.,

2013). From the 771 plant species that are traded on South African muthi markets

(Williams et al., 2011), 574 products are from the woodland and forest biome. These are taken either as self-medication or as prescription from the healer (Mander,

2004). Harvesters and traders from the muthi markets are very knowledgeable about the plant products they sell; hence, they are also able to tell whether or not the plant is scarce and where it may be found (Williams et al., 2011). South African trade of plant species used in traditional medicine is largely dependent on plant material with a long shelf life: bark, roots, whole plants, seeds and fruits. This is apparently in contrast to other regions of Africa outside southern Africa, where leaves are predominantly used (Grace et al., 2002).

The trade of indigenous plants in South Africa is part of a multi-million Rand industry. Rural households not only use medicinal plants for their health care benefits but also as a source of income (Williams, 2004; Mander, 2004). The muthi markets not only have a great impact on the traders and consumers, but also play a crucial role in the South African economy, with an estimated annual value of R2.9 billion, representing 5.6% of the National Health budget (Mander et al., 2007;

Williams et al., 2011). In the KwaZulu-Natal Province alone, over 4 000 tonnes of plant material is traded in a year, which is equivalent to a value of R60 million. At a

12 national level, 20 000 tonnes may be traded in a year, which is equivalent to a value of approximately R270 million (Cocks et al., 2004; Mander et al., 2007).

The popular areas where most research on muthi markets in South Africa is conducted include KwaZulu-Natal, Eastern Cape, Gauteng and Mpumalanga. The trade in medicinal plants is not limited to these provinces, but is carried out right through the country and its borders. Therefore, various needs of different ethnics groups and the availability of resources within each geographical region largely influences the trade of medicinal plant products (Cocks et al., 2004).

In the Eastern Cape Province, a market survey was done in six urban centres

(Port Elizabeth, King Williams Town, East London, Umtata, Uitenhage and

Queenstown) by White et al. (2004). They identified 166 medicinal plants that were popularly traded. It was also reported that an estimated 524 tonnes of plant material is traded annually in this province, at a value of approximately R27 million (White et al., 2004).

A market survey on the informal trade of medicinal plants in Mpumalanga recorded 176 species from 66 plant families, of which 62% occur in woodlands and/or forests (Botha et al., 2004). In traditional markets in Limpopo, only 70 medicinal species from 40 families were encountered, with 59% of these occurring in woodlands and/or forests (Botha et al., 2004).

Although muthi markets traders are well informed about the plant materials they sell, many problems of incorrect identification of bark have arisen due to similar vernacular names for more than one plant and in rare cases, similarities in the superficial appearances of different barks. The phenotypic appearance of bark surfaces has frequently been used for the identification of trees and for taxonomic and phylogenetic studies. This is because the external appearance of stems bearing

13 periderm or rhytidome is highly variable and of analytical value (Kotina et al., 2012).

Despite the popularity and frequent use of bark, precise descriptions of bark in the literature is negligible and relatively inaccessible (Grace et al., 2002).

2.2 Materials and methods

Eighty-two bark samples from 70 tree species were purchased at the Faraday and

Kwa Mai-Mai Muthi Markets in Johannesburg during 2016, 2017 and 2018. Each bark sample was bought for R20 (ca. $1.42) per package, which equates to a handful of bark typically with two to five solid pieces of material. Sometimes pieces of the barks sold may not necessarily come from the same plant source; some pieces may be from younger branches and stems, while others are from a mature part of the trunk. An example of a display of bark samples is shown in Figure 2.1

Figure 2.1 Examples of bark products as they are sold at the Johannesburg muthi markets. Photograph by E.L. Kotina.

2.2.1 The Muthi Markets

2.2.1.1 The Faraday Muthi Market

The Faraday Muthi Market is located in Johannesburg, Gauteng Province (Figures

2.2, 2.3 and 2.4). According to Williams and Whiting (2016), the Faraday Muthi

Market has in the past decade become the biggest market, popularly known for traditional medicine, not only in South Africa, but perhaps also the largest in southern

Africa and Africa. The trading stalls that are also used for storing or packing plant products are usually outdoors, slightly exposed or covered, with very few traders that are found selling within the old and neglected public buildings (Van Vuuren et al.,

2014). There is also an active trade of animal products at the Faraday market such as snake and crocodile skin, bones of carnivorous animals including lion, leopard and cheetah, and different kinds of birds.

Figure 2.2 Entrance to the Faraday Muthi Market. Photograph by E.L. Kotina.

Figure 2.3 Typical stall at the Faraday Muthi Market, showing bark products. Photograph by E.L. Kotina.

Figure 2.4 Processed bark products on the Faraday Muthi Market. Photograph by E.L. Kotina.

Williams and Whiting (2016) reported an estimate of 42% traders that sell animal parts (19 traders sell animals only; 74 sell animal and plant parts; 117 sell plants only; not determined for 10 traders). The local people, healers and consumers from townships, rural and urban areas around Gauteng and neighbouring provinces purchase these plant products from the muthi market for their health care benefits.

An estimated 220 muthi market traders were recorded at Faraday in October 2015, which decreased from over 300 traders recorded in October 2014 (Williams and

Whiting, 2016).

2.2.1.2 The Kwa Mai-Mai Muthi Market

The Kwa Mai-Mai Muthi Market is located south of Johannesburg under a bridge on the corner of Anderson and Berea Streets (Figures 2.5 to 2.8). The Kwa Mai-Mai

Muthi Market is known as one of the oldest markets in Johannesburg, having opened about 50 years ago. It is also known as ‘Ezinyangeni’, which means “the place of traditional healers”, as the majority of the traders are either traditional healers or sangomas. The city of Johannesburg municipality manages the muthi market. Unlike the Faraday Muthi Market, which sells mainly plant and animal products, the Kwa

Mai-Mai Muthi Market has a wider range of products such as Zulu traditional attires, traditional brooms that are often used in wedding ceremonies and traditional mats.

The trading stalls at the Kwa Mai-Mai Muthi Market are indoors and have private consultation rooms (Figure 2.5) where customers may be offered ‘medical’ service

(Figure 2.6).

Figure 2.5 Entrance to the Kwa Mai-Mai Muthi Market. Photograph by E.L. Kotina.

Figure 2.6 Typical selling stalls at the Kwa Mai-Mai Muthi Market. Photograph by E.L. Kotina.

Figure 2.7 Inside a traditional medicine stall at the Kwa Mai-Mai Muthi Market with the trader, Mr Gift Nzimande (a traditional healer), on the left. Photograph by E.L. Kotina.

There are various packaging and distribution methods adapted by traders when selling harvested bark material from the wild. These include raw, dried, solid pieces of material, or partially processed (chopped or pounded) products (Figure 2.4). Bark may be ground into fine powder, usually for use as snuffs or for incorporation into complex mixtures for use as insizi (powdered mixtures rubbed into scarification in the skin to alleviate pain). Particular bark material from plant species such as

Harpephyllum caffrum, Securidaca longepedunculata and Schotia brachypetala are often sold as chopped material. The reason given by the traders is that customers complain about the difficulties of chopping the bark by themselves at their households. Packaging of the plant material is normally in the form of newspapers, recycled liquor bottles or plastic bags (Figure 2.8). Medicines are commonly prepared with water as an extraction solvent in order to form a decoction or infusion.

Van Vuuren et al. (2014) investigated the hygienic status of plant material sold at the

Faraday Muthi Market from different traders and observed that contamination levels were above the limit required according to the World Health Organisation report. In addition, there was no variation in the contamination levels among the plant material purchased from different traders.

Figure 2.8 Inside the same trading stall shown in Figure 2.7, illustrating the way in which processed bark is stored and dispensed. Photograph by E.L. Kotina.

2.2.2 Trader information and data recorded

The traders from which products were purchased are listed in Table 2.1. Twelve out

of the 14 traders were Zulu speaking, and two were Xitsonga and Xhosa speakers

(but both communicated in Zulu). Since I am Zulu speaking, communication was

very easy and I was able to accurately record and write the Zulu vernacular names

and other data. There were no ethical issues, since only commercial information of

consumer and trader was recorded. No bark samples were collected from nature.

One sample (Warburgia salutaris) was collected from a cultivated garden tree with

permission from the owner (Prof van Wyk). Therefore, no permits were necessary.

Person Language Gender Faraday Kwa Mai-Mai Abbreviation Gift Nzimande isiZulu Male + GN Mzochithwayo IsiZulu Male + MZ Ntombi Ntuli IsiZulu Female + NN Ntombi Ntshangas IsiZulu Female + NTS Lindiwe Khumalo IsiZulu Female + LK Vamisile Gina IsiXhosa Female + VG Mkhulu Madondo IsiZulu Male + MM Nelly Tsonga Female + N Gogo Doma IsiZulu Female + DO MaNcube IsiZulu Female + MN Mkhwanazi IsiZulu Male + MK Bab’ Nqulunga IsiZulu Male + NQ Gogo Makhaza IsiZulu Female + MA S. Nkambule IsiZulu Female + SN

The selection of traders was random and based on willingness of an individual to participate, after clearly explaining in their home languages the purpose of conducting the research study. For each package of bark sample purchased, the trader was asked to provide the following information: a vernacular name or names

(‘uyalazi igama lesintu lexolo’), main uses of the product (bark) (‘lisetshenziselwani leli xolo’), method of preparation (‘lisetshenziswa ngaluphi uhlobo’) and mode of administration (‘lisebenza kanjani, uyaphuza, uyaphalaza noma uyachatha’).

Bark samples were taken to the laboratory for photographs of the external appearance of the inner and outer bark (see Appendix 1), which all were taken by

Prof van Wyk. Pieces of bark from these samples were used for further studies.

2.3 Results and discussions

2.3.1 Traders on the muthi markets

The predominant language spoken on the Faraday and Kwa Mai-Mai Muthi Markets in Johannesburg is isiZulu and it appears that most of the traders have isiZulu as their home language (Table 2.1). The dominant medicinal culture on the markets is also Zulu and very few traditional medicines from other cultures are sold. Amongst the bark products, Peltophorum africanum, Securidaca longepedunculata and

Brackenridgea zanguebarica are from the Limpopo Province; hence, they are sold with Venda vernacular names. However, the majority are used in Zulu traditional medicine (Pujol, 1990; Hutchings et al., 1996; Grace et al., 2003; Von Ahlefeldt,

2003).

In South Africa, informal trading of medicinal plants is very popular among black populations. This study indicates that 86% of traders are Zulu speaking,

22 followed by 7% of each Xhosa and Tsonga speakers. Similar results were reported by Williams (2004), who stated that nearly all the traders at the Faraday Muthi

Market are Zulu speaking. The percentage of languages of the traders recorded by

Williams (2004) were 95.8% Zulu, 2.1% Sotho, 1.1% Xhosa and 1.1% Tsonga. The majority of traders (traditional healers in particular) are from KwaZulu-Natal (the

Zululand region) as they mentioned that they harvest the plant materials from kwaNongoma, Bergville and kwa-Mhlab’ uyalingana. Language data recorded in this study clearly show that the Faraday and Kwa Mai-Mai Muthi Market traders are all

South Africans, with the majority of them coming from the KwaZulu-Natal Province.

In 1998, there was already an estimate of six million people in KwaZulu-Natal

Province who were involved in the indigenous trade of medicinal plants, either as harvesters, sellers or buyers (Mander, 1998; Ndhlala et al., 2011).

My observations confirmed that very few, if any, traders are from other South

African cultures and none was recorded from other southern African countries. In contrast, Mander et al. (2007) reported that many traders from the street markets of

Durban and Johannesburg come from Mozambique and Swaziland due to the active plant trade from these countries to South Africa. My observations agree with the data of Williams (2003) who reported that 97% of the approximately 166 traders are migrants to the Faraday Muthi Market, of whom 90% regarded the province of

KwaZulu-Natal as ‘home’. The Zulu speaking people are very proud and enthusiastic about their culture and always take pride in acknowledging and preserving their beliefs. As a result, there has been many well-documented ethnobotanical studies of the Zulu ethnic group compared to other ethnic groups in South Africa. Literature sources such as Gerstner (1938), Bryant (1966), Hutchings (1989), Cunningham

(1988), Pujol (1990) and Hutchings et al. (1996), as well as a more recent study

23 done by Grace et al. (2003) which focused on an inventory of medicinal barks in

KwaZulu-Natal, have all contributed to the fairly comprehensive documentation of

Zulu medicinal plant use.

2.3.2 Gender information on traders at the Johannesburg muthi markets

A report by Mander et al. (2007) indicated that an estimated 74% of medicinal plant harvesters, street traders and traditional healers are women, and, 80% are rural and

20% urban. Twenty six percent of the trade role players are men, of which 50% rural and 50% urban. Therefore, women are the main role players in the medicinal plant trade, predominantly those from rural areas. Their involvement in the trade creates an important source of survival, as medicinal plants are natural resources of high value that can be harvested for trade in order to generate rural incomes (Mander et al., 2007). Similar results were obtained from the present study, showing women as the dominant gender on the two Johannesburg muthi markets – 64% of the traders were females and 36 % were males.

In many ethnobotanical studies, women were found to be more active participants compared to males. Earlier records by the Department of Water and

Forestry (2005), mentioned that current market players are primarily black rural women who harvest medicinal plants in rural areas and sell them at urban street markets. Results from the present study are in accordance with Williams (2004), who reported that 70% of the Faraday Muthi Market traders in Johannesburg were women and only 30% were males.

In a muthi market survey by Ndawonde et al. (2007), carried out in Zululand, out of 63 plant traders interviewed, 77% were females. The same authors mentioned that gender imbalances might be attributed to the employment of males in the formal

24 job sector, whereas many rural women are unemployed and depend on plant products they sell to reduce poverty in their homesteads. In addition, the other reason is that males involved in the plant trade are usually at home practising as herbalists where they are often consulted by patients, rather than just being street vendors. According to the ethnobotanical study of medicinal plant use carried out by

Masevhe et al. (2015) with traditional healers as the main informants, males were generally not interested to engage in the survey regardless of the assurance that the information was for research purposes only. Many of them mentioned that they could not give away their ancestral knowledge to the researchers, and thus only 27.3% of the interviewed traditional healers were males.

2.3.3 Diversity of bark on the muthi markets

The main results of the muthi market survey are presented in Table 2.2. It includes

70 species of medicinal barks, together with their family names, vernacular names, method of preparation and medicinal uses as reported by the traders. In the present study, 35 plant families were represented by 70 medicinal barks sold at the Faraday and Kwa Mai-Mai Muthi Markets (Figure 2.9). The families with the highest numbers of species (presented as a percentage of the total) are the Fabaceae (29%),

Celastraceae (18%), Anacardaceae (15%) and Apocynaceae (12%). No medicinal barks from Asteraceae and Rubiaceae were encountered, despite these families being amongst the most species-rich in the South African flora.

Anacardiaceae Apocynaceae Apiaceae Aquifoliaceae Araucariaceae Burseraceae Caesalpinaceae Canellaceae Celastraceae Clusiaceae Combretaceae Cunoniaceae Curtisiaceae Ebenaceae Euphorbiaceae Fabaceae Lauraceae Loganiaceae Malvaceae Meliaceae Melianthaceae Moraceae Myrtaceae Ochnaceae Phyllanthaceae Pittosporaceae Polygalaceae Primulaceae Rhamnaceae Rhizophoraceae Rosaceae Rutaceae Sapotaceae Verbenacea Zygophyllaceae

Figure 2.9 Plant families of 70 medicinal bark species purchased at the Faraday and Kwa Mai-Mai Muthi Markets in Johannesburg.

According to Shai et al. (2009), the most popular plant species that are leading in

Zulu, Sotho and Xhosa ethnomedicine in South Africa are from an estimated 147 plant families, with the majority belonging to the Fabaceae, Asteraceae,

Euphorbiaceae, Rubiaceae and Orchidaceae. According to the species list published by Williams (2003), which included all plants traded and not only bark, Asteraceae

26 was the top plant family (i.e., with the highest number of species traded) represented at Faraday Muthi Market, followed by Euphorbiaceae. The inventory of bark medicines used in traditional health care in KwaZulu-Natal published by Grace and co-workers (2003) included 174 bark species, represented by 50 families. The three families with the highest number of genera were Euphorbiaceae, Anacardiaceae and

Celastraceae. These results are quite similar to my study, except for the discrepancy in Euphorbiaceae (which can be explained by the fact this family has been subdivided into the Euphorbiaceae sensu stricto and the Phyllanthaceae).

The main reasons for using bark in traditional medicine may be the presence of tannins, which are known to be non-specific protein poisons (Van Wyk and Wink,

2015, 2017). According to Hutchings (1989), tannins are frequently present in those parts of plants used in the treatment of dysentery and diarrhoea, or for respiratory ailments. Tannins are a characteristic constituent of many of the families (e.g.

Rosaceae. Fabaceae, Geraniaceae). Furthermore, tannins are most likely to be effective on account of their protein-precipitating properties which, in small doses, would form a protective, impermeable layer over mucosa and also tend to prevent the development of bacteria by denaturing the proteins within these unicellular organisms. Because they are unicellular, bacteria cannot survive, while the dead cells of the upper layers of the mucosa are rapidly replaced by cell division. In cases where activity can be ascribed to non-polar compounds, the presence of saponins may be an important consideration, as they will ensure that at least some of the non- polar constituents will be present in infusions or decoctions (which can be similar to emulsions when terpenoid glycosides are present).

Table 2.2 Seventy species of medicinal barks recorded on the Faraday (F) and Kwa Mai-Mai (M) muthi markets in Johannesburg and their ethnobotanical uses as reported by the traders (for abbreviations of the traders, see Table 2.1). An asterisk indicates exotic (non-indigenous) species*.

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Albizia adianthifolia umgadankawu + + flat-crown tree (E); Infusion The bark is soaked in hot water and used (Schumach.) platkroondoringboom (A); as a wash once a day for few days to treat W.Wight (Fabaceae) muvhada-ngoma (V); skin rashes [SN; NTS; MN; DO; VG] and umhlandlothi (X); igowane, chicken pox [LK; MA;NN;GN]. umgadankawu (Z) ‘Umgadankawu’ means ‘one who looks after the monkeys’ (the tree is known to be a suitable shelter for monkeys). Araucaria abangqongqozi + monkey puzzle (E) Infusion The bark is used in love charms [GN; MZ; heterophylla* NQ; MM; MK]. 'Abangqongqozi' means (Salisb.) Franco 'knocking on the door’, which indicates the (Araucariaceae) purpose of this medicine and its ability to knock on the door of the lost lover to remember you. Balanites maughamii iphamba + + torchwood (E); fakkelhout Decoction Powdered bark is licked from the hand Sprague (A); umnulu (S); nulu (NS) (khotha) for body pains [NQ; MM; SN; MZ; (Zygophyllaceae) MK]. The bark is taken as an emetic and enema in order to confuse a person, especially in court cases to get away with breaking the law [NTS; MK; SN; NTS]. Bark emetic is used to bring easily interpretable dreams from the ancestors [MM; MA]. Berchemia discolor uvuka + bird plum (E); bruin-ivoor Decoction The bark is used in combination with other (Klotzsch) Hemsl. (A); mogokgomo (NS); plants in order for the medication to be Rhamnaceae mokêrêtê (Tsw), effective [NQ; GN; NN; MK; LK; DO].

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai motsentsela (Tsw); Acting as an initiator or a booster. muhukhuma (V); ‘Ukuvuka’ means to be awake or bring to ubaletsheni-omkhulu (Z); life. uvuka (Z) Bersama lucens undiyaza + + glossy bersama (E); Powdered A pinch of finely powdered bark is licked (Hochst.) Szyszył. blinkblaarwitessenhout (A); and (khota) or snuffed in a raw form for the (Melianthaceae) isindiyandiya (X); undiyaza decoction treatment of stroke and headache [GN; (Z) MZ; MK; MM; NTS;]. The bark is boiled to make (imbiza) mixtures and half a cup is administered orally to relieve menstrual pain, strengthen the blood and is used for stomach ailments [MA; SN; LK; VG]. Bersama tysoniana undiyaza + common white ash (E); Powdered A pinch of finely powdered bark is licked Oliv. witessenhout (A); (khota) for treatment of stroke [GN; MZ; (Melianthaceae) mokgoba (NS); undiyaza MM; NTS; MK]. Bark is used as ‘intelezi’, to (X); undiyaza (Z) protect against witchcraft [MK; NN]. Bark is used to break spells [NQ; MA]. The Zulu praise name is umadoda ayiziwula (“stupid man”), meaning that the bark will somehow mislead a man Brackenridgea mutavhatsindi + yellow peeling plane (E); No data No data (Tsonga trader reluctant to give zanguebarica Oliv. geellekkerbreek (A); information) (Ochnaceae) mutavhatsindi (V) Bridelia micrantha umhlahle/ + coastal goldenleaf (E); Decoction Bark is used as an emetic to separate (Hochst.) Baill. umhlahla mitserie (A); motsêrê (NS); between good and bad spirits in a ritual Phyllanthaceae munzere (V); ceremony (amagobonga) of becoming a umhlahlahlungulu (X), sangoma [NQ; MA; LK; MZ; DO; N]. umhlahlangu (X); umhlahle ‘Umhlahlandlela’ means to show the way (Z) or give a clue.

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Calodendrum umemezi + + cape chestnut (E); Powdered The bark is used as a love charm, whereby capense (L.f.) omhlophe wildekastaiing (A); and paste the name of a lost lover is called Thunb. (Rutaceae) molalakgwedi (NS); ('memeza') through the powdered bark muvhaha (V); umbhaba over the palm of the hand and blow it in the (X); umemezi omhlophe direction where that person lives [SN; LK; (Z) VG; DO; GN; MM; MK]. Memeza means to call a person from afar. Finely powdered bark mixed with cold water to form a paste and applied to the face as a skin lightener [MM; VG; MA; DO; NTS; MN; MK; LK]. Carissa bispinosa umvusankunzi + num-num (E); Powdered The bark is ground into fine powder and a (L.) Desf. ex Brenan bosnoemnoem (A); small portion is licked 'khotha' for treatment (Apocynaceae) morokolo (NS); murungulu of erectile dysfunction [NQ; MM; MA; GN; (V); umvusankunzi (X); MK; NTS; LK; DO; SN; MZ] umvusankunzi (Z) Cassipourea umemezi obomvu + + Cape onionwood (E); Paste Powdered bark is mixed with water and flanaganii (Schinz) Kaapse uiehout (A); applied facially as a skin lightener and Alston ummemezi (X) umemezi used as a sunscreen [ALL, except N]. The (Rhizophoraceae) obomvu (Z) bark is used in love charms [GN; NTS; MZ].

Cinnamomum uloselina + + camphor tree (E); Infusion and Bark infusion is used as an emetic for a camphora (L.) kanferboom (A); uloselina powdered person to be admired by other people [ALL J.Presl * (Lauraceae) (Z) except N]. Powdered bark is applied on the eyebrows or a piece of solid bark is placed in the mouth in order to persuade people (usually of the opposite gender) to be attracted to you [NQ; MK].

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Combretum caffrum isidubu + Cape bushwillow (E); Infusion The bark is used to make 'izimbiza' (liquid (Eckl. & Zeyh.) Kaapse vaderlandswilg mixture) and administered orally or as an Kuntze (A); isidubu (X); umdubu enema for internal cleaning of the body. (Combretaceae) (Z) The bark is used to clean the veins [NQ; MK; SN; NTS; MZ; VG]. Pounded bark is used as ‘ikhubalo’ to chase away evil spirits [MA; MM]. Commiphora iminyela + hairy corkwood (E); harige Powdered Bark is ground into fine powder and is africana (A. Rich.) kanniedood (A); iminyela licked or applied to small razor incisions in Engl. var. africana (Z); morôka (Tsw) the body in order to prevent other (Burseraceae) medications from entering the blood or prevent/ suppress the efficacy of other medicines and bark is used to protect against enemies [NQ; MZ; GN; MM; MK; MN]. Croton sylvaticus umahlabekufeni + forest fever-berry (E); Decoction Pounded bark is boiled in water and Hochst. boskoorsbessie (A); and administered orally with a cup twice a day (Euphorbiaceae) umagwaqane (X); powdered for treatment of wounds in the stomach umhloshazane, and constipation [GN; MZ; LK; DO; SN; umahlabekufeni (Z); NN; NTS]. Powdered bark is licked or moema (NS); mula-thoho, snuffed for pain; it reaches the site of muima-vanda (V) infection faster [NQ; MA; MM; GN]. Cryptocarya latifolia umkhondweni + broad-leaved Laurel (E); Powdered Powdered bark is mixed with crocodile fat Sond. (Lauraceae) breëblaar-kwepe (A); and administered orally by licking directly umgxaleba from the palm of the hand (khotha) for (X); umkhondweni (Z) treating food poisoning [GN; MZ; MK; NN; MA; MN]. The bark is smoked for headache. Bark is used by traditional healers to detect the type of sickness in the

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai body a ‘patient’ suffers from [NQ; MA; DO; SN]; hence the Zulu meaning of the bark refers to ‘showing the way or giving a clue’ (umkhondo). Cunonia capensis L. umlulama + butterspoon tree (E); rooi- Powdered Powdered bark is mixed with powdered (Cunoniaceae) els (A); umqwashube (X); animal bones (from the “Big Five”) and umlulama (Z) used as a snuff and /or licked (khotha) for treating stroke [GN; MZ; LK; MM; MZ; NTS]. Powdered bark infusion is used for respiratory complaints and food poisoning [MM; MK; VG; MN; MA]. ‘Ukululama’ means to recover from an illness. Curtisia dentata umlahleni + + assegai-tree (E); assegai Infusion The bark is soaked in hot water and used (Burm.f.) C.A.Sm. (A); modula-tshwene (NS); as an emetic to prevent quarrel and (Curtisiaceae) mufhefhera (V); umgxina treatment of a bleeding stomach called (X); umlahleni, umlahlen’ ‘isisu segazi’ [ALL traders except N; NQ]. usefile (Z) The bark is used to trap 'ukucupha' (people who are bewitching you) [MK; NQ; MA; LK; GN]. Dichrostachys ugagane + + sicklebush (E); sekelbos Powdered Bark is powdered, mixed with paraffin and cinerea (L.) Wight & (A); mosêlêsêlê (NS); applied directly on the armpits for Arn. (Fabaceae) ugagane, umzungulu (Z) treatment of lice [GN; MZ; MN; DO; NN; MM]. The tree is planted in the yard to prevent lightning [MM; NTS]. ‘Ugagane’ refers to the hooked thorns of the tree. Dombeya unhliziyonkulu + + wild pear (E); dikbas (A); Decoction The bark is mixed with a heart of a banana rotundifolia (Hochst.) mohlabaphala (S); (seeds) and a plant known as ‘ntshungu’ Planch. (Malvaceae) motubane (NS); mulanga (Momordica balsamina) for the treatment of (V); unhliziyonkulu (Z) heart diseases, hence the plants name

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai comes from the organ it treats 'inhliziyo' (the heart) [MK; MN; DO; VG; NN; LK; NTS]. A bark decoction is used to treat heart diseases [N; MA] and gynaecological problems [NN; LK; MA; N]. The bark is used to steam ‘ukufutha’ for itchy skin problems [NQ; VG; SN; MM]. Ekebergia capensis umnyamathi/ + + Cape ash (E); essenhout Decoction Pounded bark is used for ‘ukugquma’, Sparrm. (Meliaceae) umathunzin’ (A); nyamaru (NS); facial sauna or as an emetic when a entaba mmidibidi (S); isimanaye person wants to gain dignity [ALL except (X); umnyanmathi (Z); N; SN;VG]. The bark is burnt and the mutovuma (V); smoke used to treat an unspecified nyamaru (Tsw) condition in livestock [MK; NQ; NTS]. Elaeodendron isithundu + + common saffron (E); Infusion The bark is used as intelezi; it is sprinkled croceum (Thunb.) gewone saffraan (A); (chela) in the yard (isigcawu) when a DC (Celastraceae) umbovane (X); isithundu person is getting married and protects (Z) against witchcraft. Bark is used as an emetic to clean the blood and to gain admirable dignity [MK; MA; MZ; SN]. Elaeodendron ingwavuma + + Transvaal saffronwood (E); Decoction The bark is powdered and put to boil, then transvaalensis (Burtt Transvaalsaffraan (A); taken orally (half a cup) and as an enema Davy) R.H.Archer monamane (NS) for the treatment of stomach complaints (Celastraceae) shimapana (Tso) [NN; NTS; GN; MM;NQ; GN], internal ingwavuma (Z) wounds and as emetic for treating a blocked chest [LK; N; MN; DO]. Bark is used in ‘amagobonga’ and as an enema in children with stomach complaints ‘isisu sobisi’ [MA; DO; N; NQ]. Ingwavuma is a place in Zululand.

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai

Erythrina lysistemon umnsinsi + common coral tree (E); Infusion Bark infusion is used for internal and Hutch. (Fabaceae) gewone koraalboom (A); external wounds; allergies [NN; VG; MA; muvale (NS); umnsinsi, GN; MZ; SN; LK], food poisoning [MM; umsinsi (Z); nsisimbane NTS; SN] and stomach ulcers [LK; MN; (Tso) MK; N]. Small solid pieces of the dried stem bark are used for tooth decay and earache [MN; DO; MM; MK]. The bark is used to prevent the efficacy of medication intended for witchcraft [NQ; MA]. Erythrophleum umbhemiso/ + + Swazi ordeal tree (E); Powdered A tiny pinch of finely powdered bark is lasianthum umkhwangu Swazi-oordeelboom (A); used as a snuff for headaches [ALL Corbishley umbhemise (Z) traders], sometimes mixed with powdered (Fabaceae) bark of Cunonia capensis [VG; NZ; NN]. If taken in doses just above a pinch, then it could result in bleeding from the nose [NZ; NTS; NN; MM]. Eucalyptus umdlavuza + red Ironbark (E); Decoction Decoction of the pounded bark is used in sideroxylon A.Cunn. umdlavuza (Z) the treatment of wounds and venereal ex Woolls * diseases [DO; NTS; LK; N]. The bark is (Myrtaceae) cooked and administered orally two times a day with a cup for treatment of cancer [ALL traders except N; MZ]. ‘Umdlavuza’ is a Zulu name for cancer. Euclea natalensis isizimane + Natal guarri, Natal ebony Decoction The root bark is boiled with water and A.DC. (Ebenaceae) (E); Natalghwarrie, taken orally (three cups daily) to clean the berggwarrie, (A); skin and body system [NTS; SN; NQ; NN]. umtshekisani, umkhasa Pounded stem bark is sprinkled in the yard (X); idungamuzi, – it is used to break up marriages or

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai ichitamuzi, isizimane, relationships [ALL traders]. The meaning of umshekisane, inkunzane, Zulu vernacular name (ichithamuzi) is to (Z); umhlangula (Tso) break up families. Ficus glumosa Delile inkokhokho + mountain rock fig (E); Decoction Bark decoction is used as an emetic to Moraceae berg-rotsvy (A); umthombe protect against enemies and used as a (X); inkokhokho, umnyaxa wash in the mornings and evenings for (Z); mphaya (NS); protection against witchcraft [MM; NTS; tshikululu (V) MZ]. Garcinia gerrardii umbinda forest mangosteen (E); Infusion and The bark is soaked in hot water and Harv. ex Sim bosgeelmelkhout (A); Powdered allowed to cool down; a drop is instilled in (Clusiaceae) umbande, umbindi (X); the ear to relieve pain. Powered bark isikhwelamfene, umbinda mixed with other plants such as (Z) Cassipourea flanaganii to make 'amagobonga' for connecting with your ancestors. [MA; MK; SN; LK; NQ]. Garcinia livingstonei igobandlovu + + African mangosteen (E); Powdered Dried bark is ground to a fine powder and T. Anderson Afrika-geelmelkhout (A); applied directly on the piles for their (Clusiaceae) umphimbi, ugobandlovu treatment [ALL trades except NN; NTS; (Z); mmimbi (Tso); NQ]. Powdered bark is also used to mokongono, mokononga ‘ukugcaba’ treat illnesses associated with (NS) the vertebral column running through to the head. Pounded bark is mixed with hot water and half a cup taken orally to strengthen the blood of a person with weak blood [MA; MM; MK; DO; NTS; VG]. Powdered bark is rubbed into small cuts in the skin (gcaba), to prevent danger [NQ; MZ; SN].

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Harpephyllum umgwenya + wild plum (E); wildepruim Decoction The bark is often mixed with stem bark of caffrum Bernh. (A); mothêkelê (NS); Schotia brachypetala and Syzygium (Anacardiaceae) umgwenya (Z) cordatum, ground into powder, boiled and then used as an emetic to treat respiratory complaints. [MK; MN; DO; NN; GN; MZ; VG]. Powdered bark is used for treating food poisoning [GN; MZ; MK]. Heteromorpha umbangandlala + tropical parsley (E); Decoction Decoction of the bark is used to stimulate arborescens wildepieterseliebos (A); appetite [NQ; MN; NN; LK; SN; VG; MM]. (Spreng.) Cham. & mokadala (NS); muthatha- Hence the Zulu name umbangandlala Schltdl. (Apiaceae) vhanna (V) umbangandlala refers to something that causes hunger. (Z)

Hippobromus isiphahluka + false horsewood (E); Infusion The bark is used as ‘intelezi’ to protect pauciflorus (L.f.) basterperdepisboom (A); against enemies and used by males as an Radlk. isiphahluka (Z); emetic in love charms [NQ; N; MA]. (Sapindaceae) mufhapabere (V); ulwathile (X); umfazothethayo (Z); umhlwathile (X) Ilex mitis (L.) Radlk. xidumo/ + African holly (E); Decoction The bark is used as a wash for people to (Aquifoliaceae) udumaphansi waterboom (A); admire you and to be easily attracted, monamane (NS); hence the plant name is called ‘duma’ iphuphuma, umdumo (Z), (meaning famous) [ALL traders]. umduma (X); phukgu (S); mutanzwa-khamelo (V) Kigelia africana umvongothi + + sausage tree (E); Not Bark is used for penis enlargement [SN; (Lam.) Benth. worsboom (A); modu- specified GN; MK; MM; MZ; NTS; DO; NQ]. In this (Bignoniaceae) kguhlu (NS); umvongothi case, the shape of plant part (fruits) (Z); muvevha (V) indicates the traditional use of the bark.

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Macaranga capensis unompumelelo river macaranga, false- Infusion Bark is used as a wash and emetic for (Baill.) Sim poplar (E); bringing good luck such that in everything (Euphorbiaceae) riviermakaranga, you touch turns into ‘success’ which is the valspopulier (A); Zulu meaning of the plant name umbengele, umphumeleli (unompumelelo) [ALL traders except N]. (X); iphumela, unompumelelo (Z) Maytenus undata undabulavalo koko tree (E); kokoboom Powdered The bark is ground into fine powder and (Thunb.) Blakelock (A); umnqayimpofu (X); and sprinkled over food to break or make a love (Celastraceae) undabulaluvalo (Z); decoction charm spell [N; LK; NTS]. Bark decoction is Morokgwa (NS) used to protect or fight against people who are bewitching you (abantu abakuphonsayo) [MK; MM; MZ]. ‘Uvalo’ means fear. Ochna holstii Engl. isibhanku + red ironwood (E); regte Decoction The bark decoction is used as intelezi and (Ochnaceae) rooipeerhoud, rooi- taken as a wash to protect against ysterhout (A); isibhanku enemies and chase away bad spirits [NQ; (Z);tshipfure (V) SN; MK; MN]. Ocotea bullata unukani + + black stinkwood (E); Infusion The bark is used as 'intelezi' for protection (Burch.) E. Mey. in stinkhout (A); unukani, of the traditional army during war [MK; MN; Drége (Lauraceae) umhlungulu (X); unukani SN; VG]. Bark is used as an enema, (Z) emetic and a wash to get rid of fear, bad luck and a curse resulting from witchcraft [MK; MM; MA; NN]. The Zulu name ‘unukani’ means “what are you smelling of”. Ozoroa paniculosa isifice/ isifuce + common resin tree (E); Powdered The bark is ground into powder and used (Sond.) R.Fern & A. gewone harpuisboom (A); licked (khotha) for stroke [NTS; MZ; SN]. Fern isifica (Z); monoko (NS); The bark is used to treat food poisoning (Anacardiaceae) mudumbula (V) [NQ; N; MK].

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Pappea capensis iletha, umhuluka + jacket plum, bushveld Powdered Powdered bark is licked and used for Eckl. & Zeyh. cherry (E); doppruim (A); ‘'amahlaba/ izibhobo'’ antispasmodic effect (Sapindaceae) indaba, iletha (Z); ilitye, [GN; MK; DO; NN]. The bark is boiled in umgqalutye (X); water and used as an emetic for treatment mongatane (NS); of food poisoning and to bring in luck. gulaswimbi (Tso) Powdered bark is licked ‘khotha’ to relive tshikavhavhe (V) pains in the body [MA; GN; MN; NQ]. ‘Letha’ means to bring. Peltophorum msehla/ umsenhla + African-wattle (E); Infusion Pounded bark is sprinkled in the yard to africanum Sond. huilboom (A); mosehla chase away bad spirits [NQ; MA; SN]. (Fabaceae) (NS); muparapara (Tsw); umthobo (X); isikhabamkhombe, umsehle (Z); ndzedze (Tso); musese (V) Philenoptera isihomohomo + apple-leaf (E); appelblaar Decoction The bark is used as intelezi to protect the violacea (Klotzsch) (A); isihomohomo, household from enemies [MK; N; NQ; MA]. Schrire (Fabaceae) umbandu (Z) Pittosporum umfusavu + + cheesewood (E); kasuur Infusion Infusion of the bark is used for stomach viridiflorum Sims (A); kgalagangwe (NS); complaints [ALL traders except N] and (Pittosporaceae) umkhwenkwe (X); sometimes used as a wash for skin umfusavu (Z) ailments [NTS; SN; GN]; Pleurostylia capensis umthunyelwa + bastard saffronwood (E); Powdered The bark is used to hasten dreams and to (Turcz.) Loes. bastersaffraan (A); make them clearer in cases where there is (Celastraceae) umbovane, umbovane- an urgent message that is needed, coming ontsaka (X); umngqangqa, from the ancestors. Bark is popularly used umthunyelelwa (Z) in love charm emetics and young males have a praise for the bark ‘umthunywa avume njengempaka yomthakathi’

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai (acknowledging its trustworthy use in love charms) [MA; SN; N; VG; MM]. ‘Umthunyelelwa’ means one who receives something from a messenger. Protorhus longifolia Unhlangothi + red-beech (E); Powdered A teaspoon of powdered bark is mixed (Bernh.) Engl. rooiboekenhout (A); together with “Big Five” animal bones in a (Anacardiaceae) ikhubalo (X); unhlangothi powdered form and is licked directly from (Z) the hand (khotha) for treatment of stroke [GN; MZ; MK; MN; NQ]. Sometimes powdered bark is applied onto small razor incisions in the body (ukugcaba) for stroke [MA; DO]. Prunus africana inyazangoma + + stinkwood (E); Infusion Infusion of the bark is administered orally (Hook.f.) Kalkman elimnyama rooistinkhout (A); red to treat colds and influenza [LK; MN; MA; (Rosaceae) mogohloro (NS) DO; MM; NN; NTS]. Bark is used for good inyazangoma (X); luck and for revenge [NQ; SN; N]. inyazangoma elimnyama (Z); mulala-maanga (V) Ptaeroxylon umthathe + sneeze wood (E); nieshout Decoction The bark is soaked in hot water and used obliquum (Thunb.) (A); munari (V); umthathe for steaming and also as a body wash for Radlk. (Rutaceae) (X); ubhaqa, umthathe (Z) good luck [MK; MZ; GN; MM; MA]. The bark is used to trap enermies [NQ; MK], hence ‘umthathe’ means ‘take them on’. Pterocarpus umvangazi + bloodwood (E); kiaat (A); Decoction The bark is incoparated in liquid mixtures angolensis DC. mutondo (V); mvhangazi (izimbiza) used to bring blood in the body. (Fabaceae) (Tso); mokwa (Tsw) Pounded bark is used as an emetic to bring good luck [SN; NQ; MM; GN; MK; NN]. Umvangazi means one who bleeds/ releases blood.

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Pterocelastrus usehlulabanye/ + red candlewood (E); rooi- Infusion Infusion of the bark is administered as rostratus Walp. usehlulamanye kersboom (A); Itywina (X); enemas and emetics used during war to (Celastraceae) usehlulabanye (Z) defeat opponents from another clan or group of people involved [GN; MZ; MM; MN; LK]. ‘Usehlulabanye’ means one who defeats others. Rapanea umaphipha + + Cape beech (E); Kaapse Decoction Bark decoctions are used for cleaning the melanophloeos (L.) boekenhout (A); mogono blood and are taken as a body wash for Mez (Primulaceae) (NS) isiqwane-sehlathi (X); removing bad luck (isichitho) [ALL except umaphipha (Z) NN; SN]. Rauvolfia caffra umhlambamanzi + + quinine tree (E); kinaboom Infusion The bark is used for cleansing when a Sond. (A); monadi (NS); family member had passed away; this is (Apocynaceae) umthundisa (X); done to remove 'isinyama' (“the dark umhlambamanzi (Z) shadow of the dead”). It is also used for 'ukugquma' in order to be adored by people [GN; MM; MZ;NQ; MK]. Bark emetic is used to make a clear path for the ancestor’s dreams so that a person (usually a sangoma or inyanga) can understand the message from the ancestors [SN; MA]. Schotia brachypetala umgxamu/ ihluze + + weeping boer-bean (E); Decoction The stem bark is often mixed with Sond. (Fabaceae) huilboerboon (A); molope Harpephyllum caffrum and Syzygium (NS) umutwa (Tsw); cordatum stem bark (ground into powder), umgxama, ishumnumyane boiled and then used as an emetic to treat (X); ihluze/ umgxamu (Z); respiratory complaints and food poisoning mulubi (V) [MK; MN; DO; NN; GN; MZ; VG]. Bark is soaked in cold water to make ‘ukhamba’ and used as an emetic for ‘isilungulela’

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai (heartburn) and as an enema to clean a bleeding stomach [MA; MZ; MN; N; LK]. Sclerocarya birrea umganu + marula (E); maroela (A); Infusion Bark infusion is administered orally with a (A. Rich.) Hochst. morula (NS); mufula (V); cup or sometimes used as enema for (Anacardiaceae) ukanyi (Ts) umganu (Z) stomachache [NTS; SN; VG; NQ; MZ]. The bark is used as an enema to gain dignity so that a person cannot easily be harmed. Bark is used as an emetic in mixtures for a person who is getting married [NQ; MA; NN; MM]. The name of the bark comes from ‘ukugana’ (“getting married”). Searsia chirindensis inyazangoma + red current (E); bos- Not The bark is used to protect yourself against (Baker f.) Moffett elibomvu taaibos (A); motha-thaa specified opponents who are bewitching you (and it (Anacardiaceae) (NS); umhlakothi (X); is them who will die instead) [MK; MN]. inyazangoma elibomvu (Z); Bark is mixed with cold water and sprinkled muvhadela-phanga (V) to prevent quarrel and to chase away evil spirits; it is also used to get revenge [MA; MM; SN].

Securidaca mpesu/ + violet tree (E); krinkhout Infusion The root bark is used as a drink (half a longepedunculata iphuphuma (A); mamba (NS); mpesu cup) to prevent colds and flu. The stem Fresen. (V); iphuphuma (Z) bark is sprinkled and used as a wash to (Polygalaceae) chase away bad spirits, and used as ‘umthwebulo’ (“to capture someone’s soul”). [MZ; MA; N; VG; SN; MN; DO]. Sideroxylon inerme amasethole + white milkwood (E); Decoction The bark is mixed with other plants to L. (Sapotaceae) witmelkhout (A); make up a remedy called 'izimbiza zobaba' umqwashu, (X); for male sexual problems [NTS; NN; NS; MZ; DO]. Bark is used as an emetic and

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai uMakhwelafinqane (Z); administered orally (half a cup) to treat mutaladzi-vhufa (V) chest problems associated with food poisoning, to chase away evil spirits, and treat mental illnesses [ALL traders except GN; VG; N; MM]. Bark is used in mixtures ‘izimbiza’ for treatment of kidneys and for cleaning the blood [MA; MN; DO; MZ]. ‘Amasethole’ refers to calf milk. Spirostachys umthombothi + tamboti (E); tambotie (A); Powdered The wood is ground into fine powder to africana Sond. umthombothi (Z); modiba form 'insizi' and licked directly from the (Euphorbiaceae) (NS); ndzopfori (Tso); back part of the hand (khotha) to relieve morukuru (Tsw); muonze pain and is also used as 'izithobo' (V) medication – a cloth is immersed in the infusion and dabbed onto an inflamed or swollen area [MK; MN; DO; MM; SN]. The powdered wood is applied to wounds [NQ; NTS; NN]. Strychnos henningsii umqalothi + red bitter berry (E); Powdered The bark is chewed for stomach Gilg (Loganiaceae) rooibitterbessie (A); complaints [ALL traders]. Powdered bark is umnonono, umkaloti (X) licked for ‘amahlaba’ (an antispasmodic umqalothi (Z) effect), intestinal worms and stomach complaints [MA; SN]. Syzygium cordatum umdoni + water berry (E); Decoction The bark is used for internal stomach Hochst. ex Krauss waterbessie (A); umdoni wounds. Bark of Syzygium cordatum, (Myrtaceae) (Z); umjomi (X), monthlo Schotia brachypetala and Harpephyllum (NS); mutu (V); muhlwa caffrum are powdered and mixed together, (Tso) boiled in water and taken as emetic for respiratory complaints [MK; NN; MN; DO; GN; MZ; VG]. Powdered bark is licked

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai (khotha) for body pains and used as an emetic against enemies [SN; NQ; MK; MZ]. Terminalia sericea amangwe + + silver cluster-leaf (E); Infusion The bark is used as an emetic and a wash Burch. ex DC. vaalboom (A); moxonomo to protect yourself from witchcraft [DO; VG; (Combretaceae) (NS); mogonono (Tsw); GN] and helps you to fight back [MK; LK; amangwe (Z); mususu (V) SN; NQ]. Trichilia emetica umkhuhlu + + Natal mahogany (E); Maceration The bark is used as an enema to clean the Vahl (Meliaceae) rooiessenhout (A); mamba and internal part of the body and to alleviate (NS); umkhuhlu (Z); powdered back pains. However, the bark is not boiled umkhuhlu (X); nkulu (Tso); with fire because it releases poison; it is mutuhu (V) cooked (heated) in the sun [MK; MN; DO; LK]. Raw powdered bark is sometimes mixed with petroleum jelly and applied on wounds between fingers and toes [MA; SN; MK; LK; MM; NN]. Vachellia natalitia umunga + + pale-barked sweet thorn Decoction Pounded bark is boiled in water and taken (E.Mey.) Kyal. & (E); bleekbassoetdoring, orally for wounds [NTS; MZ; N; GN; MM]. Boatwr. (=Acacia nataldoring (A); mookana The bark is used as an emetic for pimples natalitia E.Mey.) (NS); moshaoka (S); [MM; NQ; SN]. (Fabaceae) umnga (X); umunga (Z) Vachellia robusta umngamanzi + broadpod robust thorn (E); Decoction Decoctions of the bark are used for (Burch.) enkeldoring (A); mooka and infusion treatment of menstrual pains and venereal Kyalangalilwa & (NS); mvumbangwenya diseases. The bark infusion is used as a Boatwright subsp. (Tso); mokhu (T); wash and emetic to get rid of evil spirits robusta [=Acacia muvumba-ngwena (V); [NQ; MA;N;LK; SN] robusta (Burch.) umngamanzi (Z) Kyalangalilwa & Boatwright] (Fabaceae)

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai Vachellia umkhanyakude + fever tree (E); koorsboom Infusion The bark is used as a wash for good luck xanthophloea (A); mooka-kwena (NS); (as a charm); in most cases it is used when (Benth.) P.J.H. umhlosinga (Z); nkelenga a person goes for job hunting or an Hurter [=Acacia (Tso); munzhelenga (V) interview (to make a good impression on xanthophloea the employer) [ALL traders except N]. (Benth.) Umkhanyakude means ‘one who shines P.J.H.Hurter] bright from a distance’. (Fabaceae) Vitex obovata umluthu + hairy finger leaf (E); harige Decoction The bark is used as ‘intelezi’ by men who E.Mey. vingerblaar (A); umluthu use fighting sticks in order to beat up their (Verbenaceae) (Z) opponents [GN; MK; MZ; MN; DO]. Voacanga thouarsii indlabalozi/ + wild frangipani (E); Powdered The bark is licked (khotha) for pains in the Roem. & Schult) indlabaloyi indlabaloyi (Z) body [NQ; MZ; MN; DO; LK]. Powdered (Apocynaceae) bark is used to trap ‘ukucupha’ and to protect a person from witchcraft [NQ; LK]. Warburgia salutaris isibhaha + + pepper-bark tree (E); Infusion and An infusion of the bark is administered (G. Bertol.) Chiov. peperbasboom (A); powdered orally (one cup) for diarrhoea and flu [LK; (Canellaceae) isibhaha (Z); mulanga, NQ; MM; VG; MZ; NTS]. Powdered bark is manaka (V); xibaha (Tso) used is licked (khotha) for (izithobo) and to relieve or loosen the sputum in the chest and to get rid of wounds caused by colds and flu [MA; MN; NQ; LK].

Zanthoxylum umlungumabele + small knobwood (E); Powdered Dried and powdered root bark is applied or capense (Thunb.) kleinperdepram (A); rubbed directly on the mouth “to kill tooth Harv. (Rutaceae) umlungumabele (X); worm” (to treat toothache) [ALL traders]. umnungamabele (Z); Small portion of powdered bark is mixed monokwane (S); with warm water in a glass and used as a khunugumorupa (Tso) mouthwash to relieve toothache [MM; MZ;

Species (Family) Trade name Fara- Kwa Vernacular names Method of Uses as reported by the traders [praise(s)] day Mai- preparation Mai DO; LK]. Root bark is used to treat illnesses that affect the veins, lungs, blood, and kidneys; also used for backache and to treat snakebite [MA; GN; K]. Umlungumabele refers to white people’s breasts describing the shape of the thorns on the stem bark. Ziziphus mucronata umlahlankosi + + buffalo-thorn (E); A tree branch with leaves is used to carry Willd. (Rhamnaceae) blinkblaar-wag-n-bietjie the soul of a person from the place where (A); mokgalo (NS); he or she died to their place of birth where umphafa (X); umlahlankosi they will be buried. Bark is used as a wash (Z), mutshetshete (V), to remove ‘isilwane’ (dark shadow) [ALL mphasamhala (Tso) except N].

2.3.4 Method of preparation and mode of administration

Bark is mainly prepared as infusions or decoctions for oral administration, but may also be powdered for use as khotha (i.e., to be licked from the palm of the hand) or as snuff (Fig. 2.10). Surprisingly, most of the medicinal barks are not prepared as decoctions or infusions but rather as powders (25 species), which may be either taken orally, nasally as snuff or rubbed into small razor incisions on the skin.

Powders are often used for the treatment of various ailments including headaches, food poisoning, stroke, and to relieve body pains. Decoctions (23 species) and infusions (21 species) are the next most frequent methods of preparation, often associated with uses as emetics and enemas for the treatment internal disorders or sometimes added to a bath to treat skin complaints. The least frequent methods of preparation are poultices (two species), both for cosmetic purposes and cold infusions, made in cold water that is placed in sunlight (one species), used for cleaning internal parts of the body.

0 Powdered Decoction Infusion Paste/poultice Maceration

Method of preparation

Figure 2.10 Method of preparation (dosage forms) of medicinal barks according to traders on the Johannesburg muthi markets.

Bark plant material may be administered in the form of enemas, emetics, snuff, steaming inhalations, as a bath or topically as a paste /poultice) (Figures 2.10 and

2.11). The oral route is the most common (21 of the 70 barks are used in this way) followed by bathing (15), emetics (14), enemas (10), licked (10), topical as poultices

(6) and inhaled as snuff (4).

The mode of administration is a very critical aspect of traditional medicine that should be carefully handled, as there could be a thin line between toxicity and safety, depending on the dosage and method of preparation of the plant material. According to the information given by traders on the Johannesburg muthi markets, a pinch of powdered bark of Erythrophleum lasianthum is taken as snuff for headaches but anything more than a small quantity results in severe bleeding from the nostrils and causes dizziness. The bark is never recommended for oral administration because of its poisonous effects. Ndhlala et al. (2013) reported that although E. lasianthum stem bark is used in traditional medicine, most of the active principles are highly poisonous, e.g. the diterpenoids cassaine and erythrophleine (Van Wyk et al., 2009).

The bark of this species is the most famous of all southern African ordeal poisons

(as reflected in the English and Afrikaans vernacular names; trial by ordeal).

5 number of different barks different of number 0 Oral Bath Emetic Enema Licked Topical Snuff

Figure 2.11 Mode of administration of medicinal barks as recorded by the muthi market traders.

The majority of medicinal barks are taken orally as liquids or licked in a raw powdered form for the treatment of various internal ailments such as food poisoning, constipation, menstrual pains, stomach upsets and as blood cleansers. The predominant route of oral administration corresponds with Grace et al. (2003), who also reported that the majority of bark medicines are taken orally for the treatment of internal ailments. The least common method of administration is snuff, which are preparations of finely powdered, dried medicinal plants that can be drawn up into the nostrils through inhalation (Van Wyk et al., 2009).

All the powdered barks listed in Table 2.2 that are taken as snuffs are often used for headaches or stroke. Bathing is usually associated with remedies involved in the treatment of skin ailments. For example, the bark of A. adianthifolia is used as a wash for treatment of skin rashes, chicken pox and other skin infections. In this case, it can be assumed that there are some active chemical compounds involved.

On the other hand, barks are also frequently used as a bath for spiritual or magical charm purposes (to remove bad luck, for example). In this case, the efficacy probably depends on cultural and symbolic aspects rather than on the chemistry of the bark.

Enemas are aqueous or oily solutions or suspensions intended for rectal injections (Van Wyk et al., 2009) – they are administered for their anthelmintic, nutritive, purgative or sedative effect. Enemas should be freshly prepared and any solid substances should be uniformly suspended. Enemas and emetics are frequently associated with love and good luck charms and protection against enemies in the form of an intelezi or an izimbiza. Topical uses of barks include application as lotions and poultices, in most cases for the treatment of skin infections

(e.g., Erythrina lysistemon) or as cosmetics, usually applied as a skin lightener and

48 protection against sunburn (e.g., Cassipourea flanaganii). Lotions are liquid preparations intended for application to the skin. Pastes or poultices are preparations of finely powdered bark material thoroughly mixed with water, for application to inflamed or infected areas of the skin.

2.3.5 Main uses of medicinal barks

In southern Africa, bark is the most frequently used plant part in traditional medicine and is preferred by the majority of muthi market traders due to the ease of long-term storage (Williams, 1996; Mander et al. 1997). All the plant species recorded in this study (as listed in Table 2.2) have also been reported in other ethnobotanical surveys in southern Africa (Cunningham, 1988; Pujol, 1990; Hutchings et al., 1996;

Van Wyk and Gericke, 2000; Van Wyk et al., 1997, 2009; Grace et al., 2003;

Williams, 1996, 2003; Ndawonde et al., 2007; De Wet et al., 2010, 2012). This means that about 70 species account for a large proportion of the overall medicinal uses of bark.

A summary of medicinal uses of the 70 species is presented in Table 2.3, where a comparison is made between uses recorded on the muthi markets (from

Table 2.2) and in the literature (see Appendix 1). The ethnobotanical data gathered on the muthi markets show that each plant often has more than one main use. The majority of the muthi market traders (Table 2.1) reported multiple medicinal bark uses for most of the species. An analysis of the data in Table 2.2 is shown in Figure

2.12. In most cases, medicinal barks recorded with only one main use are associated with magic or charm. Such barks include Araucaria heterophylla, Bridelia micrantha, Macaranga capensis, Philenoptera violacea, Ptaeroxylon obliquum,

Pterocelastrus rostratus, Rapanea melanophloeos, Terminalia sericea and Vachellia

Table 2.3 Summary of medicinal and magical uses of the most popular medicinal barks (70 species) sold on Johannesburg muthi markets, arranged by main categories of use (see Appendix 1 for details about all uses). M = use-reports from the muthi markets – number of traders (out of 14) who reported that use; Lit = use-reports from the literature (see Appendix 1); M+Lit = number of use categories where muthi market and literature use-reports correspond.

Plant species Topical Gastro- Respira Charm Sexually Gynaeco Other Number of use /skin intestinal -tory /magical transmit- -logical uses categories ed diseases M Lit M Lit M Lit M Lit M Lit M Lit M Lit M Lit M+ Total Lit Albizia adianthifolia 9 + - - - - - + - + - + - + 1 5 1 5 Araucaria heterophylla* ------5 ------+ 1 1 0 2 Balanites maughamii - + - + - + 6 + - - - - 5 + 2 5 1 5 Berchemia discolor - + ------+ 6 + 1 3 0 4 Bersama lucens - - 4 - - - - + - + 4 + 5 + 3 4 2 5 Bersama tysoniana ------4 + - - - + 5 + 2 3 1 4 Brackenridgea zanguebarica - + - - - - - + - - - - - + 0 3 0 3 Bridelia micrantha - + - + - + 6 - - + - + - + 1 6 0 7 Calodendrum capense 8 + - - - - 7 ------2 1 1 2 Carissa bispinosa - - - + - + - - - + - - 10 + 1 4 0 5 Cassipourea flanaganii 13 + - - - + 3 ------+ 2 3 1 4 Cinnamomum camphora* - - - + - + 13 + - - - - - + 1 4 1 4 Combretum caffrum - + - + - - 2 + - - - - 6 + 2 4 1 5 Commiphora africana - + - - - + 8 - - - - - 6 + 2 3 0 5 Croton sylvaticus - + 7 + - + - - - + - + 4 2 5 1 6 Cryptocarya latifolia - - - + - + 4 - - + - + 10 2 4 0 6 Cunonia capensis - - - - 6 ------6 2 0 0 2 Curtisia dentata - + 12 + - - 5 - - + - - - - 2 3 1 4

Plant species Topical Gastro- Respira Charm Sexually Gynaeco Other Number of use /skin intestinal -tory /magical transmit- -logical uses categories ed diseases M Lit M Lit M Lit M Lit M Lit M Lit M Lit M Lit M+ Total Lit Dichrostachys cinerea 6 + - + - - 2 + - + - - + 3 4 2 5 Dombeya rotundifolia 4 + - + ------4 + 9 + 3 4 2 4 Ekebergia capensis - + - + - + 11 + - - - - 3 + 2 5 1 5 Elaeodendron croceum - - - + - + 4 - - - - - 4 2 2 0 4 Elaeodendron transvaalense - + 9 + 4 + 4 - - + - + - + 3 6 2 7 Erythrina lysistemon 7 + 7 + - - 2 + - - - + 4 + 4 5 3 5 Erythrophleum lasianthum - + - + - - - + - - - - 14 - 1 4 1 4 Eucalyptus sideroxylon* 4 + - - - - - + 4 + - + 12 + 3 5 2 5 Euclea natalensis 4 + - - - - 14 - - + - + - + 2 4 1 5 Ficus glumosa - + - - - - 3 ------1 1 0 2 Garcinia gerrardii ------5 + - - - - 5 - 2 1 1 2 Garcinia livingstonei 11 + - + - + 3 - - - - - 5 - 3 3 1 5 Harpephyllum caffrum - + 3 - 7 ------+ 2 2 0 4 Heteromorpha arborescens - + - - - + - - - - - + 7 + 1 4 0 5 Hippobromus pauciflorus ------3 ------1 0 0 1 Ilex mitis - + - + - + 14 ------1 3 0 4 Kigelia africana - + - + - + - - - + - - 8 + 1 4 0 5 Macaranga capensis - + - - - - 13 ------+ 1 2 0 3 Maytenus undata - + - + - + 6 + - - - - - 1 4 1 4 Ochna holstii ------4 ------1 0 1 1 Ocotea bullata - - - + - - 8 - - + - - - + 1 3 0 4 Ozoroa paniculosa - - - + - + ------6 - 1 3 0 4 Pappea capensis - + - - - + 4 + - + - - 8 - 1 4 0 4 Peltophorum africanum - + - + - + 3 - - + - + - - 1 5 0 6 Philenoptera violacea - + - + - + 4 ------1 3 0 4

Plant species Topical Gastro- Respira Charm Sexually Gynaeco Other Number of use /skin intestinal -tory /magical transmit- -logical uses categories ed diseases M Lit M Lit M Lit M Lit M Lit M Lit M Lit M Lit M+ Total Lit Pittosporum viridiflorum 3 + 13 + - + - - - + - - - - 2 4 2 4 Pleurostylia capensis - + - - - - 5 + ------1 2 1 2 Protorhus longifolia - - - + - - - + - - - - 7 + 1 3 0 4 Prunus africana - + - + 7 + 3 - - + - - - + 2 5 1 5 Ptaeroxylon obliquum ------7 ------+ 1 1 0 2 Pterocarpus angolensis - + - + - + 6 - - + - + 6 + 2 6 0 8 Pterocelastrus rostratus - - - - - + 5 ------+ 1 2 0 3 Rapanea melanophloeos - + - + - + 12 + - - - - - + 1 5 1 5 Rauvolfia caffra - + - - - + 7 - - - - + - + 1 4 0 5 Rhus chirindensis ------5 - - - - - + + 2 1 0 3 Schotia brachypetala - + 5 - 7 ------3 - 3 1 1 3 Sclerocarya birrea - + 5 + - - 4 + - + - + - + 2 6 2 6 Securidaca longepedunculata - + - + 7 + 7 - - + - + - + 3 6 2 3 Sideroxylon inerme - + - - 10 + 10 + - - - - 8 + 3 4 3 4 Spirostachys africana 8 + - + - + ------5 + 2 4 1 4 Strychnos henningsii - + 14 + ------+ 1 3 1 3 Syzygium cordatum - + - + 7 + 4 - - - - - 4 + 3 4 1 4 Terminalia sericea - + - - - - 7 ------+ 1 2 0 3 Trichilia emetica 6 + - + - - - - - + - - 4 + 2 4 1 4 Vachelia karroo 8 - - + - + - + - - - - - + 1 4 0 5 Vachelia robusta - + - - - + 4 - 5 - 5 - - + 3 3 0 6 Vachelia xanthophloea - + - - - - 13 + ------1 2 1 2 Vitex obovata ------5 ------+ 1 1 0 2 Voacanga thouarsii - + - + - - 2 - - + - - 5 - 2 3 0 5 Warburgia salutaris 4 + 6 + 9 + - - - + - - - + 3 5 3 5

Plant species Topical Gastro- Respira Charm Sexually Gynaeco Other Number of use /skin intestinal -tory /magical transmit- -logical uses categories ed diseases M Lit M Lit M Lit M Lit M Lit M Lit M Lit M Lit M+ Total Lit Zanthoxylon capense 3 + - + - + ------14 + 2 4 2 4 Ziziphus mucronata - + - + - + 13 ------1 3 0 4 Total no of use-reports 98 - 85 - 64 - 289 - 9 - 13 - 204 - Total no of species 15 51 11 40 9 35 47 22 2 23 3 18 32 44

53 xanthophloea. Medicinal barks used for sterility in males (Carissa bispinosa and

Kigelia africana) were also recorded with one main use. Samie and co-workers

(2010) in their ethnobotanical survey in the Limpopo Province also encountered a diversity of medicinal uses for individual plant species, with most of the plants being used for several illnesses or simultaneously for different ailments.According to the traditional uses provided by traders, potential synergism among some of the plant species was mentioned. The powdered bark of Schotia brachypetala is mixed with

Harpephyllum caffrum and Syzygium cordatum to make a decoction for treatment of respiratory ailments. Magical and charm uses encountered were often associated with ‘chasing away bad/evil spirits’, ‘protection against enemies’ as well as ‘good luck’ and ‘love charms’.

20 number of plants of number

Muthi markets literature

Figure 2.12 Main uses of the most popular bark samples on the Faraday and Kwa Mai Mai muthi markets in Johannesburg (Table 2.3), compared with literature data (see Appendix 1).

According to the data recorded from the literature, the majority of the 70 barks documented in this study are used for skin ailments (51 barks), followed by gastrointestinal tract (40 barks) and respiratory tract infections (35 barks). It is therefore surprising to note that majority of medicinal use records from the two muthi markets are associated with spiritual and charms uses. Traders recommended medicinal barks such as Araucaria heterophylla, Bridelia micrantha, Macaranga capensis, Philenoptera violacea, Ptaeroxylon obliquum, Pterocelastrus rostratus,

Rapanea melanophloeos, Terminalia sericea, Vachellia xanthophloea and Ziziphus mucronata for their magical and spiritual uses only. However, according to literature sources, these barks are used for the treatment of various illnesses associated with the skin, stomach, respiratory, sexually transmitted diseases and gynaecological disorders.

Contrary to expectation, the ethnobotanical data from the traders show only a small percentage of bark use against sexually transmitted infections (STI’s) – the traders never disclosed the type of STI ailment. Many ethnobotanical studies of plants used for sexually transmitted infections indicate that ordinary people prefer to use traditional remedies for such ailments rather than western medicine (Green,

1992; De Wet et al., 2012; Naidoo et al., 2013; Chinsembu, 2016). Traditional healers claim to have a cure for various STI’s, including HIV/AIDS, whereas in western practises there is no known cure for HIV/AIDS. In 1997, approximately 94% of traditional healers from Mpumalanga mentioned that they have treated patients with HIV/AIDS (Mander et al., 2007). The Johannesburg traders also mentioned the use of bark to treat male infertility. It is interesting that two species mentioned

(Carissa bispinosa and Kigelia africana) are used only for men (for sterility and impotence) and not for women.

According to the traders, charm and magic uses represent the most popular category of use, with at least 47 barks used (Table 2.2, Figure 2.12). This suggests that the healing systems represented at the muthi markets are largely influenced or characterised by traditional, holistic perspectives, since the majority of the traders are either herbalists, sangomas or traditional healers (izinyanga). Similar observations were reported with Mander et al. (2007) who stated that many customers choose to use traditional healers as they feel the treatment is more holistic than western medicine. Rituals combining ancestor worship, divination and plant medicines are often part of the consultation process and many customers appreciate this dual spiritual and physiological treatment. Furthermore, these findings correspond with the ethnobotanical study of Corrigan et al. (2011) that showed that the majority of remedies recorded in the KwaNibela area are associated with magical purposes such as good luck charms or warding off evil spirits.

Interesting information from one of the muthi market traders (MA, a sangoma/umthandazi) is worth mentioning in this context. She stated that the diseases of her clients do not necessarily require physical healing even though the problem may be physical. For example, she mentioned that if a child is a slow learner at school, there are mixtures of herbs that are administered orally in a powdered form before the child goes to bed. It is believed that the medicine works overnight and that there is nothing inherently wrong with the child’s brain but that it is affected by some spirits, either good (ancestral) or bad (evil), that are stuck in the brain. The same principle applies to a person suffering from epilepsy, backache and mental disturbances. Hutchings (1989) also reported diseases that are believed to be spiritual. Most of the traders mentioned that different plant species are used to treat similar spiritual illnesses. Some traditional healers may use Trichilia emetica to

56 repel bad dreams or chase away evil spirits, while others may use Vachellia karroo or Strychnos henningsii for the same purposes. The choice depends on the ancestral knowledge of the person (traditional healer, herbalist or sangoma) because these healing practises are believed to have been passed on to them through dreams, from one generation to the next.

The traders probably focus on the main uses of the bark species from their own experience, which does not necessarily correspond to the full diversity of use across the entire KwaZulu-Natal province or country (as is the case with the literature data). It is also reasonable to expect that traders would not have the in- depth knowledge of traditional healers and highly trained herbalists. Another possible explanation for the discrepancy between the literature data and the muthi market data in Table 2.2 and Figure 2.12 is that many people nowadays used modern pharmaceutical products and commercial cosmetics and no longer follow the traditional customs. It may also be that scientific studies have hitherto mainly focussed on physical ailments that can be subjected to scientific scrutiny (e.g. chemical analyses and activity studies) rather than on spiritual and charm uses that are often regarded with sceptisism by western scientists.

2.3.6 Identification of medicinal barks

According to Grace et al. (2002), dried plant products sold in the medicinal plant trade in South Africa are generally very difficult to identify, as many useful morphological characters are either absent or lost through the desiccation process.

Bark products frequently lack distinctive diagnostic characters and appear superficially similar to the untrained eye. For this reason, Grace et al. (2002) recommended the use of TLC-generated phytochemical fingerprints for bark

57 authentication. More recently, DNA barcoding has become the method of choice

(Hartvig et al. 2015; Nguyen et al. 2015). However, difficulties in plant identification are mostly due to the same or similar Zulu vernacular names being used for different plant species. In their study of medicinal plants in northern Maputaland, De Wet and

Ngubane (2014) encountered this problem and stressed the critical role of voucher specimens for accurate identification. Problems may also be due to the limitations of folk taxonomies, where all species of a genus are often given the same name (e.g. imphepho for Helichrysum species and inhlaba for Aloe species). De Boer et al.

(2014) used DNA barcoding to show the discrepancies between botanical classification systems and folk taxonomies. However, the ability of traditional healers and herbalists to identify plant materials can easily be under-estimated. With the necessary knowledge and experience, they are able to identify even closely related and strikingly similar but unrelated plant materials, including barks. Although accurate identification could easily be done through careful observation, knowledge and experience, it is also advisable to consider other methods such as anatomy, chemical profile and DNA barcoding for further accuracy.

Traders are very knowledgeable about the plant material they sell, as it is part of their daily activity. In a rare event where there was uncertainty about a sample, it turned out that it was due to similar vernacular names for different plant species or a consistent error in the literature. For example, Balanites maughamii Sprague has consistently been referred to in the literature as ugobandlovu (Watt-Breyer-

Brandwijk, 1962; Cunningham 1988; Hutchings et al., 1996, Von Ahlefeldt et al.,

2003, Williams, 2003; Grace, 2003; Van Wyk et al., 2009; Van Wyk et al., 2011a) while this name should correctly be applied to Garcinia livingstonei T. Anderson. All the traders on the Johannesburg muthi markets insisted that this is the correct

58 application of the vernacular name. Traders on the Durban muthi market confirmed that ugobandlovu is the correct name for G. livingstonei and not B. maughamii (B.-E. van Wyk, pers. comm.). The current name for the latter is iphamba. Another example is umvangazi, which has sometimes been used in the literature for Albizia versicolor

(Williams, 2003; Van Wyk et al., 2011a) while it actually refers to Pterocarpus angolensis. It is therefore clear that there is still a need for systematic documentation of the basic characters and character states of bark even though DNA barcoding and metabolomics methods may be convenient and accurate methods to confirm and verify identifications.

Mistakes may potentially also be due to traders replacing some barks with others, perhaps when their supplies of the preferred bark runs out. Since all barks have the same price, there is no motivation to replace bark and to mislead customers for making a better profit. Not a single incidence of attempted cheating was encoutered in this study.

Vernacular names of bark samples were compared to the literature sources as listed above (especially Hutchings et al., 1996). In some cases, the bark sample was compared to photographs or illustrations of bark in books (e.g. Pujol, 1990; Von

Ahlefeldt et al. 2003; Van Wyk et al., 2009, 2011; Dyer et al., 2016).

Bark samples often vary considerably, depending on the provenance or the age of the trunk from which it was sampled. This variability may result in errors and misidentifications when bark is described, and it therefore seems very difficult to create a reliable key for the identification of bark samples. In this study, there were cases whereby several bark samples of the same species were purchased from different traders in order to get a more representative sample and to account for natural variation.

As a first step towards an identification guide for commercially relevant medicinal barks of southern Africa, descriptions were prepared for all 70 barks recorded in this study and colour photographs of all 70 species (taken by B.-E. van

Wyk) are provided (Appendix 1). The descriptions include mainly the external and internal appearance, the colour and texture of the intact and powdered bark, the colour of solvent extracts, the odour of powdered bark and the presence or absence of lenticels and sclereids.

Conspicuous differences were observed when bark samples were routinely ground for extraction, using a coffee grinder. Some samples remained fibrous, while other were reduced to powder. Since the colour and texture of the ground, bark samples were so different; these characters can also be considered as a way to identify the species. For this purpose, photographs of the ground bark of all 70 species are shown in Figure 2.13. In this figure, the colour and texture or structure

(e.g. fibrous or not) can be directly compared. It is important to consider that only mature bark can be compared in this way, as the presence or absence of rhytidome will greatly influence the appearance of the powder.

Figure 2.13 Comparison of the appearance (colour and texture) of powdered bark from the 70 medicinal bark species that were purchased at the Johannesburg muthi markets. The horizontal distance of spread of all samples was 50 mm. Photographs by B.-E. van Wyk.

As a further potential approach to bark identification, a preliminary study was made of the colour of bark extracts, as shown in Figure 2.14. Powdered bark material (1 g per ml) was soaked in two solvents – one polar and one non-polar. The

Figure 2.14 Comparison of the colour of bark extracts from the 70 medicinal bark species that were purchased at the Johannesburg muthi markets (numbered as in Appendix 1). The solvents were a 75:25 mixture of water and methanol (numbered vials) and pure dichloromethane (unnumbered vials). The way in which colours was coded in Table 2.4 is shown in the bottom row. Photographs by B.-E. van Wyk. The samples are 1, Araucaria heterophylla*; 2, Albizia adianthifolia; 3, Balanites maughamii; 4, Berchemia discolor; 5, Bersama lucens; 6, Bersama tysoniana; 7, Brackenridgea zanguebarica; 8, Bridelia micrantha; 9, Calodendrum capense; 10, Carissa bispinosa; 11, Cassipourea flanaganii; 12, Cinnamomum camphora*; 13, Combretum caffrum; 14, Commiphora africana; 15, Croton sylvaticus; 16, Cryptocarya latifolia; 17, Cunonia capensis; 18, Curtisia dentata; 19, Dichrostachys cinerea; 20, Dombeya rotundifolia; 21, Ekebergia capensis; 22, Elaeodendron

62 croceum; 23, Elaeodendron transvaalense; 24, Erythrina lysistemon; 25, Erythrophleum lasianthum; 26, Eucalyptus sideroxylon*; 27, Euclea natalensis; 28, Ficus glumosa; 29, Garcinia gerrardii; 30, Garcinia livingstonei; 31, Harpephyllum caffrum; 32, Heteromorpha arborescens; 33, Hippobromus pauciflorus; 34, Ilex mitis; 35, Kigelia africana; 36, Macaranga capensis; 37, Maytenus undata; 38, Ochna holstii; 39, Ocotea bullata; 40, Ozoroa paniculosa; 41, Pappea capensis; 42, Peltophorum africanum; 43, Philenoptera violacea; 44, Pittosporum viridiflorum; 45, Pleurostylia capensis; 46, Protorhus longifolia; 47, Prunus africana; 48, Ptaeroxylon obliquum; 49, Pterocarpus angolensis; 50, Pterocelastrus rostratus; 51, Rapanea melanophloeos; 52, Rauvolfia caffra; 53, Rhus chirindensis; 54, Schotia brachypetala; 55, Sclerocarya birrea; 56, Securidaca longepedunculata; 57, Sideroxylon inerme; 58, Spirostachys africana; 59, Strychnos henningsii; 60, Syzygium cordatum; 61, Terminalia sericea; 62, Trichilia emetica; 63, Vachelia xanthophloea; 64, Vachelia karroo; 65, Vachelia robusta; 66, Vitex obovata; 67, Voacanga thouarsii; 68, Warburgia salutaris, 69, Zanthoxylon capense and 70, Ziziphus mucronata.

Polar solvent was a mixture of 75% hot water and 25% methanol, while the non- polar solvent was 100% dichloromethane at room temperature.

Further studies will be required to investigate the variability of the colour of extracts but in all cases where multiple samples of the same species were available, the colours of the extracts were similar even though the intensity of the colour may be somewhat variable. The extracts were often more or less clear (i.e., without colour) but also showed shades of yellow to orange, or red or brown. An attempt to standardise the interpretation of the colours and their varying intensities was made, as shown in the bottom row of Figure 2.14. The dichloromethane extracts generally did not show any colour, but there are noteworthy exceptions, such as samples 4, 7,

10, 11, 22, 23, 27, 30, 38 and 50 (respectively Berchemia discolor, Brackenridgea zanguebarica, Carissa bispinosa, Cassipourea flanaganii, Elaeodendron croceum,

Elaeodendron transvaalense, Euclea natalensis, Garcinia livingstonei, Ochna holstii and Pterocelastrus rostratus). Despite possible variation (both in the colours

63 obtained and the interpretation thereof, it is likely that this easy method will be useful as an additional approach to verify the identity of an unknown or unidentified bark sample or to narrow down the number of possibilities. In general, all of the morphological, anatomical and other variables should be taken into consideration when attempting to identify bark samples.

Table 2.4 Characters and character states that can be used for the identification of important and popular medicinal barks as sold at the Faraday and KwaMai-Mai muthi markets in Johannesburg (see Appendix 1 for colour photographs). Colour codes according to the Munsell Soil Chart; Ts = transverse section; RLS = radial longitudinal section. Odour probably variable, depending on the freshness of the sample. [Colour of extracts: H2O: MeOH – 3:1, DCM – see Figure 2.14 for the colour codes used in column 3 for extracts]

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Albizia Smooth in young stems or Yellowish Yes Rough and Cluster of Pale Smell like On TS (if sample adianthifolia branches; rough in brown //yellow fibrous. sclereids brown powder- wet) rays mature trunk with irregular (10YR 7/6) to Fibres come towards the (2.5Y ed maize distinguishable. cracks. Flakes sometimes strong brown off as periderm. 8/2) TS and RLS are appear rectangular. On (7.5YR 5/6) threads. sticky if wet. young stems, lenticels [Y; C] Lichens and scattered, roundish in mosses may be shape and are small to present. medium-sized. Araucaria Rough with distinct Grey with red Yes Relatively Heterogeneous, Light Sawdust Sometimes gum heterophylla horizontal striation. Flakes brown spots smooth. solid towards reddish smell recognizable. are horizontal. underneath Solid. wood and brown Lichens and the peeled off irregular (5YR mosses may be flakes // red (clusters of 6/4) present. (10R 4/6) sclereids) [B+; C] towards periderm. Balanites Young stems smooth. Grey to brown No Relatively Clusters of Greyish Sawdust Two distinct maughamii Mature bark is coarse // yellow smooth. sclereids brown smell layers: the outer with irregular shallow (10YR 7/6) Distinct ray towards (10YR with indistinct rays, cracks. Scales resemble [B; C] patterns. periderm. 5/2) the inner with

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] mosaic pattern. Lenticels broad conspicuous are not visible. rays. Berchemia Young stems smooth, Grey // red Yes Smooth, Stratified, layers Reddish Sawdust On TS scattered discolor sometimes with vertical (2.5YR 4/6) to solid. of sclereids. brown smell dots = clusters of cracks. Lenticels are not yellowish red (5YR sclereids. Lichens visible. Bark of mature (5YR 4/6) 5/3) and moss. trunk is rough with [R+; R+] rectangular scales.

Bersama Young stems smooth with Brownish Yes Smooth, No prominent Pale Sawdust Light in weight. lucens tiny vertical fissures; yellow (green solid. characters. Very brown smell Lichens. green layer is present underneath). pale brown (10 (2.5Y underneath periderm. Mature bark YR 8/4) 8/3) Lenticels scattered, small grey brown // to medium-sized and yellow (2.5Y arranged in horizontal 7/6) (sometimes-vertical) [B+; C] rows. Mature bark is rough with dominant vertical cracks. Scales are mostly irregular. Bersama Smooth in young stems. Grey brown Yes Solid, No prominent Light Dry grass On TS clusters of tysoniana Bark in mature trunk is //yellowish smooth. characters. olive smell sclereids. rough with dominant brown (10YR brown vertical cracks and 5/8) (2.5Y elongated flakes. [B+; C] 5/6) Lenticels are not visible.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Brackenridgea ROOT BARK. Bright yellow Yes Smooth No prominent Reddish Root and bark are zanguebarica Relatively rough, with and strong characters yellow thick. The bright dominant vertical cracks. brown// brown (7.5YR yellow colour is (7.5YR 4/4) 7/8) powdery. [R; Y] Bridelia Rough with mostly Scales grey Yes Fibrous. Easy to cut. Weak Dry fruit On TS distinct micrantha rectangular scales. No brown, cracks Fibres come Fibrous. Distinct red smell layers of fibres; lenticels. darker in off as layers of fibres. (7.5R tangential cracks. colour // dark threads. 4/3) Lichens and moss. reddish brown (2.5YR p/3) [R++; C] Calodendrum Smooth, even in mature Brownish grey No Smooth with Visible rays. Very Sawdust Light in weight. On capense trunk, with small shallow // very pale visible Very pale yellow pale smell TS rays straight vertical cracks. In young yellow patterns of (Munsell white yellow near cambium and stems, a green layer is (Munsell white rays in diagram 2.5 Y (white wavy towards present underneath the diagram, 2.5Y diagonal 9/2) diagram periderm. Lichens periderm. Lenticels are in 9/2) lines. 2.5Y and moss. vertical rows or scattered. [Y; C] 9.5/2) Carissa ROOT BARK. Superficial No Relatively Bark is thin with Yellow Sawdust Bright orange bispinosa Relatively smooth with appearance is smooth with distinct bright (10YR smell colour of inner part prominent scattered light brown; vertical orange layer of 7/6) of periderm. lenticels and sometimes underneath ridges. inner periderm. with pronounced dormant bright orange// buds. brownish yellow (10YR 6/6) [B+; Y++]

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Cassipourea Smooth, tiny vertical Dark reddish No Smooth Pink and yellow Pink Distinc- Bark is very thin. flanaganii cracks; lenticels on young brown (5YR, layers (5YR tive smell Lichens are stems in vertical rows, 3/3) // pink 8/4) abaundant. white-yellowish colour; on (10R 8/4) the mature stems [B+; Y] crustose lichens are present Cinnamomum Rough and deeply Dark reddish Yes Very smooth Bark is very Reddish Camphor Very light in camphora furrowed. No lenticels. brown and (slippery). thin, easy to cut. yellow smell weight. Moss is light brown in (7.5YR present, lichens cracks // 6/8) rare. reddish yellow (7.5YR 6/8) [B+; C] Combretum Relatively smooth with Greyish No Relatively Prominent light Sawdust On TS, there are caffrum tiny vertical cracks; brown//yellow smooth clusters of yellow- smell prominent clusters sometimes lenticels are (10YR 8/6) to sclereids ish of sclereids present inside the vertical dark brown towards the brown towards the cracks (they occur in (7.5YR 3/3) periderm. (10YR periderm. vertical rows with vertical [B; C] 6/4) Lichens present. apertures). Commiphora Relatively smooth with Brown to No Smooth Easy to cut. Reddish Sawdust Bark samples with africana thin superficial papery yellowish Prominent yellow smell remains of wood. layers; directly below is a (green vertical layers of (5YR Wood is reddish green (photosynthetic) underneath)// sclereids. On 6/6) yellow in colour layer; black marks or pink (7.5YR side cut if wet (7.5 YR 7/6). scales (dead or injured 8/4) to red prominent Photosynthetic (10R 4/8) stripes of yellow bark.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] parts) can be present on [R+; C] sclereids and bark samples. red other Lenticels small and tissues. scattered. Croton Smooth with green layer Yellowish grey No Smooth and No obvious Pale Sawdust Periderm is thin. sylvaticus underneath. Lenticels to brown//light solid. features. brown smell On TS, it is small to medium with yellowish (2.5Y possible, to horizontal or vertical brown (10YR 7/4) recognize rays – apertures; lenticels 6/4) to they are narrow, scattered or present brownish straight and inside cracks in mature yellow (10YR without dilatation bark samples. 6/6) towards periderm. [B; C] Bark appears to be layered, with broad inner layer with rays and narrow outer layer with sclereids. Lichens and moss may be present. Cryptocarya Smooth, lenticels small Brown (7.5YR No Smooth, Light and darker Light Aromatic Bark is thin. latifolia (<1 mm) and scattered. 4/3)// very sometimes brown layers brown smell/ Lichens foliose, dark brown with small pappery moss green. (7.5YR 2.5/3) ridges (on fresh [B+; C] cut only) Cunonia Bark smooth only in Greyish Yes; Smooth Group of Reddish Dusty Lichens present. capensis young stems. Usually brown//reddish only in sclereids brown smell rough with vertical

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] fissures and irregular black (5R mature towards the (2.5YR fissures in mature trunk. 2.5/1) bark. periderm 5/4) Lenticels are prominent, [R+;C] roundish to slightly elongated (2–6 mm) with vertical and rarely horizontal apertures. Curtisia Relatively smooth in Brownish Yes; Smooth No prominent Strong Sawdust Solid. Lichen and dentata young stems with tiny grey//strong only in characters. brown smell moss. vertical fissures; lenticels brown (7.5YR mature Slightly light (7.5YR // are of medium size and 4/6) bark. colour and 5/6) horizontal aperture. Bark [R; ±C] visible rays is rough in mature trunk towards with irregular fissures cambium. forming mosaic patterns. Dichrostachys ROOT BARK. Yellow //yellow No Smooth Root is thin; Yellow Sawdust Bark easily peels cinerea Bark looks shaggy; the (2.5Y 8/6) correspondinglyt (10YR smell off the root as long flakes are papery. [±C;C] he bark is also 7/6) flexible strips. thin. No obvious characters on side cut. Dombeya Rough with irregular Grey to dark Yes Fibrous and Easy to cut. Light Dusty Light in weight. rotundifolia cracks. brown// smooth. Tiny layers, reddish smell Fibrous on TS. reddish yellow Fibres peel colour gradually brown Lichens. (5YR 7/6) off as long changed from (5YR [B+; C] strips. light yellow 6/4) inside reddish towards outside.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Ekebergia Bark is relatively smooth Light grey with Yes Relatively Looks fibrous, Brown Distinc- Solid. Lichens and capensis in young stems with tiny yellowish smooth. but solid. (7.5YR tive smell moss. vertical fissures; lenticels lenticels in Looks 4/4) are in vertical rows 2 to young stems. fibrous, but 20 mm apart. Bark is Mature solid. rough in mature trunk with branches and irregular cracks. stems grey- brown//strong brown (7.5YR 5/8; 7.5YR 4/4) [R+; C] Elaeodendron Bark is relatively smooth Grey with No Solid and Smooth with Light red No clear Bright orange croceum with vertical fissures. prominent smooth cluster of (2.5YR smell pigment present. Lenticels are not visible. orange colour sclereids. 7/6) Solid. Lichens and underneath moss. the periderm // pinkish white (5YR 8/2) [R+; Y++] Elaeodendron Smooth with vertical Light grey to Yes; Smooth with Groups of Pink Sawdust Solid. Lichens transvaalense fissures in younger stems brown //pink only in vertical ridges sclereids in (5YR smell present. and relatively rough in (5YR 7/4) mature vertical lines 8/4) older stems with irregular [R++; Y] old (near cambium) fissures. Sometimes bark trunk. or horizontal of mature trunk has lines (towards mosaic appearance. No periderm). visible lenticels.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Erythrina Relatively smooth with Grey brown, No Smooth with Green layer Light Distinc- Photosynthetic lysistemon longitudinal grooves and green visible rays usually present brown- tive smell bark. Comma- scattered knob-like underneath underneath ish grey shaped thorns. projections with hooked the superficial superficial layer. (2.5YR and pointed prickles. layer (in very 6/2) Lenticels are prominent, old trunks in vertical rows. greenish yellow // yellow (10YR 7/8) [B; C] Erythrophleum Smooth in young stems Bark is grey Yes Smooth with Visible layers of Reddish Dusty Solid. On TS lasianthum and rough with irregular on the visible stripes sclereids brown smell visible layers of (mostly vertical) cracks in superficial that are (yellowish in (2.5YR sclereids mature trunk. parts and vertical or colour). 5/4) (yellowish in becomes sometimes colour). Lichens strong brown twisted sometimes underneath present. and is dark red further down //it may be grey or dark brown [R+; C] Eucalyptus Rough, deeply fissured Black to Yes Smooth with Fibrous Yellow- No clear Bark is very thick sideroxylon and very thick (up to 12 reddish tiny flexible ish red smell but is light in mm). Bark saturated by brown//reddish fibres on the (5YR weight. Moss small reddish black edges 5.8) sometimes

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] exudates that easily come yellow (5YR present. Lichens off as little sandy stones. 7/6) absent. [R+; C] Euclea ROOT BARK. Black (5YR No Smooth Bark is Olive No clear On TS, bark is natalensis Bark is smooth with no 2.5/1) and sometimes brown smell green towards the visible lenticels. brown green towards (2.5Y periderm. underneath the periderm. 4/3) superficial Bark can be layer //dark relatively thick. brown (7.5YR 3.4) [R++; Y++] Ficus glumosa Smooth, with tiny Reddish No Smooth On longitudinal Reddish Sawdust Bark is thick (10 to yellowish flakes; lenticels yellow (5YR section bark is yellow smell 12 mm). Light in are not visible. 6/6) or mottled with (5YR weight, easy to cut brownish brown and 7/8) with knife. yellow (10YR yellow patches 6/8) // red (2.5YR 4/8) or yellowish red (5YR 4/6) [R+; ±C] Garcinia Smooth to the touch, but Light brownish No Smooth, tiny Yellow Light Dusty Thickness 6 to 8 gerrardii heterogeneous in grey (10YR stripes are horizontal reddish smell mm. Lichens and appearance, with small 6/2) //reddish visible on a stripes (rays) brown moss present. rectangular flakes of a brown (5YR wet surface near cambium pale yellowish colour, 5/4) alternated with lenticels are small (<1 [R+; C] brown; on

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] mm), scattered and in transverse horizontal rows. section colour changes gradually from cambium to outer part Garcinia Smooth in young stems. Grey, with Yes Relatively Prominent Strong No On TS, reddish livingstonei Rough, with prominent orange smooth, layers of brown clear layers towards the vertical cracks in mature underneath saturated sclereids. (7.5YR smell cambium and stem. The bark makes a superficial with many 4/6) prominent clusters mosaic appearance. layer small dots of sclereids //yellowish red (exudates) towards the (5YR 5/8) that are dark periderm. Lichens [R++; Y++] reddish and moss present. Harpephyllum Young stems smooth with Brownish in Yes Smooth Layered Red No clear On TS and RLS caffrum vertical fissures and young stems (2.5YR smell distinctly layered. scattered lenticels. and grey in 4/8) On muthi markets, Bark from mature stem is mature bark // the bark is usually rough with dominant red available only as vertical cracks forming [R++; C] chopped samples. rectangular to irregular scales. Heteromorpha Bark is smooth and Greyish olive // No Smooth No distinct Light Distinc- Bark very thin. arborescens peeling. Underneath this pale yellowish characters olive tive Photosynthetic parchment-like superficial [B; Y] brown smell layer below the layer is a bright green (2.5Y very thin, (photosynthesising) layer. 5/4) translucent outer layer.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Lenticels with horizontal apertures scattered, but more often-in horizontal rows. Hippobromus Rough in mature stems Grey- Yes Smooth Reddish yellow Reddish Sawdust On TS, no distinct pauciflorus with prominent cracks brown//yellow layers. brown smell characters. forming rectangular (10YR 8/6) (5YR Abundant moss scales. [B+; C] 5/3) and lichens sometimes present. Ilex mitis Relatively rough with Grey to white// No Relatively Prominent Pale Sawdust Bark densely prominent, medium-sized, strong brown rough to cluster of brown smell covered with scattered lenticels. (7.5YR 5/6) to touch. sclereids. (2.5Y different types of brown (7.5YR Sometimes 8/2) lichens and traces 4/3) [Y++; C] with visible of moss. small holes. Kigelia Relatively smooth with Greyish brown Yes, in Smooth, Difficult to cut. Brown- Dry fruit On TS sclereids in africana vertical fissures. No // dark brown mature sometimes Small, scattered ish smell, tangentially visible lenticels. Mature (7.5YR 3/3) bark, if with visible group of yellow like elongated groups. trunk may have [R++; ±C] bark rays. sclereids. (10YR plums rectangular or irregular scaly. 6/6) scales. Macaranga Bark is smooth and thin. Superficial No Smooth Bark is thin. On Pink Peanuts On TS scattered capensis On young stems, lenticels appearance is thicker samples (5YR smell dots (=clusters of are small, to medium- grey with distinct vertical 7/4) sclereids). Plenty sized with horizontal reddish layer (interrupted) of lichen colonies. apertures and scattered. underneath// layers of sclereids.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] pink (2.5YR 8/3) [R+; C] Maytenus Bark is smooth. In mature Dark grey with Yes, Fibrous, peel Prominent Light Sweet Inner part is undata trunk, it can be relatively strong brown Pre- off in long clusters of reddish aromatic lighter, compared rough, with irregular layer sent strips. Fibres sclereids. brown smell to the darker outer cracks. Lenticels are not underneath // only in flexible if wet. (5YR colour. Solid. visible. yellow (10YR very 6/4) Lichens and moss 8/6) old or present. [R+; C] damag ed parts of the trunk. Ocotea bullata Relatively smooth and Grey to brown Yes, Relatively Side cut Brown Spicy Solid. Lichens and even in mature trunks. // brown Pre- smooth with sometimes with (7.5YR peppery moss may be Roundish or slightly (7.5YR 4/4) to sent vertical short vertical 5/4). smell present. elongated dark lenticels dark brown only in ridges. yellowish layers. scattered on young (7.5YR 3/2) very stems. [R+; ±C] old or damag ed parts of the trunk. Ochna holstii Relatively smooth with Brownish grey No, but Smooth. Bark is thin. No Pinkish Sawdust When broken, tiny irregular cracks. // pink (7.5YR not other prominent grey smell numerous white Lenticels are not visible. 7/4) sure features. cotton wool-like

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] [R; B+] about (5YR threads (latex old 6/2) threads?) are trunks. present. Lichens and moss present. Ozoroa Bark is more or less level Greyish black Yes Smooth. Groups of Red (5R No clear Lichens and moss paniculosa (even) to rough in old to dark brown yellowish 5/8) smell present. mature trunk, with scales and lined with sclereids; dark of irregular shape that yellow lines are resembles a mosaic underneath sometimes pattern. No lenticels. //red (5R 5/8) visible with [R++; C] exudation from secretory structures. Pappea Smooth in young stems. Grey brown // Yes Solid with Stratified with Brown Dusty On TS layers of capensis Bark of mature trunk reddish yellow prominent prominent (7.5YR smell sclereids (light relatively smooth to rough (7.5YR 6/8) to vertical layers of 4/4) yellow) alternate with irregular (mostly red (2.5YR ridges. sclereids (light with other vertical) cracks. 4/8) yellow) secondary phloem [R++; C] alternating with tissues. other secondary Lichens and moss phloem tissues. present. Peltophorum Smooth in young stems, Light grey to Yes Fibrous; Many layers of Reddish Sandy, Lichens and moss africanum rough with vertical cracks black and fibres are red fibres, brown dusty present. When you and horizontal fissures in becoming flexible when sometimes with (2.5YR smell push/pull across medium-sized branches strong brown wet. few layers of 4/4) the fibres with and very rough with and yellowish sclereids thumb, small dust irregular cracks in old underneath // clouds appear. mature trunks. Lenticels brown (7.5YR

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] are present on smooth 4/6) to red parts and their remains. (2.5YR 3/6) [R++; C] Philenoptera Young stems are smooth. Light grey, Yes Smooth and Clusters of Very No clear On TS, prominent violacea Mature stems become with brownish fibrous. visible sclereids. pale smell clusters of relatively rough and orange colour brown sclereids. flaking. Prominent vertical underneath (10YR Abundant lichens ridges/ cracks. the superficial 8/3) of different types. layers//yellow (10YR 8/6) [B; C] Pittosporum Relatively smooth. Greyish brown No Smooth, Sometimes rays Very Distinct, Lenticels often viridiflorum Lenticels small to // pale brown solid. are visible on pale white prominent, medium-sized with (10YR 6/3) side cut or brown pepper or sometimes horizontal apertures, [Y; C] breaks. (10YR pungent forming horizontal scattered or in distinct 7/3) citrus lines. On TS rays horizontal rows. smell distinct. Lichens and moss present. Pleurostylia Young stems are Grey brown // No Relatively Stratified with Pinkish Sawdust Samples brittle. capensis relatively smooth with pink (7.5YR smooth with distinct yellow gray smell When broken, rectangular scales. 8/4) vertical layers (5YR numerous white Horizontal cracks are [B; Y] ridges. alternating with 7/2) cottonwool-like sometimes dominant. pink layers. threads (latex Bark of mature trunk threads?) occur. becomes relatively rough Lichens and moss with irregular flakes. present.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Protorhus Bark is more or less level Grey to brown Yes Smooth and Dark red (10 R Dark Sweet On TS layers of longifolia (even) to rough in old and red solid with 3/4). Layers of reddish smell yellowish mature trunk, with visible underneath // scattered yellowish brown sclereids. thin fissures. Lenticels are dark red (10R ridges. sclereids up to (2.5YR Lichens may be possibly in vertical rows. 5/8) Sometimes 1.5 mm wide. 3/4) present. [R++; Y] vertical cracks occur in samples. Prunus Rough, with various Grey to black Yes Fibrous and Pink (5 Pink Faint Bark surface with africana irregular, vertical and or dark brown brittle. YR 7/4) (5YR sawdust rectangular horizontal cracks, forming to black// dark 7/4) smell adherent scales. irregular scales. No red (2.5 YR Moss and lichens lenticels. 3/6) or strong sometimes brown (7.5YR present. 4/6) [R+; C] Ptaeroxylon Relatively smooth in Greyish brown Yes Rough, very Easy to cut. Yellow Sweet Lichens and moss obliquum young stems. Bark of and yellow fibrous. Storied structure (2.5Y aromatic present. mature stems has visible inside the Fibres strong with many 8/8) smell vertical fissures and tiny vertical and flexible layers of fibres. horizontal cracks. fissures//yello when wet. w (2.5Y 8/8) [Y+; Y] Pterocarpus Rough on mature stems Dark grey to Yes Smooth with Easy to cut. Reddish Sawdust On TS, bark angolensis with prominent vertical brown dark red Alternative light brown smell saturated with cracks forming underneath//re exudates. and dark layers. (5YR layer of red sap rectangular scales. Red ddish grey 6/4) exudates. Bark is (5YR 5/2) light in weight.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] sap exudates are present [B+; Y] Lichens and moss on the bark samples. present. Pterocelastrus Bark is smooth with small, Greyish brown No Smooth with Solid or fibrous Weak Sawdust On TS fibrous rostratus scattered, yellowish //dark reddish solid fibrous with tiny vertical red (10R smell towards cambium, lenticels. brown (2.5YR appearance. yellowish lines 5/4) solid towards 3/3) Fibres brittle of fibres or periderm. Lichens [R++; Y++] and break if sclereids. present. pulled. Rapanea Relatively smooth with Greyish brown Yes Relatively The colour Reddish Sawdust Distinctive melanophloeos vertical fissures in young (10YR 5/2) smooth to the gradually brown smell appearance on stems and rough with and reddish touch. Very changes from (2.5YR inner surface due irregular cracks in mature towards the prominent pinkish inside to 4/4) to the rays. On trunk. inner surface // rays, which darker red transverse reddish black are lighter in towards the sections, (5R 2.5/1) colour than outer surface prominent light- [R++; Y] the ground coloured rays. tissues. Moss and lichens Vertical can be present. cracks often present. Rauvolfia Smooth in young stems, Yellow to light Yes Relatively Visible clusters Yellow Sandy On transverse caffra rough with short vertical brown // smooth to the of sclereids in (2.5Y smell sections, clusters cracks and horizontal yellowish touch. layers that 8/6) of sclereids lines on mature trunk. brown (10YR Sometimes easily come off scattered or in Periderm is spongy to the 5/6) with visible and are sandy layers. Bark is light tough (like polystyrene). [Y; C] rays. in texture. in weight. Lenticels are present, even in mature bark from

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] the trunk. They are medium in size and scattered. Schotia Smooth in young stems, Grey brown// Yes Smooth with The colour Pink No On TS yellowish, brachypetala coarse with vertical or light red brittle fibres. gradually (10R distinc- tangentially rarely horizontal fissures. (2.5YR 6/8) changes from 8/3) tive smell elongated patches No lenticels are visible. [R++; C] pinkish inside to (sclereids). red with Lichens and moss yellowish dots present. (sclereids) towards the outer surface Sclerocarya Smooth in young stems. Grey to black Yes Smooth, with Visible papery Dark Sawdust Gum exudates; birrea Trunk and old branches //dark reddish papery strips layers towards reddish smell prominent and with irregular or brown (5YR or layers that the cambium brown abundant sometimes roundish 3/3) peel off and a thick (2.5YR sclereids. scales; mature old trunk [B++; C] easily. scattered cluster 2.5/3) Sometimes lichens with strong and prominent of yellowish and moss present. vertical cracks. No sclereids below lenticels present. the periderm.

Searsia Smooth in young stems. Grey to strong Yes Relatively Difficult to cut. Reddish Dusty Lichens and moss chirindensis Mature bark is rough with brown in smooth, No obvious brown smell present. irregular cracks and younger stems sometimes features. (5YR irregular to rectangular and dark looks fibrous. 5/4) flakes. Lenticels scattered brown in However, on young stems or injured mature fibres are parts. They are small to very brittle

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] medium in size with trunk//red and difficult to horizontal or vertical (10R 4/6) pull off. apertures. [Y++; C] Securidaca ROOT BARK. Brown (7.5YR No Smooth Numerous Pale Sawdust On TS prominent longepeduncul Relatively smooth. 4/3)//yellow clusters of brown smell clusters of ata Lenticels present. (10YR 8/6) prominent (2.5YR (winter- sclereids. Lichens (Chopped sample) [B+; C] sclereids. 7/4) green and moss present. smell when fresh) Sideroxylon Smooth young stems. Grey to black// Yes Smooth, with Very smooth, Yellow- Sawdust Vertical and inerme Mature trunk with light red thin vertical light red. ish red smell horizontal cracks. prominent and vertically (2.5YR 6/8) lines. (5YR Lichens and moss. orientated rectangular [R++; C] 5/6) scales. Strychnos Bark smooth to rough with Greyish brown Yes Relatively Bark is thin with Brown Sawdust Bark light in henningsii very irregular, scattered (lot of lichens smooth, prominent (10YR smell weight. Groups of flakes. Small lenticels which can sometimes groups of 5/3) sclereids scattered or sometimes make stem with sclereids. prominent on TS bigger lenticels in mottled) // numerous and LS. Lichens vertically elongated yellowish black marks abundant and groups. brown (10YR of mould. moss. 5/4) [Y++; C] Syzygium Smooth in young stems. Grey to dark Yes Fibrous but Easy to cut. Red Light Faint Bark light in weight cordatum Mature bark is rough with brown // red smooth. (10 R 5/8) reddish sawdust and very thick. irregular cracks. No (10R 5/8) Fibres are colour, without brown smell Lichens and moss lenticels. [B+; C] visible patterns. are present.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] flexible when (2.5YR wet. 6/4) Terminalia Very rough with deep Greyish brown Yes Smooth, but Fibrous. Easy to Dark Sawdust Inner bark yellow, sericea vertical furrows. //brownish fibrous if cut , colour yellow- smell outer bark with yellow (10YR stripped. The gradually ish very distinct 6/8) strips are changes from brown texture (branching [R++; C] flexible, brownish yellow (10YR ridges). On TS especially to reddish 3/6) very fibrous. when wet. towards Lichens rare. periderm Trichilia Smooth in young stems. Grey-brown, No Relatively Red brown, Reddish No Solid. emetica Mature trunk relatively sometimes smooth smooth brown distinc- Bark samples may smooth to course, with yellowish.// (5YR tive smell be damaged and tiny vertical fissures. No dark reddish 5/4) Powered have different lenticels on mature trunk. brown (2.5YR bark has surface colour due 3/3). The outer a walnut to fungal growth bark colour or pecan and other abiotic can be very nut smell factors resulting mottled due to from poor drying lichens and and storing moss. practises. [B++; C] Vachellia Rough, irregularly Mottled: light Yes Fibrous (the Reddish brown, Light No Bark is very karroo fissured; no lenticels. grey fibers are sand-papery or grey distinc- fibrous. It can be superficial flexible). sandy to the (10YR tive smell dark if damaged layer and touch because 7/2) (but because of gum brown to light of sclereids causes exudates on the brown towards sneezing outer surface.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] the inner parts due to Green moss is // yellow irritation present (green (10YR 8/6) of tint) and lichens [Y; C] epithelial which give a dark tissues) tint. Vachellia Smooth to rough. Vertical Grey to brown Yes Fibrous: Pinkish red Pale red Dusty On TS and LS with robusta to irregularly cracks in old // red (2.5YR strong elastic colour with (10R smell very prominent bark. Lenticels only on 5/8) fibres, groups of 6/4) groups of smooth bark, in horizontal [B++; C] separated by yellowish yellowish rows. thin threads. sclereids. sclereids. Lichens Prominent and moss are pattern of present. rays (cf. Rapanea). Vachellia Smooth and powdery, Pale yellow No Smooth, Reddish brown Light red No The yellowish- xanthophloea lenticels are small (<1 (5Y, 8/4 )// (sometimes (2.5YR distinc- green and mm) and scattered. dark or light samples have 6/6) tive smell powdery outer brown (or wood (xylem) bark surface is yellowish if on the inner very distinctive. wood present) surface) [R+; C] Vitex obovata Smooth in young Dark grey to Yes Relatively Stratified. Light Sawdust On TS, numerous branches and stems. brown// brown smooth. grey smell spots scattered. Mature bark is coarse (7.5YR 4/3) to (10YR Lichens and moss. with dominant vertical grey (7.5YR 7/2) cracks, scaly. Scales 5/1) small (approximately [B+; C] 0.5x1 cm) look irregular

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Bark from mature trunk looks shaggy. Voacanga Relatively smooth with Pale grey to No Relatively Clusters of Olive Some- On TS clusters of thouarsii dominant shallow vertical yellow smooth. sclereids (5Y 5/4) what sclereids. cracks. Lenticels are not //brownish towards fragrant Lichens and moss visible. yellow (10YR periderm. smell are present. 6/6) Distinct rays. [Y++; Y]

Warburgia Smooth in young Grey to dark Yes Smooth. Remarkable Reddish Peppery Peppery taste. salutaris branches. Mature bark is brown // pale Sometimes colour transition, brown smell Bark has unusual rough, brittle with deep yellowish pink with visible from pale (2.5YR structures within irregular cracks. Flakes (7.5YR -/2) papery layers yellowish pink to 5/4) the cracks that irregular in shape or [B+; C] or appearing red and then resemble lenticels. vertically elongated. to be fibrous, dark brown in but the the rhytidome fibrous layers part. are very brittle. Zanthoxylum ROOT BARK. Yellow brown No Smooth and Bark is very Yellow Sweet Lenticels are capense Relatively smooth. // brown solid. thin. No obvious (2.5Y fragrance bright yellow. Lenticels abundant, (7.5YR 5/4) characters. 7/6) smell scattered, medium in size, [Y++; C] apertures horizontal. Lenticels have bright yellow colour.

Outer bark appearance Colour outer Rhyti- Inner bark Longitudinal Bark Odour Other remarkable and texture //inner dome appearance side cut or powder characters [colour of and texture break colour extracts] Ziziphus Very rough, with very Grey to brown Yes Smooth with Layers. Colour Light Dusty Layers visible on mucronata prominent vertical cracks, or dark vertical lines. red to brown. reddish smell TS and even on and some horizontal but brown// brown protruding parts of less prominent cracks. yellowish red (5YR rhytidome. Mature bark with irregular (5YR 5/8) 6/3) cracks. [Y++; C]

2.3.7 Conservation status of bark species

Bark is a problematic material from a conservation perspective because indiscriminate harvesting can cause much damage. Bark removal induces internal stress that may lead to progressive death of the plant depending on the extent of harvest. Ring-barking further reduces the survival probabilities and may result in the death of the entire tree due to the depletion of stored reserves and termination of coppice regrowth (Cocks et al., 2004; Geldenhuys, 2004; Delvaux et al., 2010;

Senkoro et al., 2014).

Due to commercial and financial pressures, traders are tempted to over- harvest bark and not adhere to customary practises and traditional methods to ensure sustainability. The systematic depletion of naturally occurring products is expected to create a constant decline in the supply of plants to the traditional medicine sub-sector (Mander et al. 2007). According to a report from the Department of Water and Forestry (2005), medicinal plant products are collected in bulk from the wild and approximately 16 000 harvesters, rural black women in particular, operate in

KwaZulu-Natal alone. According to Ndawonde et al. (2007), plant traders have not been taught conservation strategies, as they sometimes cut the entire tree or remove the bark material completely and in bulk for trading purposes. Therefore, it was suggested that plant traders be taught responsible harvesting techniques. Figure

2.15 represents the conservation status of the 70 most popular and important medicinal bark species sold at the Johannesburg muthi markets.

4% 4% 1% 1% 4% Least Concern 4% Declinging Near Threatened Vulnerable Endangered Critically endangered Not evaluated

82%

Figure 2.15 Conservation status of the 70 most popular medicinal bark species sold at the Johannesburg muthi markets.

According the Red Data List of SANBI (2017), 82% of the 70 popular medicinal bark species sold on Johannesburg muthi markets are of least concern,

4% declining, 1% vulnerable, 4% endangered, 4% near threatened, 1% critically endangered and 4% not evaluated. Brackenridgea zanguebarica is recorded under the category of critically endangered. Plant species recorded as endangered are

Cassipourea flanaganii, Ocotea bullata and Warburgia salutaris. These trees are known to have localized distribution areas and they are amongst the most popular of all southern African medicinal barks. Prunus africana is included in the category of vulnerable, facing a high risk of extinction. Plant species rated under the near threatened category include Elaeodendron transvaalense, Curtisia dentata and

Erythrophleum lasianthum. Plant species considered to be in the declining category include Ilex mitis, Rapanea melanophloeos and Pterocelastrus rostratus. The exotic

88 tree species (Cinnamomum camphora, Araucaria heterophylla and Eucalyptus sideroxylon) were obviously not evaluated.

In a study of medicinal bark harvesting and yields in southern Maputaland,

Twine (2004), 36 woodland tree species used in traditional medicine were recorded as being harvested by one or more individual harvesters for their bark. Among the tree species, Elaeodendron transvaalense was recorded as the leading species facing over-harvesting, followed by Sideroxylon inerme, Balanites maughamii,

Schotia brachypetala and Sclerocarya birrea.

Although there are many problems relating to the conservation of tree species that are harvested for the traditional medicinal plant trade, there are sustainable ways of sourcing bark material without causing extensive damage to the entire tree.

The removal of a natural resource without depleting it or compromising its ability to regenerate is defined as sustainable harvesting and it requires planning and monitoring at the species level (Struhsaher, 1998; Dzerefos, 2004). According to

Geldenhuys (2004), sustainable harvesting of bark resources requires knowledge of the entire resource area, the growing stock of those species from which bark is harvested, the response of harvested trees to bark stripping, and the market demand.

Tree species respond differently to bark harvesting, and bark regrowth varies among different plant species. Factors affecting bark regrowth include stem diameter, harvest wound shape and wound depth (Geldenhuys, 2004; Twine, 2004).

Bark is a renewable plant material if correctly harvested (Van Wyk et al., 2011b).

Sclerocarya birrea was reported to show a high capacity for bark regrowth, and harvest wounds of up to nearly 0.5 m2 in area had totally regrown bark tissue. An example of bark regrowth in this species is shown in Figure 2.16. Some species,

89 including E. transvaalense, also regrew bark from within the wound when the cambium had not been removed or seriously damaged (Twine, 2004). Preliminary results reported by Geldenhuys (2004) on a bark harvesting experiment indicated that Ocotea bullata regrows its bark relatively rapidly, while Curtisia dentata is relatively slow, and Rapanea melanophloeos shows no bark regrowth.

Figure 2.16 The trunk of a marula tree (Sclerocarya birrea) showing signs of repeated bark harvesting and subsequent wound healing (Rustenburg district). Photographs by B.-E. van Wyk.

The popularity of traditional use may directly affect the conservation status of plants species (Williams et al., 2013). A critical factor contributing to the endangered status of Elaeodendron transvaalense is the popularity of its bark for treatment of the human immune-deficiency virus and acquired immune deficiency syndrome

(HIV/AIDS) and other sexually transmitted diseases (Tshikilange et al., 2008; Samie et al., 2010; Masevhe et al., 2015; Mamba et al., 2016). In addition, Mander and co- workers (2007) reported that the HIV/AIDS epidemic fuels the demand for traditional medicine, with many consumers consulting with traditional healers who offer

HIV/AIDS treatments, or immune boosters. According to Williams (2003), Warburgia salutaris, followed by E. transvaalense, were among the most cited plant species by

Faraday Muthi Market traders as being scarce.

2.4 Summary

 The predominant culture and language spoken on the Faraday and Kwa Mai-

Mai Muthi Markets in Johannesburg is isiZulu – 86% of the traders are Zulu

speaking, followed by 7% each of Xhosa and Tsonga speakers.

 Women were found to be the dominant gender on both the Faraday and Kwa

Mai-Mai Muthi Markets in Johannesburg.

 The 70 most popular medicinal barks on the markets were from species

belonging to 35 plant families. The most important families were Fabaceae

(29% of the species), Celastraceae (18%), Anacardaceae (15%) and

Apocynaceae (12%). Surprisingly, no records of medicinal barks from large

families such as Asteraceae and Rubiaceae were encountered.

 Most of the studied barks are used for more than one ailment. According to

the literature record, the majority of the barks are used for the treatment of

skin infections (51 barks), followed by gastrointestinal tract (40 barks), and

respiratory ailments with 35 barks. In contrast, according to the uses reported

by the muthi markets traders, the majority of the barks are used for charm or

magic (47 barks), followed by skin ailments (15 barks) and gastrointestinal

tract ailments (11 barks).

 The majority of the bark remedies are administered orally as liquid extracts or

they are used in the form of raw powdered material that is licked from the

back of the hand in the traditional way, known as khotha.

 Out of the 70 medicinal barks recorded, 57 species (82%) are least concern;

three species (4%) are declining – I. mitis, R. melanophloeos and P. rostratus;

three (4%) are near threatened – E. transvaalense, C. dentata and E.

lasianthum; one species is vulnerable – P. africana; three species (4%) are

endangered –C. flanaganii, O. bullata and W.salutaris and one species is

critically endangered– B. zanguebarica.

2.5 Conclusions

The correct identification of medicinal barks sold on the muthi market remains a challenge due to various factors, including similar vernacular names used for unrelated species, the absence of useful morphological characters, natural geographic variation of individual species and most importantly, a lack of proper documentation in the form of published descriptions and photographs. The study showed that bark still plays an important role in primary health care and that there is a need to accurately record the diversity of traditional uses.

CHAPTER 3. ANTIMICROBIAL ACTIVITY OF SELECTED EXTRACTS

3.1 Introduction

The human body is regularly exposed to potentially dangerous bacteria and other harsh environmental factors, which contribute to the development of many infectious diseases (Twilley and Lall, 2014). Medicinal plants have been used for the treatment of various infectious diseases since ancient times and they represent a rich source of antimicrobial agents (Dias et al., 2012). In addition, Oyedemi et al. (2012) mentioned that medicinal plants remain a reservoir of biologically active compounds with biochemical and therapeutic properties. Hence, by 2004, about 122 drugs were already discovered through ethnobotanical research from 94 plant species (Fennell et al., 2004).

Bark is a frequently used plant part in traditional medicine, particularly in southern Africa (Williams, 1996; Grace et al., 2003; Mander et al., 2007). The occurrence of different natural products in specific plant organs is one factor that contributes to variations encountered in the biological or pharmacological activity between different plant parts of the same species. Chemical constituents that are produced in bark may therefore differ quantitatively or qualitatively from those of other plant parts (McGaw et al., 2002). Bark is well-known to contain a rich diversity of secondary metabolites (mainly tannins, alkaloids, flavonoids, terpenoids, saponins and polysaccharides) that are of major benefit to human health (Hutchings, 1989;

Van Wyk and Wink, 2015). Investigation of the antimicrobial activity of bark is critical, considering the frequency and popularity of its use in traditional medicine.

According to Dias et al. (2012), traditional medicinal practices have formed the basis of many of the early medicines, followed by subsequent clinical,

93 pharmacological and chemical studies. Furthermore, a number of studies have been conducted on the antimicrobial screening of medicinal plants from ethnobotanical leads (Rios and Reico, 2005; Van Vuuren, 2008; Ndhlala et al., 2013; Van Vuuren and Holl, 2017) but very few have focused on the screening of medicinal barks in particular. Bark plant material has been shown to possess noteworthy antimicrobial activity against infections associated with the skin, gastrointestinal tract and respiratory tract. Barks that have been involved in the treatment of wounds include species such as Bridelia ferruginea Benth., Garcinia polyantha Oliv. and Kigelia africana (McGaw et al., 2008; Agyare et al., 2016); sores have been treated with bark of Ekebergia capensis and Erythrina lysistemon, skin rashes with bark from

Elaeodendron transvaalense and Rauvolﬁa caffra and eczema with bark from

Harpephyllum caffrum and Albizia adianthifolia (Mabona and Van Vuuren, 2013;

Twilley and Lall, 2014). Some bark materials (Ziziphus mucronata, Pittosporum viridiflorum, Alstonia boonei De Wild.) have been used for other infectious diseases caused by parasites such as malaria (Okoye et al., 2014; Zinginga et al., 2017).

Gastrointestinal ailments that are commonly treated in traditional medicine include dysentery and diarrhoea. Popular barks screened for gastrointestinal complaints include Schotia brachypetala (McGaw et al., 2002), Curtisia dentata (Shai et al.,

2009), Brachylaena transvaalensis Hutch. ex E.Phillips & Schweick. (De Wet et al.,

2010), Sclerocarya birrea (Moyo et al., 2011), Adansonia digitata L. and Kigelia africana (Ndhlala et al., 2013), as well as Terminalia sericea and Garcinia livingstonei (Van Vuuren et al., 2015). Activity against respiratory ailments, including tuberculosis, have been tested using barks from species such as Warburgia salutaris, Vachellia xanthophloea, Berchemia discolor, Bridelia micrantha,

Cryptocarya latifolia and Ekebergia capensis (Lall and Mayer, 1999; McGaw et al.,

2008; Green et al., 2010). Barks for colds and coughs include Commiphora africana,

Combretum molle R.Br. ex G.Don, Rauvolfia caffra, Warburgia salutaris and

Syzygium cordatum (McGaw et al., 2008; Van Wyk, 2011; York et al., 2012). Other antimicrobial studies of barks that have been undertaken include sexually transmitted diseases (Tshikilange et al., 2008; Samie et al., 2010; Mamba et al.,

2016). Using the search engines Scopus, Science Direct and Google Scholar, all published antimicrobial studies that could be found for the 70 popular bark species recorded in this study are listed in the inventory (Appendix 1).

3.1.1 Role of bark for skin ailments

The skin is the largest organ in the human body and the primary defence mechanism against the external environment (Lee et al., 2006). Diseases associated with skin infections account for approximately 34% of all diseases worldwide including both developed and developing countries (Kishore and Lall, 2014). The popularity of traditional medicinal plants used for dermatological infections has resulted in many skin products that are supplemented with plant extracts. According to the ethnobotanical literature, over 100 plant species have been considered for use in traditional medicine for the treatment of dermatological disorders (Mabona and Van

Vuuren, 2013; Kishore and Lall, 2014). The majority of southern African medicinal plants used for dermatological ailments are reported to promote wound healing

(Mabona and Van Vuuren, 2013). Cutaneous wound healing is a serious public health concern as many characteristic diseases may arise due to the compromised immunity of the skin. Bark preparations that are often used in traditional healthcare for the treatment of various skin diseases include powders, poultices, pastes, lotions, infusions and decoctions. As a powder, the bark is applied directly to the skin on the

95 infected area. Such is the case for the powdered bark of Erythrina lysistemon, used to disinfect wounds (Pujol, 1990; Rabe and Van Staden, 1997; Pillay et al., 2001). As a poultice (directly to the skin), bark of Erythina lysistemon is applied on swellings and boils (Pujol, 1990, Hutchings et al., 1996; Grace et al., 2003). As lotions, pounded bark of Albizia adianthifolia is ground into a fine powder and used in aqueous lotions for the treatment of itchy skin complaints such as eczema

(Hutchings et al., 1996; Grace et al., 2003). As infusions and decoctions,

Elaeodendron transvaalense bark is administered as an infusion for skin inflammation and rashes (Van Wyk and Gericke, 2000, 2018). Even though many antimicrobial studies have been conducted, antimicrobial activity focusing only on bark are poorly documented compared to leaves. Infections associated with the skin include burns, dermatitis (eczema), psoriasis, skin rashes, acute erythema, vitiligo and deep wounds. Frequently encountered bacterial pathogens that may cause these skin disorders include Staphylococci and Streptococci (impetigo and open wound infections), as well as Pseudomonas aeruginosa (furunculosis and open wound infection), amongst others (Mabona and Van Vuuren, 2013).

3.1.2 Role of bark for respiratory ailments

The respiratory system is the most vulnerable site for bacterial infections; with primary influenza infection, being frequently encountered (Low, 2008). Ecological disturbances and frequent antimicrobial use are reported as major contributors to bacterial accumulation in the respiratory tract (Prat and Lacoma, 2016). In most cases, such infections occur during winter and in the early spring months (Richter et al., 2016). Respiratory tract infections continue to be a serious threat to populations of all ages throughout the world due to their ease of transmission and substantial

96 morbidity and mortality. However, differences in the pathogen spectra between the upper and lower respiratory tract remains debatable (Richter et al., 2016; Lin et al.,

2017). In addition, acute respiratory tract infections, especially acute sinusitis, acute pharyngitis, acute bronchitis and non-specific upper respiratory tract infection, including the common cold, are the major cause of antibiotic prescriptions in hospitals and clinics (Low, 2008; Prat and Lacoma, 2016).

In South Africa, infections of the respiratory tract that are associated with high mortality rates include inﬂuenza, pneumonia and tuberculosis (York et al., 2011) the frequently used medicinal plants in traditional healthcare to treat these bacterial infections still require scientific validation (York et al., 2012). There are many South

African plants that are traditionally used for the treatment of respiratory ailments –

McGaw et al. (2008) documented almost 180 medicinal plants used for treatment of tuberculosis and related symptoms such as chronic cough, respiratory complaints and fever. In all the studies focusing on plants traditionally used for treatment of respiratory ailments, little attention has been given to medicinal barks (Lall and

Meyer, 1999; McGaw et al., 2008; York et al., 2012; Fomogne-Fodjo et al., 2014;

Dzoyem et al., 2016).

A few popular bark species exhibiting noteworthy antimicrobial activity against respiratory ailments include Prunus africana, Warburgia salutaris, Cinnamomum camphora, Terminalia sericea and Sclerocarya birrea (Rabe and Van Staden, 1997;

York et al., 2012; Ndhlala et al., 2013). These barks are either taken as infusions or decoctions (Musanga cecropioides) (Fomogne-Fodjo et al., 2014), as smoke inhalants (Warburgia salutaris) (Mohagheghzadeh et al., 2006; Van Wyk et al., 2009) or used in steam inhalation (Albizia ferruginea [(Guill. & Perr.) Benth.)] (Fomogne-

Fodjo et al., 2014). The antimicrobial screening of medicinal plants used traditionally

97 for the treatment of respiratory ailments other than TB has been neglected. Hence, many studies associated with treatment of infectious diseases of the respiratory tract have given attention to traditional medicinal plants with potential antimycobacterial properties (York et al., 2012; Van Vuuren and Holl, 2017). The common pathogens associated with respiratory tract infections include Haemophilus influenzae,

Klebsiella pneumoniae, Moraxella catarrhalis, Streptococcus pneumoniae,

Staphylococcus aureus and Mycobacterium species (McGaw et al., 2008; York et al.,

2012; Fomogne-Fodjo et al., 2014; Van Vuuren and Holl, 2017).

3.1.3 Role of bark for gastrointestinal tract ailments

Gastrointestinal infections are predominantly associated with pathogens transmitted from faecal-contaminated water, food or environment, as well as poor hygiene associated with poverty and a poor nutritional status (Ashbolt, 2004; Pandey et al.,

2014). In the year 2000, there were already an approximate 2.4% reported cases of diarrhoea worldwide ascribed to climate change. The World Health Organisation states that an estimated, 2.5 billion people globally lack improved sanitation facilities, and nearly one billion people do not have access to safe drinking water. The vast majority of diarrhoeal diseases in the world (88%) is attributed to unsafe water, sanitation and hygiene (Ashbolt, 2004; Samie et al., 2005; WHO, 2009) with annual death cases due to these conditions reported globally approximately 3.7 % or 54.2 million (Ashbolt, 2004; WHO, 2009).

More than 200 known diseases of the gastrointestinal tract are transmitted through food. The causes of foodborne illness include viruses, bacteria, parasites, toxins, metals, and prions (Mead et al., 1999) and these illnesses are associated with symptoms that vary from mild gastroenteritis to life-threatening neurologic,

98 hepatic, and renal syndromes. In 2005, there were more than 25 000 deaths caused by diarrhoeal diseases in South Africa (Basson, 2009). Medicinal plants used traditionally for the treatment of diarrhoea are reported to have antispasmodic properties; they delay gastrointestinal transit, suppress gut motility, stimulate water adsorption and/or reduce electrolyte secretion (Palombo, 2006). Hence, different

South African populations in various communities depend on a wide range of different medicinal plants for the treatment of gastrointestinal disorders such as diarrhoea and infections caused by intestinal parasites (Samie et al., 2005). South

African rural communities, including northern Maputaland and Bizana, have been reported to lack access to safe drinking water, good hygiene, and sanitation (De Wet et al., 2010; Madikizela et al., 2012). Therefore, antimicrobial studies of traditional medicinal plants used for treatment of gastrointestinal tract ailments are still very relevant in South Africa.

Bark preparations for gastrointestinal tract infections are usually in the form of decoctions and infusions, administered either orally, or as enemas and emetics

(Mabogo, 1990; Pujol, 1990; Hutchings et al., 1996; Van Wyk and Gericke, 2000,

2018; Grace et al., 2003; Van Wyk et al., 2009; De Wet et al., 2010). Considering the fact that barks are rich in tannins (Hutchings, 1989) compared to other plant part, it is the logical choice for the treatment of gastrointestinal ailments (Van Wyk et al., 2009;

Van Wyk and Wink, 2017). Tannins act by denaturing proteins and enzymes, thereby killing all the cells invading the intestines, including bacterial cells. The cells of the mucosa are also killed but because they are part of a multicellular organism, the layer is rapidly regenerated. De Wet and co-workers (2010) reported the frequent use of trees, particularly bark or leaves, for the treatment of diarrhoea in the ethnobotanical survey they conducted in northern Maputaland. Popular medicinal

99 barks with noteworthy antimicrobial activity against a wide range of gastrointestinal tract pathogens include Garcinia livingstonei, Sclerocarya birrea, Terminalia sericea,

Kigelia africana and Syzygium cordatum (Samie et al., 2005; Ndhlala et al., 2013;

Van Vuuren et al., 2015; Van Vuuren and Holl, 2017). The commonly known bacteria associated with gastrointestinal tract infections include Salmonella typhi (typhoid fever), Salmonella paratyphi (paratyphoid fever), Shigella species (bacillary dysentery), Vibrio cholerae (cholera), and enteropathogenic E. coli (gastroenteritis).

Considering the vast potential of barks as possible sources for antimicrobial medication, an investigation was undertaken to screen a selection of 32 medicinal barks purchased from the Johannesburg muthi markets for antibacterial activity.

These barks were chosen because of their traditional use against infectious diseases. According to a review by Van Vuuren and Holl (2017), most studies have focused on collecting plant material in situ or from botanical gardens, while another vital source that has been neglected is the muthi markets. Barks samples purchased from muthi markets are rarely fresh, so that the result may differ from studies where fresh materials are used. Many of the South African populations in townships and peri-urban areas frequently prefer the use of muthi market plant products for their health care benefits.

3.2 Materials and methods

3.2.1 Plant extraction

Out of 70 medicinal barks purchased from the Faraday and Kwa Mai-Mai Muthi

Markets in Johannesburg, 32 barks and 64 crude extracts of methanol and dichloromethane were subjected to antimicrobial screening. The chosen bark samples for antimicrobial screening were based on the traditional medicinal uses

100 supplied by traders at the muthi markets. The bark material was partially cleaned with a brush to remove the lichens, moss and sand or dust particles, which would normally be the case with customers. Pathogens associated with skin, gastrointestinal and respiratory tract infections were selected. Dried bark samples were ground to a fine powder using a blender. Approximately 28 g of each powdered bark material was soaked in methanol and dichloromethane solvent for 48 hours at room temperature. Methanol was chosen as solvent because it is likely to extract a wider diversity of polar compounds than water. It is often suggested that water extracts should be used when studying traditional medicine, because this is the traditional dosage form. However, bark often contains high amounts of triterpenoid glycosides (saponins) and other compounds that may act as emulsifying agents. As a result, traditional decoctions will also contain non-polar compounds. The presence of substantial quantities of non-polar compounds in a water decoction was demonstrated in this study using Elaeodendron transvaalense as an example. DCM was selected to extract non-polar components. Extracts were filtered using a

Whatman No. 1 filter paper and left for evaporation. The total dry yields of the bark extracts are shown in Table 3.1.

3.2.2 Minimum inhibitory concentration (MIC) technique

For antimicrobial testing, crude plant extracts were prepared to a starting concentration of 32 mg/ml dissolved in either acetone or dimethyl sulfoxide (DMSO).

When plant extracts did not dissolve in acetone, sonication was used for approximately 10 min until thoroughly dissolved.

101

Table 3.1 Bark sample yields and selected pathogens for further study.

Yield in grams (g)/ 28 g Bark samples Pathogens Methanol Dichloromethane Albizia adianthifolia Skina and stomachb 0.422 0.051 Albizia versicolor Respiratoryc 0.507 0.074 Balanites maughamii Stomach 0.765 0.138 Bersama lucens Stomach 0.589 0.052 Calodendrum capense Skin 0.276 0.068 Cinnamomum camphora Respiratory 0.253 0.062 Croton sylvaticus Stomach 0.446 0.062 Cryptocarya latifolia Respiratory 0.386 0.079 Dombeya rotundifolia Stomach 0.311 0.065 Ekebergia capensis Skin and stomach 0.422 0.091 Elaeodendron transvaalense Skin and stomach 0.429 0.082 Erythrina lysistemon Skin and stomach 0.334 0.076 Erythrophleum lasianthum Stomach 0.391 0.100 Harpephyllum caffrum Skin and stomach 0.579 0.060 Kigelia africana Stomach 0.208 0.575 Ocotea bullata Stomach 0.495 0.095 Peltophorum africanum Respiratory 0.516 0.061 Pittosporum viridiflorum Skin and stomach 0.494 0.074 Prunus africana Respiratory 0.472 0.046 Pterocelastrus rostratus Respiratory 0.318 0.227 Rapanea melanophloeos Skin and respiratory 0.264 0.094 Rauvolfia caffra Skin and stomach 0.246 0.049 Schotia brachypetala Stomach 0.661 0.051 Sclerocarya birrea Stomach 0.487 0.049 Securidaca longepedunculata Skin and stomach 0.275 0.511 Strychnos henningsii Skin and stomach 0.179 0.053 Syzygium cordatum Skin and stomach 0.225 0.059 Trichilia emetica Stomach 0.484 0.176 Warburgia salutaris Respiratory 0.632 0.134

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Yield in grams (g)/ 28 g Bark samples Pathogens Methanol Dichloromethane Vachellia karroo Stomachb 0.388 0.047 Vachellia robusta Respiratoryc 0.528 0.068 Ziziphus mucronata Skin and stomach 0.413 0.052 aPseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis; bBacillus cereus ATCC 11175, Enterococcus faecalis ATCC 29121, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028 and Shigella sonnei ATCC 9290; cKlebsiella pneumoniae ATCC 13883, Moraxella catarrhalis ATCC 23246, Pseudomonas aeruginosa ATCC 743971 and Staphylococcus aureus ATCC 25923).

The micro-titre plate technique was used to determine the minimal inhibitory concentration (MIC) values of crude plant extracts. The technique involves the use of

96-well micro-titre plates and tetrazolium salts to indicate microbial growth (Eloff,

1998). Ciprofloxacin, dissolved in sterile water, was used as a positive control at a starting concentration of 0.01 mg/ml. The positive control was selected because of its well-known broad-spectrum activity against bacteria and was used in the present study to confirm the antimicrobial susceptibility of the bacterial strains screened.

Negative controls were acetone and DMSO. The growth medium was included as a culture control to confirm the presence of microbial growth in the absence of plant extracts.

Sterile 96-well micro-titre plates were prepared under a laminar flow cabinet unit using an aseptic technique by insertion of 100 µl of Tryptone Soya broth (TSB) into each well. A volume of 100 µl of each plant extract (32 mg/ml), and the positive and negative controls were transferred into the wells in the first row of the micro-titre plates. Serial dilutions were performed by transferring 100 µl of the well content and subsequently diluting the extracts and controls by 50% each time. Crude extracts were initially tested at 8.00 mg/ml (when diluted in broth and culture) in 96-well microplates and serially diluted two-fold to 0.06 mg/ml. A 100 µl of the sub-culture

(bacteria) was added to all the wells of each micro-titre plate. The culture was first

103 diluted in broth (0.5 McFarland standard) and thereafter diluted 1:100 to give a density of approximately 1×106 colony forming units/ml (CFU/ml). Each micro-titre plate was then sealed with a sterile adhesive seal and incubated at 37°C for 24 hours. To confirm purity of the culture, each diluted pathogen-broth mixture was also streaked onto Tryptone Soya agar (TSA) and incubated overnight. After incubation,

40 µl of p-iodonitrotetrazolium (INT) dissolved in sterile water was added to all micro- titre plate wells and used as a microbial growth indicator. The screening test was repeated three times.

3.3 Results and discussions

An MIC value of 1.00 mg/ml or less has here been considered as noteworthy antibacterial activity of medicinal bark extracts, as proposed by various authors, including Van Vuuren (2008).

3.3.1 Skin ailments

A recommendation by Van Vuuren (2008) indicates that when investigating the efficacy of traditional medicinal plants for antimicrobial activity, it is very critical to consider the relevant pathogen in question. Results obtained from the present study

(Table 3.2) reveals the antimicrobial potential of bark extracts against skin pathogens with MIC value ranging from 0.004 mg/ml to 4.00 mg/ml. Sixty-four percent of the medicinal barks showed noteworthy activity (MIC value of ≤ 1 mg/ml) against the

Gram-negative Pseudomonas aeruginosa, 55% against Gram-positive

Staphylococcus epidermidis and 36% against Staphylococcus aureus . These results are quite surprising, as most antimicrobial studies report that Gram-positive bacteria are more susceptible to plant extracts compared to the Gram-negative bacteria due to the impermeable membrane (Rabe and van Staden 1997; Tshikalange et al.

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2005). However, Olajuyigbe and Afolayan (2012), focusing on the in vitro antibacterial and time-kill assessment of crude methanolic stem bark extract of

Acacia mearnsii against bacteria in shigellosis, also noted Gram-negative bacteria being more susceptible to plant extracts compared to the Gram-positive bacteria.

Medicinal barks that demonstrated the most potent antimicrobial activity were recorded from the following plant species: Elaeodendron transvaalense, Erythrina lysistemon, Rapanea melanophloeos and Ziziphus mucronata.

Table 3.2 Antimicrobial activities of medicinal barks that were tested against skin pathogens (mean value for three repetitions). Values are given in mg/ml.

Pseudomonas Staphylococcus Staphylococcus aeruginosa aureus epidermidis Bark samples ATCC 27853 ATCC 25923 ATCC 12228

MeOH DCM MeOH DCM MeOH DCM

Albizia adianthifolia 2.66 2.66 4.00 3.33 4.00 1.33 Calodendrum capense 4.00 2.66 5.33 5.33 4.00 4.00 Ekebergia capensis 1.00 1.00 2.00 5.33 2.00 4.00 Elaeodendron transvaalense 0.83 0.37 1.66 0.33 1.66 0.26 Erythrina lysistemon 0.50 0.50 0.20 0.20 0.12 0.004 Harpephyllum caffrum 0.66 1.66 3.33 4.00 1.00 1.00 Pittosporum viridiflorum 1.33 1.00 3.33 1.66 4.00 4.00 Rapanea melanophloeos 0.66 0.66 1.33 0.50 0.25 0.66 Rauvolfia caffra 1.66 1.66 3.33 4.00 0.50 1.00 Securidaca longepedunculata 1.33 1.66 8.00 2.00 4.00 1.33

Ziziphus mucronata 0.83 0.83 0.50 0.83 0.50 1.33 Ciprofloxacin positive control 0.07 0.05 (µg/ml) 0.02 Acetone negative control 8.00 ˃8.00 ˃8.00 DMSO negative control 8.00 8.00 8.00 Noteworthy activity given in bold

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Elaeodendron transvaalense: The stem bark of E. transvaalense is used traditionally for the treatment of skin rashes and skin infections (Van Wyk and

Gericke, 2000, 2018; Grace et al., 2003). Noteworthy activity against P. aeruginosa

(MIC of 0.37 mg/ml) and against S. aureus (MIC of 0.33 mg/ml) were demonstrated from the dichloromethane extract and the lowest MIC value of 0.26 mg/ml was noted against S. epidermidis. The antimicrobial screening of E. transvaalense stem bark against skin pathogens has been previously neglected, with many studies focusing on screening of pathogens associated with sexually transmitted diseases. In a study by Tshikilange et al. (2005), focused on antimicrobial activity of plants used to treat sexually transmitted diseases, very poor activity was reported, with MIC values of

20.00 mg/ml against S. aureus exhibited by the water extract of the roots. Various authors (Tshikilange et al., 2008; Samie et al., 2010; Mamba et al., 2016; Sigidi et al.

2017) have screened bark extracts, reported noteworthy activity against HIV/AIDS, and selected Candida species.

Erythrina lysistemon: Bark extracts demonstrated the best antimicrobial activity against the bacterial pathogens screened; with MIC values between 0.004 mg/ml and 1 mg /ml. Powdered, bark of E. lysistemon is widely used in traditional medicine for the treatment of sores, wounds and abscesses (Hutchings et al., 1996;

Van Wyk et al., 1997; Grace et al., 2003). Similar MIC values from both methanol and DCM extracts were noted against P. aeruginosa (MIC of 0.50 mg/ml) and S. aureus (MIC of 0.20 mg/ml). Staphylococcus epidermidis was the most susceptible bacteria against both extracts, yielding the lowest MIC value of 0.12 mg/ml

(methanol) and 0.004 mg/ml (DCM).

Bacterial inhibition against S. aureus has demonstrated the efficacy of the traditional use against wounds, since S. aureus is a bacterial strain associated with

106 wound infection (Mabona and Van Vuuren, 2013). Reports from earlier studies

(Rabe and Van Staden, 1997) recorded the methanol bark extract of E. lysistemon to exhibit no bacterial inhibition against S. aureus. Furthermore, Pillay et al. (2001), focused on cyclooxygenase inhibition and anti-bacterial activities of South African

Erythrina species. They reported that ethyl acetate and ethanol extracts from the stem bark of E. lysistemon displayed cyclooxygenase inhibition of more than 90% for both the 50 and 500 µg/ml extracts.

In addition, no antimicrobial activity was observed for E. lysistemon stem bark from the water, ethyl acetate, and ethanol extracts tested against S. epidermidis, P. aeruginosa and E. coli using the disc diffusion assay. However, activity was noted against S. aureus. Pillay and co-workers (2001) observed that bark of Erythrina species is more active in terms of antibacterial activity than the leaves. In this case, the possible reasons for such a huge difference in the antimicrobial activities is the type of solvent extract used and the different antimicrobial techniques used.

However, more recent results by Mabona et al. (2013) demonstrated noteworthy activity against S. aureus with MIC of 0.20 mg/ml from the leaf

(methanol/dichloromethane) (1:1) extract when tested on the same strains as in this study. In addition, Mukandiwa et al. (2012) reported similar MIC values against S. aureus (0.31 mg/ml) from methanol and dichloromethane extracts of the leaves from

E. lysistemon.

Rapanea melanophloeos: The stem bark of Rapanea melanophloeos is used in traditional medicine for the treatment of wounds and as a skin lightener

(Grace et al., 2003). Noteworthy activity against S. aureus with a mean MIC value of

0.50 mg/ml was recorded from the dichloromethane extract. Noteworthy activity with an MIC value of 0.66 mg/ml was noted against P. aeruginosa by both extracts. The

107 lowest MIC value of 0.25 mg/ml against S. epidermidis was demonstrated from the methanol extract. In a study by Madikizela and co-workers (2012), the dichloromethane leaf extract of R. melanophloeos exhibited weak activity against

S.aureus, with an MIC value of 3.12 mg/ml. Variation in reported MIC values could be due to differences in the selected strains of S. aureus (ATCC 25923) and S. aureus (ATCC 12600). Variation observed in antimicrobial screening of crude plant extracts may be attributed to a variety of factors, including the assay technique, the culture medium and the bacterial strain used (Fennell et al, 2004; Reico and Rios,

2005; Van Vuuren, 2008).

Ziziphus mucronata: The stem bark of Z. mucronata is applied topically for treating sores (Mabona and Van Vuuren, 2013) and septic swellings of the skin, boils and wounds (Dzoyem et al., 2016). Pseudomonas aeruginosa demonstrated noteworthy activity with MIC values of 0.83 mg/ml noted from both extracts. The lowest MIC value of 0.50 mg/ml was observed against S. aureus and S. epidermidis from the methanol extract. Results from the present study are in accordance with those reported by Mabona et al. (2013), against S. aureus (1.00 mg/ml) and P. aeruginosa (1.00 mg/ml) using a ratio of 1:1 (methanol/ dichloromethane) solvent extract of the stem barks. Earlier studies by Rabe and Van Staden (1997), reported no activity for the methanol bark extract of Z. mucronata against S. aureus.

3.3.2 Gastrointestinal tract ailments

The medicinal barks that exhibited the most potent antibacterial activity (Table 3.3) against gastrointestinal tract bacteria include Garcinia livingstonei, Erythrina lysistemon, Elaeodendron transvaalense, Bersama lucens, Schotia brachypetala,

Sclerocarya birrea and Ziziphus mucronata.

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Table 3.3 Antimicrobial activities of medicinal bark extracts tested against gastrointestinal tract pathogens (mean value for three repetitions). Values are given in mg/ml.

Bark samples Bacillus cereus Enterococcus Escherichia Salmonella Shigella sonnei

ATCC 11175 faecalis coli typhimurium ATCC 9290

ATCC 29121 ATCC 8739 ATCC 14028

Solvent extracts MeOH DCM MeOH DCM MeOH DCM MeOH DCM MeOH DCM

Bersama lucens 0.33 0.50 0.50 0.50 2.00 1.00 0.66 1.33 1.33 2.00

Croton sylvaticus 0.75 0.50 2.00 1.00 4.00 1.00 2.00 1.66 ˃8.00 3.00

Dombeya rotundifolia 1.33 0.50 0.75 0.66 1.50 1.66 1.00 1.00 2.00 2.66

Ekebergia capensis 0.25 0.50 2.00 2.00 1.00 ˃8.00 2.00 2.00 1.00 2.66

Elaeodendron transvaalense 0.41 0.20 0.50 0.26 0.66 0.41 1.33 1.00 1.00 0.75

Erythrina lysistemon 0.20 0.12 0.16 0.10 1.00 0.41 1.00 1.00 0.16 0.16

Erythrophleum lasianthum 1.33 0.83 1.66 1.33 1.00 2.00 0.83 3.00 1.66 ˃8.00

Garcinia livingstonei 0.06 0.12 0.18 0.75 0.41 2.00 0.25 2.00 0.83 0.50

Kigelia africana 1.66 0.16 2.66 0.83 1.50 1.33 2.00 2.66 4.00 1.66

Ocotea bullata 0.66 0.66 1.33 1.50 1.66 2.66 1.50 ˃8.00 2.66 4.00

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Bacillus Enterococcus Escherichia Salmonella Shigella Bark samples cereus faecalis coli typhimurium sonnei

ATCC 11175 ATCC 29121 ATCC 8739 ATCC 14028 ATCC 9290

MeOH DCM MeOH DCM MeOH DCM MeOH DCM MeOH DCM

Schotia brachypetala 1.00 0.50 0.83 0.66 1.00 0.33 0.83 0.66 0.83 1.33

Sclerocarya birrea 1.00 0.50 0.66 0.50 1.00 0.50 0.83 2.00 0.83 2.00

Strychnos henningsii 2.66 1.00 1.50 ˃8.00 ˃8.00 2.66 ˃8.00 2.00 ˃8.00 8.00

Trichilia emetica 3.33 1.66 0.83 1.66 5.33 1.66 1.66 1.66 3.33 ˃8.00

Vachellia karroo 2.00 0.66 2.00 0.66 8.00 1.66 4.00 1.00 ˃8.00 4.00

Ziziphus mucronata 1.66 0.66 1.00 1.00 0.66 1.66 0.25 1.00 0.50 1.33

Ciprofloxacin positive control ˂0.02 ˂0.02 0.06 ˂0.02 0.07

(µg/ml)

Acetone negative control ˃8.00 ˃8.00 ˃8.00 ˃8.00 ˃8.00

DMSO negative control 4.00 4.00 6.66 6.66 6.66

Noteworthy activity given in bold

110

Garcinia livingstonei: The methanol extract of G. livingstonei demonstrated the best antimicrobial activity against all five gastrointestinal tract bacteria screened.

Garcinia livingstonei is used traditionally for the treatment of diarrhoea (De Wet et al., 2010; Van Vuuren et al., 2015). Good activity with MIC values of 0.25 mg/ml against S. typhimurium and 0.41 mg/ml against E. coli were noted. Excellent activity with the lowest MICs of 0.06 mg/ml against B. cereus and 0.18 mg/ml against E. faecalis were noted.

A recent study by Van Vuuren and co-workers (2015), focusing on the antimicrobial evaluation of plants used for the treatment of diarrhoea in a rural community in northern Maputaland, KwaZulu-Natal, South Africa, yielded similar results from the organic bark extract (1:1 methanol: dichloromethane). The MIC values include 0.12 mg/ml against B. cereus, 0.19 mg/ml against S. typhimurium,

0.34 mg/ml against E. faecalis and 0.38 mg/ml against Shigella flexneri.

Furthermore, isolated chemical compounds from the leaf extract of G. livingstonei were reported to demonstrate excellent activity with MICs of 6.00 and 8.00 μg/ml against Enterococcus faecalis and Escherichia coli respectively (Kaikabo et al.,

2009; Kaikabo and Eloff, 2011).

Bersama lucens: Stem bark of B. lucens is often used traditionally for the treatment of various pharmacological ailments that are not of infectious origin, with the exception of sexually transmitted diseases (Pujol, 1990; Hutchings et al., 1996;

Van Wyk et al., 1997, 2009; Grace et al., 2003; Stafford et al., 2008; Philander,

2011). Its traditional use against stomach ailments is apparently reported here for the first time. The best antimicrobial activity with the lowest MIC value of 0.33 mg/ml was noted against B. cereus from the methanol extract. Noteworthy activity with MIC of

0.50 mg/ml against E. faecalis was demonstrated by both the solvent extracts. The

111 methanol extract exhibited noteworthy activity against the Gram-negative S. typhimurium (MIC of 0.66 mg/ml). Satisfactory activity against S. sonnei (MIC of

1.33 mg/ml) and noteworthy activity against E. coli (MIC of 1.00 mg/ml) were recorded. However, earlier studies by Buwa and Van Staden (2006), on the antibacterial and antifungal activity of traditional medicinal plants used against venereal diseases in South Africa. They reported poor activity of B. lucens stem bark against E. coli. In addition, the ethanol extract exhibited MIC value of 3.12 mg/ml whereas the water extract yielded a MIC value of ˃12.5 mg/ml.

Schotia brachypetala: Bark and roots of S. brachypetala are widely used in traditional medicine as a remedy for dysentery and diarrhoea (McGaw et al., 2002;

Van Wyk et al., 2009). Noteworthy activity was demonstrated for the DCM extract against B. cereus (MIC of 0.50 mg/ml), and against E. faecalis and S. typhimurium with similar activity (MIC of 0.66 mg/ml). Best activity was observed against E. coli with the lowest MIC value of 0.33 mg/ml. The dichloromethane extract from the present study showed potent antibacterial activity against E. coli, compared to the ethanol bark extract that yielded poor inhibition with MIC of 3.31 mg/ml from earlier studies by McGaw et al. (2002). The methanol extract exhibited noteworthy activity against three pathogens.

The methanol extract of the bark has also been screened against other

Shigella species and demonstrated noteworthy activity against Shigella dysenteriae

(MIC of 0.15 mg/ml) and against Shigella flexneri (MIC of 0.31 mg/ml) in a study conducted by Mathabe et al. (2006) focusing on the antimicrobial activity of medicinal plants used to treat diarrhoea. The organic extracts from the present study demonstrated noteworthy activity, which differed with results obtained by Van

Vuuren and co-workers (2015). The (methanol: dichloromethane) extracts from their

112 study exhibited poor antimicrobial activity against B. cereus, E. coli and S. typhimurium with MIC values of 8 mg/ml. In addition, corresponding activity was only observed against E. faecalis with MIC of 0.63 mg/ml. The major reason for such variation in the MIC values could be attributed to the different choice of solvent extract (Eloff, 1998). Successful detection of phytochemicals from plant extracts could vary significantly depending on the type of solvent extract used (Muthee et al.,

2016).

Sclerocarya birrea: Stem bark of S. birrea is widely used in traditional medicine for the treatment of stomach ailments, including diarrhoea (Van Wyk et al.,

2009; De Wet et al. 2010; Van Vuuren et al., 2015), and gastritis, peptic ulcers and stomach cancer (Njume et al., 2011). Bark extracts demonstrated overall good antimicrobial activity against five bacterial strains screened with MIC values ≤1 mg/ml. Noteworthy activity with a similar MIC value of 0.83 mg/ml against S. typhimurium and S. sonnei was recorded from the methanol extract. The dichloromethane extract exhibited similar noteworthy activity with an MIC value of

0.50 mg/ml against B. cereus, E. faecalis and E. coli.

The results from this study differ with those obtained by Moyo et al. (2011) against E. coli (MIC of 3.12 mg/ml) reported from the dichloromethane extract of twig bark from S. birrea. These differences in the MIC activities may be attributed to the age of the plant, thus a high preference of (83%) for mature bark has been noted from traders (Moyo et al., 2011). Higher MIC values demonstrated from the present study on mature bark rather than low MIC values reported from the twig bark further supports the reason why traders would prefer mature bark, simply because of the efficacy.

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3.3.3 Respiratory tract ailments

The majority of the South African population, from rural areas in particular, depend on traditional medicinal plants for the treatment of respiratory disorders (Van Vuuren and Holl, 2017). Many antimicrobial studies of plant species used traditionally for the treatment of respiratory ailments have focused on screening of Mycobacterium bacterial strains associated with tuberculosis (TB) infection, with very little attention given to screening of bacterial strains responsible for other respiratory ailments.

Meanwhile there is a possibility that plants can be effective against colds, coughs or chest complaints, which are caused by bacterial infections other than TB (Lall and

Mayer, 1999; McGaw et al., 2008; York et al., 2012; Van Vuuren and Holl, 2017).

Noteworthy results have been obtained from the present study on the antimicrobial screening of medicinal barks against respiratory tract pathogens.

Staphylococcus aureus was the most susceptible bacteria with 80% inhibition from medicinal barks at a concentration of 1.00 mg/ml. Accumulation of S. aureus in the upper airways may be either random or persistent. Hence, the ability to scavenge iron, co-ordinate gene expression, and horizontal acquisition of useful genetic elements makes S. aureus a successful component of the respiratory flora. The way in which P. aeruginosa colonises the respiratory tract is poorly understood (Prat and

Lacoma, 2016).

Medicinal barks screened from the present study (Table 3.4) exhibited noteworthy activity with the lowest MIC of 0.20 mg/ml against P. aeruginosa. These include Pterocelastrus rostratus, Rapanea melanophloeos, Warburgia salutaris and

Prunus africana.

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Table 3.4 Medicinal bark extracts tested against respiratory tract pathogens (mean value of three replicates). Values are given in mg/ml.

Bark samples Klebsiella Moraxella Pseudomonas Staphylococcus pneumoniae catarrhalis aeruginosa aureus ATCC 13883 ATCC 23246 ATCC 743971 ATCC 25923 Solvent extracts MeOH DCM MeOH DCM MeOH DCM MeOH DCM Cinnamomum camphora 1.66 2.00 1.33 2.00 1.66 2.66 1.00 1.00 Cryptocarya latifolia 1.66 1.66 2.00 0.83 2.00 1.00 1.66 0.83 Peltophorum africanum 1.00 0.66 2.00 2.00 1.66 1.66 2.00 0.83 Prunus africana 1.66 0.50 1.33 1.00 0.83 0.50 1.33 0.83 Pterocelastrus rostratus 0.66 0.83 0.66 0.41 0.25 0.20 0.50 0.50 Rapanea melanophloeos 0.50 0.50 0.50 0.41 0.50 0.50 0.41 0.41 Syzygium cordatum 1.00 0.66 1.66 2.66 2.00 3.33 1.66 2.66 Vachellia robusta 1.00 1.66 1.00 1.00 0.83 1.66 1.66 2.66 Warburgia salutaris 1.00 1.33 2.00 0.41 1.33 0.25 1.66 0.50 Ciprofloxacin (+) control (µg/ml) 0.03 0.05 0.03 0.07 Acetone negative control ˃8.00 ˃8.00 ˃8.00 ˃8.00 DMSO negative control 4.00 4.00 8.00 8.00 Noteworthy activity given in bold

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Pterocelastrus rostratus: Bark decoctions are taken as emetics for respiratory ailments and administered orally for a blocked and wheezing chest (Pujol, 1990;

Grace et al., 2003). The best antimicrobial activity was demonstrated from the extracts against the four bacterial strains tested with MIC values ranging from 0.20 mg/ml to 0.83 mg/ml. Activity against K. pneumoniae and M. catarrhalis with MIC value of 0.66 mg/ml was noted from the methanol extract and 0.50 mg/ml against S. aureus from both extracts. Excellent activity with MIC values of 0.25 mg/ml

(methanol) and 0.20 mg/ml (dichloromethane) were detected against P. aeruginosa.

The broad-spectrum noteworthy antimicrobial activity of P. rostratus is not surprising, as Pterocelastrus bark species have been previously reported to be used for respiratory ailments in traditional medicine (Pujol, 1990; McGaw et al., 2008).

Rapanea melanophloeos: The bark of R. melanophloeos is used to treat respiratory ailments (Dzoyem et al., 2016). Dried powdered bark or fresh bark is chewed for sore throats (Grace et al., 2003). The stem bark of R. melanophloeos is used in many parts of sub-Saharan Africa for the treatment of respiratory tract infections (Amenya et al., 2014). Bark extracts exhibited broad-spectrum antimicrobial activity against the bacterial respiratory tract pathogens tested.

Noteworthy activity with MIC values of 0.50 mg/ml was detected from selected extracts against K. pneumoniae, M. catarrhalis, and P. aeruginosa. The lowest MIC value of 0.41 mg/ml was noted from the dichloromethane extract against M. catarrhalis and from both extracts against S. aureus. Rapanea melanophloeos bark was reported to be used for TB related symptoms, and acetone bark extracts exhibited an MIC value of 5 mg/ml but no activity was observed from the water extracts against Mycobacterium tuberculosis (Lall and Mayer, 1999; McGaw et al.,

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2008). From the results presented here, it is observed that R. melanophloeos could possibly be used for chest ailments other than TB in particular.

Warburgia salutaris: A pinch of powdered bark is taken in a spoonful of cold water or smoked for colds and dry cough. Bark decoctions and infusions are used for chest complaints (Mabogo, 1990; Hutchings et al., 1996; Van Wyk et al., 2009; Rabe and Van Staden, 1997; Mohagheghzadeh et al., 2006; Grace et al., 2003). The dichloromethane extract demonstrated noteworthy activity against S. aureus with an

MIC value of 0.50 mg/ml. Noteworthy activity of 1.00 mg/ml against K. pneumoniae

(methanol extract) and 0.41 mg/ml against M. catarrhalis (dichloromethane extract) were recorded. The lowest MIC value of 0.25 mg/ml against P. aeruginosa was detected from the dichloromethane extract.

Earlier studies by Rabe and Van Staden (1997) reported the activity of a methanol extract, which demonstrated a similar MIC value of 1.33 mg/ml against S. aureus, but no activity was observed against K. pneumoniae. In addition, the aqueous stem bark extracts of W. salutaris exhibited activity against S. aureus, S. epidermidis and B. subtilis with MIC ranging from 0.15 mg/ml to 0.38 mg/ml, thus validating the efficacy of the water extract of stem bark used in traditional medicine.

Prunus africana: Stem bark is used in traditional medicine for treatment of prostate cancer, typhoid, ulcers, malaria, skin rashes, coughing and fever (Bii et al.,

2010; Koros et al., 2016). There were no significant differences in antimicrobial activity between methanol and dichloromethane crude extracts. Dichloromethane extract demonstrated noteworthy activity with an MIC value of 0.83 mg/ml against S. aureus. The results are in agreement with Mwitari et al. (2013) where methanol extract from stem bark exhibited antimicrobial activity with an MIC value of 0.78 mg/ml against S. aureus. In the present study, the highest MIC value recorded was

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1.66 mg/ml against K. pneumoniae from the methanol extract. Noteworthy activity of

1 mg/ml against M. catarrahalis was noted from the dichloromethane extract. A lowest MIC value of 0.50 mg/ml against K. pneumoniae and P. aeruginosa was also observed from the dichloromethane extract.

3.3.4 The influence of solvent extract on the antimicrobial activity of the barks

The methanol extract of Ekebergia capensis demonstrated noteworthy activity with

MIC value of 1.00 mg/ml against E. coli, whereas no antibacterial activity (MIC of ˃8 mg/ml) was noted from the dichloromethane extract. The bark of Vachellia karroo is commonly used traditionally as a remedy for stomach complaints. However, the methanol extract exhibited very poor activity with an MIC value of 8.00 mg/ml, while the dichloromethane extract demonstrated noteworthy antimicrobial activity with an

MIC value of 1.00 mg/ml against E. coli. The stem bark extracts of Trichilia emetica exhibited a similar MIC value of 1.66 mg/ml against B. cereus, E. faecalis, E. coli and

S. typhimurium from the dichloromethane extract. Antimicrobial activity reported by

Van Vuuren and co-workers (2015) on medicinal plants used against diarrhoea at northern Maputaland were more potent compared to this study. A (MeOH/DCM) 1:1 extract from the bark yielded noteworthy activity against B. cereus (MIC of 0.29 mg/ml), against E. faecalis (MIC of 0.29 mg/ml), against E. coli (0.95 mg/ml) and against S. typhimurium (MIC of 0.20 mg/ml).

3.3.5 Overall antimicrobial activity of medicinal barks

In general, the medicinal barks studied here demonstrated great activity against skin, gastrointestinal and respiratory tract pathogens (Figure 3.1) thereby validating the

118 traditional uses. Bacillus cereus was the most susceptible bacterium with 88% of medicinal barks demonstrating noteworthy activity ranging from 0.06 mg/ml to 1.00 mg/ml, followed by K. pneumoniae and M. catarrahalis, with 70% of the barks.

Bacillus cereus is a Gram-positive bacterium that is a common cause of food poisoning with symptoms of nausea, vomiting and diarrhoea. In addition, the ability of B. cereus to cause food poisoning is associated with its resistence to low pH, as the production of enterotoxins is crucial for the survival of B. cereus spores.

Therefore, vegetative bacterium cells ingested with food through the stomach reaches the small intestine alive (Sastalla et al., 2013; Diao et al., 2018). The least susceptible bacteria was a Gram-negative Shigella sonnei, which was inhibited by

23% of the medicinal barks at a ≤ 1 mg/ml concentration. Shigellosis is a severe and life-threatening diarrhoeal infection in both developing and developed countries with annual cases of 91 million reported worldwide. Approximately 95% of shigellosis cases are caused by Shigella sonnei and S. flexneri (Baseer et al., 2017). Much more research is needed in terms of antimicrobial screening of traditional medicinal plants for scientific validation of their efficacy against such pathogens. Some of the medicinal barks were reported by traders to be used for more than one ailment.

Barks such as Elaeodendron transvaalense, Ekebergia capensis, Erythrina lysistemon and Ziziphus mucronata were reported to be used for treatment of skin and gastrointestinal tract ailments. These barks demonstrated noteworthy activity in both categories.

Poorer bacterial inhibition of skin pathogens was anticipated against Gram- negative bacteria (P. aeruginosa in particular) due to their well-known impermeable membranes to plant extracts. However, the opposite is true from the results obtained in the present study.

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100

Percentage Percentage of medicinal barks 20

Figure 3.1 Medicinal barks (32 species) with noteworthy activity (≤ 1 mg/ml) against the tested bacterial strains.

Staphylococcus aureus, a Gram-positive bacterium, was found to be more resistant to bark extracts compared to other bacterial strains of the skin that were tested.

According to Mead et al. (1999), three pathogens, Salmonella, Listeria, and

Toxoplasma species, are responsible for 1 500 deaths each year, more than 75% of those caused by known pathogens, while unknown agents account for the remaining

62 million illnesses, 265 000 hospitalizations, and 3 200 deaths. Bacillus cereus, E. coli and K. pneumoniae have been reported to produce toxins and proteins that promote gastroenteritis and diarrheal diseases (Cock and Van Vuuren, 2015). No less than 54% of the medicinal barks tested inhibited growth of S. typhumirium with

MIC value between 0.25 mg/ml and 1 mg/ml. As a result, medicinal plant research plays a critical role in understanding the value of natural products as antimicrobial agents.

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3.4 Summary

 Popular medicinal barks sold at the Johannesburg muthi markets are a good

source of antimicrobial agents.

 Bacillus cereus was the most susceptible bacterium to medicinal barks screened

for gastrointestinal tract ailments, with inhibition of ≤1 mg/ml from 88% of the 32

medicinal barks tested.

 Pseudomonas aeruginosa was the most susceptible bacterium to the medicinal

barks screened for skin ailments

 The lowest MIC value demonstrated against skin ailments was 0.004 mg/ml for

Staphylococcus epidermidis by Erythrina lysistemon.

 The most active barks for skin pathogens were Elaeodendron transvaalense,

Erythrina lysistemon, Rapanea melanophloeos and Ziziphus mucronata.

 The lowest MIC value demonstrated for barks used for stomach ailments was

0.06 mg/ml against Bacillus cereus by Garcinia livingstonei.

 The most active medicinal barks for stomach pathogens were Garcinia

livingstonei, Erythrina lysistemon, Elaeodendron transvaalense, Bersama lucens,

Schotia brachypetala, Sclerocarya birrea and Ziziphus mucronata.

 Klebsiella pneumoniae and Moraxella catarrahalis were the most susceptible

respiratory tract pathogens with 70% of the medicinal barks inhibiting bacterial

growth at concentration of ≤1 mg/ml.

 Pterocelastrus rostratus demonstrated the lowest MIC value of 0.20 mg/ml

against Pseudomonas aeruginosa.

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The most active medicinal barks for respiratory tract pathogens were Pterocelastrus rostratus, Rapanea melanophloeos, Warburgia salutaris and Prunus africana

3.5 Conclusions

The results of the antimicrobial tests performed on medicinal barks sold on the

Johannesburg muthi markets showed that the bark and/or bark extracts have antimicrobial properties against pathogens associated with the skin, stomach and respiratory tract. These results agree with the anecdotes supplied by the traders.

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CHAPTER 4. CYTOTOXICITY OF SELECTED EXTRACTS AND COMPOUNDS

4.1 Introduction

The long term use and efficacy of traditional medicinal plants as antibacterial agents

(Pillay et al., 2001; Buwa and van Staden, 2006; Samie et al., 2010; Nielsen et al.,

2012; Mabona et al., 2013; Mosa et al., 2014; Van Vuuren and Holl, 2017), has contributed to the perception of these plant species to be relatively safe (Fennell et al. 2004; Verschaeve and Van Staden, 2008). On the other hand, recent scientiﬁc research has shown that many plants used as food or in traditional medicine are potentially toxic, mutagenic and carcinogenic (Verschaeve and Van Staden, 2008).

Cytotoxicity studies of southern African medicinal plants used traditionally for various infectious diseases, has recently gained popularity (Coe et al., 2010; Ndhlala et al.,

2011; Ndhlala et al., 2013; McGaw et al., 2014). However, many cytotoxicity assays of plant extracts have focused on poisonous plants that are lethal against cancerous cells rather than normal human cells (McGaw et al., 2014).

An estimated 750 poisonous substances have been identified from 150 000 plant secondary metabolites, which are present in an estimated 1000 plant species

(Wink and Van Wyk, 2008). Plant toxins are associated with life-threatening conditions in humans, affecting critical organs and key functions such as the central nervous system (CNS), thus interfering with the co-ordination of nerve functions of the body. The most common toxins reported from plant species include neurotoxins, those affecting the brain and CNS, followed by cytotoxins and metabolic toxins that affect organs such as the kidney, liver, heart and lungs (Ndhlala et al., 2013). Groups of phytochemicals that are reported to be extremely poisonous are alkaloids, cardiac

123 glycosides, phorbol esters, lectins and cyanogenic glycosides (Wink and Van Wyk,

2008; Van Wyk et al., 2009; Ndhlala et al., 2013; Naidoo et al., 2013).

The importance of cytotoxicity assays conducted on plant extracts as well as isolated pure compounds is to test their safety as pharmaceuticals or cosmetics, in which case minimal to no toxicity is of major importance (McGaw et al., 2014). Some

African medicinal barks that have been subjected to cytotoxicological studies include

Albizia schimperiana (Oliv.), Alstonia boonei (De Wild.), Berchemia zeyheri (Sond.)

Grubov, Bersama engleriana Gürke, Commiphora edulis (Klotzsch) Engl., Euclea divinorum Hiern, Kigelia africana, Mangifera indica L., Prunus africana, Pterocarpus angolensis, Rauvolfia macrophylla, Searsia lancea (L.f.) F.A.Barkley, Spirostachys africana, Syzygium cordatum, Tetrapleura tetraptera (Schum. & Thonn.) Taub.

Warburgia stuhlmannii Engl. and Withania somnifera (L.) Dunal (Camacho et al.,

2003; Musuyu Muganza et al., 2012; Mwitari et al., 2013; Ndhlala et al., 2013;

McGaw et al., 2014).

Popular southern African medicinal barks with toxic phytochemical properties include Erythrophloeum lasianthum, which is an important traditional medicine used as a snuff for headaches. The active toxic principles include two diterpenoid alkaloids, cassaine and erythrophleine. The bark has been harvested for muthi market trade from as early as 1946 [Gerstner (1946), as cited by Williams et al.

(2013)] and it is still actively traded on the Johannesburg muthi markets. Erythrina caffra stem bark contains toxic alkaloids such as erythraline and erysotrine, with symptoms of intoxication that include paralysis of the skeletal muscles, disturbance of the gastrointestinal (GI) tract and CNS; trembling, unrest, spasm, dyspnoea and cardiac arrest (Ndhlala et al., 2013). Spirostachys africana has a poisonous stem and wood, with symptoms including destruction of mucous membranes, blindness if

124 in contact with the eyes, and death (Ndhlala et al., 2013). Regardless of its deadly effects, pieces of wood are still being actively sold on the Johannesburg muthi markets up to date.

Scientific evaluation of the cytotoxic properties of traditional remedies is of major public concern, as large populations in developing countries depend on or prefer the use of traditional medicines. A brine shrimp toxicity assay of selected medicinal barks sold at the Johannesburg muthi markets was undertaken in this study. The brine shrimp, Artemia species, are small invertabrates occurring in seawater and other saline ecosystems. Artemia is commonly used in laboratory assays as an agent to detect toxic effects by estimating LC50 values (Median lethal concentration). Artemia nauplii have been subjected to a wide range of toxicological studies including antibiotic drugs, engineered nanomaterials, herbicides, insecticides, mycotoxins, pharmaceuticals, anticorrosive agents, plant extracts, oil, wastewater and marine discharge (Meyer et al., 1982; Carballo et al., 2002;

Bussmann et al., 2011; Libralato et al., 2016; Muthee et al., 2016).

The brine shrimp assay is quick, easy and cost effective. It is normally used as a preliminary screening for assessment of toxicity and requires further additional laboratory validation. The brine shrimp test may however, give possible indications of the cytotoxicity of the tested material (Meyer et al., 1982; Pimenta et al., 2003;

Bussmann et al., 2011; Gian et al., 2015).

4.2 Materials and methods

A total of 20 medicinal barks and three isolated compounds were screened for cytotoxicity using the brine shrimp assay. The choice of barks to study was

125 influenced by the antimicrobial activity (Chapter 3) of the extracts. For those that exhibited noteworthy activity, the aim was to determine if the activity is not influenced by the toxic properties of the extracts. In some cases, extracts that demonstrated moderate activity were also selected.

4.2.1 Sample preparation for the brine shrimp assay

Dried powdered stem bark, approximately (1 g), was soaked in 2–3 ml of methanol and dichloromethane. After the solvent extracts had evaporated, the dried solid material was dissolved in 10% aqueous dimethylsulfoxide (DMSO) to obtain a concentration of 2 mg/ml.

4.2.2 The brine shrimp assay

The brine shrimp eggs were incubated at 25°C for 24 hrs. A mass of 16 g sea salt was dissolved in 500 ml distilled water for the preparation of artificial seawater. A sealed conical shaped plastic container was placed in an inverted position in an empty glass beaker. The dried brine shrimp (Artemia franciscana) eggs (1 g) were then added to the prepared artificial seawater on the inverted plastic bottle. A rotary pump was placed at the bottom of the container to create aeration, in the presence of a constant light source. This was done in order to mimic the natural seawater environment to ensure suitable conditions for the eggs to hatch. This mixture was then poured on the micro-titre well lid, placed upside down with full exposure to the light source, to channel or promote accumation of the nauplii in clusters. This was done to increase the number of brine shrimp for collecting (pipetting). Then 48-well micro-titre plates were prepared by adding 400 μl of the salt-water solution

126 containing about 40–60 live nauplii to each well along with 400 μl of the bark extracts. The same volume of 400 μl was used for addition of positive control, solvent control and negative control on the brine shrimp eggs. The positive control used was 1.6 mg/ml potassium dichromate, a highly toxic, frequently used and recommended reference compound for aquatic organisms. The solvent control was

DMSO, which was kept at 10%. The number of dead brine shrimp was obtained by viewing the plates under a light microscope at 40-x magnification following 24 and 48 hrs of exposure to the test samples at room temperature. After counting at 48 hrs, a lethal dose of acetic acid (Saarchem; 100% (v/v); 50 μl) was added to each well, and then a final death count was undertaken in order to calculate the percentage mortality. To calculate the percentage mortality, the number of dead brine shrimp was divided by the initial number of brine shrimps (40–60) and then multiplied by 100

(Mortality = no. of dead nauplii / initial no. of live nauplii x 100). A mortality percentage of 50% and above was regarded as toxic (Bussmann et al., 2011).

4.3 Results and discussions

A recent concern to limit the use of higher animals for toxicological studies arose from the animal rights’ advocates. Hence, the use of brine shrimp is rapidly gaining value as a quick and simple test for detecting the bioactivity/toxicity of plant extracts and guiding phytochemical fractionation in the search for novel compounds useful in health care. In addition, the brine shrimp lethality test can be used as a convenient tool for screening and fractionation in the discovery and monitoring of natural products (Muthee et al., 2016). Table 4.1 displays the brine shrimp lethality activity of selected methanol and dichloromethane extracts from 20 medicinal barks as well as

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three isolated compounds from Erythrina lysistemon bark. All the tested extracts and

isolated compounds exhibited cytotoxic effects with less than 50% mortality. The

negative control demonstrated 0% mortality in every plate. The solvent control

demonstrated a maximum mortality rate of 5.34%. The positive control resulted in a

100% mortality rate of brine shrimp on all the plates within 48 hrs of exposure.

Table 4.1 Cytotoxicity of 20 medicinal bark extracts and some isolated compounds that were tested in the brine shrimp assay.

Solvent Percentage Percentage Total dry extract mortality @ mortality @ yield (g) Plant species 24 hrs 48 hrs

Albizia versicolor Methanol 22.55 37.97 0.0044 Croton sylvaticus Methanol 13.64 32.71 0.0157 Cryptocarya latifolia DCM 18.17 30.88 0.0117 Dombeya rotundifolia DCM 7.84 7.84 0.0034 Ekebergia capensis Methanol 8.97 19.17 0.0028 Elaeodendron transvaalense DCM 22.37 43.00 0.0041 Erythrina lysistemon Methanol 26.51 37.05 0.0147 Erythrina lysistemon DCM 10.21 16.68 0.0188 Garcinia livingstonei Methanol 19.12 46.85 0.0199 Harpephyllum caffrum Methanol 9.87 13.61 0.0067 Pittosporum viridiflorum DCM 12.57 17.94 0.0126 Prunus africana DCM 22.00 32.00 0.0029 Pterocelastratus rostratus DCM 12.99 26.33 0.0185 Rapanea melanophloeos Methanol 14.42 23.09 0.0092 Rapanea melanophloeos DCM 11.84 17.33 0.0039 Schotia brachypetala DCM 24.00 36.10 0.0025 Sclerocarya birrea Methanol 6.38 12.46 0.0026 Securidaca longependunculata Methanol 18.33 30.89 0.0058 Syzygium cordatum Methanol 8.22 20.00 0.0091

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Solvent Percentage Percentage Total dry extract mortality @ mortality @ yield (g) Plant species 24 hrs 48 hrs

Vachellia karroo Methanol 7.99 18.21 0.0084 Warburgia salutaris DCM 13.61 22.33 0.0062 Ziziphus mucronata DCM 8.33 15.18 0.0093 E. lysistemon compound 7 8.59 23.56 0.0033 E. lysistemon compound 8 12.99 22.11 0.0029 E. lysistemon compound 10 6.75 14.05 0.0037

CONTROLS Negative (salt water 32 mg/ml) 0 0

Negative(10% DMSO) 2.11 5.34

Positive (Potassium 89.65 100 dichromate)

The organic bark extracts from the present study did not demonstrate toxicity in the

brine shrimp assay, as the mortality rate displayed by medicinal barks was less than

50%. Barks from plant species such as Ziziphus mucronata, Sclerocarya birrea,

Harpephylllum caffrum and Dombeya rotundifolia displayed minimal toxicity with less

than 15% mortality after the 48-hour exposure period. Garcinia livingstonei methanol

extract displayed higher levels of toxicity amongst the other barks with 46.85%

mortality. The dichloromethane extract from Dombeya rotundifolia exhibited the least

activity with the lowest mortality of 7.84%.

Differences in polarities of solvent extracts used for similar plant species will

yield a diverse range of quantitative differences in phytochemicals, which may result

in a different response to the brine shrimp assay. The methanol and DCM extract of

the same species displayed varying toxicity values as observed for Erythrina

129 lysistemon and Rapanea melanophloeos. Bussmann et al. (2011) also observed similar results of varying toxicity values between the same plants species because of different solvent extracts.

Erythrina lysistemon dichloromethane extracts demonstrated potent antibacterial activity with the lowest MIC value of 0.004 mg/ml against

Staphylococcus epidermidis and ≤ 1 mg/ml for all tested bacteria associated with the skin and gastrointestinal tract. The isolated E. lysistemon compounds (Table 4.1) displayed low toxic levels in the brine shrimp assay with mortality rates ranging from

14.05% to 23.56% while these individual compounds exhibited excellent antimicrobial activity with the lowest MIC of 1.20 µg/ml against B. cereus.

From the lethality test results of this study, it can be concluded that the noteworthy activity exhibited by the bark extracts is not strongly influenced by the presence or absence of toxic properties of the bark material, since they displayed mortality rates of less than 50%, which is regarded as non-toxic. These results support the safety and efficacy of the relevant traditional medicinal uses. Various authors have done screening of many medicinal plants for various infectious diseases as well as isolated compounds and proceeded to use the brine shrimp lethality test in order to assess the safety of these natural products (Pimenta et al.,

2003; Bussmann et al., 2011; Nguta and Mbaria, 2013; Tanamatayarat, 2016).

The ethyl acetate and methanol extracts from the stem bark of Garcinia livingstonei was examined for cytotoxicity in the human melanoma Cell line MeWo, and the extracts demonstrated toxicity with 100 µg/ml causing greater than 80% cell death. However, isolated pure compounds from the bark (morrelloflavone, morrelloflavone-7’’-sulphate and sargoal) only caused between 10% and 20% cell death under 25 µM exposure and were therefore considered non-toxic. At the same

130 time, they demonstrated the desired efficacy for the traditional use as skin lighteners by inhibiting melanin production (Mulholland et al., 2013).

Cock and Van Vuuren (2015) evaluated the toxicity of aqueous and methanolic leaf and selected bark extracts of South African plants using a modiﬁed

Artemia franciscana nauplii lethality assay. In this review, it was reported that methanol and water extracts from the stem bark of Warburgia salutaris and

Syzygium cordatum exhibited less than 50% mortality rate in the brine shrimp test.

Furthermore, the leaf methanol and water extracts of Vachellia karroo, Pittosporum viridiflorum, Schotia branchypetala, Sclerocarya birrea and Ziziphus mucronata demonstrated low toxicity in the brine shrimp assay, with less than 50% mortality, while some of these plants induced mortality below 20% following 24 hrs and 48 hrs of exposure, indicating that the extracts are of low toxicity. Therefore, the reported results are in accordance with the results for bark extracts, indicating the overall low lethality activity for both the leaves and barks. In some cases, only a particular part of a plant is toxic and not the entire plant. For example, Bersama lucens was found to contain toxic properties only in the leaves and roots and not in the bark (Ndhlala et al., 2013). The cytotoxicity assay of the acetone leaf extract of Pittosporum viridiflorum was considered non-toxic against Vero monkey kidney cells which was determined by using the 3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

(MTT) reduction assay (Elisha et al., 2016).

In a review by Maroyi (2017), focusing on the ethnomedicinal uses, phytochemistry and pharmacology of Vachellia karroo, acute toxicity of the shoots was evaluated. Extracts caused deaths in rats at higher doses. However, oral treatments in rats with an extract at 800 mg/kg did not cause any signiﬁcant change in the red blood cell count, packed cell volume, haemoglobin concentration, mean

131 corpuscular volume, mean corpuscular haemoglobin concentration, mean corpuscular haemoglobin, white blood cells and its differentials. The author recommended that the plant should be used with great caution for traditional medicinal purposes.

Toxic medicinal plants continue to be used widely in traditional medicine, regardless of their poisonous effect (Ndhlala et al., 2013). Traders from the

Johannesburg muthi markets were very aware of the toxic effects of Erythrophleum lasianthum bark and gave a warning of the symptoms if a person exceeds the recommended dosage. Hence, it is critical to adhere to the correct dosage in order to avoid harmful side effects. A review by Ndhlala et al. (2013) focusing on the toxicology of some important southern African medicinal plants, recorded that 80% of the plant species used in traditional medicine contain toxic properties. It is clear that when a traditional medicine is effective and biologically active, it has the potential to be harmful at very high doses. The same is true (or even more so) for pure chemical compounds that are used in Western biomedicine.

In most cases, the factors contributing to acute poisoning from traditional medicinal plants are attributed to misidentification, incorrect preparation or inappropriate administration and dosage. This is largely influenced by self- administration rather than the innate risks of using traditional health care (Fennell et al., 2004) and the severity depends on the route of administration, growth stage or plant part, amount utilised, the plant species, solubility of the toxins and susceptibility of the victim (Ndhlala et al., 2013). According to Verschaeve and Van Staden (2008), most of the traditional medicinal plants have never been the subject of exhaustive toxicological tests such as are required for modern pharmaceutical compounds.

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Hence, toxicological studies are vital in the developmental process of botanical products to safe and effective drugs (Amenya et al., 2014).

4.4 Summary

 Medicinal bark extracts subjected to the brine shrimp lethality assay were

found not to be toxic, despite the noteworthy MICs exhibited by some of the

extracts.

 The outcome of screening encourages further projects aimed at isolation and

elucidation of active metabolites in antimicrobial outcomes, where possible

synergisms and antagonisms can also be considered.

 If poisonous metabolites are present in the extracts, their concentrations are

too low to have noticeable toxic effects on the brine shrimp.

4.5 Conclusions

All the medicinal barks that were screened in the current study were non-toxic.

Therefore, the antimicrobial activity of medicinal bark extracts was apparently not due to toxic compounds. There are many instances where bark extracts showed noteworthy antimicrobial activity against a number of pathogens, while displaying low mortality rates values, e.g. Erythrina lysistemon, Rapanea melanophloeos, and

Sclerocarya birrea.

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CHAPTER 5. ANTIMICROBIAL ACTIVITY OF ISOLATED COMPOUNDS

5.1 Introduction

Historically, pharmacological screening of natural or synthetic compounds has been the basis of countless therapeutic agents. At least 130 drugs currently in use globally, are pure compounds extracted from higher plants or modified further synthetically (Mahesh and Satish, 2008). Natural products continue to provide unique structural diversity in comparison to standard combinatorial chemistry.

Medicinal plants are the richest bio-resource of drugs of traditional system of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediaries and chemical entities for synthetic drug (Muthee et al.,

2016).

Biological activities of natural products depend on the presence of certain chemical compounds, in particular secondary metabolites, and the presence of these compounds may correlate with plant classification (McGaw et al., 2002).

The increase of antibiotic resistance of microorganisms to conventional drugs has required the search for new, efficient and cost effective ways to manage infectious diseases (Samie et al., 2005; Oyedemi et al., 2012). According to the review by Van Vuuren and Holl (2017), antimicrobial data of traditional medicinal plants become valuable only when included with the chemistry, toxicity and/or other biological properties.

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5.2 Materials and methods

5.2.1 Compound isolation and structural elucidation of Elaeodendron transvaalense

Isolated compounds 1, 2 and 3: Dried and powdered bark (427 g) was soaked in 2 L of dichloromethane: methanol (1:1) for 48 hrs. The solvent was then filtered through celite and evaporated completely. The extract was subjected to flash chromatography over silica gel using 20% ethyl acetate in cyclohexane (v/v) as mobile phase. Three compounds were isolated and identified as lup-20(30)-ene-3α,

29-diol (1) (23.3 mg), 6β-hydroxylup-20(29)-ene-3-one (2) (67.1 mg) and 30- hydroxylup-20(29)-ene-3-one (3) (94.4 mg). NMR spectra for the three compounds isolated were generated on a 500 Mhz Bruker Avance (Bruker, Germany) and the

13C spectra were matched by Dr N.J. Sadgrove to published values; 1 was matched to spectra reported by Ullah et al. (1999), 2 was matched to spectra by Hisham et al.

(1996), and 3 to spectra by Tinto et al. (1992). The 13C spectra of the three compounds are provided as Appendix 2.

Isolated compound 4: Dried and powdered bark (327 g) was soaked in 2 L of distilled water for 48 hrs. The aqueous phase was separated by filtration and the more lipophilic fraction was partitioned into ethyl acetate using three consecutive washes of 100 ml. The organic phase was separated and evaporated to dryness and triterpenes were partitioned into DCM from the residue. Isolated compound 4 remained in the residue at >90% purity. The residue was subjected to column chromatography using a 20:80 ratio of hexane: ethyl acetate to afford 1.1g of pure 4, which was identified by Dr Sadgrove by comparing NMR spectra to that provided by

Drewes and Mashimbye (1993).

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5.2.2 Compound isolation and structural elucidation of Erythrina lysistemon

Dried and powdered bark (291.7 g) was soaked in 1500ml of 1:1 mixture of dichloromethane: methanol for 48 hrs. The solvent was then filtered through celite and evaporated completely to afford 9.33 g of extract. Dry flash chromatography over silica gel (v/v of 20% acetone, 20% ethyl acetate, 60% pet ether) was used to reduce the crude extract to 0.58 g, where the majority of the polar compounds adhered to the silica gel. The 0.58 g was then loaded onto a silica gel column with starting mobile phase of 10% ethyl acetate in 90% toluene (v/v). This was incrementally increased to 30% ethyl acetate in 70% toluene (v/v). Thin layer chromatography on aluminium-backed plates was used to guide fraction combining

(20% ethyl acetate, 80% pet ether) and seven isolated compounds were collected.

NMR spectra for the seven compounds were generated on a 500 MHz Bruker

Avance (Bruker, Germany) and the 13C and 1H spectra were matched by Dr

Sadgrove to published values. 1 (erybraedin A, 93.7 mg) and 2 phaseollidin were matched to spectra reported by Mitscher et al. (1988), 3 (abyssinone V-4’ methyl ether, 236 mg) was matched to spectra by Yenesew et al. (1998), 4 (eryzerin C, 32.6 mg) to spectra by Tanaka et al. (2003), 5 (alpumisoflavone, 164 mg) to spectra by

Juma and Majinda (2006), 6 (lysisteisoflavanone, 176 mg) to spectra by El-Masry et al. (2002) and 7 (cristacarpin, 116 mg) to spectra by Dagne et al. (1993). The 1H

NMR and 13C NMR spectra of these compounds are provided as Appendix 3 and

Appendix 4, respectively).

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5.2.3 Compound isolation and structural elucidation of Warburgia salutaris

Bark of W. salutaris was obtained from cultivated specimens and purchased from the muthi market. Bark was hydrodistilled using a Clevenger-type apparatus. Vouchers specimens of the cultivated trees were lodged at the University of Johannesburg

Herbarium (JRAU).

Extraction, isolation and chemical assignment

For antimicrobial testing, methanol and dichloromethane were used as extraction solvents to extract dry plant material. The outcome of antimicrobial testing influenced the decision to use a ratio of 1: 1 methanol: dichloromethane in 1500ml as the extraction solvent for further isolation (717.72 g of powdered bark). Essential oils were extracted by hydrodistillation using a Clevenger-type apparatus.

Isolation of components from essential oils and bark extract (MeOH: DCM) used flash column chromatography exclusively over silica gel with cyclohexane and ethyl acetate as the mobile phase. Essential oils were subjected to column chromatography with 1:9 of ethyl acetate: .cyclohexane. The bark extract was chromatographed with 1:9 of ethyl acetate: cyclohexane (10% EthOAc) as eluent but the ratio was gradually increased to 2:8.

The identities of all isolated compounds were determined by Dr Sadgrove by comparison of 1H and/or 13C NMR spectra with published values using a Bruker

Avance 500Mhz NMR. Compounds that were identified included drimenol 1 (24 mg)

(Urban and Capon, 1996), E-nerolidol 2 (114 mg) (Machado et al., 1998), 12α- acetal-polygodial 3 (102.6 mg) (Ying et al., 1995), polygodial 4 (72.4 mg)

(Mashimbye et al., 1999), ugandensidial 5 (188 mg) (Cortes et al., 1990) and warburganal 6 (57.6 mg) (Mashimbye et al., 1999).

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5.2.4 Antimicrobial screening of isolated compounds

The antimicrobial testing was undertaken as per Chapter 3, Section 3.2.2. However, for isolated compounds, starting concentrations varied from 1 mg/ml to 5 mg/ml, depending on the yield of isolated compounds. The final starting concentration of isolated compounds ranged from 0.25 to 1.25 mg/ml or 0.002 to 0.009 mg/ml in the last row.

5.3 Results and discussion (chemical structure of isolated compounds)

Dr Sadgrove elucidated the chemical structures of seventeen isolated pure compounds from the present study.

5.3.1 Elaeodendron transvaalense

A dichloromethane extract yielded three major triterpenes and a polyphenol as shown in Figure 5.1. The identity of compounds 1─3 were assigned as follows; lup-

20(30)-ene-3α, 29-diol (1), 6β-hydroxylup-20(29)-ene-3-one (2) and 30-hydroxylup-

20(29)-ene-3-one (3) and 4’-O-methyl epigallocatechin (4).

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Figure 5.1 Structures of chemical compounds isolated from the bark of Elaeodendron transvaalense. Chemical structures drawn by N.J. Sadgrove.

The study by Tshikalange and Hussein (2010) assigned 1, 3 and 4 but the current study is the first report of 2 in the stem bark of E. transvaalense, although it was previously isolated from root bark of the same species under its previous name

Cassine transvaalensis (Drewes et al., 1991). The three compounds are lupenol derivatives but 1 is a diol. Compounds 1 and 3 are 3-oxo-lupenol derivatives and so may be of relevance in the anticancer activity that has been described anecdotally for this species in traditional medicine, since the greatest cytotoxicity was ascribed to this form in Tshikalange and Hussein (2010), as well as Sheng-Ding et al. (1984).

Aldehyde lupane derivatives demonstrated even greater cytotoxicity (Sheng-Ding et al. 1984), but these were not isolated in the current study.

In the previous study by Tshikalange and Hussein (2010), the diol 1 was least cytotoxic. However, in the current study the same diol 1 demonstrated the most

139 pronounced antimicrobial activity. Thus, structure activity relationships suggest that reduction of the 3-oxo group on 2 and 3 enhances antimicrobial activity and reduces cytotoxicity. The structural similarity of 1 to lupeol, a dietary 3-oxo lupenol triterpene, is evident. Lupeol is well-known for its powerful anti-inflammatory and anti-cancer activities (Saleem, 2009) and it will be of interest to investigate possible anti- inflammatory effects of the lupenol triterpenes in E. transvaalense.

5.3.2 Erythrina lysistemon

Figure 5.2 Structures of chemical compounds isolated from the bark of Erythrina lysistemon. Chemical structures drawn by N.J. Sadgrove.

Seven major flavonoid derivatives were isolated and identified, as shown in

Figure 5.2. The identities of the isolated compounds were determined by Dr

140

Sadgrove as follows: erybraedin A (1), phaseollidin (2), abyssinone V-4’ methyl ether

(3), eryzerin C (4), alpum-isoflavone (5), lysisteisoflavanone (6) and cristacarpin (7), as shown in Figure 5.2.

One flavonoid (3), two isoflavonoids (5, 6), three pterocarpans (1, 2, 7) and one isoflavan (4) were isolated in the current study. Four of the derivatives are di- prenylated (1-4), two are prenylated (6, 7) and one is pyrano-cyclised over the phenolic hydroxyl group (5), where the prenyl group is closed into a heterocycle with reduced hydrophobicity.

Compound 4 is reported from E. lysistemon for the first time. Only compounds

1, 3 and 5 have been previously studied for antimicrobial activity (Chukwujekwu et al., 2011; Wanjala et al., 2002) using the accepted method of Eloff (1998).

Compounds 1–7 are widely distributed across the genus. For example, cristacarpin (7) has also been isolated from E. burana Chiov. (Dagne et al., 1993) and was demonstrated to be potentially active against cancer through their selective activity against DNA damage repair deficient yeasts. Both 1 and 2 are also present in extracts from the Nigerian medicinal plant E. mildbraedii (Mitscher et al., 1988).

Compound 3 is also present in the Kenyan species E. burttii Baker.f. (Yenesew et al., 1998), which together with 5 demonstrated in vitro estrogenic (Nde et al., 2012) and anti-cholesterol gallstone formation activity (Mvondo et al., 2015).

The genus Erythrina is well known for its alkaloids, often termed ‘Erythrina alkaloids’, with over 110 known compounds documented (Parsons and Palframan

2010). A number of various flavonoid derivatives has also been previously reported from the stem bark of E. lysistemon. More than 50 flavonoids have been isolated from the Erythrina genus over the past two decades (El-Masry et al., 2002). In addition, El-Masry et al. (2002) isolated three prenylated flavonoid derivatives from a

141 dichloromethane stem bark extract of E. lysistemon in Egypt. Pillay et al. (2001) isolated the antibacterial isoflavone weighteone from an ethanol extract of the stem bark of E. lysistemon.

5.3.3 Warburgia salutaris

Figure 5.3 Structures of chemical compounds isolated from the bark of Warburgia salutaris. Chemical structures drawn by N.J. Sadgrove.

Six compounds were isolated (Figure 5.3) and their chemical structures determined by Dr Sadgrove. The two major compounds isolated from the bark essential oils of a cultivated tree were drimenol (1) and E-nerolidol (2). Further components isolated from the bark purchased from muthi market by solvent extraction included the known non-volatile sesquiterpenes 12α-acetal-polygodial (3), polygodial (4), ugandensidial

(5) and warburganal (6).

The essential oil component E-nerolidol 2 is the only non-drimane compound assigned in the current study and is reported in Warburgia for the first time. This is

142 not surprising since the chemistry of essential oils is poorly documented for

Warburgia salutaris. Furthermore, drimenol 1, 12α-acetal-polygodial 3 and ugandensidial 5 were also identified for the first time in W. salutaris. Previous chemical investigations on W. salutaris revealed the presence of drimane sesquiterpenes such as isopolygodial, warburganal, polygodial, mukaadial, salutarisolide (Mashimbye et al., 1999), muzigadial (Rabe and Van Staden, 2000) and 11α-hydroxycinnamosmolide (Madikane et al., 2007). Another study identified furans and furanones (Mohanlall and Odhav, 2009), but these were not detected in the current study. Drimane sesquiterpenes are known to possess antimycobacterial, antibacterial, antifeedent, antifungal, anti-inflammatory and antimycotoxigenic activity

(Maroyi, 2014; Leonard and Viljoen, 2015). Other prominent international botanical sources of drimane sesquiterpenoids are Polygonum (Fukuyama et al., 1982),

Drymis winteri (Montenegro et al., 2014), Canella (Ying et al., 1995) and Tasmannia

(Mathie et al., 2017), to name a few. The drimane-type sesquiterpenes confer a pepper-type aroma or flavour to these species (Mathie et al., 2017), which obviously influences vernacular and botanical names such as Tasmanian pepper (Tasmannia lanceolata), water pepper (Polygonum hydropiper) and pepperbark tree (Warburgia salutaris).

It is evident from the current study that chemical variability can be expected in

W. salutaris. This is not a surprise since the three previous chemical studies of W. salutaris also demonstrated chemical variation. A study of specimens from the

Zoutpansberg Mountains of the Venda region (Limpopo province) identified warburganal, polygodial, mukaadial, isopolygodial and a new lactone, which the authors named salutarisolide (Mashimbye et al., 1999). Another study of cultivated specimens from Silverglen Nature Reserve near Durban reported muzikadial (Rabe

143 and Van Staden, 2000), which was not isolated in the earlier study. A separate study from the cultivated trees at Silverglen identified 11α-hydroxycinnamosmolide

(Madikane et al., 2007), which they described as having potent anti-mycobacterial activity. The crude extract itself demonstrated this anti-mycobacterial activity

(Leonard et al., 2010).

In the current study, further chemical variation was observed, from bark of cultivated tree and purchased from the muthi market. Both 3 and 5 are not only reported in W. salutaris for the first time, but they also constitute the dominant components in the solvent extracts. Ugandensidial 5 was previously isolated from W. ugandensis and W. stuhlmannii (Leonard and Viljoen, 2015). However, the current study constitutes the first reported occurrence of 12α-acetal-polygodial 3 in the genus Warburgia. It was previously isolated from another member of the family

Canellaceae, Canella winterana (Ying et al., 1995).

5.4 Results and discussion (Antimicrobial screening of isolated compounds)

5.4.1 Antimicrobial activity of compounds from Elaeodendron transvaalense

Column chromatography of the dichloromethane crude extract afforded three main compounds (1–3) (Figure 5.1). Compound 1 demonstrated the most pronounced activity by inhibiting all bacterial strains selected (Table 5.1), with the lowest MIC value of 0.104 mg/ml against P. aeruginosa and S. typhimurium. Poor bacterial inhibition was observed against S. sonnei by most of the plant extracts.

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Table 5.1 Minimum inhibitory concentrations (mg/ml) of solvent extracts and isolated compounds from Elaeodendron transvaalense bark

E. coli P. S. aureus S. S. S. ATCC aeruginosa ATCC epidermi- sonnei typhimu- 8739 ATCC 25923 dis ATCC ATCC rium 27853 12228 9290 ATCC 14028 E. transvaalense 1.33 1.00 1.33 1.66 1.00 1.33 MeOH

E. transvaalense 0.66 0.41 0.50 0.25 0.75 1.00 DCM

Compound 1 0.23 0.10 0.15 0.20 0.31 0.10

Compound 2 0.26 0.26 0.52 0.52 0.83 0.15

Compound 3 0.52 1.04 0.31 0.31 0.62 0.31

Compound 4 0.09 0.18 0.18 0.18 0.18 0.09 Ciprofloxacin 0.15 0.07 0.10 0.15 0.10 ˂0.02 (µg/ml)

Acetone ˃8.00 ˃8.00 ˃8.00 ˃8.00 ˃8.00 ˃8.00

DMSO 8.00 8.00 4.00 8.00 5.33 4.00

The isolated compounds 1 and 2 exhibited moderate activity against E. coli with MIC values of 0.23 mg/ml and 0.26 mg/ml respectively. The lowest MIC values were noted from compounds 1 and 2 against S. typhimurium at 0.10 mg/ml and 0.15 mg/ml respectively. The identity of compound 4 was assigned as 4’-O-methyl epigallocatechin, which was identified in the extracts of E. transvaalense in an earlier study (Tshikalange and Hussein, 2010). The higher yield in aqueous extracts and the generally lower MIC values (Table 5.1) provide a clear indication that this compound is of importance in the antimicrobial outcomes in traditional practice. However,

145 combination studies of the triterpenes and 4 should be conducted to provide a clearer picture of such antimicrobial activity in practice.

Thus, the results show that the stem bark of E. transvaalense contains three main lupenol triterpenes and one polyphenol with moderate antimicrobial activity against the selected pathogens. They also provide a plausible scientific rationale for the popularity of the bark in African traditional medicine.

5.4.2 Antimicrobial activity of compounds from Erythrina lysistemon

The antimicrobial activities presented in Table 5.2 clearly demonstrate that this class of compound (prenylated isoflavonoid derivative) has noteworthy antimicrobial activity as defined by the criteria set out by Van Vuuren and Holl (2017). The more potent activity of the DCM extract means that the active antimicrobial metabolites are of mid-range polarity and not likely to be glycosylated conjugates. Thus compounds

1–7 are the most likely to be important in antimicrobial outcomes.

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Table 5.2 Minimum inhibitory concentrations (µg/ml) of solvent extracts and isolated compounds from Erythrina lysistemon bark

S. P. S. B. cereus E.coli epidermi- aeruginosa aureus ATCC ATCC dis ATCC ATCC 11175 8739 ATCC 27853 25923 12228

125 1000 1000 125 125 E. lysistemon MeOH

E. lysistemon DCM 125 500 500 125 4.80

Compound 1 1.20 2.40 19.50 2.40 2.40

Compound 2 9.70 19.50 19.50 9.70 4.80

Compound 3 19.50 313 313 39 156

Compound 4 9.70 4.80 4.80 4.80 2.40

Compound 5 31 125 150 31 125

Compound 6 2.00 7.00 31 62 31

Compound 7 156 625 78 156 312

Ciprofloxacin 0.02 0.07 0.07 0.07 0.07

Acetone 8.00 8.00 8.00 8.00 8.00

Erybraedin A (1) was identified in extracts of E. latissima and was one of the most active compounds tested in the study by Wanjala et al. (2002). Abyssinone IV was the other most potent compound, which is related to 3 of the current study, differing only by the presence of a methoxy group on the 4’ carbon of 3. The MIC

147 value for 1 by Wanjala et al. (2002) was lower than the outcome achieved in the present study, which is not surprising since there is no consistency of values across the literature. However, the general trend is that compounds 1, 3 and 5 demonstrated more potent antimicrobial activity in other studies (Chukwujekwu et al.,

2011; Wanjala et al., 2002) as compared to the results obtained in this study. For example, 1 gave 0.55 μg/mL against S. aureus in the study by Wanjala et al. (2002), whereas 2.4 μg/mL was achieved in the current study (Table 5.2). In the study by

Chukwujekwu et al. (2011), 5 gave 3.9 μg/mL against both S. aureus and E. coli, but here it gave 31 and 125 μg/mL respectively. For the same two pathogens, 3 gave 31 and 3.9 μg/mL respectively (Chukwujekwu et al., 2011), whereas 59 and 260 μg/mL was demonstrated in this study.

However, from a structure activities perspective, the relative MIC values in

Table 5.2 make sense. It appears that prenylation enhances activity and diprenylation further enhances it. For example, compound 5, which has no prenyl group, demonstrated lesser activity compared to the other compounds (except 3 and

7). It is hypothesized that prenylation enhances microbial membrane penetration by the attachment of a strongly lipophilic arm to the molecule (Araya-Coutier et al.,

2017). From Table 5.2, it appears that methoxylation of an OH group on the same ring as the prenyl moiety reduces the activity against microbes. This is evident because the least activity is derived from compounds 3 and 7. This observation is generally supported by other works on methoxylation in chalcones (Cushnie and

Lamb, 2011) and the study by Rukachaisirikul et al., (2007) which screened the same class of compounds against Mycobacterium tuberculosis. However, the enhanced antimicrobial activity of 6 may be explained by the presence of another

148

OH group at position 6‘, on the same ring as the prenyl and methoxy group

(positions 3‘and 4‘respectively).

Table 4.1 demonstrates that three of the isolated compounds (1–3) and the crude extracts have low cytotoxicity against brine shrimp and no cytotoxicity at 1 mg/mL. Unfortunately, compounds 4–7 were exhausted in antimicrobial testing and were not screened for brine shrimp lethality in the current study. However, crude extracts demonstrated no cytotoxicity, again with no cytotoxicity at 1 mg/mL (Table

4.1), so it is feasible that screening of compounds 1–3 is a reflection of the general lethality of the other metabolites. However, in a separate study lethality (10 μg/mL) was measured for compound 5 (Juma and Majinda, 2006), which is the most chemically unique compound isolated in this study, since the prenyl group is pyranocyclised. Thus, this compound needs to be examined further to confirm the published result. Furthermore, in a separate study the compound phaseollidin was demonstrated to be toxic against three cell lines (Dagne et al., 1993).

5.4.3 Antimicrobial activity of compounds from Warburgia salutaris

The antimicrobial activities of compounds extracted from Warburgia salutaris is summarised in Table 5.3. Warburganal is probably the best known of the compounds isolated in the current study for biological activities. It has potent antifungal, molluscicidal and antifeedant activity (Kubo et al. 1977). However, polygodial and muzikadial have also been examined by Kubo and Taniguchi (1988) and showed significant antifungal activity.

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The antimicrobial activity of muzikadial was also significant, with values ranging from 12.5 – 100 ug/ml (Rabe and van Staden, 2000), which is more active than the isolated compounds tested in the current study.

In the study by Rabe and Van Staden (2000), muzikadial was also tested against the respiratory pathogen Klebsiella pneumoniae but had low activity.

Warburganal, polygodial and muzikadial have also been demonstrated to exhibit strong cytotoxicity against cancer cell lines (Leonard and Viljoen, 2015; Montenegro et al., 2014). Polygodial was demonstrated to produce no mutagenicity at concentrations below toxicity (Anke and Sterner, 1991).

Table 5.3 Minimum inhibitory concentrations (mg/ml) of solvent extracts, essential oils and isolated compounds from Warburgia salutaris bark.

Klebsiella Moraxella Pseudomo Staphylo-

pneumoniae catarrhalis nas coccus

ATCC ATCC aeruginosa aureus

13883 23246 ATCC ATCC

743971 25923

W. salutaris bark (MeOHl) 1.00 2.00 1.66 1.66

W. salutaris bark (DCM) 1.00 0.42 0.25 0.50

EO bark (cultivated tree) 0.50 1.00 0.5 1.00

EO bark (muthi market) 0.83 0.50 0.25 0.50

Drimenol (1) 0.20 0.06 0.31 0.06

E-nerolidol (2) 0.31 0.03 0.26 0.41

12α-Acetal-polygodial (3) 0.15 0.62 0.62 0.52

Polygodial (4) 0.02 ˃0.25 ˃0.25 ˃0.25

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Ugandensidial (5) 0.13 0.10 0.07 0.13

Warburganal (6) 0.13 0.20 0.10 0.15

Ciprofloxacin positive 0.07 0.07 0.06 0.06

control (µg/ml)

Acetone negative control ˃8.00 ˃8.00 ˃8.00 ˃8.00

DMSO negative control 4.00 4.00 4.00 4.00

The other compounds isolated in the current study are less known for their biological activities. Not much is known of the biological activities of drimenol, 12α- acetal-polygodial and ugandensidial (also known as cinnamodial). The antimicrobial activities demonstrated in the current study are therefore relevant.

Since W. salutaris has its strongest tradition in smoke inhalation therapy, it is necessary to consider the biological activities of metabolites that are capable of stability in the gaseous phase. The essential oil components are obviously of relevance in this context. The dialdehyde molecules risk derivatisation at higher temperatures, but there is a chance that some will be exposed to the mucous membranes of the lungs in such smoke therapies. However, it may be of more relevance to consider drimenol in this scenario. In the current study, drimenol had the greatest antimicrobial activity and so it is considered as one of the more important compounds involved in therapeutic activity in smoke inhalation therapy.

Otherwise, in applications where poultices and lipophilic extracts are applied topically, or where ingestion of an extract is involved, the other compounds may be considered. For example, both polygodial and mukadial displayed significant anti- inflammatory activities in vitro by lipoxygenase inhibition (Frum et al., 2005). Such activity is of relevance where intestinal inflammation is a targeted symptom. In

151 addition, where insecticidal activity is the objective it is self-evident that the insecticidal drimanes, such as warburganal, are the active compounds.

5.5 Summary

 The outcome of the antimicrobial testing gave insight into structure activity

relationships.

 Previously unreported compounds from Erythrina lysistemon (2 and 4) and

Warburgia salutaris (1, 2, 3 and 5) were isolated and assigned.

 E. lysistemon: Prenylation of flavonoids enhanced antimicrobial activity, due

to the lipophilic nature of the prenyl group enabling solubility into bacterial cell

walls. Double prenylation further enhances MIC activity, and further still if the

prenyl group is on the same aromatic ring as an –OH group; activity is

reduced if the –OH group is methoxylated.

 E. transvaalense: the presence of the ketone group reduces activity and

increases cytotoxicity, therefore, reducing the ketone group into a hydroxyl

group increases antimicrobial activity of the compound and reduces toxicity.

 W.salutaris: methoxylation reduces the antimicrobial activity of compounds.

5.6 Conclusions

It is quite surprising that even well studied medicinal plant species yielded

previously unreported compounds and produced interesting new antimicrobial

data. This means that the chemical compositions and antimicrobial effects of

medicinal barks are still in need of further scientific investigation.

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CHAPTER 6. GENERAL SUMMARY AND CONCLUSIONS

6.1 Study of bark on the muthi markets

The study of medicinal barks on the Johannesburg muthi markets revealed that 70 species of bark are the most popular and widely used, and of these, about 55 species are of major commercial relevance. The study showed that even the most important commercial barks used for medicinal application often have charm or magic uses. The commercial ethnobotanical information recorded from the traders showed that 67% of medicinal barks have spiritual uses. The majority of the barks are administered orally, and the dominant method of preparation is powdered material taken as a snuff, licked (khota), rubbed on small razor incisions on the skin and incoparated into traditional herbal remedies (izimbiza, intelezi) in the form of infusions and decoctions. The study also revealed that medicinal barks often have more than one main use. The uses according to the traders sometimes differ from those recorded in the literature.

Three of the 70 barks appear to have no recorded history of use (Cunonia capensis, Eucalyptus sideroxylon and Voacanga thouarsii) in the South African literature. No biological activity studies of the following medicinal barks seem to be available in the literature: Bersama tysoniana, Cassipourea flanaganii, Cunonia capensis, Ochna holstii and Pterocelastrus rostratus. No information on the main chemical compounds of the following medicinal barks could be found in the literature:

Balanites maughamii, Bersama lucens, Bersama tysoniana, Carissa bispinosa,

Cassipourea flanaganii, Cunonia capensis, Heteromorpha arborescens,

Hippobromus pauciflorus, Ilex mitis, Macaranga capensis, Ozoroa paniculosa,

Pleurostylia capensis, Pterocelastrus rostratus and Searsia chirindensis.

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No literature data could be found on any isolated chemical componds from the bark of the following well-studied medicinal plants of commercial importance:

Commiphora africana, Curtisia dentata, Pittosporum viridiflorum and Vachellia karroo. The chemical compositions of the leaves of these species (and bark species in general) are much better known than that the barks.

The study also revealed that some barks have been consistently misidentified in the scientific literature. The first example is igobandlovu, which has frequently been incorrectly identified as Balanites maughamii instead of Garcinia livingstonei.

The second example is umvangazi, which has been identified as Albizia versicolor instead of Pterocarpus angolensis. The correct names for Balanites maughamii and

Albizia versicolor are iphamba and mpesu, respectively.

The predominant language spoken on the Faraday and Kwa Mai-Mai Muthi

Markets in Johannesburg is isiZulu – 86% of the traders are Zulu speaking, followed by 7% each of Xhosa and Tsonga speakers. The bark materials therefore reflect largely the Nguni and Zulu healing traditions. As anticipated, the dominant gender was women on both muthi markets.

The 70 most popular medicinal barks on the markets were from species belonging to 35 plant families. The most important families were Fabaceae (29% of the species), Celastraceae (18%), Anacardiaceae (15%) and Apocynaceae (12%).

Surprisingly, no records of medicinal barks from large, species-rich families such as

Asteraceae and Rubiaceae were encountered. Harvesting of bark can be destructive if traditional methods of conservation and sustainable use are not followed. An assessment of the conservation status of the 70 medicinal barks showed that 82% are of least concern; 4% are declining; 4% are near threatened; 1% is vulnerable;

4% are endangered and 1% is critically endangered.

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The existing documentation of medicinal bark in various literature sources has limited value as an aid to identification. An inventory of the 70 most popular species is therefore presented (Table 2.4 and Appendix 1), with the correct vernacular names, photographs, diagnostic characters and detailed morphological descriptions.

Characters such as the outer and inner bark appearance or texture, colour of the inner and outer bark, presence or absence of the rhytidome, descriptions of longitudinal and transverse sections, colour and odour of powdered bark, typical presence or absence of lichens and moss on some barks and other diagnostically valuable traits were recorded.

6.2 Antimicrobial activity

Sixty-eight extracts of MeOH and DCM from 32 selected medicinal barks has shown antimicrobial activity against infectious diseases associated with skin, gastrointestinal tract and respiratory tract. Positive results were obtained against

Pseudomonas aeruginosa ATCC 743971, Staphylococcus aureus ATCC 25923;

Staphylococcus epidermidis ATCC 12228; Bacillus cereus ATCC 11175; Escherichia coli ATCC 8739; Enterococcus faecalis ATCC 29121; Salmonella typhimurium ATCC

14028; Shigella sonnei ATCC 9290; Klebsiella pneumoniae ATCC 13883 and

Moraxella catarrhalis ATCC 23246. The overall noteworthy antimicrobial activity of the medicinal barks often supported the recorded traditional medicinal uses.

Pseudomonas aeruginosa was the most susceptible bacteria to the medicinal barks screened for skin ailments. The most active barks with MIC values ≤1 mg/ml for skin pathogens were Elaeodendron transvaalense, Erythrina lysistemon, Rapanea melanophloeos and Ziziphus mucronata. Bacillus cereus was the most susceptible bacteria to medicinal barks screened for gastrointestinal tract ailments. Medicinal

155 barks with potent antimicrobial activity against stomach pathogens were Garcinia livingstonei, Erythrina lysistemon, Elaeodendron transvaalense, Bersama lucens,

Schotia brachypetala, Sclerocarya birrea and Ziziphus mucronata. Klebsiella pneumoniae and Moraxella catarrahalis were the most susceptible respiratory tract pathogens with 70% of barks inhibiting bacterial growth at concentration of ≤1 mg/ml.

Medicinal barks that exhibited noteworthy activity against respiratory tract pathogens were Pterocelastrus rostratus, Rapanea melanophloeos, Warburgia salutaris and

Prunus africana.

6.3 Cytoxicity

A preliminary assay of cytotoxicity, using the brine shrimp lethality assay showed that none of the active or moderately active bark extracts was toxic, despite the noteworthy MICs exhibited by some of the extracts and isolated compounds. If poisonous metabolites are present in the extracts, their concentrations are too low to have noticeable toxic effects on the brine shrimp.

6.4 Antimicrobial activity and chemistry of selected bark species

The study revealed that even well documented and well-studied medicinal plant species might produce new data (new records of compounds and previously unrecorded antimicrobial activity). It was noted that antimicrobial activity of isolated compounds was influenced by the chemical structures. For example, prenylated flavonoids from Erythrina lysistemon exhibited potent antimicrobial activity with the lowest MIC value of 2.40 µg/ml against Staphylococcus aureus and S. epidermidis for erybraedin A. This can be ascribed to the lipophilic nature of the prenyl group of

156 the compound, enabling solubility into bacterial cell walls. In the case of

Elaeodendron transvaalense, the presence of a ketone group reduced the antimicrobial potential of the compounds; reducing the ketone group into a hydroxyl group increased the antimicrobial activity of the compound and reduced its toxicity.

The average MIC values yielded by some of the compounds from Warburgia salutaris are a result of the presence of a methoxy group; hence, methoxylation seems to reduce the antimicrobial activity of individual compounds. The study revealed previously unreported compounds from Erythrina lysistemon

(erythrobyssusin-11 and eryzerin C), and from Warburgia salutaris (drimenol, E- nerolidol, 12α-acetal-polygodial and ugandensidial). Isolated compounds were invariably found to be more active to all the bacterial strains when compared to the crude bark extracts.

6.5 Conclusions

The study has shown that a wide diversity of medicinal barks are still popularly used in trational medicine. It also showed that medicinal barks and their uses are relatively poorly studied. The results show that important new phytochemical and antimicrobial data can be generated even from species that have apparently been well studied by previous researchers.

6.6 Recommendations for future studies

More ethnobotanical surveys are required to generate comprehensive and accurate information on the full diversity of uses of medicinal barks. The existing literature

157 gives no direct data on the popularity and relative importance of the various species that are commonly sold. It usually also does not distinguish between main uses and secondary uses, and may be biased towards those uses that seems to be scientifically plausible (magical and ritual uses are rarely reported). This may partly explain the differences between the uses recorded on the muthi markets and those reported in the literature.

Much research work remains to be done to gain a better understanding of the cultural importance of medicinal barks, their main active constituents and their potential efficacy in treating various ailments.

158

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APPENDIX 1. INVENTORY OF THE MOST POPULAR MEDICINAL BARKS SOLD ON MUTHI MARKETS IN JOHANNESBURG

The following bark samples (including a few root and wood samples) were purchased at the Faraday and Kwa Mai-Mai Muthi Markets in Johannesburg. The trade names were recorded, as well as the main uses of these medicinal barks as supplied by the traders, who were all experienced herbalists. It is important to note that the bark samples presented here reflect to full diversity of medicinal barks that were available for sale on the two muthi markets during 2016 to 2018. Several visits were made during 2016 and the first half of 2017 to find previously unrecorded bark samples and it can therefore be assumed that all the most important and popular medicinal barks are presented here. The photographs are inserted at maximum size in order to show the subtle differences in texture and colour that are often typical or diagnostic of the species. An attempt is made to provide accurate descriptions for all the samples. A summary of the results of antimicrobial activity of selected methanol and dichloromethane extracts of the barks against pathogens associated with the skin, gastrointestinal tract and respiratory tract infections are presented. The results for antimicrobial screening are given as per type of bacterial pathogens. Biological activities reported in various literature sources are also briefly listed. A total of 17 chemical compounds were isolated and identified from selected species with noteworthy activity. Chemical compounds reported specifically from bark in various literature sources are also briefly listed. The results show that much basic chemical exploration work on bark remains to be done. Various search engines were used for the literatures studies, including Google, Google Scholar, UJoole (a search engine from the University of Johannesburg research portal), Scopus and ScienceDirect, as well as the African Journal Archives (SABINET) for a better coverage of grey literature such as MSc dissertations and PhD theses. The search words used for antimicrobial and chemical studies included the current scientific binomial name of the species (sometimes the genus, if no data was found for the species), followed by the following selection of words and phrases: chemistry; phytochemistry; chemical constituents; bark; bioactive compounds; biological activity; antimicrobial activity; antibacterial; antifungal.

Albizia adianthifolia Species: Albizia adianthifolia (Schumach.) W.Wight Family: Fabaceae Trade name: umgadankawu Vernacular names: flat-crown tree (Eng); platkroondoringboom (Afr); muvhada-ngoma (Ven); umhlandlothi (Xho); igowane, umgadankawu (Zul) Method of preparation: Infusion (topical) Uses according to traders: The bark is soaked in hot water and used as a wash 'ukugeza' once a day for a couple of days to treat skin rashes and chicken pox. Uses according to the literature: Bark is used for cleansing of blood. In Swaziland, bark and roots are used for skin diseases and scabies. Pounded bark is used as an aqueous lotion for the treatment of eczema and other itchy skin complaints, as a body wash 'ukugeza' or as a facial sauna 'ukugquma'. Bark is used in the treatment of bronchitis, in love charms, as emetics and enemas administered to pregnant women to clear urine. Powdered bark is used as a snuff for headaches and sinusitis. The bark is also used for epilepsy, gonorrhoea and eye sight problems. Maceration of stem bark and root is used as an antidote against poison [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Corrigan et al. (2011); Tamakou et al. (2012)]. Outer bark appearance: Yellowish brown. Smooth in young stems; rough in mature trunk with irregular cracks. Flakes sometimes appear rectangular. On young stems, lenticels are small to medium-sized, roundish in shape and scattered. Inner bark appearance: Yellow (10YR 7/6) to strong brown (7.5YR 5/68). Rough and fibrous. Fibres come off as threads. Habitat and distribution: Woodland often associated with coastal and montane forest. North of Eastern Cape, KwaZulu-Natal, Limpopo, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin pathogens––(Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa). Literature report: anthelmintic and anti-amoebic activity (Fennell et al., 2004). Antibacterial activity against sensitive and resistant strains of P. aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli and Providencia stuartii (Tchinda et al., 2016). Biological activities of n-hexadecanoic acid isolated from the bark include, anti-inﬂammatory, antioxidant, hemolytic, and mosquito larvicide (Abubakar and Majinda, 2016). Chemistry: Ethyl acetate stem bark extract yielded, lupeol, aurantiamide acetate, mixtures of two fatty acids: oleic acid and n-hexadecanoic acid, octadecanoic acid and docosanoic acid (Tamakou et al., 2012). Reported compounds from the n-hexane, methanol and chloroform extracts of the heartwood are n-hexadecanoic acid (34.85%), stigmasterol (28.64%), oleic acid (6.28%), 24S 5α- stigmast-7-en-3β-ol 5 (4.37%), chondrillasterol (18.23%), 9, 12-octadecadienoic acid (Z, Z)-, methyl ester (17.58%) and trans-13-octadecenoic acid, methyl ester (37.23%). 5-methoxyluteolin, melacacidin, and isookanin, 2,3-epoxy-3-[4-hydroxy-3,4- dimethoxyphenyl] propan-1-ol, D-chiro inositol (pinitol) and protocatechuic acid (Abubakar and Majinda 2015, 2016). Conservation status: Least concern

Araucaria heterophylla Species: Araucaria heterophylla (Salisb.) Franco Family: Araucariaceae Trade name: abangqongqozi Vernacular names: Monkey puzzle (Eng); abangqongqozi (Zul) Method of preparation: Infusion Uses according to traders: The bark is used for love charms. The meaning of the vernacular name 'abangqongqozi' is 'knocking on the door’, which indicates the purpose of this medicine in its ability to knock on the door of the lost lover to remember you. Uses according to the literature: No records of southern African traditional medicinal uses have been reported. It is used for timber and for decorations as a Christmas tree (Schans et al. 2004). Outer bark appearance: Grey with red brown spots underneath the peeled off flakes. Rough with distinct horizontal striation and flakes. Inner bark appearance: Red (10R 4/6). Relatively smooth and solid. Habitat and distribution: Endemic to Norfolk Island, in the Pacific Ocean. Biological activity: The resin extract showed dose-dependent antiulcerogenic activity against ethanol-induced stomach ulcers in Sprauge Dawely rat and contains gastroprotective activity (Abdel-Sattar et al., 2009). Chemistry: The resin exudates from the stem contains diterpenes and lignans (Caputo and Mangoni, 1974; Abdel-Sattar et al., 2009). Chloroform extract of the resin exudates yielded three labdane diterpenes namely; labda-8(17),14-diene, 13-epicupressic acid, and 13-Oacetyl-13- epicupressic acid (Abdel-Sattar et al., 2009). Conservation status: Not evaluated Notes: A. heterophylla is a Christmas tree (Schans et al., 2004) and was popular on the muthi markets for its magical use in love charms.

25 mm

Balanites maughamii Species: Balanites maughamii Sprague Family: Zygophyllaceae Trade name: iphamba Vernacular names: torchwood (Eng); fakkelhout (Afr); umnulu (Sot); nulu (NSo); igobandlovu (Zul) Method of preparation: Decoction (emetic, enema) Uses according to traders: Powdered bark is licked from the hand (khotha) for body pains. The bark is taken as an emetic and enema and is used to confuse a person, especially in court cases to get away with breaking the law. Bark emetic is used to bring dreams that are easy to interpret from the ancestors. Uses according to the literature: Bark infusion is used in protective rituals against evil spirits. Bark is used as an emetic to treat parasitic worms, coughs and to make an exhilarating bath. It is applied in the form of cutaneous implantations to strengthen the body. In Mozambique, a paste of the bark is cooked and taken orally as a general tonic, or cooked together with beans to treat haematuria. Toxic characteristics of the bark have been reported [Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Van Wyk and Gericke (2000) Grace et al. (2003); Philander (2011); Van Wyk et al. (2011)]. Outer bark appearance: Grey to brown. Young stems smooth. Mature bark is coarse with irregular shallow cracks. Scales resemble a mosaic pattern. Lenticels are not visible. Inner bark appearance: Yellow (10 YR 7/6). Relatively smooth. Distinct ray patterns. Habitat and distribution: Hot, low-altitude bushveld and sand forest. KwaZulu-Natal, Mpumalanga, Limpopo, Zimbabwe and Mozambique Biological activity: Dichloromethane stem bark extract was one of the two most active antimalarial agent against chloroquine-resistant (PfUP1) strain of Plasmodium falciparum using the flow cytometric method (Pillay et al., 2008). The bark exhibited moderate results in the bitterness value test. Bitter tasting plant substances have been observed to contain antibacterial, antifungal, antioxidant and hepatoprotective properties (Olivier and Van Wyk, 2013). The bark tested negative for larvicidal activity against Anopheles arabiensis mosquitoes (Mavundza et al., 2013). Chemistry: Data on the chemical constituents of the bark could not be found in the literature. However, Balanites species have been reported to contain several steroidal glycosides derived from diosgenin and structurally related sapogenins (Van Wyk et al., 2009). Conservation status: Least concern

Berchemia discolor Species: Berchemia discolor (Klotzsch) Hemsl. Family: Rhamnaceae Trade name: uvuka Vernacular names: bird plum (Eng); bruin-ivoor (Afr), mogokgomo (NSo); mokêrêtê, motsentsela (Tsw); muhukhuma (Ven); Ubaletsheni-omkhulu; uvuka (Zul) Method of preparation: Decoction Uses according to traders: The bark is used in combination with other plants in order for the medication to be effective. Acting as an initiator or a booster. Uses according to the literature: The bark is used as a poultice for treatment of wounds, for infertility and to relieve toothache [Mabogo (1990); Hutchings et al. (1996); Van Wyk and Gericke (2000); Grace et al. (2003); Green et al. (2010); Van Wyk et al. (2011)] Outer bark appearance: Grey. Young stems smooth, sometimes with vertical cracks. Lenticels are not visible. Bark of mature trunk is rough with rectangular scales. Inner bark appearance: Red (2.5 YR 4/6) to yellowish red (5 YR 4/6). Smooth and solid. Habitat and distribution: Bushveld at low altitudes. Mpumalanga, Limpopo, Zimbabwe, Mozambique, Botswana and Namibia. Biological activity: The bark exhibited antitubeculous activity against Mycobacterium tuberculosis H37Ra (Green et al., 2010). Antifungal activity against Candida albicans, Candida krusei and Cryptococcus neoformans (Samie et al., 2010). Chemistry: The methanol root bark extract yielded five new prenylated flavonoids; (6aS, 11aS)-1- hydroxyleiocarpin, discoloranone A, isodiscoloranone A, discoloranone B, isodiscoloranone B and other known compounds nitidulin, amorphigenin, dabinol, heminitidulan, 3-hydroxy-4′-O- methylglabridin, 4′-hydroxycabenegrin A-I, leiocarpin, leiocin, leiocinol, and nitidulan (Chin et al., 2006). Conservation status: Least concern

Bersama lucens Species: Bersama lucens (Hochst.) Szyszył. Family: Melianthaceae Trade name: undiyaza Vernacular names: glossy bersama (Eng); blinkblaarwitessenhout (Afr); isindiyandiya (Xho); undiyaza (Zul) Method of preparation: Powdered (oral; snuff) Uses according to traders: A pinch of finely powdered bark is licked (khotha) or snuffed in a raw form for the treatment of stroke and headache. The bark is boiled to make (imbiza) mixtures and half a cup is administered orally to relieve menstrual pain, strengthen the blood and is used for stomach ailments. Uses according to the literature: The bark is used for barrenness and impotence, to relieve menstrual pain, leprosy, venereal diseases and as a protective charm against evil spirits and lightning strikes. Powdered bark is taken as a snuff to treat congestive headaches, strokes and apoplexy. A tincture of the bark is used as an emetic to calm nervous disorders [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Starfford et. al. (2008); Philander (2011)]. Outer bark appearance: Brownish yellow (green layer underneath). Young stems smooth with tiny vertical fissures; green layer is present underneath periderm. Lenticels scattered, small to medium-sized and arranged in horizontal (sometimes-vertical) rows. Mature bark is rough with dominant vertical cracks. Scales can be vertical and irregular. Inner bark appearance: Yellow (2.5 Y 7/6). Smooth and solid. Habitat and distribution: It grows in various habitats ranging from swamps, coastal forests, high inland forests, and forest margins. Eastern Cape, KwaZulu-Natal, Mpumalanga, Swaziland and Southern Mozambique. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: ethanol extract of the bark exhibited antibacterial activity and good antifungal activity against Candida albicans (Buwa and Van Staden, 2006). Chemistry: No literature data seems to be available on the active constituents of the bark. However, related species of Bersma has been shown to contain cytotoxic bufadienolides (Kupchan et al., 1971; Vanhaelen et al., 1972) and glucuronide triterpene saponins (Tapondjou et al., 2006). Conservation status: Least concern Notes

Bersama tysoniana Species: Bersama tysoniana Oliv. Family: Melianthaceae Trade name: undiyaza Vernacular names: common white ash (Eng); witessenhout (Afr); mokgoba (NSo); undiyaza (Xho); undiyaza (Zul) Method of preparation: Powdered (oral) Uses according to traders: A pinch of finely powdered bark is licked (khota) for stroke. The bark is used as (intelezi), to protect against from witchcraft. Uses according to the literature: The bark is used for barrenness and impotence, to relieve menstrual pain, to treat leprosy, hysteria and as a protective charm. Decoction of the bark is used for treatment of livestock diseases (gall sickness) [Hutchings et al. (1996); Grace et al. (2003); Masika and Afolayan (2003)]. Outer bark appearance: Grey brown. Smooth in young stems. Bark in mature trunk is rough with dominant vertical cracks and elongated flakes. Lenticels are not visible. Inner bark appearance: Yellowish brown (10YR 5/8). Smooth and solid. Habitat and distribution: Montane forest and associated with woodland. Eastern Cape, KwaZulu- Natal, Limpopo, Mpumalanga and Swaziland. Biological activity: No literature data seems to be available available. However, the genus Bersama is known to exhibit antitumor, spamolytic, cardiotonic, antibacterial and antiviral activities (Tapondjou et al., 2006). The overall pharmacological activity of B. tysoniana and bark in particular is poorly documented. Chemistry: No literature data seems to be available on the active constituents of the bark. However, related species of Bersma has been shown to contain cytotoxic bufadienolides (Kupchan et al., 1971; Vanhaelen et al., 1972) and glucuronide triterpene saponins (Tapondjou et al., 2006). Conservation status: Least concern

Brackenridgea zanguebarica

Species: Brackenridgea zanguebarica Oliv. Family: Ochnaceae Trade name: Mutavhatsindi Vernacular names: yellow peeling plane (Eng); Geellekkerbreek (Afr); Mutavhatsindi (Ven) Method of preparation: No data Uses according to traders: No data (Tsonga trader reluctant to give information). Uses according to the literature: The root and stem bark are used in magic charms by the VhaVenda to protect the homesteads from enemies. Root and bark are used for treatment of wounds, swollen ankles, amenorrheoea and worms. The bark is used for treatment of wounds [Mabogo (1990); Möller et al. (2006)]. Outer bark appearance: Bright yellow and strong brown. Relatively rough, with dominant vertical cracks. Inner bark appearance: brown (7.5YR 4/4). Smooth. Habitat and distribution: Bushveld and deciduous woodlands. Limpopo, Zimbabwe and Mozambique. Biological activity: An ethanol extract of the bark has shown antiviral activity against Herpes simplex virus type 1 and potent antibacterial activity against Gram-positive bacteria associated with skin infection (Möller et al., 2006). The root bark extracts tested negative against Candida albicans (Steenkamp et al., 2007). Chemistry: Bark extract yielded a dimeric dihydrochalcone and a dimeric chalcone which were present in significant amounts (Drewes and Hudson, 1984). Conservation status: Critically endangered

Bridelia micrantha

Species: Bridelia micrantha (Hochst.) Baill. Family: Phyllanthaceae Trade name: umhlahle Vernacular names: coastal goldenleaf (Eng); mitserie (Afr); motsêrê (NSo); munzere (Ven); umhlahlangu, umhlahlahlungulu (Xho); umhlahle (Zul) Method of preparation: Decoction Uses according to traders: The bark is used as an emetic to separate between good and bad spirits in a ritual ceremony (amagobonga) of becoming a sangoma. Uses according to the literature: The bark is used for treatment of wounds, to relieve toothache and for abortion during one to two months of pregnancy. Powdered bark is applied on the skin for burns to enhance the healing process. The bark decoction is used for stomach ache, tapeworm, diarrhoea, to treat gonorrhoea and other sexually transmitted diseases. The bark is also used as a cough expectorant, laxative and treatment of diabetes [Mabogo (1990); Hutchings et al. (1996); Grace et al. (2003); Green et al. (2010); Mabona et al. (2013); Van Wyk et al. (2011)]. Outer bark appearance: Scales are grey brown, cracks darker in colour. Bark is rough with mostly rectangular scales. No lenticels. Inner bark appearance: Reddish brown (2.5YR p/3). Fibrous, fibres come off as threads. Habitat and Distribution: Bushveld, coastal, riverine and swamp forest, often in wet areas. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland, Zimbabwe and Mozambique. Biological activity: The bark demonstrated antitubercolosis activity against Mycobacterium tuberculosis H37Ra (Green et al., 2010). Antifungal activity against Candida albicans and Candida krusei, Trichophyton mentagrophytes and Microsporum canis (Samie et al., 2010; Mabona et al., 2013). Antibacterial activity against, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, gentamycin methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Brevibacillus agri and Propionibacterium acnes (Mabona et al., 2013). Chemistry: Friedelin, taraxerone, epifriedelinol, taraxerol, gallic acid and ellagic acid were isolated from the bark; the presence of leucodelphinidin and caffeic acid was also noted (Pegel and Rogers, 1968; Ngueyem et al., 2009). Conservation status: Least concern

Calodendrum capense Species: Calodendrum capense (L.f.) Thunb. Family: Rutaceae Trade name: umemezi omhlophe Vernacular names: Cape chestnut (Eng); wildekastaiing (Afr); molalakgwedi (NSo); muvhaha (Ven); umbhaba (Xho); umemezi omhlophe (Zul) Method of preparation: Powdered/ paste (topical) Uses according to traders: The bark is used as a love charm, a name of the lost lover is ‘called out’ (memeza) through the powdered bark over the palm of the hand and blown in the direction of that person. Powdered bark is mixed with cold water as a paste and applied to the face as a skin lightener. Uses according to the literature: The bark is used as an ingredient for skin ointments, as a moisturiser and to treat pimples [Van Wyk et al. (1997, 2009); Grace et al. (2003); Philander (2011); Kishore and Lall (2014); Van Wyk et al. (2011)]. Outer bark appearance: Brownish grey. Smooth, even in mature trunk, with small shallow vertical cracks. In young stems, a green layer is present underneath the periderm. Lenticels are in vertical rows or scattered. Inner bark appearance: Very pale yellow (Munsell white diagram 2.5 Y 9/2). Smooth with visible patterns of rays in diagonal lines. Habitat and distribution: Montane forest and riverine thicket. Eastern Cape, Gauteng, KwaZulu- Natal, Limpopo, Mpumalanga, North West, Western Cape, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin pathogens ‒ (Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa). Literature report: the root bark and calodendroline demonstrated larvicidal activity against Aedes aegypti (Kiprop et al., 2005). Chemistry: The acetone root bark extract yielded calodendrolide, limonin and limonin diosphenol (Kiprop et al., 2005). The hexane and dichloromethane extracts of the stem bark yielded, 7-O- dimethylallyl demethylenedictamnine, confusameline, limonin and bergapten (Okwemba, 2012). Conservation status: Least concern

Carissa bispinosa Species: Carissa bispinosa (L.) Desf. ex Brenan Family: Apocynaceae Trade name: umvusankunzi Vernacular names: num-num (Eng); bosnoemnoem (Afr); morokolo (NSo); murungulu (Ven); umvusankunzi (Xho); umvusankunzi (Zul) Method of preparation: Powdered (oral) Uses according to traders: The bark is ground into fine powder and a small portion is used licked (khotha) for treatment of erectile dysfunction. Uses according to the literature: The bark and root are used in calving difficulties in cattle. Stem bark is used for pulmonary diseases, as an aphrodisiac and for sexually transmitted diseases. Root is used for toothache. Root extracts are administered orally for coughs and for diarrhoea [Mabogo (1990); Van Wyk and Gericke (2000); Luseba and Tshisikhawe (2013); Maroyi et al. (2013)]. Outer bark appearance: Superficial appearance is light brown and presence of a bright orange colour underneath. Bark is relatively smooth with prominent scattered lenticels and sometimes with pronounced dormant buds. Inner bark appearance: Brownish yellow (10 YR 6/6). Relatively smooth with vertical ridges. Habitat and distribution: It grows in wooded areas. Eastern Cape, Western Cape, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Eastern parts of Free State, Lesotho, Swaziland, Zimbabwe and Mozambique, extending westwards to Botswana and Namibia. Biological activity: The root extracts contains analgesic, antiviral and diuretic properties (Patel, 2013; Maroyi, 2013. Sesquiterpenes isolated from Carissa species have been shown to possess antimicrobial, antimalarial, anticancer and anti-inﬂammatory effects (Kaunda and Zhang, 2017). Chemistry: No data could be found on chemical constituents of the bark in the literature. Ursolic acid has been isolated from the leaves. Related species of the genus Carissa contains lupane, ursane, oleanane, D:C-friedoleane triterpene and isohopane type triterpene from the roots (Kaunda and Zhang, 2017). Conservation status: Least concern +

Cassipourea flanaganii Species: Cassipourea flanaganii (Schinz) Alston Family: Rhizophoraceae Trade name: umemezi obomvu Vernacular names: Cape onionwood (Eng); Kaapse uiehout (Afr); ummemezi (Xho) umemezi obomvu (Zul) Method of preparation: Powdered/ paste (topical) Uses according to traders: Powdered bark is mixed with water and applied facially as a skin lightener and sun screen. Uses according to the literature: The bark is used for skin diseases, as a cosmetic, for sunburn, as a skin lightener, to alleviate pimples, for cancer, chest ailments and hypertension. Powdered bark is used to soften hair [Pujol (1990); Hutchings et al. (1996); Philander (2011); Otang et al. (2012); Afolayan et al (2014)]. Outer bark appearance: Dark reddish brown. Smooth, tiny vertical cracks; lenticels on young stems in vertical rows, white-yellowish colour; on the mature stems crustose lichens are present. Inner bark appearance: Pink (10 R, 8/4) and smooth. Habitat and distribution: Scarp Forest, Southern Mistbelt Forest, Thornveld, Eastern Valley Bushveld. Eastern Cape and KwaZulu-Natal. Biological activity: Pharmacological activity of the cosmetic use of the bark is poorly documented. Chemistry: No literature data could be found on chemical constituents of the bark. However, related species such as C. gerrardii and C. gummiflua stem bark have yielded flavanol glycoside and proanthocynidins namely; afzelechin-3-O-X-L-rhamnopyranoside, epiafzelechin- (4β→8,2β→O→7)-ent-afzelechin, afzelechin, kaempferol-3-α-L-rhamnopyranoside and epiafzelechin (Drewes et al., 1992a; Drewes and Taylor, 1994; Hou et al., 2010). Conservation status: Endangered

Cinnamomum camphora Species: Cinnamomum camphora (L.) J.Presl Family: Lauraceae Trade name: uloselina Vernacular names: camphor tree (Eng); kanferboom (Afr); uloselina (Zul) Method of preparation: Infusion (emetic) and powdered (topical) Uses according to traders: Bark infusion is used as an emetic (phalaza) for a person to be admired by other people. Bark is used as a powder and applied on the eyebrows or a piece of bark is placed in the mouth to persuade people in order to be attractive to the opposite gender. Uses according to the literature: The bark is used as a love charm, as an emetic and as a perfume. The bark is used for treatment of fevers, colds and influenza. The bark is used to relieve abdominal discomfort [Hutchings et al. (1996); Van Wyk (1997, 2009); Van Wyk and Gericke (2000); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Dark reddish brown and light brown in cracks. Bark is rough and deeply furrowed. No lenticels. Inner bark appearance: Reddish yellow (7.5 YR 6/8). Very smooth (slippery). Habitat and distribution: Native to Asia; invades coastal forest margins and riverbanks. Western Cape, Eastern Cape, KwaZulu-Natal and Limpopo. Biological activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa and Staphylococcus aureus). Literature report: essential oil from the bark exhibited antibacterial activity against Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogens and S. aureus (Inouye et al., 2001). Chemistry: The methanol bark extract yielded two new monoglycerides, l-(28- hydroxyoctacosanoyl) glycerol and l-(24-hydroxytetracosanoyl) glycerol, and two flavanols, 5,7- dimethoxy-3’,4’-methylenedioxyflavan-3-ol and 4’-hydroxy-5,7,3’-trimethoxyflavan-3-ol. The known compounds isolated include obtusilactone and isoobtusilactone (Mukherjee et al., 1994). Camphor oil from the stem contains safrole, borneol, heliotropin, terpineol and vanillin (Hutchings et al., 1996). The major essential constituent isolated from the bark is cinnamaldehyde (Inouye et al., 2001). Conservation status: Not evaluated (exotic)

Combretum caffrum Species: Combretum caffrum (Eckl. & Zeyh.) Kuntze Family: Combretaceae Trade name: isidubu Vernacular names: Cape bushwillow (Eng); Kaapse vaderlandswilg (Afr); isidubu (Xho); umdubu (Zul) Method of preparation: Infusion (enema) Uses according to traders: Bark is used to make (izimbiza) (liquid mixture) and administered as an enema for internal cleaning of the body. The bark is used to clean the veins. Pounded bark is used as (ikhubalo) to chase away evil spirits. Uses according to the literature: Root bark is used in magic charm to harm enemies. The roots are used in Zulu traditional medicine as a tonic, appetite stimulant and as a wound dressing. Combretum species are used for coughs, infertility, sexually transmitted diseases, stomach ailments, and applied topically for treating sores, wounds and cancer [Van Wyk et al. (1997, 2009); Grace et al. (2003); Mabetha and McGaw 2018]. Outer bark appearance: Greyish brown. Relatively smooth with tiny vertical cracks. Sometimes lenticels present inside vertical cracks (they occur in vertical rows with vertical apertures). Inner bark appearance: Yellow (10YR 8/6) to dark brown (7.5 YR 3/3). Relatively smooth. Habitat and distribution: Forest and associated woodland. KwaZulu-Natal, Eastern Cape and Western Cape. Biological activity: Extracts of the bark has shown antifungal activity against Alternaria alternaria, Aspergillus niger, Mucor hiemalis and Schizophyllum commune, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schencki (Afolayan et al., 2002; Masoko et al., 2007). The isolated compounds mentioned below were reported to show powerful inhibition on the growth of murine lymphocytic leukemia cell line and in colon cancer cells (Pettit et al., 1982; Mabeta and McGaw, 2018). Chemistry: The stem wood yielded combretastatin, combretastatin A-1, dihydrophenanthrenes and dihydrophenanthrenes (Pettit et al., 1986; Orsini et al., 1997; Eloff et al., 2008; Mabeta and McGaw, 2018). Conservation status: Least concern

Commiphora africana Species: Commiphora africana (A.Rich.) Engl. var. africana Family: Burseraceae Trade name: iminyela Vernacular name: hairy corkwood (Eng); harige kanniedood (Afr); morôka (Tsw); iminyela (Zul) Method of preparation: Powdered Uses according to traders: Bark is ground into a fine powder and is licked (ncinda) or applied to small razor incisions (gcaba) to prevent other medication from entering the blood or prevent the efficacy of other medicines. Uses according to the literature: Bark decoction is used for treatment of pneumonia, bronchitis, whooping cough, diabetes, heartburn, stomach pains and dysentery [Van Wyk and Gericke (2000); Grace et al. (2003); Kareru et al. (2008); Johnson et al. (2012); Ohadoma et al. (2016)]. Outer bark appearance: Brown yellowish (green layer underneath). Relatively smooth with thin superficial papery layers; directly below is a green (photosynthetic) layer; black marks or scales (dead or injured parts) can be present on bark samples. Lenticels are small and scattered. Inner bark appearance: Pink (7.5 YR 8/4) to red (10 R 4/8). Smooth. Habitat and distribution: Dry bush and woodland, usually in rocky places. KwaZulu-Natal, Limpopo, Mpumalanga, North West, Zimbabwe, Swaziland, Namibia and Botswana. Biological activity: Bark extract has shown activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis in the disc diffusion assay (Kareru et al., 2008). Mixture of chloroform: methanol extract of the stem exhibited good antioxidant and anti-cancer activity as well as antimicrobial activity against Klebsiella pneumoniae, Bacillus cereus, Staphylococcus aureus, Candida albicans and Cryptococcus neoformans (Paraskeva et al., 2008). Aqueous extracts of the stem bark and root bark demonstrated anthelmintic activity against earthworm (Gbolade and Adeyemi, 2008). Aqueous and ethanolic stem bark extracts were reported to exhibit hypoglycemic properties in normoglycemic Wistar rats (Goji et al., 2009). The methanol stem bark extract demonstrated potent anti-ulcer activity in albino rats (Nuhu et al., 2016). The ethanol extract of the stem bark showed antibacterial activity against Pseudomonas aeruginosa, Proteus mirabilis, E. coli, K. pneumoniae and S. aureus (Ohadoma et al., 2016). Chemistry: The chemical constituents of the bark in particular is largely neglected. However, the chemical constituents of the myrrh, an exudate produced from the secretory tissue in the bark is largely documented. It is commonly used in traditional medicine in some parts of the world including China, India and Egypt other than southern Africa. Numerous phytochemicals have been detected from the resinous exudates of Commiphora species, these include terpenoids such as α- pinene, camphene, β-pinene, myrcene and limonene. More than 20 furanosesquiterpenoids covering furanogermacrane, furanoeudesmanes, furanoguaiane, furanocadinane and furanoelemane have been identified (Shen et al., 2012). Conservation status: Least concern

Croton sylvaticus Species: Croton sylvaticus Hochst. Family: Euphorbiaceae Trade name: umahlabekufeni Vernacular names: forest fever-berry, woodland croton (Eng); boskoorsbessie (Afr); moema, moema-tswetsi (NSo); mula-thoho (Ven); umfeze, umagwaqane (Xho); umhloshazane, umahlabekufeni (Zul) Method of preparation: Decoction and powdered Uses according to traders: Pounded bark is cooked with water and administered orally twice a day for the treatment of wounds, stomach ailments and used for constipation. Powdered bark is licked or snuffed for pain, it reaches site of infection faster. Uses according to the literature: The bark is used as a remedy for oral administration and used in the treatment of tuberculosis (Ndunda et al., 2015). Decoction of the bark is used for abdominal disorders, rheumatism, fever, digestive problems, internal inflammation, and intestinal complaints and for treatment of uterine diseases (Maroyi et al., 2017). Finely ground bark is rubbed into incisions on the skin as an irritant for inflammation and pain in the chest [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (2009); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Yellowish grey to brown. Smooth with green layer underneath. Lenticels small to medium with horizontal or vertical apertures; lenticels scattered or present inside cracks in mature bark samples. Inner bark appearance: Light yellowish brown (10YR 6/4) to brownish yellow (10 YR 6/6). Smooth and solid. Habitat and Distribution: Bushveld and grassland. KwaZulu-Natal, Mpumalanga, Limpopo, Eastern Cape, Mozambique, Zimbabwe, Botswana and Namibia. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: isolated compounds by Ndunda et al. (2015) were inactive against Bacillus subtilis, Xanthomonas campestris and Candida albicans (Ndunda et al., 2015). Aqueous extracts of the root and stem bark showed antifungal activity against Candida albicans, ethanolic extact of the bark exhibited anti-inflammatory activity (Maroyi et al., 2017). Chemistry: The root bark contain crotin (McGaw et al., 2008). The GC-MS analysis of the stem bark yielded, hardwickiic acid, β-sitosterol, tigmasterol, trans-phytol, 5, 16-dihydroxy-trans-ent- cleroda-3, 13-diene; 15-acetoxy-2-oxo-trans-ent-cleroda-3, 13-diene; lupenone; 3 β-acetoxylup- 20(29)-ene, β-amyrin, trans-annonene, (+)-[5R, 6S, 9R]-4, 5-dihydroblumenol A, lignoceryl trans- ferulate and (+)-syringaresino (Langat, 2009; Maroyi et al., 2017). The root bark yielded four clerodane diterpenoids, the new ent-3,13E-clerodadiene-15-formate, the known 15-acetoxy-ent3, 13E-clerodadiene; ent-3,13E-clerodadien-15-ol; and hardwickiic acid, two known halimane diterpenoids, penduliflaworosin and crotohalimaneic acid and one labdane diterpenoid, labda-13E- ene-8α,15-diol (Ndunda et al., 2015; Maroyi et al., 2017). Conservation status: Least concern

Cryptocarya latifolia Species: Cryptocarya latifolia Sond. Family: Lauraceae Trade name: umkhondweni Vernacular names: broad-leaved laurel (Eng); breëblaar-kweper (Afr); umgxaleba (Xho); umkhondweni (Zul) Method of preparation: Powdered (oral) Uses according to traders: Powdered bark is mixed with crocodile fat and administered by licking directly from the palm of the hand (khotha) for treating food poisoning. The bark is smoked for headache. Bark is used by traditional healers to detect type of sickness in the body a ‘patient’ suffers from. Uses according to the literature: Powdered bark mixed with crocodile fat is used to treat chest ailments. The bark is used to treat internal pains, allergy and tuberculosis. An infusion of the powdered bark is used for muscular and stomach cramps taking a cup in the mornings and evenings. Decoctions are used as enemas for the treatment of urinary tract diseases, internal pains, uterine spasms and menstrual pains [Pujol (1990); Hutchings et al. (1996); Lall and Meyer (1999); Grace et al. (2003)]. Outer bark appearance: Brown (7.5 YR, 4/3). Smooth, lenticels small (<1 mm) and scattered. Inner bark appearance: Very dark brown (7.5 YR, 2.5/3). Smooth, sometimes with small ridges. Habitat and distribution: Eastern Cape and KwaZulu-Natal Biological activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa and Staphylococcus aureus). Literature report: hexane extract of the bark has shown anti-inflammatory activity evaluated using the cyclooxygenase assay (Zschocke and Van Staden, 2000). The acetone extract of the bark showed antimycobacterial activity against Mycobacterium tuberculosis (Lall and Mayer, 1999; McGaw et al., 2008). Chemistry: A major compound isolated from the bark is 6- substituted 5, 6-dihydro-pyrones (cryptofolione), followed by isolation of cryptocaryalactone, (2'-acetoxy)-6-hept-4-enyl-5, 6- dihydro-2-H- pyran-2-one, cryptocaryolone, (2',4'-diacetoxy)-6-Pentyl-5,6-dihydro-2-H-pyran-2- one, cryptocaryolone diacetate and (2',4',6'-triacetoxy)-6-Heptyl-5,6-dihydro-2-H-pyran-2- one (Drewes et al., 1995; 1996). Conservation status: Declining

Cunonia capensis Species: Cunonia capensis L. Family: Cunoniaceae Trade name: umlulama Vernacular names: butterspoon tree (Eng); rooi-els (Afr); umqwashube (Xho); umlulama (Zul) Method of preparation: Powdered (snuff; oral) Uses according to traders: Powdered bark is mixed with animal bones (from the Big Five) in a powdered form and used as a snuff and /or licked for treating stroke. Powdered bark infusion is used for respiratory complaints and food poisoning. Uses according to the literature: The bark in particular has no specific records of medicinal uses. Outer bark appearance: Greyish brown. Bark smooth only in young stems. Usually rough with vertical fissures and irregular fissures or cracks in mature trunk. Lenticels prominent, roundish to slightly elongated (2-6mm) with vertical and rarely horizontal apertures Inner bark appearance: Reddish black (5R 2.5/1) and smooth. Habitat and distribution: Afromontane forests and moist areas, especially along the streams. Western Cape, Eastern Cape, KwaZulu-Natal and Mozambique. Biological activity: No data could be found in the literature. Chemistry: No data seems to be avaible on the bioactive chemical constituents of the bark in the literature. Conservation status: Least concern

Curtisia dentata Species: Curtisia dentata (Burm.f.) C.A.Sm. Family: Curtisiaceae Trade name: umlahleni Vernacular names: assegai (Eng); assegaai (Afr); modula-tshwene (NSo); mufhefhera (Ven); umgxina (Xho); umlahleni (Zul) Method of preparation: Infusion Uses according to traders: The bark is soaked in hot water and used as an emetic to prevent quarrel and bleeding stomach called ‘isisu segazi’. Bark is used to trap 'ukucupha' people who are bewitching you. Uses according to the literature: The bark is used for a bleeding stomach, diarrhoea, to strengthen the blood, for pimples and to promote sexual activity. It is used in skin-lightening ingredients in the Eastern Cape [Pujol (1990); Hutchings et al. (1996); Van Wyk (1997, 2009); Grace et al. (2003); Shai et al. (2008); Otang et al. (2012)]. Outer bark appearance: Brownish grey. Relatively smooth in young stems with tiny vertical fissures; lenticels are of medium size and horizontal aperture. Bark is rough in mature trunk with irregular fissures forming mosaic patterns. Inner bark appearance: Strong brown (7.5 YR 4/6) and smooth. Habitat and distribution: Coastal and montane forest. Western Cape, Eastern Cape, KwaZulu- Natal, Mpumalanga, Limpopo and Mozambique. Biological activity: Antifungal activity against Aspergillus fumigatus, Candida albicans, Sporothrix schenckii and Cryptococcus neoformans. Antibacterial activity against Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa. Mycobacterium tuberculosis H37Rv, methicillin-resistant S. aureus, ampicillin resistant Klebsiella pneumoniae, carbenicillin-resistant Pseudomonas aeruginosa, chloramphenicol-resistant Citrobacter and Microsporum audouinii (Shai et al., 2009; Nielsen et al., 2012; Soliman et al., 2017). Chemistry: No available data seems to be available for the bark. The chemical constituents and pharmacological activity of the leaves is largely documented. Conservation status: Near threatened

Dichrostachys cinerea Species: Dichrostachys cinerea (L.) Wight & Arn. Family: Fabaceae Trade name: ugagane Vernacular names: sicklebush (Eng); sekelbos (Afr); mosêlêsêlê (NSo); ugagane, umzungulu (Zul) Method of preparation: Powdered (topical) Uses according to traders: Bark is powdered, mixed with paraffin and applied directly on the armpits for treatment of lice. The tree is planted in the yard to prevent lightning. Uses according to the literature: The bark is used for snake and scorpion bites, skin infections, malaria, elephantiasis, and as a ritual cleanser. Dried powdered bark is applied topically to sores or skin eruptions for the treatment of syphilis. It is also used as a pain relief in Swaziland. Bark is also used for ulcers and bleeding stomach. Stem is used as a toothache eremedy [Pujol (1990); Hutchings et al. (1996); Corrigan et al. (2011); Muthaura et al. (2015); Chinsembu (2016); Akhalwaya et al. (2018)]. Outer bark appearance: Yellow. Root bark looks shaggy; the flakes are papery. Inner bark appearance: Yellow (2.5 Y 8/6) and smooth Habitat and distribution: Bushveld usually invasive and thicket-forming, especially in overgrazed areas. KwaZulu-Natal, Mpumalanga, Gauteng, Free State, North West, Limpopo, Swaziland, Namibia, Botswana, Zimbabwe and Mozambique. Biological activity: The stem aqueous extract and 1:1 mixture of (DCM and MeOH) exhibited activity on oral pathogens namely, Gram-positive cariogenic bacterial strains; Streptococcus mutans, Streptococcus sanguis, and Lactobacillus casei, two Gram-negative periodontal pathogens, Porphrymonas gingivalis and Fusobacterium nucleatum and finally, three yeast strains namely; Candida albicans, Candida glabrata and Candida krusei, these are in connection with oral candidiasis (Akhalwaya et al., 2018). Neutralization test to determine non-cytotoxic concentration (ID50) that inhibits or protects 50% of the monolayer cells against destruction by the virus compared to uninfected cells was active from the methanol root bark extract (Prinsloo et al., 2018). Chemistry: The root and stem bark both contain friedelan-3-one and friedelan-3-ol. The stem bark extract yielded, betulinic acid and 3-O-β-d-glucopyranosyl-β-sitosterol, apigenin, luteonin, monoglycerides of tetracosanoic and 26-hydroxyhexacosanoic acids, 7,4´-dihydroxyflavon and 7,3´,4´-trihydroxyflavon (Fotie et al., 2004). Active constituents with anti-HIV properties from the root bark and leaves are friedelin; friedlan-3-ol; sitosterol and amyrin; octacosanol; hentricontanol; coumarins imperatorin; marmesin and aesculetin (Prinsloo et al., 2018). Conservation status: Least concern

Dombeya rotundifolia Species: Dombeya rotundifolia (Hochst.) Planch. Family: Malvaceae Trade name: unhliziyonkulu Vernacular names: wild pear (Eng); dikbas (Afr); mohlabaphala (Sot); motubane (NSo); mulanga (Ven); unhliziyonkulu (Zul) Method of preparation: Decoction (oral) Uses according to traders: The bark is mixed with a heart of a banana (seeds) and another plant known as ‘ntshungu’ (Momordica balsamina) for the treatment of heart diseases hence the plants name comes from the organ that it treats 'inhliziyo' mean the heart. A bark decoction is used to treat heart diseases and gynaecological problems. The bark is used to steam ‘ukufutha’ for itchy skin problems. Uses according to the literature: The inner bark is used for a weak heart and nausea in pregnant women. Bark or wood infusions are administered orally or as enemas for intestinal ulceration, stomach complaints, haemorrhoids, diarrhoea, fevers and headache. The bark is boiled in water and taken as a tea or infusions for palpitations, nausea, to induce abortion and for irregular menstruation. The tough, inflexible fibers of the bark are used to cover wounds, or act as splints for broken limbs in humans and livestock [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Reid et al. (2001); Grace et al. (2003)].

Outer bark appearance: Grey to dark brown. Rough with irregular cracks. Inner bark appearance: Reddish yellow (5 YR 7/6). Fibrous but smooth. Fibres peel off as long strips. Habitat and distribution: Bushveld and wooded grassland. KwaZulu-Natal, Mpumalanga, Gauteng, Limpopo, Swaziland, Botswana, Namibia, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: anti-inflammatory activity was reported from the bark extracts, investigated using the prostaglandin-synthesis inhibitor assay (COX-1) (Reid et al., 2001). Chemistry: The chloroform extract of the stem bark yielded two compounds, lupeol and - sitosterol identified by spectroscopic methods (Ndwingah et al., 2005). Conservation status: Least concern

Ekebergia capensis Species: Ekebergia capensis Sparrm. Family: Meliaceae Trade name: umnyamathi Vernacular names: Cape ash (Eng); essenhout (Afr); nyamaru (NSo); mmidibidi (Sot); nyamaru (Tsw); mutovuma (Ven); isimanaye (Xho); umnyanmathi (Zul) Method of preparation: Decoction Uses according to traders: Pounded bark is used for 'ukugquma' facial sauna when a person wants to gain dignity. The bark is burn and the smoke is used to treat an unspecified condition in livestock. Uses according to the literature: Decoctions of the bark are taken as emetics, for heartburn and for respiratory and chest complaints. The bark is traditionally used to protect chiefs against witchcraft and is also taken as love charms and emetics. The ground bark is used in flour and water poultice for abscesses and boils and in hot water infusions for pimples, administered both as a blood purifying emetic and as a wash, and it is also used to treat slowness. The stem bark is used to treat malaria. The powdered bark and roots are mixed together to make tea for gastritis [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Van Wyk et al. (2011); Muthaura et al. (2015)]. Outer bark appearance: Light grey with yellowish lenticels in young stems. Grey-brown in mature stems. Bark is relatively smooth in young stems with tiny vertical fissures; lenticels are in vertical rows 2 to 20 mm apart. Bark is rough in mature trunk with irregular cracks. Inner bark appearance: Strong brown (7.5 YR 5/8; 7.5 YR 4/4). Relatively smooth. Looks fibrous, but solid. Habitat and distribution: Coastal and montane forest. Western Cape, Eastern Cape, KwaZulu- Natal, Mpumalanga, Limpopo, Swaziland, Mozambique, Zimbabwe and Botswana. Biological activity: Antimicrobial activity against skin and stomach pathogens ‒ (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: organic extracts of the bark has shown activity against S. aureus, methicillin resistant Staphylococcus aureus, gentamycin methicillin resistant Staphylococcus aureus, Bacillus subtilis, Candida albicans, Brevibacillus agri, Propionibacterium acnes, Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, Klebsiella pneumoniae and Neisseria gonorrhoeae as well as HIV-1 RT inhibitory activity (Mulaudzi et al., 2011; Mabona et al., 2013; Ngeny et al., 2013). The root bark extracts was toxic against Vero cells, and its constituents exhibited moderate antiplasmodial activity (Irungu et al., 2014). Chemistry: The hexane extract of the bark yielded, atraric acid, -sitosterol, palmitate and oleate esters of -sitosterol lupeol and oleanonic acid. The chloroform extract yielded lupeol and the methanol extract yielded 3- epioleanolic acid. Only lupeol was isolated from the wood. Limonoids and coumarins were not detected (Mulholland et al., 1997). Murata et al. (2008) isolated 10 new triterpenoids (ekeberins) and 17 known compounds from the stem bark. Conservation status: Least concern

Elaeodendron croceum

Species: Elaeodendron croceum (Thunb.) DC. Family: Celastraceae Trade name: isithundu Vernacular names: common saffron (Eng); gewone saffraan (Afr); umbovane (Xho); isithundu (Zul) Method of preparation: Infusion Uses according to traders: The bark is used as intelezi, it is sprinkled (chela) in the yard (isigcawu) when a person is getting married and protects against witchcraft. Bark is used as an emetic to clean the blood and to gain admirable dignity. Uses according to the literature: The bark is used to clean the digestive tract, and get rid of chest congestion [Pujol (1990); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Grey with prominent orange colour underneath the periderm. Bark is relatively smooth with vertical fissures. Lenticels are not visible. Inner bark appearance: Pinkish white (5YR 8/2). Solid and smooth Habitat and distribution: Margins of coastal and montane forests. Western Cape, Eastern Cape, KwaZulu-Natal, Limpopo, and Zimbabwe. Biological activity: Bark extracts showed antibacterial and antifungal activity against Bacillus anthracis, Candida albicans, Gardnerella vaginalis, Neisseria gonorrhoeae and Oligella ureolytica (Elisha et al., 2016; Mamba et al., 2016). Aqueous extract of the stem bark exhibited good antibacterial activity against Staphylococcus aureus, Shigella flexneri, Enterococcus faecalis and Salmonella typhimurium. In addition, the aqueous bark extract showed good antioxidant and in vtro anti-inflammatory activity (Odeyemi and Afolayan, 2017). Chemistry: The stem bark extracts of hexane, ethyl acetate and ethanol yielded, (+ )-6R, 13R-11, 11-dimethyl-1, 3, 8, 10-tetrahydroxy-9-methoxypeltogynan and (-)-4’-O- methoxyepigallocatechin. Along with ouratea proanthocyanidin, galactitol, tingenone, tingenin B, and three pentacyclic triterpenes, canophyllol, 3-hydroxyIupeo1 and 30-hydroxylup 20(29)-en-3- one (Drewes and Mashimbye, 1993; Yelani et al., 2010). Ethanolic extract of E. croceum yielded a well-known digitoxigenin-glucoside (Prinsloo et al., 2010). Conservation status: Least concern

Elaeodendron transvaalense Species: Elaeodendron transvaalense (Burtt Davy) R.H.Archer Family: Celastraceae Trade name: ingwavuma Vernacular names: Transvaal saffronwood (Eng); Transvaalsaffraan (Afr); monamane (NSo); shimapana (Tso); ingwavuma (Zul) Method of preparation: Decoction (oral, enema, emetic) Uses according to traders: The bark is powdered and put to boil, then administered orally (half a cup) or taken as an enema for the treatment of stomach complaints, internal wounds and as an emetic for treating a blocked chest. The bark is used in ‘amagobonga’, and as an enema in children with stomach complaints ‘isisu sobisi’. Uses according to the literature: An infusion of the bark is used as an enema for stomach-ache, kidney problems and fever. Decoction and/or powdered bark is licked directly from the palm of the hand followed by water and is used treatment of diarrhoea, intestinal worms and intestinal cramps. The bark is used for piles, kidney, and bladder infections, to relieve body pain, heavy menstruation, skin rashes and skin infections. It is also used for tanning and as a tea [Pujol (1990); Hutchings et al. (1996); Van Wyk (2000); Van Wyk et al. (1997, 2009); Grace (2003); Samie et al. (2010); Mthethwa et al. (2014)]. Outer bark appearance: Light grey to brown. Smooth with vertical fissures in younger stems and relatively rough in older steams with irregular fissures. Sometimes bark of mature trunks has mosaic appearance. No visible lenticels. Inner bark appearance: Pink (5YR 7/4). Smooth with vertical ridges. Habitat and distribution: Bushveld. KwaZulu-Natal, Mpumalanga, Limpopo, North West, Swaziland, Namibia, Zimbabwe, Mozambique and Botswana. Biological activity: Antimicrobial activity against skin and stomach pathogens ‒ (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: crude bark extracts and isolated compounds inhibited the growth of both Vero and MCF-7 cell lines (Tshikalange and Hussei, 2010). The bark extract tested negative for anti-inflammatory activity (Sigidi et al., 2017). Crude extracts of the bark has shown HIV-1RT activity, isolated compounds from the bark were additionally evaluated on HIV-1IN and viral proteins (Sigidi et al., 2017; Prinsloo et al., 2018). Chemistry: In the present study, a mixture of 1:1 (MeOH and DCM) extract yielded three major triterpenes, lup-20(30)-ene-3α, 29-diol (1), 6β-hydroxylup-20(29)-ene-3-one (2) and 30- hydroxylup-20(29)-ene-3-one (3) and a polyphenol assigned as 4’-O-methyl epigallocatechin (4) from an aqueous extract. Literature report: the ethanol extract of the stem bark yielded five known pure compounds (lup-20(30)-ene-3α,29-diol), lup-20(29)-ene-30-hydroxy-3-one, taraxastanonol, β-sitosterol and 4’ –O-methylepigallocatechin (Tshikalange and Hussei 2010). Active constituents isolated from the bark include, 3-Oxo-28-hydroxylbetuli-20(29)-ene and 3, 28- dihydroxylbetuli-20(29)-ene (Mthethwa et al., 2014; Prinsloo et al., 2018). Conservation status: Near threatened

Erythrina lysistemon Species: Erythrina lysistemon Hutch. Family: Fabaceae Trade name: umnsinsi Vernacular names: common coral tree (Eng); gewone koraalboom (Afr); muvale (NSo); nsisimbane (Tso); umnsinsi, umsinsi (Zul) Method of preparation: Infusion Uses according to traders: The bark infusion is used for internal and external wounds, allergies and stomach ulcers. The bark is used as an emetic for food poisoning. Small dried pieces of the bark are used for tooth decay. The bark is used to prevent the efficacy of medication intended for witchcraft. Uses according to the literature: The bark is used as a poultice for swellings and abscesses. Ash of burnt bark is used to disinfect wounds and burnt fomentation used on sprains and strained ligaments. Chiefs traditionally use soaked water from the bark as a wash. It is used as a purifying emetic and to facilitate childbirth. The Vhavenda use the bark for toothache and as a mouthwash. Strips of bark are used to soothe sore hands and in a tea to relieve labour pains [Mabogo (1990); Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Pillay et al. (2001); Grace et al. (2003); Mabona and Van Vuuren (2013); Van Wyk et al. (2011); Masevhe et al. (2015)]. Outer bark appearance: Grey brown, green underneath the superficial layer (in very old trunks greenish yellow). Relatively smooth with longitudinal grooves and scattered knob-like projections with hooked pointed thorns. Lenticels are prominent in vertical rows. Inner bark appearance: Yellow (10 YR 7/8). Smooth with visible rays Habitat and distribution: Bushveld and coastal bush, frequently on warm, north-facing slopes of rocky ridges. Eastern Cape, KwaZulu-Natal, Mpumalanga, Gauteng, North West, Limpopo, Swaziland, Botswana, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin and stomach pathogens ‒ (Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Enterococcus faecalis). Literature report: the acetone bark extract showed antimalarial activity against Plasmodium falciparum (Prozesky et al., 2001). Prenylated flavonoids from the bark showed cytotoxicity against colon cancer cell line HCC 2998 (El-Masry, 2002); Methanol extract of the bark inhibited protein tyrosine phosphate 1B (PTP1B) (Dao et al., 2009). The bark aqueous extract and 1:1 mixture of (DCM and MeOH) exhibited activity against Gram-positive cariogenic bacterial strains, Gram-negative periodontal pathogen and three yeast strains; Candida albicans, C. glabrata and C. krusei, these are in connection with oral candidiasis (Akhalwaya et al., 2018). Chemistry: In the present study, a mixture of 1:1 (MeOH and DCM) yielded seven major prenylated flavonoid derivatives which were isolated and identified as follows: erybraedin A (1), phaseollidin (2), abyssinone V-4’ methyl ether (3), eryzerin C (4), alpumisoflavone (5), lysisteisoflavanone (6) and cristacarpin (7). Isolates 4 is reported from E. lysistemon for the first time. Literature report: Pillay et al. (2001) isolated the antibacterial isoflavone weighteone from an ethanol bark extract. El-Masry et al. (2002) isolated three prenylated flavonoid derivatives from a dichloromethane stem bark extract. Conservation status: Least concern

Erythrophleum lasianthum Species: Erythrophleum lasianthum Corbishley Family: Fabaceae Trade name: umbhemiso/ umkhwangu Vernacular names: Swazi ordeal tree (Eng); Swazi-oordeelboom (Afr); umbhemise (Zul) Method of preparation: Powdered (snuff) Uses according to traders: A tiny pinch of finely powdered bark is used as a snuff for headaches sometimes mixed with powdered bark of Cunonia capensis. If taken in doses just above a pinch, then it could cause bleeding from the nose. Uses according to the literature: Powdered bark is taken as a snuff to relieve a person from hallucinations and spells and/ or licked (khotha) for a persistent migraine, body pain and hysteria. It is sometimes mixed with the powdered bark of Warburgia salutaris. Decoctions of the bark are taken for intestinal spams, abdominal pains, as a powerful purgative. Ground bark is taken in limited doses against internal spasms. The bark is also used for snakebite, as an anthelmintic and as a magic/ spiritual charms. The powdered bark is snuffed to relieve headache, colds and for lung sicknesses in cattle [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003)]. Outer bark appearance: Bark is grey on the superficial part and becomes strong brown underneath and is dark red further down. Smooth in young stems and rough with irregular (mostly vertical) cracks in mature trunk. Inner bark appearance: The bark may be grey or dark brown. Smooth with visible stripes that are vertical or sometimes twisted. Habitat and distribution: Sand forest, grassland and Savanna. KwaZulu-Natal, Swaziland and Mozambique. Antimicrobial activity: Stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Chemistry: The bark is popularly known to be toxic. Poisonous substances include diterpenoid alkaloid, cassaine, erthrophleine (Van Wyk et al., 2009; Ndhlala et al., 2013). Conservation status: Near threatened

Eucalyptus sideroxylon

Species: Eucalyptus sideroxylon A.Cunn. ex Woolls Family: Myrtaceae Trade name: umdlavuza Vernacular names: red Ironbark (Eng); umdlavuza (Zul) Method of preparation: Decoction Uses according to traders: Decoction of the pounded bark is used in the treatment of wounds and sexually transmitted infections. The bark is cooked and administered orally two times a day with a cup for treatment of cancer. ‘Umdlavuza’ is a Zulu name for cancer. Uses according to the literature: No traditional medicinal uses of the bark seems to be available in the literature on southern Africa region. Outer bark appearance /texture: Black to reddish brown. Rough, deeply fissured and very thick (up to 12mm). Bark is saturated by small reddish black exudates that easily come off as little sandy stones. Inner bark appearance /texture: Reddish yellow (5YR 7/6). Smooth with tiny flexible fibres on the edges. Habitat and distribution: Native to Australia (Soliman et al., 2017). Biological activity: Essential oils from the stem bark as well as aqueous and methanol extracts exhibited antibacterial and antifungal activity against Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis, aphylococcus epidermidi and Candida albicans (Hossam, 2008). Ethanol- water extract of the bark exhibited potent antioxidant activity (Miranda et al., 2016; Soliman et al., 2017). Chemistry: The wood extract contains resveratrol and another hydroxystilbene and their glucosides (Hillis and Isoi, 1965). The bark contains high yields of catechin and gallic acid (Miranda et al., 2016). Conservation status: Not evaluated

Euclea natalensis Species: Euclea natalensis A.D.C Family: Ebenaceae Trade name: isizimane Vernacular names: hairy guarri (Eng); harige ghwarrie (Afr); ichitha, isizimane (Zul) Method of preparation: Decoction (oral) Uses according to traders: Root bark is boiled with water and administered orally (three cups a day) to clean the skin and body system. Pounded stem bark is sprinkled in the yard- it is used to break up marriages or relationships. Uses according to the literature: The powdered bark is boiled in water and administered orally twice a day to treat the sexually transmitted disease 'drop' and menstrual pains. The bark is chewed as a mouth wash. The root bark is used against scrofulous swellings and a pounded decoction mixed with root bark of Zanthoxylum capense and other species is taken orally for purifying the blood. Ash of burnt powdered bark is mixed with crocodile fat or petroleum jelly for the treatment of abnormal growths. Moisturised root bark is applied on the lips as a cosmetic. The stem bark is used as an ingredient in preparations to treat urinary tract diseases and susceptibility to sores [Pujol (1990); Hutchings et al. (1996); Van Wyk and Gericke (2000); Grace et al. (2003)]. Outer bark appearance: Black (5YR 2.5/1) and brown underneath superficial layer. Bark smooth with no visible lenticels. Inner bark appearance: Dark brown (7.5YR 3.4) and smooth. Habitat and Distribution: Arid rocky scrub to dune bush, open grassveld, woodland, forest, forest margin, riverine fringes, bushveld and in swamps. Eastern Cape, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Western Cape, Swaziland and Mozambique. Biological activity: Acetone and water extracts of the root bark demonstrated potent activity against H37Rv Mycobacterium tuberculosis, mutagenic activity (Lall and Meyer, 1999; McGaw et al., 2008; Verschaeve and Van Staden, 2008). The root bark crude extract as well as shinanolone (isolated compound) exhibited potent activity against Gram-positive bacterial strains and antimycobacterial activity against a drug-sensitive strain of Mycobacterium tuberculosis (Weigenand et al., 2004). Ethanol extract inhibited the growth of both Gram-positive and Gram- negative oral pathogens namely Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Actinomyces israelii, Candida albicans, Porphyromonus gingivalis, Privotella intermedia and Streptococcus mutans (More et al., 2008). The DCM extract of the stem bark and root bark has shown larvicidal properties against Anopheles arabiensis (Maharaj et al., 2012). Ethanol root bark extract was active against Propionibacterium acnes (Sharma and Lall, 2014). Chemistry: The ethanol root bark extract yielded two new compounds, octahydroeuclein and 20(29)-lupene-3 beta-isoferulate and three known compounds, shinanolone, lupeol, and betulin (Weigenand et al., 2004). The root contains pentacyclic terpenoids, diospyrin and octahydroeuclein (McGaw et al., 2008). Conservation status: Least concern

Ficus glumosa Species: Ficus glumosa Delile Family: Moraceae Trade name: inkokhokho Vernacular names: mountain rock fig (Eng); berg-rotsvy (Afr); umthombe (Xho); inkokhokho, umnyaxa (Zul); mphaya (NSo); tshikululu (Ven) Method of preparation: Decoction (emetic; topical) Uses according to traders: Decoction of the bark is used as an emetic to protect against enemies and also used as a wash 'ukugeza' in the mornings and evenings to prevent witchcraft. Uses according to the literature: Decoctions of the bark are used to treat anaemia and is given to cows to increase milk production. The latex is applied to wounds as a disinfectant [Hutchings et al. (1996); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Reddish yellow (5YR, 6/6) or brownish yellow (10 YR, 6/8). Smooth, with tiny yellowish flakes, lenticels are not visible. Inner bark appearance: Red (2.5 YR, 4/8) or yellowish red (5 YR, 4/6) and Smooth. Habitat and distribution: Bushveld and frost-protected sites in grassland; often a rock-splitter. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Gauteng, North West, Zimbabwe and Mozambique. Biological activity: Methanol stem bark extract demonstrated antidiabetic properties on the alloxan-induced diabetic mice, and best antioxidant activity in DPPH spectrophotometric assay (Madubunyi et al., 2012). Chemistry: Organic extract of the stem bark was subjected to repeated column chromatography and yielded two new ceramides together with twenty one known compounds namely; (lanosta- 7,24-dien-3-one), (lanosta-8,24-dien-3-one), β-amyrine, lupeol, 6-prenylpinocembrin, bergapten, chiricanine A, genistein, wighteone, 6-prenylapigenin, 3,4-dihydroxybenzoic acid, 2,4,5- trihydroxybenzoic acid, alpinumisoflavone, 4'-O-methylalpinumisoflavone, luteolin, catechine, β- sitosterol-3-O-(6'-O-heptadecanoyl)-β-d-glucopyranosyl, polystachyol, lyoniresinol-2a-O-β-d- xylopyranoside, β-sitosterol-3-O-β-d-glucopyranoside and dongnoside E (Nana et al., 2012). Conservation status: Least concern

Garcinia gerrardii Species: Garcinia gerrardii Harv. ex Sim Family: Clusiaceae Trade name: umbinda Vernacular names: forest mangosteen (Eng); bosgeelmelkhout (Afr); umbande, umbindi (Xho); isikhwelamfene, umbinda (Zul) Method of preparation: Powdered and infusion Uses according to traders: The bark is soaked in hot water and allowed to cool down; a drop is instilled in the ear to relieve pain. The bark is ground into powder and mixed with other plants such as Cassipourea flanaganii to make 'amagobonga' for connecting with ancestors. Uses according to the literature: The bark is used as “intelezi”, and in sprinkling charms to prevent lightening [Pujol (1990); Grace et al. (2003)]. Outer bark appearance: Light brownish grey. Smooth to the touch, but heterogeneous appearance with small rectangular flakes of a pale yellowish colour, lenticels small (<1 mm, horizontal lines), scattered and in horizontal rows. Inner bark appearance: Reddish brown (5YR, 5/4). Smooth, tiny stripes are visible on a wet surface. Habitat and distribution: Forests, in kloofs, on forest margins, sometimes on stream banks. Eastern Cape, KwaZulu-Natal, Mpumalanga and Swaziland. Biological activity: Isolated compound xanthone from the root bark has shown fungicidal activity against Cladosporium cucumerinum (Sordat‐ Diserens et al., 1989). The dichloromethane extract of the root bark and stem bark exhibited fungicidal activity against Cladosporium cucumerinurn (Cepleanu et al., 1994). Chemistry: The DCM and MeOH extacts of the root bark yielded three prenylated xanthones; garcigerrin A and garcigerrin B are pyrano derivatives of xanthone and 12b-hydroxy-des-d- garcigerrin A (Sordat‐ Diserens et al., 1989). Conservation status: Least concern

Garcinia livingstonei Species: Garcinia livingstonei T.Anderson Family: Clusiaceae Trade name: igobandlovu Vernacular names: African mangosteen (Eng); afrikageelmelkhout (Afr); mokongono (NSo); muphiphi (Ven); igobandlovu, umpimbi (Zul) Method of preparation: Powdered (topically) Uses according to traders: Dried bark is ground to a fine powder and applied directly on the piles for their treatment. Powdered bark is also used to (ukugcaba) treat illnesses associated with the vertebral column running through to the head. Pounded bark is mixed with hot water and drink half a cup to strengthen the blood of a person with weak blood. Powdered bark is applied on small razor incisions on the skin (ukugcaba) to prevent danger. Uses according to the literature: The bark is used for treatment of coughs, fever, internal parasites and diarrhoea. The bark is used in cosmetics as a skin lightening agent [Hutchings et al. (1996); De Wet et al. (2010); Van Wyk et al. (2011); Mulholland et al. (2013)]. Outer bark appearance: Grey, but orange underneath superficial layer. Smooth in younger stems. Rough, with prominent vertical cracks in mature stem. The bark resembles a mosaic appearance. Inner bark appearance: Yellowish red (5YR 5/8). Relatively smooth, saturated with many small dots (exudates) that are dark reddish in colour. Habitat and Distribution: Low-altitude bushveld and subtropical coastal grassland. KwaZulu- Natal, Limpopo, Mpumalanga, Swaziland, Mozambique, Zimbabwe, Botswana and Namibia. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: activity of morelloflavone isolated from the bark include, anti-inflammatory (Gil et al., 1997), anti-HIV (Lin et al., 1997), potent tyrosinase inhibitor, antimalarial and analgesic properties (Mulholland et al., 2013). The root bark extract and isolated compounds showed activity against Plasmodium falciparum (Mbwambo et al., 2006). Bark extracts exhibited potent antimicrobial activity against gastrointestinal tract bacteria (Van Vuuren et al., 2015). Bark extract has shown antioxidant activity (Muriithi et al., 2016). Chemistry: The dichloromethane extract of the stem bark yielded betulin (2.5 g), betulin aldehyde (5 mg), lupeol (5 mg), lupenone (5 mg), euphol (50 mg) and stigmasterol (120 mg). While a mixture of ethyl acetate/methanol extracts yielded morelloflavone (20 mg) and morelloflavone-7″- sulphate (20 mg) (Mulholland et al., 2013). The root bark extract yielded ent-naringeninyl-(I-3α,II- 8)-4‘-O-methylnaringenin, along with five known xanthones and two known biflavonoids, 1,4,5- trihydroxy-3-(3-methylbut-2-enyl)-9H-xanthen-9-one, dimeric xanthone garcilivin A and its diastereoisomer garcilivin C (Mbambwo et al., 2006). The crude chloroform, ethyl acetate/acetone and methanol extracts of the twigs and stem wood yielded two new benzophenone derivatives; 4,30,40 trihydroxy-2,6-dimethoxybenzophenone and 30-β-d-glucosyloxy-4,40-dihydroxy-2,6- dimethoxy-benzophenone along with known compounds; 3,4,4,6-tetrahydroxy-2- methoxybenzophenone, montixanthone, 6-deoxyisojacareubin, lanosta-8,24-diene-3β-ol, lupeol, botulin, stigmasterol, morelloflavone, podocarpusflavone, volkensiflavone (Muriithi et al., 2016). Conservation status: Least concern

Harpephyllum caffrum Species: Harpephyllum caffrum Bernh. Family: Anacardiaceae Trade name: umgwenya Vernacular names: wild plum (Eng); wildpruim (Afr); mothêkelê (NSo); umgwenya (Zul) Method of preparation: Decoction (emetic) Uses according to traders: The bark of Harpephyllum caffrum is often mixed with the bark of Schotia brachypetala and Syzygium cordatum and ground into a powder, boiled and taken as an emetic to treat respiratory complaints. Powdered bark is used for treatment of food poisoning. Uses according to the literature: The bark decoctions are taken as emetics to purify blood, strengthen the body and also used as washes for skin complaints such as acne and eczema. The bark is used to purify the skin for beauty purposes by means of facial steam bath. Powdered burnt bark is rubbed into scarification, around sprains and fractures [Pujol (1990); Hutchings et al. (1996); Van wyk et al. (1997, 2009); Van wyk and Gericke (2000); Grace et al. (2003); Van Wyk et al (2011); Kishore and Lall (2014);]. Outer bark appearance: Brownish in young stems and grey in mature bark. Young stems smooth with vertical fissures and scattered lenticels. Bark from mature stem is rough with dominant vertical cracks forming rectangular to irregular scales. Inner bark appearance: Red and smooth. Habitat and distribution: Coastal and inland forest. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland and Mozambique. Biological activity: Antimicrobial activity against skin pathogens (Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa). Literature report: the bark extracts exhibited hypoglycemic and hypotensive properties (Buwa and Van staden, 2006). Bark aqueous and organic extract has shown activity Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Gentamycin methicillin resistant Staphylococcus aureus, S. epidermidis, P. aeruginosa, Bacillus sabtilis, Candida albicans, Brevibacillus agri, Propionibacterium acnes, Trichophyton mentagrophytes, Microsporum canis (McGaw et al., 2000; Mabona et al., 2013). The methanol and dichloromethane stem bark extract exhibited dose-dependent acetylcholinesterase inhibitory activity and high antioxidant activity (Moyo et al., 2010). Chemistry: A mixture of cardanols, an alkyl p-coumaric acid ester, and (+)-catechin were isolated from the stem bark (Moodley et al., 2014). Chemistry of the leaves is more dominant. Conservation status: Least concern

Heteromorpha arborescens Species: Heteromorpha arborescens (Spreng.) Cham. & Schltdl Family: Apiaceae Trade name: umbangandlala Vernacular names: tropical parsley (Eng); wildepieterseliebos (Afr); mokadala (NSo); muthatha- vhanna (Ven) umbangandlala (Zul) Method of preparation: Decoction Uses according to traders: Decoction of the bark is administered orally to stimulate appetite. Uses according to the literature: The bark is used against acute abdominal pain, scrofula and in ethno veterinary medicine. It is used to treat nervous and mental disorders. A decoction of the root bark used to treat shortness of breath, coughs and dysentery. The smoke from the burning plant is inhaled to treat headaches [Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003)]. Outer bark appearance: Greyish olive. Bark is smooth and peeling. Underneath this parchment- like superficial layer is a bright green (photosynthesising) layer. Lenticels with horizontal apertures scattered, but more often-in horizontal rows. Inner bark appearance: Pale yellowish and smooth. Habitat and distribution: Wooded grassland, bushveld and on forest margins. Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Northern Cape, Western Cape, Swaziland, Lesotho, Botswana and Zimbabwe. Biological activity: Ethanol and DCM extracts of the root bark showed antiviral activity against poliovirus and rhinovirus respectively (Beuscher et al., 1994). Aqueous and organic extracts of the root and stem bark showed anti-inflammatory properties by inhibing COX-1 activity (Lundgaard et al., 2008). The bark has antimycobacterial properties (McGaw et al. 2008). Organic extract 1:1 (MeOH: DCM) of the bark has shown antimicrobial activity against Candida albicans, Cryptocarya neoformans, Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus and Moraxella catarhalis (Suliman, 2010). Chemistry: Major essential oil components are α-pinene, germacrene D and sabinene (Mwangi et al., 1994; McGaw et al., 2008; Suliman, 2010). The chemical constituents of the leaves is more dominant compared to bark. Some chemical studies are conducted on syn. Heteromorpha trifoliata. Conservation status: Least concern

Hippobromus pauciflorus Species: Hippobromus pauciflorus (L.f.) Radlk. Family: Sapindaceae Trade name: isiphahluka Vernacular names: false Horsewood (Eng); basterperdepisboom (Afr); isiphahluka (Zul); mufhapabere (Ven); ulwathile (Xho); umhlwathile (Xho) Method of preparation: Infusion Uses according to traders: The bark is used as ‘intelezi’ to protect against enemies and used by males as an emetic in love charms. Uses according to the literature: The bark in particular has no specific traditional medicinal uses. However, the antimicrobial activity of the leaves, bark and roots was conducted see Pendota et al. (2009). Outer bark appearance: Grey-brown. Rough in mature stems with prominent cracks forming rectangular scales. Inner bark appearance: Yellow (10YR 8/6) and Smooth. Habitat and distribution: Riverine thickets, along stream banks and at the margins of evergreen forests. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga. Biological activity: Extracts of aerial parts has shown anthelmintic activity (McGaw et al. 2007). The bark extracts exhibited antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Micrococcus kristinae, Shigella flexneri, Serratia marcescens, Aspergillus niger, Aspergillus flavus and Penicillium notatum (Pendota et al., 2009). Chemistry: No data could be found in the literature regarding the chemical constituents of the bark. Conservation status: Least concern

Ilex mitis Species: Ilex mitis (L.) Radlk. Family: Aquifoliaceae Trade name: umdumo Vernacular names: African holly (Eng); waterboom (Afr); monamane (NSo); phukgu (Sot); mutanzwa-khamelo (Ven); umduma (Xho); iphuphuma, umdumo (Zul) Method of preparation: Decoction (topical) Uses according to traders: The bark is used as a wash for people to admire you; hence, the plant name is called ukuduma (meaning famous). Uses according to the literature: A paste or decoction of the bark is applied to treat sores and skin rashes. The bark infusions are used to reduce fever and for the treatment of diarrhoea. The pounded bark and leaves are prepared to a foamy lather that is used as a body wash to treat flu. The bark is used as a wash inpatients with influenza. The bark is used as an enema to treat colic in children [Hutchings et al. (1996); Grace et al. (2003); Mabona and Van Vuuren (2013); Van Wyk et al. (2011)]. Outer bark appearance: Grey to white. Relatively rough with prominent, medium-sized, scattered lenticels. Inner bark appearance: Brown. Relatively rough to touch. Sometimes with visible small, scattered holes. Habitat and distribution: Forest and Bushveld, on the banks of rivers and streams. Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Western Cape, Swaziland, Lesotho, Zimbabwe and Mozambique. Biological activity: The bark aqueous extract and mixture of 1:1 (MeOH and DCM) exhibited antimicrobial activity against Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), gentamycin– methicillin-resistant Staphylococcus aureus (GMRSA), Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, Brevibacillus agri, Propionibacterium acnes, Trichophyton mentagrophytes and Microsporum canis (Mabona et al., 2013). Chemistry: The bark chemical constituents could not be found in the literature. However, the presence of pelargonidin 3-glucoside, pelargonidin 3-xylosylglucoside, cyanidin 3-glucoside and also cyanidin 3-xylosylglucoside was reported in the fruits of various Ilex species including Ilex mitis (Santamour, 1973; Alikaridis, 1987). Compounds such as α-amyrin, baurenol, neoilexonol, lupeol and triterpene saponins have been isolated from the bark of related Ilex species (Alikaridis, 1987 Fan et al., 2015). Conservation status: Declining

Kigelia africana Species: Kigelia africana (Lam.) Benth. Family: Bignoniaceae Trade name: umvongothi Vernacular names: sausage tree (Eng); worsboom (Afr); modukguhlu (NSo); muvevha (Ven); umvongothi (Zul) Method of preparation: Not specified Uses according to traders: The bark is used for penis enlargement. In this case, the shape of plant part (fruits) indicates the traditional use of the bark. Uses according to the literature: The ground bark decoctions are administered as enemas to children with stomach ailments. The bark is used for syphilis, gonorrhoea, rheumatism, ulcers and pneumonia. The stems and twigs are used for sores, wounds, snakebites and dysentery. The bark has also been reported to treat HIV. In Zimbabwe, decoctions are used to rinse the mouth in order to relieve pain and inflammation caused by toothache [Hutchings et al. (1996); Van wyk et al. (1997, 2009); Grace et al. (2003); Shai et al. (2008); Corrigan et al. (2011)]. Outer bark appearance: Greyish brown. Relatively smooth with vertical fissures, no visible lenticels. Mature trunk can be with rectangular or irregular scales. Inner bark appearance: Dark brown (7.5 YR 3/3). Smooth, sometimes with visible rays. Habitat and distribution: River banks and open woodlands. Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Zimbabwe, Botswana and Mozambique. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: best antimicrobial activity reported against Bacillus subtilis, E. coli and Klebsiella pneumoniae (Ndhlala et al., 2013). Ethyl acetate extract of the bark demonstrated potent antimalarial activity (Zofou et al., 2011). Isolated compounds from the bark p-hydroxy-cinnamic acid, specicoside and atranorin were reported with anti-malarial activity (Ntie-Kang et al., 2014). Methanol and water extract of the stem bark has potent anti-HIV activity (Prinsloo et al., 2018). Chemistry: A mixture of hexane/ethyl acetate stem bark extract yielded; atranorin, 2β, 3β, 19α- tryhydroxy-urs-12-ene-28-oic acid, 2β-hydroxyoleanolic acid, specicoside, p-hydroxy-cinnamic acid (Zofou et al., 2011; Ntie-Kang et al., 2014). A review of K. africana reported the following chemical compounds isolated from the bark, quercetin, luteolin, 6-hydroxy liteolin, luteolin-7-O- glycoside, 6-hydroxyluteolin-7-O-glycoside, 6-methoxymellein, kigelin and 3-demethylkigelin, 6- demethylkigelin, atranorin (Bello et al., 2016). Naphthoquinones, iridoids, sterols, coumarins, flavonoids and alkaloids kigelin, β-sitosterol, 1,3dimethylkigelin and ferulic acid have been isolated from the bark (Prinsloo et al., 2018). Conservation status: Least concern

Macaranga capensis Species: Macaranga capensis (Baill.) Sim Family: Euphorbiaceae Trade name: unompumelelo Vernacular names: river macaranga, false-poplar (Eng); riviermakaranga, valspopulier (Afr); umbengele, umphumeleli (Xho); iphumela, unompumelelo (Zul) Method of preparation: Infusion (emetic, topical) Uses according to traders: The bark is used as a wash and emetic in good luck charms in order for a person to become successful. Hence, the meaning of the plant’s name is success (unompumelelo). Uses according to the literature: Root and bark extracts are taken orally as an aphrodisiac. The bark is used to treat skin diseases and sunburn [Pujol (1990); Hutchings et al. (1996); Grace et al. (2003); Maroyi et al. (2013); Van Wyk et al. (2011)]. Outer bark appearance: Superficial appearance is grey with reddish layer underneath. Bark is smooth and thin. Lenticels on young branches. They are small to medium in size with horizontal apertures and are scattered. Inner bark appearance: Pink (2.5 YR 8/3) and Smooth. Habitat and distribution: Low-altitude forest, along streams and in wet areas. Eastern Cape, KwaZulu-Natal, Zimbabwe and Mozambique. Biological activity: The extract showed potent antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa (Kaikabo et al., 2009). Chemistry: No data from the literature seems to be available regarding chemical constituents from the bark. The major components present in Macaranga species include epitaraxerol and taraxerone with small amounts of taraxerol, β-amyrin and friedelin (Markstӓdter et al., 2000). Conservation status: Least concern

Maytenus undata Species: Maytenus undata (Thunb.) Blakelock Family: Celastraceae Trade name: undabulavalo Vernacular names: koko tree (Eng); kokoboom (Afr); Morokgwa (NSo); tshibvukahalwa (Ven); umnqayimpofu (Xho); undabulaluvalo (Zul) Method of preparation: Powdered and decoction (oral) Uses according to traders: The bark is ground into a fine powder and sprinkled over cooked meal/ food to break or make a love charm. The bark is used as an emetic to protect or fight against people who are bewitching you (abantu abakuphonsayo). Uses according to the literature: The bark is used for the treatment of malaria, ulcers and other stomach problems. The branches are used in witchcraft by the VhaVenda. It is used for inflammation, pain and chest pain [Mabogo (1990); Grace et al. (2003); Muthaura et al. (2007); Mokoka et al. (2013); Veloso et al. (2017)]. Outer bark appearance: Dark grey with strong brown layer underneath. Bark is smooth. On mature trunk can be relatively rough, with irregular cracks. Lenticels are not visible. Inner bark appearance: Yellow (10YR 8/6). Fibrous, peel off in long strips. Fibres flexible when wet. Habitat and distribution: Savannah and rocky outcrops. Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern Cape, North West and Zimbabwe. Biological activity: The methanol and water extracts of the rootbark exhibited potent antiplasmodial activity against Plasmodium falciparum and antifungal activity (Clarkson et al., 2004; Muthaura et al., 2015; Veloso et al., 2017). Chemistry: Various extracts from aerial parts including the stem bark yielded four new 12- oleanene and 3,4-seco-12-oleanene triterpene acids; namely (3-oxo-11α-methoxyolean-12-ene-30- oic acid), (3-oxo-11α-hydroxyolean-12-ene-30-oic acid), (3-oxo-olean-9(11),12-diene-30-oic acid) and 3,4-seco-olean-4(23),12-diene-3,29-dioic acid (20-epi-koetjapic acid), along with the known 3,11-dioxoolean-12-ene-30-oic acid (3-oxo-18β-glycyrrhetinic acid), koetjapic acid, and the 12- oleanene artifact 3-oxo-11α-ethoxyolean-12-ene-30-oic acid (Muhammad et al., 2000; Mokoka et al., 2013; Veloso et al., 2017). Conservation status: Least concern

Ochna holstii Species: Ochna holstii Engl. Family: Ochnaceae Trade name: isibhanku Vernacular names: real red pear, red ironwood (Eng), regetrooipeerhout, regterooipeer (Afr), tshipfure (Ven) isibhanku (Zul) Method of preparation: Decoction (topical) Uses according to traders: The bark decoction is used as intelezi and taken as a wash to protect against enemies and chase away bad spirits. Uses according to the literature: The bark in particular has no recorded traditional medicinal uses. However, the shoots are soaked in water and used in ethno-veterinary medicine in cattle, unspecified part of the plant is used for gastrointestinal disorders in Tanzania [Schlage et al. (2000); Grace et al. (2003); Luseba and Tshisikhawe, (2013)]. Outer bark appearance: Brownish grey. Relatively smooth with tiny irregular cracks. Lenticels are not visible. Inner bark appearance: Pink (7.5 YR 7/4) and smooth. Habitat and distribution: Forests. Limpopo, Mpumalanga, KwaZulu-Natal, Eastern Cape, and also in Swaziland, Zimbabwe and Mozambique. Biological activity: No literature data is available on the biological activity of the bark. The pharmacology and phytochemistry of the bark and the whole plant is poorly documented. Information of the isolated compounds below was obtained from a PhD thesis. Chemistry: The methanol extract of the stem bark yielded; afzelone D, calodenone, lophhirone, 5,4'-dimethoxy-6,7-methylenedioxyisoflavone, 5,3',4'-trimethoxy-6,7-methyIenedioxyisoflavone, (±)-catechin, and 2,4-dihydroxyphenylmethyl acetate (Tsegaye, 2015). Conservation status: Least concern

Ocotea bullata Species: Ocotea bullata (Burch.) E. Meyer in Drege Family: Lauraceae Trade name: unukani Vernacular names: black stinkwood (Eng); stinkhout (Afr); unukani, umhlungulu (Xho); unukani (Zul) Method of preparation: Infusion Uses according to traders: The bark is used as an 'intelezi' for protection of the traditional army during war. The bark is used as an enema, emetic and wash to get rid of fear, bad luck or a curse as a result of witchcraft. Uses according to the literature: The bark is used as a general tonic, for the treatment of nervous disorders and diarrhoea in children. The powdered bark is mixed with that of Croton gratissimus and Zingiber officinale root. It is blown through a hollow reed through the penis into the bladder to treat urinary complaints. Snuff or smoke from the bark is inhaled to treat headaches. It is used for a “bleeding stomach”, and as an emetic for emotional and nervous disorders [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Grey to brown. Relatively smooth and even in mature trunks. Roundish or slightly elongated dark lenticels scattered on young stems. Inner bark appearance: Brown (7.5 YR 4/4) to dark brown (7.5 YR 3/2). Relatively smooth with vertical ridges. Habitat and distribution: Montane forest. Western Cape, Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga and Swaziland. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: the bark extracts exhibited good prostaglandin synthesis inhibitory activity (Jäger et al., 1996). Stem bark extracts exhibited potent antidiabetic, anti-inflammatory and antioxidant activity which was screened using α-amylase, α-glucosidase, 5-lipoxygenase enzymes and DPPH radical- scavenging activity respectively (Ogundajo et al., 2018). Chemistry: The heaxane, chloroform and ethyl acetate extracts of the stem bark yielded; neolignan ketone, namely ocobullenone, bullatone, iso –ocobullenone and ∆8‵ -3,4- methylenedioxy-3‵ ,4‶ -methylenedioxy-l‵ ,2‵ ,3‶ 6-tetrahydro-6‵ -oxo-8,3‵ -neolignan (Drewes et al., 1995) and sibyllenone (Fennel et al., 2004; Steenkamp et al., 2005; Ogundajo et al., 2018). Conservation status: Endangered

Ozoroa paniculosa Species: Ozoroa paniculosa (Sond.) R.Fern & A. Fern Family: Anacardaceae Trade name: isifica Vernacular names: common resin tree (Eng); gewone harpuisboom (Afr); monoko (NSo); mudumbula (Ven); isifica (Zul) Method of preparation: Powdered (oral) Uses according to traders: The bark is ground into powder and licked (khotha) for the treatment of stroke. The bark is also used to treat food poisoning. Uses according to the literature: Powdered bark is administered orally or as an enema to treat acute inflammation in the chest and dysentery. The bark is used in ethno-veterinary medicine for abdominal ailments [Hutchings et al. (1996); Grace et al. (2003); McGaw et al. (2008); Van Wyk et al. (2011)]. Outer bark appearance: Greyish black to dark brown and lined with yellow underneath. Bark is more or less even to rough in old mature trunk, with scales of irregular shape that resembles a mosaic pattern. No lenticels. Inner bark appearance: Red (5R 5/8) and smooth. Habitat and distribution: Prevalent in deciduous woodlands and in bushveld. Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Zimbabwe, Namibia, Botswana and Mozambique. Biological activity: The acetone extract of the bark showed potent activity against Enterococcus faecalis, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Mycobacterium aurum (Seaman, 2006). The pharmacological activity of the bark in particular is poorly documented. However, the roots have various biological activities reported. Chemistry: The acetone extract of the bark yielded two anacardic acids namely; 6-[8(z)- pentadecenyl] salicylic acid and 6-pentadecylsalicyclic acid (Seaman, 2006). Conservation status: Least concern

Pappea capensis Species: Pappea capensis Eckl. &Zeyh Family: Sapindaceae Trade name: iletha, umhuluka Vernacular names: jacket plum, bushveld cherry (Eng); doppruim (Afr); ilitye, umgqalutye (Xho); mongatane (NSo); gulaswimbi (Tso); tshikavhavhe (Ven); indaba, iletha (Zul) Method of preparation: Powdered (oral) Uses according to traders: The powdered bark is licked (khotha) for 'amahlaba/ izibhobo' antispasmodic effect. The bark is boiled in water and used as an emetic for the treatment of food poisoning and to bring in luck. The powdered bark is used licked to relieve pains in the body. Uses according to the literature: The bark infusion is used to gain courage, strengthen the body and is also used in protective sprinkling charms. The bark is used to treat whooping cough, chest complaints, sore throat, wounds, eye infections and sexually transmitted diseases [Mabogo (1990); Hutchings et al. (1996); Van Wyk and Gericke (2000); Grace et al. (2003); Van Wyk et al. (2011); Kareu et al. (2012)]. Outer bark appearance: Grey brown. Smooth in young stems. Bark of mature trunk relatively smooth to rough with irregular (mostly vertical) cracks. Inner bark appearance: Reddish yellow (7.5YR 6/8) to red (2.5YR 4/8). Solid with prominent vertical ridges. Habitat and distribution: Bushveld, riverine thicket, wooded grassland and karroid vegetation. It is dispersed within the nine provinces of South Africa, through to Mozambique and Zimbabwe. Biological activity: Twigs showed antimalarial activity against Plasmodium falciparum (Bapela et al., 2014).The leaf extracts have been screened against various biological activities. Data on the pharmacological properties of bark extacts is largely neglected. Chemistry: Isolation of biological compounds from the leaf is more dominant. Data on the bark extacts is neglected. Conservation status: Least concern

Peltophorum africanum Species: Peltophorum africanum Sond. Family: Caesalpinaceae Trade name: msehla/ umsenhla Vernacular names: African-wattle (Eng); huilboom (Afr); mosehla (NSo); muparapara (Tsw); ndzedze (Tso); musese (Ven); umthobo (Xho); isikhabamkhombe, umsehle (Zul) Method of preparation: Infusion Uses according to traders: Pounded bark is sprinkled in the yard to chase away bad spirits. Uses according to the literature: The bark and roots are used for sterility and backache. The Sotho people chew fresh bark for colic. The Vhavenda use the bark for coughs, sore throats, tuberculosis, intestinal parasites, fever, ulcers, and blisters in the mouth. A decoction of the stem bark is used to treat general sexually transmitted infections (STI's), wounds, human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS)diarrhoea, dysentery and as a general tonic [Mabongo (1990); Huchings et al. (1996); Van wyk and Gericke (2000); Grace et al. (2003); Samie et al. (2005); Theo et al. (2009); Van Wyk et al. (2011); Maroyi et al. (2013); Chinsembu (2016)]. Outer bark appearance: Light grey to black and becomes strong brown and yellowish underneath. Smooth in young stems, rough with vertical cracks and horizontal fissures in medium- sized stems and very rough with irregular cracks in old mature trunks. Lenticels are present on smooth parts and their remains. Inner bark appearance: Brown (7.5 YR 4/6) to red (2.5 YR 3/6). Fibrous, fibres flexible are when wet. Habitat and distribution: Bushveld, usually on sandy soils. Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Swaziland, Namibia, Botswana, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa and Staphylococcus aureus). Literature report; The ethyl acetate and butanol extracts of the stem bark exhibited inhibitory activity against HIV-1, CXCR4 (X4) and CCR5 (R5) tropic viruses (Theo et al., 2009). Petroleum ether, dichloromethane, 80% ethanol and water extracts of the stem bark demonstrated antimicrobial activity against Bacillus subtilis, Escherichia coli, K. pneumoniae, S. aureus, Neisseria gonorrhoeae and Candida albicans. Methanol and water extracts exhibited HIV-1 RT inhibitory activity (Mulaudzi et al., 2011; Prinsloo et al., 2018). Chemistry: The ethyl acetate and butanol bark extracts yielded; (+)-catechin, a flavonoid, and bergenin, a C-galloylglycoside, respectively. Betulinic acid from the ethyl acetate fraction and was identified for the first time on the plant species (Theo et al., 2009). A review by Mazimba (2014) on P. africanum reported; isolated compounds from the bark, heartwood and roots as follows: berginin, norbegenin, 11-O-(E)-p-coumaroylbergin, 11-O-galloylbegenin, gallic acid, methylgallate, 3-O-methylgallic acid, chlorogenic acid, from the bark and leaves as follows: betulinic acid, β-amyrin, β-sitosterol, stigmasterol, from the bark and roots: contain red coloured gallotannin, epigallocatechin-3-O-gallate, (+)-gallocatechin-3-O-gallate, robinetinidol-3-O-gallate, catechin-3-O-rhamnoside, flava-3-ol, fisetinidol, robinetinidol, catechin (Mazimba, 2014; Prinsloo et al., 2018). Conservation status: Least concern

Philenoptera violacea Species: Philenoptera violacea (Klotzsch) Schrire Family: Fabaceae Trade name: isihomohomo Vernacular names: apple-leaf (Eng); appelblaar (Afr); isihomohomo, umbandu (Zul) Method of preparation: Decoction Uses according to traders: The bark is used as ‘intelezi’ to protect the household from enemies. Uses according to the literature: The stem bark is used as a laxative, for treatment of skin diseases, reduce fevers, and in therapy for convulsions. An infusion of the bark is used to treat diarrhoea and dysentery. The powdered bark and root-bark is used to treat colds and snakebite [Hutchings et al. (1996); Grace et al. (2003); Van Wyk et al. (2011); Ntsoelinyane et al. (2014)]. Outer bark appearance: Light grey with brownish orange colour underneath the superficial layers. Younger branches and stems are smooth. Mature stems become relatively rough and flaking. Prominent vertical rigdes/ cracks. Inner bark appearance: Yellow (10YR 8/6). Smooth and fibrous Habitat and distribution: Bushveld and woodland, often at low altitude along rivers. KwaZulu- Natal, Limpopo, Mpumalanga, Eastern Cape, Swaziland, Namibia, Botswana, Zimbabwe and Mozambique. Biological activity: The DCM extract of the stem bark has shown larvicidal properties against Anopheles arabiensis (Maharaj et al., 2012). The methanol extract of the twigs exhibited poor antioxidant free radical scavenging activity, the extracts were inactive as anticancer agents (Ntsoelinyane et al., 2014). The methanol extract of the twigs exhibited antibacterial activity against Salmonella typhimurium and did not show mutagenic properties (Ntsoelinyane-Mfengwana and Mashele, 2016). The pharmacological investigation of the plant is poorly documented. Chemistry: Only data regarding the total phenolic content and class of secondary metabolites could be found. Conservation status: Least concern

Pittosporum viridiflorum Species: Pittosporum viridiflorum Sims Family: Pittosporaceae Trade name: umfusavu Vernacular names: cheesewood (Eng); kasuur (Afr); kgalagangwe (NSo); umkhwenkwe (Xho); umfusavu (Zul) Method of preparation: Infusion (oral, topical) Uses according to traders: An infusion of the bark is used for stomach complaints, and rarely used as wash for skin ailments. Uses according to the literature: The bark decoctions induce febrile complaints, taken for back pains and administered as emetics or enemas for stomach problems. The stem bark is also used to treat chest complaints, for fever, inflammation, malaria and as antidote for insect bites. The powdered bark is used to for treating toothache in Swaziland. The bark is used as a chewing stick for the management of opportunistic oral fungal infections in HIV/AIDS patients. Decoction or infusion of the bark is taken orally for several weeks in the Eastern Cape, to cure cancer, while the root powder is taken orally for cancer [Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Van Wyk and Gericke (2000); Grace et al. (2003); Van Wyk et al. (2011); Okoye et al. (2014); Elisha et al. (2016); Madikizela and McGaw (2017)]. Outer bark appearance: Greyish brown. Relatively smooth. Lenticels small to medium-sized with horizontal apertures scattered or in distinct horizontal rows. Inner bark appearance: Pale brown (10 YR 6/3). Very smooth and solid. Habitat and distribution: Forest margins, bush clumps and bushveld, often in rocky places. Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Western Cape, Swaziland, Lesotho, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin pathogens‒ (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa). Literature report; the acetone extract of the bark showed antioxidant activity in the DPPH, nitric oxide (NO), and hydrogen peroxide (H2O2) radical scavenging assays (Otang et al., 2012). The ethanol bark extract showed antibacterial activity against P. aeruginosa, S. aureus, P. mirabilis, K. pneumonia and E. coli (McGaw et al., 2000; Madikizela and McGaw, 2017). Isolated compound from the bark showed potent antimalarial activity against Plasmodium falciparum and Leishmania donovani parasites (Nyongbela et al., 2013), methanol extract of the bark showed anticancer activity (Madikizela and McGaw, 2017), Chemistry: The methanol stem bark from Pittosporum mannii (synonym) yielded, 1-0- {alpha-L- rhamnopyranosyl}-23-acetoxyimberbic acid 29-methyl ester (Nyongbela et al., 2013; Madikizela and McGaw, 2017). The chemical isolation and characterisation of the leaf extracts is more dominant. Conservation status: Least concern

Pleurostylia capensis

Species: Pleurostylia capensis (Turcz.) Loes. Family: Celastraceae Trade name: umthunyelwa Vernacular names: bastard saffronwood (Eng), bastersaffraanhout (Afr), murumelelwa (Ven); umbovane (Xho), umngqangqa, umthunyelelwa (Zul) Method of preparation: Powdered Uses according to traders: The bark is used make dreams clear, hasten the dreams in cases where there is an urgent message coming from the ancestors and to chase away bad dreams. The bark is popularly used as a love charm. Uses recorded in the literature: The bark is used in magic charms. The bark is used as a cosmetic, in steam bath and for cleansing [Mabogo (1990); Hutchings et al. (1996); Grace et al. (2003); Razwinani et al. (2014)]. Outer bark appearance: Grey brown. Young stems relatively smooth with rectangular scales, sometimes horizontal cracks are dominant. Bark of mature trunk becomes relatively rough with irregular flakes. Inner bark appearance: Pink (7.5 YR 8/4). Relatively smooth with vertical ridges. Habitat and distribution: Scrub, wooded ravines, along rivers and streams, and in coastal and mountain forest. Western Cape, Eastern Cape, Kwazulu-Natal, Mpumalanga, Swaziland and Limpopo Province. Biological activity: The bark extracts has shown antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Mycobacterium smegmatis (Clinical isolates), Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Streptococcus pyogenes, Pseudomonas aeruginos, Salmonella typhimurium and Candida albicans. Antioxidant activity, evaluated using 1, 1 diphenyl-2-picryl- hydraxyl (DPPH) technique, and anti- inflammatory activity, evaluated using the enzyme-based cyclooxygenase assays; COX-1 and COX-2 (Razwinani et al., 2014). Chemistry: The isolation and identification of active compounds of both the root bark and stem bark could not be found in the literature. Conservation status: Least concern

Protorhus longifolia Species: Protorhus longifolia (Bernh.) Engl. Family: Anacardiaceae Trade name: unhlangothi Vernacular names: red-beech (Eng); rooiboekenhout (Afr); ikhubalo (Xho); unhlangothi (Zul) Method of preparation: Powdered (oral) Uses according to traders: A teaspoon of powdered bark is mixed together with “Big Five” animal bones in powdered form and is licked (khotha) directly from the hand for treatment of stroke. Sometimes powdered bark is applied onto small razor incisions in the body (ukugcaba) for stroke. Uses according to the literature: Pulverised bark is injected to cure hemiplegic paralysis believed to be caused by the same bark through witchcraft. Bark decoctions are taken as emetics to relieve heartburn and a bleeding stomach by the Zulu culture. The bark is used traditionally to ‘thin’ blood. The bark is reported to be poisonous [Pujol (1990); Hutchings et al. (1996); Grace et al. (2003); Van Wyk et al. (2011); Mosa et al. (2014)]. Outer bark appearance: Grey to brown and red underneath. Bark is more or less level (uniform) to rough in old mature trunk, with dominant vertical cracks. Lenticels are probably in vertical rows. Inner bark appearance: Dark red (10 R 5/8). Smooth and solid with scattered ridges. Sometimes vertical cracks occur in samples. Habitat and distribution: Coastal and montane forest, on rocky outcrops and in riverine vegetation. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga and Swaziland. Biological activity: The chloroform crude extract of the bark and isolated lanostane-type triterpenes exhibited good anti-platelet aggregation activity on rats, evaluated on thrombin, adenosine diphosphate (ADP), epinephrine and arachidonic acid. The methanol extract of the bark exhibited potent antioxidant activity. Isolated compound, 3β-hydroxylanosta-9, 24-dien-24-oic acid from the bark has shown significant anti-inflammatory and in vivo antihyperglycemic activity (Mosa et al., 2011a, 2011b, 2015). Chemistry: The chloroform extract of the bark yielded two lanostane-type triterpenes namely, 3- Oxo-5α-lanosta-8, 24-dien-21-oic acid and 3β-hydroxylanosta-9, 24-dien-24-oic acid (Mosa et al., 2011; 2015). Conservation status: Least concern

Prunus africana Species: Prunus africana (Hook.f.) Kalkman Family: Rosaceae Trade name: inyazangoma elimnyama Vernacular names: stinkwood (Eng); rooistinkhout (Afr); red mogohloro (NSo) inyazangoma (Xho); mulala-maanga (Ven); inyazangoma elimnyama (Zul) Method of preparation: Infusion (oral) Uses according to traders: Infusion of the bark is administered orally to treat colds and influenza. Uses according to the literature: Bark decoction is used to treat intercostal pains. Medication from the stem bark has been reported to be active against prostatic hypertrophy. The bark is used for the treatment of stomach ache, urinary tract disorders, kidney disease, chest pain, malaria, inflammation and in wound dressings [Pujol (1990); Van Wyk et al. (1997, 2009); Grace et al. (2003); Bodeker et al. (2014); Van Wyk et al. (2011); Muthaura et al. (2015)]. Outer bark appearance: Grey to black or dark brown to black. Very rough, with various irregular vertical and horizontal cracks, forming irregular scales. No lenticels. Inner bark appearance: Dark red (2.5 YR 3/6) or strong brown (7.5 YR 4/6). Fibrous and brittle. Habitat and distribution: Montane forest, particularly in mist-belt areas. Eastern Cape, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Swaziland, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Staphylococcus aureus). Literature report: Isolated compounds from the bark extracts such as phytosterols, particularly β- sitosterol have anti-inflammatory properties that inhibit the swelling of the prostate gland, pentacyclic triterpenoids that provide anti-edematous activity and ferulic acid esters, or their chemical derivatives, which have a powerful hypocholesterolemic activity in the prostate (Kadu et al., 2012). Best antimicrobial activity against Bacillus subtilis, S. aureus and K. pneumoniae (Ndhlala et al., 2013). The methanol stem bark extract showed antimicrobial activity against Staphylococcus aureus and methicilin resistance Staphylococcus aureus (Mwitari et al., 2013). Ethanol extracts of the stem bark has anti-HIV properties (Prinsloo et al., 2018). Chemistry: Triterpenic acids including derivatives of ursolic and oleanolic acids were detected (Fourneau et al., 1996). Neolignan (Van Wyk et al., 1997) and β-sitosterol (McGaw et al., 2008). The bark extracts of P. africana sampled from 20 different trees throughout Africa yielded avarage concentrations of lauric acid (18), myristic acid, (22), n-docosanol (25), ferulic acid (49), β- sitostenone (198), β-sitosterol (490), and ursolic acid (743) evaluated using the GC–MSD (Kadu et al., 2012). The bark contains daucosterol, oleanolic acid, docosyl ferulate (Jena et al., 2016). Ferulicacid, n-docosanol, lauric acid, myristic acid, β-sitostenone and β-sitoreol are active constituents isolated from the bark (Prinsloo et al., 2018). Conservation status: Vulnerable species facing a high risk of extinction.

Ptaeroxylon obliquum Species: Ptaeroxylon obliquum (Thunb.) Radlk. Family: Rutaceae Trade name: umthathe Vernacular names: sneeze wood (Eng); nieshout (Afr); munari (Ven); umthathe (Xho); ubhaqa, umthathe (Zul) Method of preparation: Decoction (topical) Uses according to traders: The bark is soaked in hot water and used for steaming and also as a body wash for good luck. The bark is used to trap enemies. Uses according to the literature: Bark infusions are used to treat fever, for rheumatism, arthritis and heart disease. The powdered bark or heartwood is traditionally used as a snuff to relieve headaches [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Philander (2011); Van Wyk et al. (2011)] Outer bark appearance: Greyish brown and yellow inside the vertical fissures. Relatively smooth in young stems. Bark of mature stems with prominent vertical fissures and tiny horizontal cracks. The fissures are not too deep. Inner bark appearance: Yellow (2.5 Y 8/8). Rough, very fibrous. Fibres are strong and flexible when wet. Habitat and distribution: Woodland, scrub forest, evergreen montane and sand forest. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Western Cape, Swaziland, Namibia, Botswana, Zimbabwe and Mozambique. Biological activity: Mixture of 1:1 (DCM and MeOH) bark extracts has shown antimalarial activity against Plasmodium falciparum (Clarkson et al., 2004). Bark extracts exhibited activity against Mycobacterium tuberculosis H37Rv, methicillin-resistant Staphylococcus, Escherichia coli, Klebsiella pneumoniae, carbenicillin-resistant Pseudomonas aeruginosa, chloramphenicol-resistant Citrobacter freundi, Candida albicans and Microsporum audouinii (Nielsen et al., 2012; Cock and Van Vuuren, 2015). Chemistry: The ethyl acetate extract of the heartwood yielded a major compound peucenin. The early fractions yielded a new chromone, desoxykarenin, two new isomeric coumarins; 7-0-(3, 3- dimethylallyl) scopoletin and neishoutin, and the dominant β-sitosterol. Later fractions were, peucenin, nieshoutol (a third new coumarin), and a novel chromone, karenin. The petrol extract also yielded a new coumarin, identified as 7-O-(l, l-dimethylallyl) scopoletin (McCabe et al., 1967). The heartwood extract also yielded seven coumarins, which were isolated and identified as follows: obliquin, preyletin, obliquetin, cyclo-obliquetin, obliquetol, obliquol and scopoletin, which was present in trace amounts (Dean et al., 1967). The chloroform extract of the roots yielded ptaerobliquol, ptaeroxylinol acetate, peucenin, prenyletin and scopoletin (Agostinho et al., 2013). Conservation status: Least concern Notes

Pterocarpus angolensis

Species: Pterocarpus angolensis DC. Family: Fabaceae Trade name: umvangazi Vernacular names: bloodwood (Eng); kiaat (Afr); mutondo (Ven); mvhangazi (Tso); mokwa (Tsw); umvangazi (Zul) Method of preparation: Decoction (oral) Uses according to traders: Pounded bark is used as an emetic to bring luck. Uses recorded in the literature: The bark is used for treating piles, amenorrhoea, sexually transmitted diseases, infertility, haematuria, bilharzia, bleeding nose, stomach disorders, headaches and tuberculosis. Red sap from the inner bark is used to treat of sores and ringworms. Infusion of the bark is used for treatment of diarrhoea and menorrhagia [Mabogo (1990); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Dark grey to brown underneath. Rough on mature stems with prominent vertical cracks forming rectangular scales. Red sap exudates are present on the bark samples. Inner bark appearance: Reddish grey (5YR 5/2) Smooth with dark red exudates. Habitat and distribution: Deep sandy soils or well-drained rocky slopes and in woodlands. Biological activity: Dichloromethane extract of the bark has shown antimalarial activity against Plasmodium falciparum (Clarkson et al., 2004). The bark extracts showed antibacterial and antifungal activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae and Candida albicans, and HIV-1 RT inhibitory activity (Mulaudzi et al., 2011). The dichloromethane and petroleum ether extracts of the bark showed potent anti-inflammatory activity in both COX-1 and -2 assays (Mulaudzi et al. 2013). Isolated compounds showed antibacterial activity and low cytotoxicity (Samie et al. 2009). The biological activity of isolated compounds from the bark below (Abubakar and Majinda, 2016) include, antifungal„ antioxidant, cytotoxic, anti-inﬂammatory, analgesic, antipyretic, antihypertensive and mosquito larvicide (Abubakar and Majinda, 2016). Aqueous extract of the bark exhibited anti-tuberculosis activity and moderate inhibition of the HIV replication (Sigidi et al., 2016). Chemistry: Ethanol extract of the stem bark yielded, phthalate and four derivatives of epicatechin [(-)-epicatechin, epicatechin-3-O-galate, epicatechin (4b–8)-epicatechin (B2), and a hexamer of epicatechin] (Samie et al., 2009). The n-hexane and chloroform extracts of the stem bark yielded the following: tetratriacontane (31.67%), n-hexadecanoic acid (10.29%), 7-dehydrodiosgenin (9.58%), stigmasta-3, 5-dien-7-one (7.13%), lupeol (6.54%), octadecanoic acid (5.89%), friedelan- 3-one (2.56%), hexadecanoic acid, methyl ester (1.84%), and tetradecanoic acid (1.84%), 1- octacosanol (9.87%) (Abubakar and Majinda, 2016). The 1:1 mixture of DCM and MeOH yielded caffeic acid, catechin, epicatechin, gallic acid, protocatechuic acid, taxifolin and rutin (Zininga et al., 2017). Conservation status: Least concern

Pterocelastrus rostratus Species: Pterocelastrus rostratus Walp. Family: Celastraceae Trade name: usehlulabanye Vernacular names: red candlewood (Eng); rooi-kersboom (Afr); Itywina (Xho); usehlulabanye (Zul) Method of preparation: Infusion (enema, emetic) Uses according to traders: Infusion of the bark is administered as enemas and emetics used during war to defeat opponents from another clan or group of people involved. Uses according to the literature: Bark decoctions are taken as emetics for respiratory ailments. In Swaziland, a tablespoon of powdered bark mixed with Rapanea melanophloeos bark is added to a litre of warm water, and a tablespoon of this is taken three times a day to treat general body pains. The bark is also used for chest block and a wheezing chest [Pujol (1990); Grace et al. (2003)]. Outer bark appearance: Greyish brown. Bark is smooth with small scattered yellowish lenticels. Inner bark appearance: Dark reddish brown (2.5 YR 3/3). Smooth with solid fibrous appearance. Fibres brittle break if pulled. Habitat and distribution: Forest and montane scrub in forest margins and on mountain sides. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga and Swaziland. Biological activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa and Staphylococcus aureus). Chemistry: No data in the literature seems to be available on the isolated chemical constituents of the bark. Conservation status: Declining Notes +

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Rapanea melanophloeos Species: Rapanea melanophloeos (L.) Mez Family: Primulaceae Trade name: umaphipha Vernacular names: Cape beech (Eng); Kaapse boekenhout (Afr); mogono (NSo) isiqwane- sehlathi (Xho); umaphipha (Zul) Method of preparation: Decoction (oral, topical) Uses according to traders: Bark decoction is used for cleaning the blood and taken as a body wash for removing bad luck (isichitho). Uses according to the literature: The bark is used for strengthening the heart, acidity, stomach ailments, muscular pain, and fevers and also in sprinkling charms against lightening. Bark decoctions are taken for hematemesis and stomachache. Fresh pieces of dried powdered bark are chewed to relieve sore throats and to treat wounds. The bark is used as a skin lightener in the Eastern Cape [Hutchings et al. (1996); Van wyk et al. (1997, 2009); Grace et al. (2003); Van Wyk et al. (2011); Madikizela et al. (2012)]. Outer bark appearance: Greyish brown (10YR 5/2) and reddish towards the inner surface. Relatively smooth with vertical fissures in youngn and rough with irregular cracks in mature trunk. Inner bark appearance: Reddish black (5R 2.5/1).Relatively smooth in touch with visible rays, which lighter in colour then ground tissues. Often vertical cracks occur in samples. Habitat and distribution: Forests and bush clumps, often on moist areas. Western Cape, Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin and respiratory pathogens (Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Moraxella catarrhalis). Literature report: ethyl acetate extract of the bark exhibited potent antioxidant activity with the highest reduction potential and chelating activity on Fe2+. In addition, the bark extracts showed an anti-platetlet aggregation activity induced by thrombin, ADP and epinephrine (Mosa et al., 2011). Chemistry: benzoquinones, embelin/ rapanone (Midiwo, 2002; Muthee et al., 2011; Amenya et al., 2014). Conservation status: Declining

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Rauvolfia caffra Species: Rauvolfia caffra Sond. Family: Apocynaceae Trade name: umhlambamanzi Vernacular names: quinine tree (Eng); kinaboom (Afr); monadi (NSo); umthundisa (Xho); umhlambamanzi (Zul) Method of preparation: Infusion (topical: wash and facial sauna) Uses according to traders: The bark is used for cleansing when a family member had passed away; this is done to remove 'isinyama' (“the dark shadow of the dead”). It is also used for ‘ukugquma’ in order to be adored by people. Bark emetic is used to make a clear path for the ancestor’s dreams so that a person (usually a sangoma or inyanga) can understand the message from the ancestor. Uses according to the literature: Bark preparations are applied to measles, urticarial, and other skin rashes. The bark is chewed to relieve coughs. Bark decoctions are used for abdominal and pelvic complaints, fever, as a tranquiliser for hysteria, insomnia, and malaria. It is used for general body swelling, rheumatism, fever, uterine complaints and pneumonia, while infusions are taken to kill maggots in wounds by the VhaVenda [Mabongo (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Yellow to light brown. Smooth in young stems, rough with short vertical cracks and horizontal lines on mature trunk. Periderm is spongy to the tough (like polystyrene). Lenticels are present, even in mature bark from the trunk. They are medium in size and scattered. Inner bark appearance: Yellowish brown (10YR 5/6). Relatively smooth and spongy to the touch. Sometimes with visible rays. Habitat and distribution: Coastal forest and forest margins. Eastern Cape, Gauteng, KwaZulu- Natal, Limpopo, Mpumalanga, North West, Western Cape, Swaziland, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin pathogens (Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa). Literature report: the bark extract exhibited potent antimalarial activity against Plasmodium falciparum (Bapela et al., 2014). Chemistry: Thirty-two alkaloids were isolated from the stem bark and 28 were identified as follows: corynane, strictamine, sarpagan, akuammicine, pleiocarpamine, indolenine, dihydroindole, peraksine, heteroyohimbine, hydroxyheteroyohimbine, oxindole, 2-acyl-indole, and suaveoline and yohimbine types. The presence of anhydronium base serpentine was noted. The major alkaloids were identified as follows: ajmaline and norajmaline (dihydroindoles), ajmalicinine and ajmalicine (heteroyohimbines), geissoschizol (E-seco indole) and pleiocarpamine (Nasser and Court, 1984). Rauvolfine, reserpine and rescinnamine are active constituents of the bark (McGaw et al., 2008) Conservation status: Least concern

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Schotia brachypetala Species: Schotia brachypetala Sond. Family: Fabaceae Trade name: umgxamu Vernacular names: weeping boer-bean (Eng); huilboerboon (Afr); molope (NSo) umutwa (Tsw); umgxama, ishumnumyane (Xho); mulubi (Ven); ihluze/ umgxamu (Zul) Method of preparation: Decoction (emetic; enema) Uses according to traders: The stem bark of Schotia brachypetala is often mixed with Harpephyllum caffrum and Syzygium cordatum stem bark ground into powder, boiled and then used as an emetic to treat respiratory complaints and food poisoning. The bark is soaked in cold water to make ‘ukhamba’ and used as emetic for ‘isilungulela’ (heartburn) and as an enema to clean bleeding stomach. Uses according to the literature: Bark infusions are taken as emetics for pimples and decoctions are taken for heartburn and a ‘hangover headache’ occurring after excessive consumption of alcohol. The bark is also used to wash and reduce swellings on the body. In Venda, the bark is used to treat nervous and cardiac conditions [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Van Wyk et al. (2011)]. Outer bark appearance: Grey brown. Smooth in young stems, coarse with vertical or rarely horizontal fissures. No lenticels are visible. Inner bark appearance: Light red (2.5 YR 6/8). Smooth with brittle fibres. Habitat and distribution: Warm dry areas in bushveld, deciduous woodland and scrub forest. Eastern Cape, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland, Mozambique and Zimbabwe. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: the bark extracts has antimicrobial activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus (McGaw et al., 2002). Methanol extract of the bark showed antioxidant activity and promote protection against cellular lipid peroxidation (Steenkamp et al., 2005). Aqueous and organic extracts of the bark exhibited activity against seven bacterial strains associated with diarrhoea (Van Vuuren et al., 2015). Chemistry: Chemical composition of the bark in particular is poorly documented. The heartwood contains polyhydroxystilbenes, with the main constituent being trans-3, 3’, 4, 5, 5’- pentahydroxystilbene. Compounds that are also present in small amounts are; trans-3, 3’, 4, 5’- tetrahydroxystilbene, catechin, epicatechin, and traces of cis-3, 3’, 4, 5, 5’ pentahydroxystilbene (McGaw et al., 2002; Steenkamp et al., 2005; Sobeh et al., 2016). Conservation status: Least concern

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Sclerocarya birrea Species: Sclerocarya birrea (A. Rich.) Hochst. Family: Anacardiaceae Trade name: umganu Vernacular names: marula (Eng); maroela (Afr); morula (NSo); mufula (Ven); ukanyi (Tso); umganu (Zul) Method of preparation: Infusion (oral; enema) Uses according to traders: Bark infusion is administered orally with a cup or sometimes used as enema for stomach ache. The bark is used as an enema to gain dignity so that a person cannot easily get harmed. Bark is used as an emetic in mixtures for person who is getting married. Uses according to the literature: Bark decoctions are administered as enemas for treatment of malaria and diarrhoea, administered orally in small dosages for the treatment of gonorrhoea and abdominal complaints. It is also taken as tea twice a day to strengthen the heart. The inner bark is boiled and applied as poultice to ulcers, smallpox and skin complaints. The Venda use the bark for fevers and stomach ailments, headaches, toothache, backache and infertility. The plant species is well-known as a fertility tree and used in marriage ceremonies by the Zulu culture [Mabogo (1990); Pujol (1990); Hutchings et al. (1996); Van Wyk et al (1997, 2009); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Grey to black. Smooth in young stems. Trunk and old branches with irregular or sometimes roundish scales; mature old trunk with strong and prominent vertical cracks. No lenticels present. Inner bark appearance: Dark reddish brown (5YR 3/3). Smooth, with papery strips or layers that peel off easily. Habitat and distribution: Bushveld and various types of woodland, on sandy soil or occasionally sandy loam. KwaZulu-Natal, Limpopo, Mpumalanga, Gauteng, North West, Swaziland, Namibia, Botswana, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report: a review by Fennell et al. (2004) reported potent anthelmintic and anti-amoebic activity of the bark. The methanol and dichloromethane young stem extract exhibited dose-dependent acetylcholinesterase inhibitory activity and high antioxidant activity (Moyo et al., 2010). Noteworthy activity against Cryptococcus neoformans, Klebsiella pneumoniae, Moraxella catarrahalis, Mycobacterium smegmatis, and Staphylococcus aureus (York et al., 2012). Ethanol extract of the bark was active against Propionibacterium acnes and excellent antioxidant activity (Sharma and Lall, 2014). Methanol extract of the bark has anti-HIV properties (Prinsloo et al., 2018). Chemistry: Active constituents isolated from the bark and leaves include, proanthocyanidins, gallotannins and flavonoids such as quercetin 3-O-alpha-l-(5''-galloyl) arabinofuranoside, quercetin 3-O-β-d-(6′′-galloyl) glucopyranoside, quercetin 3-O-α-L-rhamnopyranoside, kaempferol 3-O-β-d- (6′′-galloyl)glucopyranoside, gallic acid and (-)-epigallocatechin 3-O-galloyl ester (Braca et al. 2003; Prinsloo et al., 2018). Conservation status: Least concern

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Searsia chirindensis Species: Searsia chirindensis Baker f. Family: Anacardiaceae Trade name: inyazangoma elibomvu Vernacular names: red current (Eng); bos-taaibos (Afr); motha-thaa (NSo); umhlakothi (Xho); muvhadela-phanga (Ven); inyazangoma elibomvu (Zul) Method of preparation: Not specified Uses according to traders: The bark is used to protect yourself against opponents who are bewitching you, and it is they who die instead. Bark is mixed with cold water to chase away quarrel and evil spirits; it is also used to get revenge. Uses according to the literature: The bark is used to strengthen the body, stimulate circulation and for rheumatism. Bark decoctions are taken for mental disturbances in Transkei [Pujol (1990); Hutchings et al. (1996); Grace et al. (2003); Madikizela et al. (2013); Van Wyk et al. (2011)]. Outer bark appearance: Grey to strong brown in younger stems and dark brown in mature trunk. Smooth in young stems. Mature bark is rough with irregular cracks and irregular to rectangular flakes. Lenticels scattered on young stems or injured parts. They are small to medium in size with horizontal or vertical apertures. Inner bark appearance: Red (10 R 4/6). Relatively smooth, sometimes looks fibrous. However, fibres are very brittle and difficult to pull off. Habitat and distribution: Occurs naturally in forests, along forest margins, scrub forest and rocky hillsides. Eastern Cape, KwaZulu-Natal, Mpumalanga, Limpopo, Western Cape, Swaziland, Zimbabwe and Mozambique. Biological activity: The methanol stem bark extract showed potent free radical scavenging activity and anti-hyperglycaemia properties. The extract decreased the blood glucose levels without altering functions of the liver and kidney in rats, which was reflected by the absence of electrolyte imbalances and lipid peroxidation damage (Moosa and Mabandla, 2017). Chemistry: No data on the active constituents of the bark could be found. However, Madikizela et al. (2013) isolated and identified four ﬂavonol glycosides and methyl gallate from the leaf extract; these were screened for antibacterial activity. Conservation status: Least concern

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Securidaca longepedunculata Species: Securidaca longepedunculata Fresen. Family: Polygalaceae Trade name: mpesu Vernacular names: violet tree (Eng); krinkhout (Afr); mamba (NSo); mpesu (Ven); iphuphuma (Zul) Method of preparation: Infusion Uses according to traders: The stem bark is used as a drink (half a cup) to prevent colds and flu. The root bark is soaked in cold water and is sprinkled in the yard and also used as a wash to chase away bad spirits, and used as ‘umthwebulo’(“to capture someone’s soul”). Uses according to the literature: The stem bark is used in arrow poisons and the plant is known as an ordeal poison. Dried bark is used for treatment of epilepsy. The root is applied to wounds and sores, used to treat tuberculosis, gonorrhoea, for relief of rheumatism, backache, and impotency, as an anthelmintic, purgative and as a contraceptive. The root bark is used to treat coughs and chest complaint [Van Wyk et al. (1997, 2009); Samie et al. (2010); Luo et al. (2013); Van Wyk et al. (2011)]. Outer bark appearance: Brown (7.5YR 4/3). Relatively smooth. Lenticels are present. (Chopped sample) Inner bark appearance: Yellow (10YR 8/6) and Smooth. Habitat and distribution: Bushveld, woodland and arid Savanna soils. Limpopo, North West, Gauteng, Namibia, Botswana, Zimbabwe and Mozambique. Biological actictivity: Antimicrobial activity against skin pathogens (Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa). Literature report: good scavenging antioxidant activity from the root bark, antioxidant activity was evaluated using the DPPH radical scavenging test and ABTS radical scavenging test. Anti-inflammation activity was evaluated using the ability to inhibit nitrate production in activated macrophages (Muanda et al., 2010). Extracts reported to contain anti-HIV properties (Prinsloo et al., 2018) Chemistry: The major essential oil component form the root is methyl salicylate (methyl 2- hydroxybenzoate) (Jayasekara et al., 2005). The root bark of is mainly composed of polyphenolic and ﬂavonoid compounds, with trace amounts of total tannin and total anthocyanin (Muanda et al., 2010). Active constituents against HIV are caffeoylquinic acids: 3, 4, 5-tri-o-caffeoylquinic (Prinsloo et al., 2018). Conservation status: Least concern Notes

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Sideroxylon inerme Species: Sideroxylon inerme L. Family: Sapotaceae Trade name: amasethole Vernacular names: white milkwood (Eng); witmelkhout (Afr); umqwashu, (Xho); Mutaladzi- vhufa (Ven); umakhwelafinqane; amasethole (Zul) Method of preparation: Decoction Uses according to traders: The stem bark is mixed with other plants to make up a remedy called 'izimbiza zobaba’ for male sexual problems. Bark is used as an emetic and administered orally (half a cup) to treat chest problems associated with food poisoning, to chase away evil spirits, and for mental illnesses. Bark is used in mixtures ‘izimbiza’ for treatment of kidneys and for cleaning the blood. Uses according to the literature: The root bark is taken as an enema to induce excessive sweat. An infusion is used to chase away bad dreams. The stem bark is used as a tonic, to treat broken bones, for coughs and paralysis. The bark is used traditionally for skin diseases and widely used as a skin lightener [Hutchings et al. (1996); Grace et al. (2003); Van Wyk et al. (2011); Lall and Kishore (2014)]. Outer bark appearance: Grey to black. Smooth young stems. Mature trunk with prominent and vertically orientated rectangular scales. Inner bark appearance: Light red (2.5YR 6/8). Smooth, with thin vertical lines. Habitat and distribution: Coastal dune thicket and forests, also in littoral forests and in bushveld. Western Cape, Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Gauteng, Swaziland, Zimbabwe and Mozambique. Biological activity: The crude extracts and isolated compounds from the bark exhibited potent antioxidant DPPH scavenging activity and anti-tyrosine activity. Methanol and acetone extracts of the bark showed signiﬁcant inhibition of monophenolase enzyme. The methanol extract also exhibited reduction of melanin content in melanocytes without being potentially toxic to the cells (Momtaz et al., 2008). Anti-oxidant and anti-tyrosinase activity was observed from the bark (Lall and Kishore, 2014). Ethanol bark extract was active against Propionibacterium acnes (Sharma and Lall, 2014). Chemistry: Methanol extract of the bark yielded, epigallocatechin gallate and procyanidin B1 (Momtaz et al., 2008). Conservation status: Least concern

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Spirostachys africana Species: Spirostachys africana Sond Family: Euphorbiaceae Trade name: umthombothi Vernacular names: tamboti (Eng); tambotie (Afr); modiba (NSo); ndzopfori (Tso); morukuru (Tsw); muonze (Ven); umthombothi (Zul) Method of preparation: Powdered (oral) Uses according to traders: The wood is ground into fine powder to form 'insizi' and licked directly from the back part of the hand (khotha) to relieve pain and is also used as 'izithobo' medication – a cloth is immersed in the infusion and dabbed onto an inflamed or swollen area. The powdered wood is applied to wounds Uses according to the literature: The wood or bark is used for headaches, bleeding of the nose and gums, for colds, flu and fever. The Zulu culture use finely powdered stem bark mixed with other plants and this is put to boil for the treatment of stomach ulcers and acute gastritis and administered orally by taking three tablespoons per day. Dried bark is used as an ointment for rashes in infants. Infusions of the bark are taken as purgatives for constipation, renal ailments, blood purification and kidney disease. The stem is used as a remedy for toothache [Mabogo (1990); Hutchings et al. (1996); Grace et al. (2003); Van Wyk et al. (2011); Akhalwaya et al. (2018)]. Habitat and distribution: Bushveld, often on low altitude and on heavy soils along rivers and streams. Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Swaziland, Mozambique, Zimbabwe and Namibia. Biological activity: The bark extract as well as isolated compounds from the bark (3-acetyl aleuritolic acid) showed potent antimicrobial activity against Staphylococcus aureus; Salmonella typhy; Vibrio cholerae, Escherichia coli; Shigella dysenteriae, Shigella flexneri and Shigella boydii (Mathabe et al., 2006; Mathabe et al., 2008). The bark aqueous extract and 1:1 mixture of (DCM and MeOH) exhibited activity on oral pathogens, namely Gram-positive cariogenic bacterial strains; Streptococcus mutans, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus casei, Gram-negative periodontal pathogens two; Porphrymonas gingivalis and Fusobacterium nucleatum and lastly, three yeast strains; namely Candida albicans, Candida glabrata and Candida krusei, these are in connection with oral candidiasis. The extracts of the bark displayed the greatest reduction of adherent S. mutans cells (Akhalwaya et al., 2018) Chemistry: The ethanol extract of the stem bark yielded four known compounds, two triterpenoids, [d-Friedoolean-14-en-oic acid (3-acetyl aleuritolic acid)] and Lupeol, and two diterpenes, [ent-2,6alpha-dihydroxy-norbeyer-1,4,15-trien-3-one (diosphenol 2)] and (ent-3beta- hydroxy-beyer-15-ene-2-one) (Mathabe et al., 2008). Conservation status: Least concern

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Strychnos henningsii Species: Strychnos henningsii Gilg Family: Loganiaceae Trade name: umqalothi Vernacular names: red bitter berry (Eng); rooibitterbessie (Afr); umnonono (Xho); umqalothi (Zul) Method of preparation: Powdered and Raw (chewed) Uses according to traders: The bark is chewed for stomach complaints. The powdered bark is licked for ‘amahlaba’ (an antispasmodic effect), intestinal worms and stomach complaints. Uses according to the literature: The powdered bark is mixed with cold water and administered orally for nausea. The bark is used as an anthelmintic, a general health tonic, to treat dysmenorrhea, schistosomiasis, as an emetic to get rid of food poison, for complaints of the gastrointestinal tract, snakebites, erectile dysfunction and diabetes mellitus. The bark is chewed for stomach complaints and decoction is used to treat malaria [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Otang et al. (2012)]. Outer bark appearance: Greyish brown (lot of lichens, which can make the bark mottled). Bark smooth to rough with very irregular, scattered flakes. Small lenticels scattered or sometimes bigger lenticels in vertically elongated groups. Inner bark appearance: Yellowish brown (10YR 5/4). Relatively smooth, sometimes with numerous black marks of mould. Habitat and distribution: Dry and moist forests, wooded hillsides, thickets, on rocky hills, coastal forests and stream banks. Eastern Cape, KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland and Mozambique. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei, and Enterococcus faecalis). Literature report: aqueous extract of the bark showed both in -vivo and in-vitro antioxidant activities, evaluated using spectroscopic method against 1,1-diphenyl-2picrylhydrazyl (DPPH), superoxide anions, hydrogen peroxide (H2O2), nitric oxide (NO), 2,2’- azinobis [3-ethylbenzothiazoline-6- sulfonic acid] diammonium salt (ABTS) and the ferric reducing agent (Oyedemi et al., 2010). Aqueous stem bark extract contains anti-hyperglycaemia properties (Oyedemi et al., 2013). Chemistry: The bark contains crystalline alkaloids (Rindl et al., 1929).The chloroform and ether extracts of the bark yielded alkaloids identified as rindline, henningsoline, and the main alkaloid diaboline. In addition, present in small quantities was henningsamine (Grossert et al., 1965). Compounds isolated and identified from the root bark and stem bark include holstiine, diaboline, strychnochromine and guianensine (Oyedemi et al., 2013). Conservation status: Least concern

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Syzygium cordatum Species: Syzygium cordatum Hochst. ex Krauss Family: Myrtaceae Trade name: umdoni Vernacular names: water berry (Eng); waterbessie (Afr); umjomi (Xho), monthlo (NSo); muhlwa (Tso); mutu (Ven); umdoni (Zul) Method of preparation: Decoction Uses according to traders: The bark is used for internal stomach wounds. Bark of Syzygium cordatum, Schotia brachypetala and Harpephyllum caffrum are powdered and mixed together, boiled in water and taken as emetic for respiratory complaints. Powdered bark is licked (khotha) for body pains. Uses according to the literature: The bark is used for headaches, amenorrhoea, diarrhoea, stomach complaints and wounds. In Zimbabwe, the stem bark is crushed to make a decoction to treat tuberculosis. The powdered bark is used as fish poison [Mabogo (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Van Wyk and Gericke (2000); Grace et al. (2003); Maroyi et al. (2013)]. Outer bark appearance: Grey to dark brown. Smooth in young stems. Mature bark is rough with irregular cracks. No lenticels. Inner bark appearance: Red (10 R 5/8). Fibrous but smooth. Fibres are flexible when wet. Habitat and distribution: Forest margins, in bush, in riverine thicket and forest, and in open grasslands. Eastern Cape, KwaZulu-Natal, Mpumalanga, Limpopo, Swaziland, Botswana, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against respiratory tract pathogens ‒; (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa and Staphylococcus aureus). Literature report: the bark extracts exhibited potent antifungal activity against Candida albicans (Steenkamp et al., 2007). Noteworthy activity against Cryptococcus neoformans, Klebsiella pneumoniae, Moraxella catarrahalis, Mycobacterium smegmatis, and Staphylococcus aureus (York et al., 2012). Aqueous and methanol extract of the bark exhibited antioxidant activity for use as a potential supplement for oxidative stress-related disorders. The extracts decreased the cytotoxic parameters such as cytotoxixity, ROS generation, lipid peroxidation and apoptosis (Cordier et al., 2013). Chemistry: The hexane extract of the bark yielded the major components, which were isolated and identified as friedelin, epi-friedelinol and β-sitosterol. The same compounds were present in the ether extract along with arjunolic acid. The acetone bark extract yielded, Gallic and ellagic acids. Prolonged hot mineral-acid treatment yielded delphinidin as the only anthocyanidin, probably derived from leucodelphinidin or related polymers (Candy et al., 1968; McGaw et al., 2008). Conservation status: Least concern

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Terminalia sericea Species: Terminalia sericea Burch. ex DC. Family: Combretaceae Trade name: amangwe Vernacular names: silver cluster-leaf (Eng); vaalboom (Afr); moxonomo (NSo); mogonono (Tsw); mususu (Ven); amangwe (Zul) Method of preparation: Infusion (emetic; topical) Uses according to traders: Bark is used as an emetic and a wash to protect yourself from witchcraft and helps you fight back. Uses according to the literature: Powdered bark is taken with mealie meal for treating diabetes by the Sotho culture. The bark is used in Mozambique for anal wounds and stomachache. The powdered bark of T. sericea is mixed with Combretum kraussii powdered roots for treating wounds. The bark is used to treat diarrhoea, skin infections, blocked and runny nose [Pujol (1990); Mabogo (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Masoko et al. (2005); De wet et al. (2010, 2012); Cock and Van Vuuren (2015); Mongalo et al. (2016)]. Outer bark appearance: Greyish brown. Very rough with deep vertical furrows Inner bark appearance: Brownish yellow (10YR 6/8). Smooth, but fibrous if stripped. The strips are flexible, especially when wet. Habit and distribution: Open woodlands particularly on sandy soils and in mixed deciduous forests. Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, Northern Cape, Namibia, Botswana, Zimbabwe, Mozambique and Swaziland. Biological activity: The bark extracts has shown antibacterial activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and anti-inflammatory activity against cyclooxygenase (COX-1) enzyme (Eldeen et al., 2005). Noteworthy activity against Cryptococcus neoformans, Klebsiella pneumoniae, Moraxella catarrahalis, Mycobacterium smegmatis, and Staphylococcus aureus (York et al., 2012). Organic and aqueous extract of the bark contains antimycobacterium activity against Mycobacterium smegmatis (York et al., 2012; Fyhrquist et al., 2014). Bark extracts showed activity against Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis, Sporothrix schenkii, Neisseria gonorrhoeae and Klebsiella pneumoniae. (Mamba et al., 2016). Acetone stem bark extract contains an acetylcholinesterase eﬀ ect (Mongalo et al., 2016). Chemistry: The acetone stem bark extract yielded, β-sitosterol, β-sitosterol-3-acetate, lupeol and 3-onestigmasterol and two sets of mixtures of isomers (epicatechin-catechin; MI1 and epigallocatechingallocatechin; MI2) (Nkoboke, 2011). The ethanol root bark extract contains 3′5'- dihydroxy-4-(2hydroxy-ethoxy) resveratrol-3-O-βrutinoside and resveratrol-3-β-rutinoside (Joseph et al., 2007; Mongalo et al., 2016). Triterpenes, sericoside and arjunglucoside occur in roots and stem bark, stigmasterol and β-sitosterol were isolated in the root bark (Fyhrquist et al., 2014). Conservation status: Least concern

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Trichilia emetica Species: Trichilia emetica Vahl Family: Meliaceae Trade name: umkhuhlu Vernacular names: Natal mahogany (Eng); rooiessenhout (Afr); mamba (NSo); nkulu (Tso); mutuhu (Ven); umkhuhlu (Xho); umkhuhlu (Zul) Method of preparation: Maceration, infusion (heated by the sun, not boiled) (enema) Uses according to traders: The bark is used as an enema to clean the internal part of the body and to alleviate back pains. However, the bark is not boiled with fire because it releases poison; it is cooked (heated) in the sun. Raw powdered bark or sometimes mixed with petroleum jelly is applied on wounds between fingers and toes. Uses according to the literature: Bark decoctions are taken for stomach and intestinal complaints. Bark infusions are used for lumbago, rectal ulceration in children, dysentery and backache. Enemas made from the bark are administered for kidney ailments, as blood cleansers and intestinal worms. Infusions of the bark mixed with the same quantity of Spirostachys africana bark are used to treat constipation in Swaziland. The stem bark is used against malaria. The bark is used in wound healing, for syphilis and cirrhosis [Mabogo (1990); Hutchings et al. (1996); Van Wyk et al (1997, 2009); Grace et al. (2003); Shai et al. (2008); Van Wyk et al. (2011)]. Outer bark appearance: Outer colour not useful for identification of this bark, because it can be very mottled due to lichens and moss. Grey-brown, sometimes yellowish. Smooth in young stems. Mature trunk relatively smooth to course, with tiny vertical fissures. No lenticels on mature trunk. Inner bark appearance: Dark reddish brown (2.5 YR 3/3). Relatively smooth Habitat and distribution: Riverine forest and bushveld. KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland, Mozambique, Botswana and Zimbabwe Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei, and Enterococcus faecalis). Literature report; the dichloromethane stem bark extract showed potent antitrypanosomal and antiplasmodial activity against Plasmodium falciparum. Isolated liminoids from the bark showed to inhibit insect growth and DNA damage repair (Prozesky et al., 2001; Komane et al., 2011). Chemistry: The following compounds: sendanin, trichilinin, trichilin A, trichilin B, trichilin C, trichilin D, trichilin E, dregeana 4, nymania 1, rohituka 3, rohituka 5, rohituka 7, Trichilia substance Tr-A, Trichilia substance Tr-B, Trichilia substance Tr-C and seco-A-protolimonoid have been isolated from the bark (Komane et al., 2011). Conservation status: Least concern

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Vachellia natalitia Species: (Vachellia natalitia E.Mey.) (=Vachellia karroo (Hayne) Banfi & Galasso s.l.) Family: Fabaceae Trade name: umunga Vernacular names: pale-barked sweet thorn (Eng); bleekbassoetdoring (Afr); umnga (Xho); umunga (Zul) Method of preparation: Decoction (oral; emetic) Uses according to traders: Pounded bark is boiled and drunk for wounds. The bark is used as an emetic for pimples. Uses according to the literature: Bark is used as an emetic. Ground bark infusions are used for stomach-ache, diarrhoea, dysentery, haemorrhage and also as antidote for cattle poisoning. Bark is mixed with leaves in a tea for coughs, colds and conjunctivitis. Bark decoctions are used to eliminate symptoms of witchcraft. Bark and roots are boiled together and used as mouthwash [Mabogo (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Grace et al. (2003); Corrigan et al. (2011); Masevhe et al. (2015)]. Outer bark appearance: Mottled: light grey in the superficial layer and brown to light brown towards the inner parts. Rough, irregularly fissured, no lenticels. Inner bark appearance: Yellow (10YR 8/6). Fibrous (the fibers are flexible). Habitat and distribution: Bushveld, grassland, and coastal dune forest; All nine provinces of South Africa, Namibia, Botswana and Zimbabwe. Biological activity: Antimicrobial activity against stomach pathogens ‒ (Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report; bark extracts exhibited activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Neisseria gonorrhoeae, Candida albicans and HIV-1 RT inhibitory activity (Mulaudzi et al., 2011). Aqueous extract of the bark has shown anti- inflammatory and analgesic activity, while methanol bark extract has shown good HIV-1 reverse transcriptase inhibition (Adedapo et al., 2008; Maroyi, 2017). The bark aqueous extract and 1:1 mixture of (DCM and MeOH) exhibited activity against Gram-positive cariogenic bacterial strains, Gram-negative periodontal pathogens and the following three yeast strains; Candida albicans, Candida glabrata and Candida krusei, these are in connection with oral candidiasis (Akhalwaya et al., 2018). Chemistry: No data on chemical compounds of the bark could be found in the literature. The gum exudates from bark has pharmacological properties and contains L-arabinose units namely; 3-O-β- L-arabino-pyranosyl-L-arabinose and 3-O-α- D-galacto- pyranosyl-L-arabinose (Van Wyk et al., 2009). Chemical constituents of the wood, leaves and roots are widely studied. See a review by Maroyi et al., 2017. Conservation status: Least concern

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Vachellia robusta Species: Vachellia robusta (Burch.) Kyalangalilwa & Boatwright Family: Fabaceae Trade name: umngamanzi Vernacular names: broadpod robust thorn (Eng); enkeldoring (Afr); mooka (NSo); mvumbangwenya (Tso); mokhu (Tsw); muvumba-ngwena (Ven); umngamanzi (Zul) Method of preparation: Decoction and infusion (oral) Uses according to traders: Decoctions of the bark are used for treatment of menstrual pains and sexually transmitted infections. The bark infusion is used as a wash and emetic to get rid of evil spirits. Uses according to the literature: Steam from the crushed boiled bark is inhaled for chest complaints and also for the treatment of skin disorders. Ground bark is mixed with water to repel snakes [Hutchings et al (1996); Grace et al. (2003); Corrigan et al. (2011)]. Outer bark appearance: Grey to brown. Smooth to rough. Vertical to irregularly cracks in old bark. Lenticels only on smooth bark, in horizontal rows Inner bark appearance: Light red (2.5 YR 5/8). Fibrous: strong elastic fibres, separated by thin threads. Visible patterns of radial rays. Habitat and distribution: Bushveld and grassland. Eastern Cape, Free State, Gauteng, KwaZulu- Natal, Limpopo, Mpumalanga, North West, Northern Cape, Botswana, Zimbabwe and Mozambique. Biologigal activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa and Staphylococcus aureus). Chemistry: The bark contains cyanogenic glycosides and hydrocyanic acid (McGaw et al., 2008). Conservation status: Least concern

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Vachellia xanthophloea Species: Vachellia xanthophloea (Benth.) P.J.H.Hurter Family: Fabaceae Trade name: umkhanyakude Vernacular names: fever tree (Eng); koorsboom (Afr); mooka-kwena (NSo); nkelenga (Tso) munzhelenga (Ven) umhlosinga (Zul) Method of preparation: Infusion (topical) Uses according to traders: The bark is used as a wash for good luck (as a charm); in most cases it is used when a person goes for job hunting or an interview (to make a good impression on the potential employer). Uses according to the literature: Dried powdered bark is used as a preventative, or an emetic for the treatment of malaria. A small portion of ground bark is mixed with water and used for bathing so that people may not have bad judgements about you (e.g. thief). The bark is also used as a good luck charm [Hutchings et al. (1996); Grace et al. (2003); Corrigan et al. (2011)]. Outer bark appearance: Pale yellow (5Y, 8/4). Smooth and powdery, lenticels are small (<1 mm) and scattered. Inner bark appearance: Dark or light brown (or yellowish if wood present). Smooth (sometime samples can be with wood). Habitat and distribution: Bushveld mainly restricted to riverbank and low-lying swampy areas. Kwazulu-Natal, Mpumalanga, Limpopo, Swaziland, Zimbabwe and Mozambique.

Biological activity: The bark extracts has shown antimycobacterial activity against H37Rv Mycobacterium tuberculosis (Lall and Meyer, 1999; McGaw et al., 2008). Aqueous extract of the bark exhibit antimalarial activity against Plasmodium falciparum (Pillay et al., 2008). Chemistry: The bark contains hydroxyproline, serine, dimethyl-triptamine, β-amyrin and botulin (McGaw et al., 2008). Conservation status: Least concern

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Vitex obovata Species: Vitex obovata E.Mey. Family: Lamiaceae Trade name: umluthu Vernacular names: hairy finger leaf (Eng); harige vingerblaar (Afr); umluthu (Zul) Method of preparation: Decoction Uses according to traders: Men who use fighting sticks in order to beat up their opponents use the bark as ‘intelezi’. Uses according to the literature: The bark infusions are used as purifying emetics when a kraal member is dying. Vitex has been reported to be used in traditional medicine for treatment of depression, sexually transmitted diseases, malaria, asthma, allergy, wounds, skin diseases, snake bite and body pains [Hutchings et al. (1996); Grace et al (2003); Nyiligira et al. (2008)] Outer bark appearance: Dark grey to brown. Smooth in young stems. Mature bark is coarse with dominant vertical cracks, scaly. Scales are small (approximately 0.5x1 cm) look irregular Bark from mature trunk looks shaggy. Inner bark appearance: Brown (7.5 YR 4/3) to grey (7.5 YR 5/1). Relatively smooth Habitat and distribution: Bushveld, coastal scrub and riverine bush. Eastern Cape, KwaZulu- Natal, Limpopo, Mpumalanga and Swaziland. Biological activity: Essential oils from the aerial parts has shown weak activity against Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Salmonella typhimurium and the yeast, Cryptococcus neoformans. The essential oils exhibited anti-inflammatory activity, which was evaluated using using a 5-lipoxygenase assay. The methanol and acetone extracts of the aerial parts exhibited antioxidant activity and potent antimalarial activity against Plasmodium falciparum chloroquine-resistant FCR-3 (Nyiligira et al., 2004; Nyiligira et al., 2008). Chemistry: The essential oils showed high yields of 1,8-cineole (13.5% and 8.7%), α-copaene (8.5% and 14.2%), caryophyllene oxide (6.9% and 5.6%) and γ-muurolene (5.7% and 6.1%). Occurring in trace amounts were p-cymene, α-pinene, (Z)-3-hexen-1-ol, linalool, and trans-p- menth-2-en-1-ol (Nyirigila et al., 2004). Conservation status: Least concern

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Voacanga thouarsii Species: Voacanga thouarsii Roem. & Schult. Family: Apocynaceae Trade name: indlabalozi/ indluyabaloyi Vernacular names: wild frangipani (Eng); umthomfi (Xho); indlabaloyi (Zul) Method of preparation: Powdered Uses according to traders: The bark is licked (khotha) to relieve pains in the body. Powdered bark is used to trap ‘ukucupha’ and to protect a person from witchcraft. Uses according to the literature: The medicinal uses of the bark seem not to be available in the southern African literature sources. However, the genus Voacanga has been used in the treatment of leprosy, diarrhea, and generalized edema, convulsions in children as well as to treat cases of orchitis, ectopic testes and gonorrhoea (Hussain et al., 2012). Outer bark appearance: Pale grey to yellow. Relatively smooth with dominant shallow vertical cracks. Lenticels are not visible. Inner bark appearance: Brownish yellow (10 YR 6/6). Relatively smooth. Habitat and distribution: Moist areas, riverine, savanna and in swamp forests. Eastern Cape, KwaZulu Natal and Mozambique. Biological activity: Voacamine, an isolated compound from the bark has shown to possess potent cardiotonic activity with less toxicity than the existing cardenolide-based drugs. It also exhibited potent antibacterial activity against Gram-positive bacteria, antimalarial activity against in vitro chloroquine-sensitive and resistant strains of Plasmodium falciparum and exhibited in vivo activity (Hussain et al., 2012). Chemistry: A mixture of different solvent extracts from the bark yielded voacangine, ibogaine, voacamine, vobtusine, voacristine, iboluteine, vobasine, 18’-decarbomethoxyvoacamine and voaluteine (Goldblatt et al., 1970; Koroch et al., 2009; Hussain et al., 2012) Conservation status: Least concern

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Warburgia salutaris Species: Warburgia salutaris (G. Bertol.) Chiov. Family: Canellaceae Trade name: isibhaha Vernacular names: pepper-bark tree (Eng); peperbasboom (Afr); xibaha (Tso) mulanga, manaka (Ven); isibhaha (Zul) Method of preparation: Infusion and powdered Uses according to traders: Infusion of the bark is taken orally (one cup) for diarrhoea and flu. The powdered bark is licked (khotha) to relieve or loosen the sputum in the chest and to get rid of wounds caused by colds and flu. Uses according to the literature: Powdered bark is taken with cold water or a pinch is smoked, sometimes mixed with Cannabis sativa leaf for colds and a dry cough. The bark is used in emetics for febrile complaints and for rheumatism. The bark is used for toothache in Tanzania. The Vhavenda use the bark for chest complaints, malaria, sexually transmitted infections, purgatives, backache, and stomach ulcers. In Kenya, the bark is used for constipation and stomach ache. The powdered bark is mixed with fats and applied on the skin to treat sores, inflammation, and skin eruptions. In West Africa, the bark is used for influenza, fevers, pains, and other gastro-intestinal disorders [Mabogo (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Rabe and Van Staden (2000); Grace et al. (2003); Van Wyk et al. (2011)]. Outer bark appearance: Grey to dark brown. Smooth in young branches. Mature bark is rough, brittle with deep irregular cracks. Flakes irregular in shape or vertically elongated. Inner bark appearance: Pale yellowish pink (7.5 YR -/2). Smooth, sometimes with visible papery layers or may look fibrous, but the fibery layers are very brittle. Habitat and distribution: Occurs in savanna woodland, coastal forest and Afromontane forest. KwaZulu-Natal, Limpopo, Mpumalanga, Swaziland, Mozambique and Zimbabwe. Biological activity: Antimicrobial activity against respiratory tract pathogens ‒ (Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Staphylococcus aureus). Literature report; bark extracts have shown antibacterial and antifungal activity against Bacillus subtilis, Staphylococcus aureus, methicillin-resistant S. aureus, gentamycin methicillin-resistant S. aureus, S. epidermidis, P. aeruginosa, Candida albicans, Brevibacillus agri, Propionibacterium acnes, Trichophyton mentagrophytes, Microsporum canis (Mabona et al., 2013; Ndhlala et al., 2013). Aqueous and 1:1 mixture of (DCM and MeOH) extracts exhibited activity against Gram- positive cariogenic bacterial strains, Gram-negative periodontal pathogens and three yeast strains; namely Candida albicans, C. glabrata and C. krusei, these are in connection with oral candidiasis (Akhalwaya et al., 2018). The 11α-hydroxycinnamosmolide has anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and M. bovis (Madikane et al., 2007). Chemistry: In the present study; the two major essential oil components isolated were drimenol and E-nerolidol, along with the known non-volatile sesquiterpenes 12α-acetal-polygodial, polygodial, ugandensidial and warburganal. Previous literature studies reported; the presence of drimane sesquiterpenes such as isopolygodial, warburganal, polygodial, mukaadial, salutarisolide (Mashimbye et al., 1999), muzigadial (Rabe and Van Staden, 2000) and 11α- hydroxycinnamosmolide (Madikane et al., 2007). Furans and furanones were identified (Mohanlall and Odhav, 2009). Conservation status: Endangered

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Zanthoxylon capense

Species: Zanthoxylon capense (Thunb.) Harv. Family: Rutaceae Trade name: umlungumabele Vernacular names: small knobwood (Eng); kleinperdepram (Afr); monokwane (Sot); khunugumorupa (Tso); umlungumabele (Xho); umnungamabele (Zul) Method of preparation: Powdered or infusion (topical, mouth rinse, gargle) Uses according to traders: Dried powdered stem bark is applied or rubbed directly on the mouth ‘’to kill tooth worm’’ (to treat toothache). Small amount of powdered bark is mixed with warm water in a glass and used as a mouthwash to relieve toothache. Root bark is used to treat illnesses that affects the veins, lungs, blood and kidneys; also used for backache and to treat snakebites. Uses according to the literature: The powdered bark is rubbed into incisions over two days to treat paralyzed limbs. The stem is inserted into a cavity for toothache and used as a gargle in Transkei. The bark is commonly used for snakebite, skin eruptions and as a tonic. The bark and/or root decoctions and infusions are taken for influenza and colds, bronchitis, pleurisy, blood poisoning, constipation, acne and sores and as mouthwashes and an anthelmintic and also used to treat tuberculosis in Mozambique. Dried raw powdered stem bark is applied directly on the tooth to relieve ache. Both the leaves and bark are mixed together to treat anthrax (skin, lung and bowel disease caused by Bacillus anthracis) [Pujol (1990); Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Van Wyk and Gericke (2000); Grace et al. (2003)]. Outer bark appearance: Yellow brown. Relatively smooth. Lenticels are abundant, bright yellow, and of medium size with horizontal apertures. Inner bark appearance: Brown (7.5 YR 5/4). Smooth and solid. Habitat and distribution: Bushveld, forest margins and grassland, often associated with bush clumps and rocky places. Western Cape, Eastern Cape, KwaZulu-Natal, Free State, Gauteng, North West, Limpopo, Mpumalanga, Swaziland, Mozambique and Zimbabwe. Biological activity: Hexane and dichloromethane crude extracts as well as isolated compounds has shown antimycobacterial activity against Mycobacterium smegmatis and two reference strains of Mycobacterium tuberculosis, H37Ra (avirulent) and H37Rv (virulent) (Luo et al., 2013). The stem aqueous extract and 1:1 mixture of (DCM and MeOH) exhibited activity against Gram-positive cariogenic bacterial strains, Gram-negative periodontal pathogens and three yeast strains; namely Candida albicans, C. glabrata and C. krusei, these are in connection with oral candidiasis (Akhalwaya et al., 2018). Chemistry: The petrol extract of both the root and stem bark yielded (+)-sesamin (Fish et al., 1973). The methanol extract of the roots yielded two new 2-arylbenzofuran neolignans and a new benzophenanthridine alkaloid, along with six known benzophenanthridine alkaloids; namely decarine, norchelerythrine, dihydrochelerythrine, 6-acetonyldihydrochelerythrine, tridecanonchelerythrine, and 6-acetonyldihydronitidine (Luo et al., 2012; 2013). The same extract yielded; oxychelerythrine, oxynitidine, arnottianamide, syringaresinol, tembamide, (+)- ailanthoidiol, (+)-tembamide acetate, (+)-ailanthoidiol diacetate, (+)-ailanthoidiol dibutanoate, (+)- ailanthoidiol di-2-methylbutanoate, (+)-ailanthoidiol di-2-ethylbutanoate, (+)-ailanthoidiol didodecanoate, and (+)-ailanthoidiol dibenzoate (Cabral et al., 2015). Conservation status: Least concern

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Ziziphus mucronata Species: Ziziphus mucronata Willd. Family: Rhamnaceae Trade name: umlahlankosi Vernacular names: buffalo-thorn (Eng); blinkblaar-wag-n-bietjie (Afr); mokgalo (NSo); mphasamhala (Tso); mutshetshete (Ven); umphafa (Xho); umlahlankosi (Zul) Method of preparation: Decoction (topical: wash) Uses according to traders: In the Zulu culture, a tree branch with leaves is used to carry the soul of a person from the place where he or she died to their place of birth where they will be buried. Bark is used as a wash to remove ‘isilwane’ (dark shadow). Uses according to the literature: The stem bark is used to treat boils, skin infections, dysentery, tubercular gland swelling and measles. Bark infusions are taken as emetics for respiratory ailments and for chronic coughs. Steam baths from the bark are used to purify skin texture. Bark decoctions are used for rheumatism and stomach ailments [Hutchings et al. (1996); Van Wyk et al. (1997, 2009); Van Wyk and Gericke (2000); Grace et al. (2003); Samie et al. (2010); Van Wyk et al. (2011)]. Outer bark appearance: Grey to brown or dark brown. Very rough, with very prominent vertical cracks, and some horizontal but less prominent cracks. Mature bark with irregular cracks. Inner bark appearance: Yellowish red (5YR 5/8). Smooth with vertical lines. Habitat and distribution: Grows in areas dominated by thorny vegetation. It is also found in woodlands, open scrubland, on rocky koppies, open grasslands, and forest margins. Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern Cape, North West, Swaziland, Namibia, Botswana, Zimbabwe and Mozambique. Biological activity: Antimicrobial activity against skin and stomach pathogens ‒ (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Shigella sonnei and Enterococcus faecalis). Literature report; acetone extract of the bark showed antimalarial activity against Plasmodium falciparum (Prozesky et al., 2001). The bark extract showed potent antioxidant activity and free radical scavenging capability (Olajuyigbe Afolayan, 2011). Bark extracts showed activity against Staphylococcus aureus, S. epidermidis, P. aeruginosa, Candida albicans, Brevibacillus agri, Propionibacterium acnes, Trichophyton mentagrophytes, Microsporum canis (Mabona et al., 2013). The root bark extract showed potent antioxidant activity (Masehla et al., 2016). Aqueous extract of the bark exhibited anti-tuberculosis activity and moderate inhibition of the HIV replication (Sigidi et al., 2016). The bark aqueous extract and 1:1 mixture of (DCM and MeOH) exhibited activity against Gram- positive cariogenic bacterial strains, Gram-negative periodontal pathogens and three yeast strains namely; Candida albicans, Candida glabrata and Candida krusei, these are in connection with oral candidiasis (Akhalwaya et al., 2018). Chemistry: The bark containts alkaloids (Dictionary of Natural Products, 1996 cited in McGaw et al., 2008). Ethanolic extract of the bark yielded, caffeic acid, catechin, epicatechin, gallic acid, protocatechuic acid, taxifolin and rutin (Zininga et al., 2017). Conservation status: Least concern

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Appendix 2 – 13C NMR spectra of the four compounds isolated Elaeodendron transvaalense bark

13C NMR spectrum of Compound 1

13C NMR spectrum of Compound 1 (continued)

13C NMR spectrum of Compound 2

13C NMR spectrum of Compound 3

13C NMR spectrum of Compound 4

Figure 1 - alpumisoflavanone

Figure 2 - abyssinone v4

Figure 3 - cristacarpin

Figure 4 - lysisteisoflavanone

Figure 5 - phaseollidin

Figure 6 - erybraedin A

Figure 7 - eryzerin C 13C NMR Abyssinone - 4 - 13C NMR Cristacarpin 13C NMR Erybraedin A methyl ether Ours Published Difference Ours Published Difference Ours Published Difference 17.9 17.7 0.2 17.99 17.93 0.06 18.1 17.9 0.2 22.6 22.7 -0.1 17.99 18.03 -0.04 18.1 17.9 0.2 25.9 25.6 0.3 22.53 22.45 0.08 25.9 25.7 0.2 56.1 56.2 -0.1 23.32 23.25 0.07 25.9 25.7 0.2 69.7 69.8 -0.1 25.89 25.86 0.03 28.6 28.4 0.2 77.4 77.3 0.1 25.89 25.96 -0.07 28.6 28.7 -0.1 84.4 84.6 -0.2 40.24 40.1 0.14 43.3 43 0.3 103.7 103.8 -0.1 67.03 66.94 0.09 61.1 60.9 0.2 104.1 104.5 -0.4 79.01 78.93 0.08 79.5 79.3 0.2 110.4 110.4 0 108.24 108.18 0.06 95.7 95.6 0.1 113 113.2 -0.2 109.88 109.79 0.09 96.8 96.6 0.2 113.9 114.3 -0.4 110.45 110.41 0.04 103 103 0 120.8 120.8 0 112.89 112.76 0.13 122.8 122.5 0.3 120.8 120.8 0 115.09 115.07 0.02 122.8 122.5 0.3 122.1 122.3 -0.2 118.98 118.91 0.07 126 125.9 0.1 131.9 131.5 0.4 121.59 121.56 0.03 126 125.9 0.1 132.5 132.4 0.1 121.87 121.85 0.02 133.1 133 0.1 155.8 155.9 -0.1 122.5 122.43 0.07 133.1 133 0.1 157.2 157.3 -0.1 129.5 129.46 0.04 133.9 133.7 0.2 158.7 158.8 -0.1 134.6 134.52 0.08 135.4 135.4 0 160 160.2 -0.2 135.12 135.1 0.02 135.5 135.4 0.1 154.13 154.03 0.1 156.8 156.4 0.4 155.81 155.16 0.65 163.5 163.3 0.2 155.98 155.82 0.16 164.5 164.2 0.3 158.66 158.53 0.13 165.1 165.3 -0.2 196.3 196.3 0

13C NMR Eryzerin C 13C NMR Lysisteisoflavanone Ours Published Difference Ours Published Difference 17.97 17.8 0.17 17.9 17.7 0.2 18 17.8 0.2 25.9 25.7 0.2 22.72 22.5 0.22 28.1 27.9 0.2 25.9 25.8 0.1 45.3 45.3 0 29.2 28.8 0.4 55.6 55.4 0.2 29.8 29 0.8 69.6 69.6 0 31.1 30.8 0.3 69.6 69.9 -0.3 32 31.7 0.3 95.6 95.5 0.1 70.1 69.9 0.2 101.1 100.7 0.4 103.3 103.1 0.2 101.9 101.8 0.1 108.1 107.8 0.3 113.6 113.3 0.3 113.9 113.9 0 122.8 122.6 0.2 114.9 114.8 0.1 123.1 122.9 0.2 119.7 119.6 0.1 128.1 128.3 -0.2 120.54 120.3 0.24 132.6 132.4 0.2 122.6 122.4 0.2 154.2 153.8 0.4 122.9 122.7 0.2 158.3 158.1 0.2 127.8 127.6 0.2 163.4 163.3 0.1 128.6 128.3 0.3 165.2 164.8 0.4 133.6 133.5 0.1 166.1 166.3 -0.2 133.8 133.7 0.1 197.2 197.2 0 150.9 150.7 0.2 151.8 151.6 0.2 154.6 154.5 0.1 155.4 155.1 0.3

13C NMR Phaseollidin 13C NMR Alpumisoflavanone Ours Published Difference Ours Published Difference 18 17.9 0.1 28.6 28.7 -0.1 23.32 23.1 0.22 28.6 28.7 -0.1 25.9 25.6 0.3 79.2 78.5 0.7 40.2 40.1 0.1 95.8 95.3 0.5 66.7 66.6 0.1 106.5 106 0.5 78.3 78.2 0.1 106.9 106.5 0.4 103.8 103.7 0.1 116.1 115.9 0.2 108.4 108.2 0.2 116.3 116.1 0.2 109.8 110 -0.2 116.3 116.1 0.2 110.5 110.9 -0.4 123.2 123.3 -0.1 113.2 112.6 0.6 124.8 123.9 0.9 118.8 118.7 0.1 129.6 128.6 1 121.5 121.6 -0.1 131.4 130.7 0.7 122.5 122.3 0.2 131.4 130.7 0.7 132.5 132.4 0.1 154.8 153 1.8 135.3 134.4 0.9 157.8 156.4 1.4 156 155.5 0.5 158.8 157.2 1.6 156.9 156.5 0.4 158.9 157.7 1.2 157.1 157.4 -0.3 160.9 160 0.9 158.6 158.5 0.1 182.4 181.4 1

No solvent specified in paper We used MeOD